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The respiratory tract is one of the main portals of entry into the human body for microbial pathogens. On average, a human takes about 20,000 breaths each day. This roughly corresponds to 10,000 liters, or 10 cubic meters, of air. Suspended within this volume of air are millions of microbes of terrestrial, animal, and human origin—including many potential pathogens. A few of these pathogens will cause relatively mild infections like sore throats and colds. Others, however, are less benign. According to the World Health Organization, respiratory tract infections such as tuberculosis, influenza, and pneumonia were responsible for more than 4 million deaths worldwide in 2012.1
At one time, it was thought that antimicrobial drugs and preventive vaccines might hold respiratory infections in check in the developed world, but recent developments suggest otherwise. The rise of multiple-antibiotic resistance in organisms like Mycobacterium tuberculosis has rendered many of our modern drugs ineffective. In addition, there has been a recent resurgence in diseases like whooping cough and measles, once-common childhood illnesses made rare by effective vaccines. Despite advances in medicine and public health programs, it is likely that respiratory pathogens will remain formidable adversaries for the foreseeable future.
• 22.1: Anatomy and Normal Microbiota of the Respiratory Tract
The upper respiratory tract is colonized by an extensive and diverse normal microbiota, many of which are potential pathogens. Few microbial inhabitants have been found in the lower respiratory tract, and these may be transients. Members of the normal microbiota may cause opportunistic infections, using a variety of strategies to overcome the innate nonspecific defenses (including the mucociliary escalator) and adaptive specific defenses of the respiratory system.
• 22.2: Bacterial Infections of the Respiratory Tract
The respiratory tract can be infected by a variety of bacteria, both gram positive and gram negative. Although the diseases that they cause may range from mild to severe, in most cases, the microbes remain localized within the respiratory system. Fortunately, most of these infections also respond well to antibiotic therapy.
• 22.3: Viral Infections of the Respiratory Tract
Viruses cause respiratory tract infections more frequently than bacteria, and most viral infections lead to mild symptoms. The common cold can be caused by more than 200 viruses, typically rhinoviruses, coronaviruses, and adenoviruses, transmitted by direct contact, aerosols, or environmental surfaces. Due to its ability to rapidly mutate through antigenic drift and antigenic shift, influenza remains an important threat to human health. Two new influenza vaccines are developed annually.
• 22.4: Respiratory Mycoses
Fungal pathogens are ubiquitous in the environment. Serological studies have demonstrated that most people have been exposed to fungal respiratory pathogens during their lives. Yet symptomatic infections by these microbes are rare in healthy individuals. This demonstrates the efficacy of the defenses of our respiratory system. In this section, we will examine some of the fungi that can cause respiratory infections.
• 22.E: Respiratory System Infections (Exercises)
Footnotes
1. 1 World Health Organization. “The Top Ten Causes of Death.” May 2014. http://www.who.int/mediacentre/factsheets/fs310/en/
Thumbnail: Coronaviruses are a group of viruses known for causing the common cold. They have a halo or crown-like (corona) appearance when viewed under an electron microscope. (Public Domain; CDC/Dr. Fred Murphy).
22: Respiratory System Infections
Learning Objectives
• Describe the major anatomical features of the upper and lower respiratory tract
• Describe the normal microbiota of the upper and lower respiratory tracts
• Explain how microorganisms overcome defenses of upper and lower respiratory-tract membranes to cause infection
• Explain how microbes and the respiratory system interact and modify each other in healthy individuals and during an infection
Clinical Focus: Part 1
John, a 65-year-old man with asthma and type 2 diabetes, works as a sales associate at a local home improvement store. Recently, he began to feel quite ill and made an appointment with his family physician. At the clinic, John reported experiencing headache, chest pain, coughing, and shortness of breath. Over the past day, he had also experienced some nausea and diarrhea. A nurse took his temperature and found that he was running a fever of 40 °C (104 °F).
John suggested that he must have a case of influenza (flu), and regretted that he had put off getting his flu vaccine this year. After listening to John’s breathing through a stethoscope, the physician ordered a chest radiography and collected blood, urine, and sputum samples.
Exercise \(1\)
Based on this information, what factors may have contributed to John’s illness?
The primary function of the respiratory tract is to exchange gases (oxygen and carbon dioxide) for metabolism. However, inhalation and exhalation (particularly when forceful) can also serve as a vehicle of transmission for pathogens between individuals.
Anatomy of the Upper Respiratory System
The respiratory system can be conceptually divided into upper and lower regions at the point of the epiglottis, the structure that seals off the lower respiratory system from the pharynx during swallowing (Figure \(1\)). The upper respiratory system is in direct contact with the external environment. The nares (or nostrils) are the external openings of the nose that lead back into the nasal cavity, a large air-filled space behind the nares. These anatomical sites constitute the primary opening and first section of the respiratory tract, respectively. The nasal cavity is lined with hairs that trap large particles, like dust and pollen, and prevent their access to deeper tissues. The nasal cavity is also lined with a mucous membrane and Bowman’s glands that produce mucus to help trap particles and microorganisms for removal. The nasal cavity is connected to several other air-filled spaces. The sinuses, a set of four, paired small cavities in the skull, communicate with the nasal cavity through a series of small openings. The nasopharynx is part of the upper throat extending from the posterior nasal cavity. The nasopharynx carries air inhaled through the nose. The middle ear is connected to the nasopharynx through the eustachian tube. The middle ear is separated from the outer ear by the tympanic membrane, or ear drum. And finally, the lacrimal glands drain to the nasal cavity through the nasolacrimal ducts (tear ducts). The open connections between these sites allow microorganisms to move from the nasal cavity to the sinuses, middle ears (and back), and down into the lower respiratory tract from the nasopharynx.
The oral cavity is a secondary opening for the respiratory tract. The oral and nasal cavities connect through the fauces to the pharynx, or throat. The pharynx can be divided into three regions: the nasopharynx, the oropharynx, and the laryngopharynx. Air inhaled through the mouth does not pass through the nasopharynx; it proceeds first through the oropharynx and then through the laryngopharynx. The palatine tonsils, which consist of lymphoid tissue, are located within the oropharynx. The laryngopharynx, the last portion of the pharynx, connects to the larynx, which contains the vocal fold (Figure \(1\)).
Exercise \(2\)
1. Identify the sequence of anatomical structures through which microbes would pass on their way from the nares to the larynx.
2. What two anatomical points do the eustachian tubes connect?
Anatomy of the Lower Respiratory System
The lower respiratory system begins below the epiglottis in the larynx or voice box (Figure \(2\)). The trachea, or windpipe, is a cartilaginous tube extending from the larynx that provides an unobstructed path for air to reach the lungs. The trachea bifurcates into the left and right bronchi as it reaches the lungs. These paths branch repeatedly to form smaller and more extensive networks of tubes, the bronchioles. The terminal bronchioles formed in this tree-like network end in cul-de-sacs called the alveoli. These structures are surrounded by capillary networks and are the site of gas exchange in the respiratory system. Human lungs contain on the order of 400,000,000 alveoli. The outer surface of the lungs is protected with a double-layered pleural membrane. This structure protects the lungs and provides lubrication to permit the lungs to move easily during respiration.
Defenses of the Respiratory System
The inner lining of the respiratory system consists of mucous membranes (Figure \(3\)) and is protected by multiple immune defenses. The goblet cells within the respiratory epithelium secrete a layer of sticky mucus. The viscosity and acidity of this secretion inhibits microbial attachment to the underlying cells. In addition, the respiratory tract contains ciliated epithelial cells. The beating cilia dislodge and propel the mucus, and any trapped microbes, upward to the epiglottis, where they will be swallowed. Elimination of microbes in this manner is referred to as the mucociliary escalator effect and is an important mechanism that prevents inhaled microorganisms from migrating further into the lower respiratory tract.
The upper respiratory system is under constant surveillance by mucosa-associated lymphoid tissue (MALT), including the adenoids and tonsils. Other mucosal defenses include secreted antibodies (IgA), lysozyme, surfactant, and antimicrobial peptides called defensins. Meanwhile, the lower respiratory tract is protected by alveolar macrophages. These phagocytes efficiently kill any microbes that manage to evade the other defenses. The combined action of these factors renders the lower respiratory tract nearly devoid of colonized microbes.
Exercise \(3\)
1. Identify the sequence of anatomical structures through which microbes would pass on their way from the larynx to the alveoli.
2. Name some defenses of the respiratory system that protect against microbial infection.
Normal Microbiota of the Respiratory System
The upper respiratory tract contains an abundant and diverse microbiota. The nasal passages and sinuses are primarily colonized by members of the Firmicutes, Actinobacteria, and Proteobacteria. The most common bacteria identified include Staphylococcus epidermidis, viridans group streptococci (VGS), Corynebacterium spp. (diphtheroids), Propionibacterium spp., and Haemophilus spp. The oropharynx includes many of the same isolates as the nose and sinuses, with the addition of variable numbers of bacteria like species of Prevotella, Fusobacterium, Moraxella, and Eikenella, as well as some Candida fungal isolates. In addition, many healthy humans asymptomatically carry potential pathogens in the upper respiratory tract. As much as 20% of the population carry Staphylococcus aureus in their nostrils.1 The pharynx, too, can be colonized with pathogenic strains of Streptococcus, Haemophilus, and Neisseria.
The lower respiratory tract, by contrast, is scantily populated with microbes. Of the organisms identified in the lower respiratory tract, species of Pseudomonas, Streptococcus, Prevotella, Fusobacterium, and Veillonella are the most common. It is not clear at this time if these small populations of bacteria constitute a normal microbiota or if they are transients.
Many members of the respiratory system’s normal microbiota are opportunistic pathogens. To proliferate and cause host damage, they first must overcome the immune defenses of respiratory tissues. Many mucosal pathogens produce virulence factors such as adhesins that mediate attachment to host epithelial cells, or polysaccharide capsules that allow microbes to evade phagocytosis. The endotoxins of gram-negative bacteria can stimulate a strong inflammatory response that damages respiratory cells. Other pathogens produce exotoxins, and still others have the ability to survive within the host cells. Once an infection of the respiratory tract is established, it tends to impair the mucociliary escalator, limiting the body’s ability to expel the invading microbes, thus making it easier for pathogens to multiply and spread.
Vaccines have been developed for many of the most serious bacterial and viral pathogens. Several of the most important respiratory pathogens and their vaccines, if available, are summarized in Table \(1\). Components of these vaccines will be explained later in the chapter.
Table \(1\): Some Important Respiratory Diseases and Vaccines
Disease Pathogen Available Vaccine(s)2
Chickenpox/shingles Varicella-zoster virus Varicella (chickenpox) vaccine, herpes zoster (shingles) vaccine
Common cold Rhinovirus None
Diphtheria Corynebacterium diphtheriae DtaP, Tdap, DT,Td, DTP
Epiglottitis, otitis media Haemophilus influenzae Hib
Influenza Influenza viruses Inactivated, FluMist
Measles Measles virus MMR
Pertussis Bordetella pertussis DTaP, Tdap
Pneumonia Streptococcus pneumoniae Pneumococcal conjugate vaccine (PCV13), pneumococcal polysaccharide vaccine (PPSV23)
Rubella (German measles) Rubella virus MMR
Severe acute respiratory syndrome (SARS) SARS-associated coronavirus (SARS-CoV) None
Tuberculosis Mycobacterium tuberculosis BCG
Exercise \(4\)
1. What are some pathogenic bacteria that are part of the normal microbiota of the respiratory tract?
2. What virulence factors are used by pathogens to overcome the immune protection of the respiratory tract?
Signs and Symptoms of Respiratory Infection
Microbial diseases of the respiratory system typically result in an acute inflammatory response. These infections can be grouped by the location affected and have names ending in “itis”, which literally means inflammation of. For instance, rhinitis is an inflammation of the nasal cavities, often characteristic of the common cold. Rhinitis may also be associated with hay fever allergies or other irritants. Inflammation of the sinuses is called sinusitis inflammation of the ear is called otitis. Otitis media is an inflammation of the middle ear. A variety of microbes can cause pharyngitis, commonly known as a sore throat. An inflammation of the larynx is called laryngitis. The resulting inflammation may interfere with vocal cord function, causing voice loss. When tonsils are inflamed, it is called tonsillitis. Chronic cases of tonsillitis may be treated surgically with tonsillectomy. More rarely, the epiglottis can be infected, a condition called epiglottitis. In the lower respiratory system, the inflammation of the bronchial tubes results in bronchitis. Most serious of all is pneumonia, in which the alveoli in the lungs are infected and become inflamed. Pus and edema accumulate and fill the alveoli with fluids (called consolidations). This reduces the lungs’ ability to exchange gases and often results in a productive cough expelling phlegm and mucus. Cases of pneumonia can range from mild to life-threatening, and remain an important cause of mortality in the very young and very old.
Exercise \(5\)
Describe the typical symptoms of rhinitis, sinusitis, pharyngitis, and laryngitis.
Smoking-Associated Pneumonia
Camila is a 22-year-old student who has been a chronic smoker for 5 years. Recently, she developed a persistent cough that has not responded to over-the-counter treatments. Her doctor ordered a chest radiograph to investigate. The radiological results were consistent with pneumonia. In addition, Streptococcus pneumoniae was isolated from Camila’s sputum.
Smokers are at a greater risk of developing pneumonia than the general population. Several components of tobacco smoke have been demonstrated to impair the lungs’ immune defenses. These effects include disrupting the function of the ciliated epithelial cells, inhibiting phagocytosis, and blocking the action of antimicrobial peptides. Together, these lead to a dysfunction of the mucociliary escalator effect. The organisms trapped in the mucus are therefore able to colonize the lungs and cause infections rather than being expelled or swallowed.
Key Concepts and Summary
• The respiratory tract is divided into upper and lower regions at the epiglottis.
• Air enters the upper respiratory tract through the nasal cavity and mouth, which both lead to the pharynx. The lower respiratory tract extends from the larynx into the trachea before branching into the bronchi, which divide further to form the bronchioles, which terminate in alveoli, where gas exchange occurs.
• The upper respiratory tract is colonized by an extensive and diverse normal microbiota, many of which are potential pathogens. Few microbial inhabitants have been found in the lower respiratory tract, and these may be transients.
• Members of the normal microbiota may cause opportunistic infections, using a variety of strategies to overcome the innate nonspecific defenses (including the mucociliary escalator) and adaptive specific defenses of the respiratory system.
• Effective vaccines are available for many common respiratory pathogens, both bacterial and viral.
• Most respiratory infections result in inflammation of the infected tissues; these conditions are given names ending in -itis, such as rhinitis, sinusitis, otitis, pharyngitis, and bronchitis.
Footnotes
1. 1 J. Kluytmans et al. “Nasal Carriage of Staphylococcus aureus: Epidemiology, Underlying Mechanisms, and Associated Risks.” Clinical Microbiology Reviews 10 no. 3 (1997):505–520.
2. 2 Full names of vaccines listed in table: Haemophilus influenzae type B (Hib); Diphtheria, tetanus, and acellular pertussis (DtaP); tetanus, diphtheria, and acellular pertussis (Tdap); diphtheria and tetanus (DT); tetanus and diphtheria (Td); diphtheria, pertussis, and tetanus (DTP); Bacillus Calmette-Guérin; Measles, mumps, rubella (MMR) | textbooks/bio/Microbiology/Microbiology_(OpenStax)/22%3A_Respiratory_System_Infections/22.01%3A_Anatomy_and_Normal_Microbiota_of_the_Respiratory_Tract.txt |
Learning Objectives
• Identify the most common bacteria that can cause infections of the upper and lower respiratory tract
• Compare the major characteristics of specific bacterial diseases of the respiratory tract
The respiratory tract can be infected by a variety of bacteria, both gram positive and gram negative. Although the diseases that they cause may range from mild to severe, in most cases, the microbes remain localized within the respiratory system. Fortunately, most of these infections also respond well to antibiotic therapy.
Streptococcal Infections
A common upper respiratory infection, streptococcal pharyngitis (strep throat) is caused by Streptococcus pyogenes. This gram-positive bacterium appears as chains of cocci, as seen in Figure \(1\). Rebecca Lancefieldserologically classified streptococci in the 1930s using carbohydrate antigens from the bacterial cell walls. S. pyogenes is the sole member of the Lancefield group A streptococci and is often referred to as GAS, or group A strep.
Similar to streptococcal infections of the skin, the mucosal membranes of the pharynx are damaged by the release of a variety of exoenzymes and exotoxins by this extracellular pathogen. Many strains of S. pyogenes can degrade connective tissues by using hyaluronidase, collagenase and streptokinase. Streptokinase activates plasmin, which leads to degradation of fibrin and, in turn, dissolution of blood clots, which assists in the spread of the pathogen. Released toxins include streptolysins that can destroy red and white blood cells. The classic signs of streptococcal pharyngitis are a fever higher than 38 °C (100.4 °F); intense pharyngeal pain; erythema associated with pharyngeal inflammation; and swollen, dark-red palatine tonsils, often dotted with patches of pus; and petechiae (microcapillary hemorrhages) on the soft or hard palate (roof of the mouth) (Figure \(2\)). The submandibular lymph nodes beneath the angle of the jaw are also often swollen during strep throat.
Some strains of group A streptococci produce erythrogenic toxin. This exotoxin is encoded by a temperate bacteriophage (bacterial virus) and is an example of phage conversion (see The Viral Life Cycle). The toxin attacks the plasma membranes of capillary endothelial cells and leads to scarlet fever (or scarlatina), a disseminated fine red rash on the skin, and strawberry tongue, a red rash on the tongue (Figure \(2\)). Severe cases may even lead to streptococcal toxic shock syndrome (STSS), which results from massive superantigen production that leads to septic shock and death.
S. pyogenes can be easily spread by direct contact or droplet transmission through coughing and sneezing. The disease can be diagnosed quickly using a rapid enzyme immunoassay for the group A antigen. However, due to a significant rate of false-negative results (up to 30%1), culture identification is still the gold standard to confirm pharyngitis due to S. pyogenes. S. pyogenes can be identified as a catalase-negative, beta hemolytic bacterium that is susceptible to 0.04 units of bacitracin. Antibiotic resistance is limited for this bacterium, so most β-lactams remain effective; oral amoxicillin and intramuscular penicillin G are those most commonly prescribed.
Sequelae of S. pyogenes Infections
One reason strep throat infections are aggressively treated with antibiotics is because they can lead to serious sequelae, later clinical consequences of a primary infection. It is estimated that 1%–3% of untreated S. pyogenes infections can be followed by nonsuppurative (without the production of pus) sequelae that develop 1–3 weeks after the acute infection has resolved. Two such sequelae are acute rheumatic fever and acute glomerulonephritis.
Acute rheumatic fever can follow pharyngitis caused by specific rheumatogenic strains of S. pyogenes (strains 1, 3, 5, 6, and 18). Although the exact mechanism responsible for this sequela remains unclear, molecular mimicry between the M protein of rheumatogenic strains of S. pyogenes and heart tissue is thought to initiate the autoimmune attack. The most serious and lethal clinical manifestation of rheumatic fever is damage to and inflammation of the heart (carditis). Acute glomerulonephritis also results from an immune response to streptococcal antigens following pharyngitis and cutaneous infections. Acute glomerulonephritis develops within 6–10 days after pharyngitis, but can take up to 21 days after a cutaneous infection. Similar to acute rheumatic fever, there are strong associations between specific nephritogenic strains of S. pyogenes and acute glomerulonephritis, and evidence suggests a role for antigen mimicry and autoimmunity. However, the primary mechanism of acute glomerulonephritis appears to be the formation of immune complexes between S. pyogenes antigens and antibodies, and their deposition between endothelial cells of the glomeruli of kidney. Inflammatory response against the immune complexes leads to damage and inflammation of the glomeruli (glomerulonephritis).
Exercise \(1\)
1. What are the symptoms of strep throat?
2. What is erythrogenic toxin and what effect does it have?
3. What are the causes of rheumatic fever and acute glomerulonephritis?
Acute Otitis Media
An infection of the middle ear is called acute otitis media (AOM), but often it is simply referred to as an earache. The condition is most common between ages 3 months and 3 years. In the United States, AOM is the second-leading cause of visits to pediatricians by children younger than age 5 years, and it is the leading indication for antibiotic prescription.2
AOM is characterized by the formation and accumulation of pus in the middle ear. Unable to drain, the pus builds up, resulting in moderate to severe bulging of the tympanic membrane and otalgia (ear pain). Inflammation resulting from the infection leads to swelling of the eustachian tubes, and may also lead to fever, nausea, vomiting, and diarrhea, particularly in infants. Infants and toddlers who cannot yet speak may exhibit nonverbal signs suggesting AOM, such as holding, tugging, or rubbing of the ear, as well as uncharacteristic crying or distress in response to the pain.
AOM can be caused by a variety of bacteria. Among neonates, S. pneumoniae is the most common cause of AOM, but Escherichia coli, Enterococcus spp., and group B Streptococcus species can also be involved. In older infants and children younger than 14 years old, the most common bacterial causes are S. pneumoniae, Haemophilus influenzae, or Moraxella catarrhalis. Among S. pneumoniae infections, encapsulated strains are frequent causes of AOM. By contrast, the strains of H. influenzae and M. cattarhalis that are responsible for AOM do not possess a capsule. Rather than direct tissue damage by these pathogens, bacterial components such as lipopolysaccharide (LPS) in gram-negative pathogens induce an inflammatory response that causes swelling, pus, and tissue damage within the middle ear (Figure \(3\)).
Any blockage of the eustachian tubes, with or without infection, can cause fluid to become trapped and accumulate in the middle ear. This is referred to as otitis media with effusion (OME). The accumulated fluid offers an excellent reservoir for microbial growth and, consequently, secondary bacterial infections often ensue. This can lead to recurring and chronic earaches, which are especially common in young children. The higher incidence in children can be attributed to many factors. Children have more upper respiratory infections, in general, and their eustachian tubes are also shorter and drain at a shallower angle. Young children also tend to spend more time lying down than adults, which facilitates drainage from the nasopharynx through the eustachian tube and into the middle ear. Bottle feeding while lying down enhances this risk because the sucking action on the bottle causes negative pressure to build up within the eustachian tube, promoting the movement of fluid and bacteria from the nasopharynx.
Diagnosis is typically made based on clinical signs and symptoms, without laboratory testing to determine the specific causative agent. Antibiotics are frequently prescribed for the treatment of AOM. High-dose amoxicillin is the first-line drug, but with increasing resistance concerns, macrolides and cephalosporins may also be used. The pneumococcal conjugate vaccine (PCV13) contains serotypes that are important causes of AOM, and vaccination has been shown to decrease the incidence of AOM. Vaccination against influenza has also been shown to decrease the risk for AOM, likely because viral infections like influenza predispose patients to secondary infections with S. pneumoniae. Although there is a conjugate vaccine available for the invasive serotype B of H. influenzae, this vaccine does not impact the incidence of H. influenzae AOM. Because unencapsulated strains of H. influenzae and M. catarrhalis are involved in AOM, vaccines against bacterial cellular factors other than capsules will need to be developed.
Bacterial Rhinosinusitis
The microbial community of the nasopharynx is extremely diverse and harbors many opportunistic pathogens, so it is perhaps not surprising that infections leading to rhinitis and sinusitis have many possible causes. These conditions often occur as secondary infections after a viral infection, which effectively compromises the immune defenses and allows the opportunistic bacteria to establish themselves. Bacterial sinusitis involves infection and inflammation within the paranasal sinuses. Because bacterial sinusitis rarely occurs without rhinitis, the preferred term is rhinosinusitis. The most common causes of bacterial rhinosinusitis are similar to those for AOM, including S. pneumoniae, H. influenzae, and M. catarrhalis.
Exercise \(2\)
1. What are the usual causative agents of acute otitis media?
2. What factors facilitate acute otitis media with effusion in young children?
3. What factor often triggers bacterial rhinosinusitis?
Diphtheria
The causative agent of diphtheria, Corynebacterium diphtheriae, is a club-shaped, gram-positive rod that belongs to the phylum Actinobacteria. Diphtheroids are common members of the normal nasopharyngeal microbiota. However, some strains of C. diphtheriae become pathogenic because of the presence of a temperate bacteriophage-encoded protein—the diphtheria toxin. Diphtheria is typically a respiratory infection of the oropharynx but can also cause impetigo-like lesions on the skin. Although the disease can affect people of all ages, it tends to be most severe in those younger than 5 years or older than 40 years. Like strep throat, diphtheria is commonly transmitted in the droplets and aerosols produced by coughing. After colonizing the throat, the bacterium remains in the oral cavity and begins producing the diphtheria toxin. This protein is an A-B toxin that blocks host-cell protein synthesis by inactivating elongation factor (EF)-2 (see Virulence Factors of Bacterial and Viral Pathogens). The toxin’s action leads to the death of the host cells and an inflammatory response. An accumulation of grayish exudate consisting of dead host cells, pus, red blood cells, fibrin, and infectious bacteria results in the formation of a pseudomembrane. The pseudomembrane can cover mucous membranes of the nasal cavity, tonsils, pharynx, and larynx (Figure \(4\)). This is a classic sign of diphtheria. As the disease progresses, the pseudomembrane can enlarge to obstruct the fauces of the pharynx or trachea and can lead to suffocation and death. Sometimes, intubation, the placement of a breathing tube in the trachea, is required in advanced infections. If the diphtheria toxin spreads throughout the body, it can damage other tissues as well. This can include myocarditis (heart damage) and nerve damage that may impair breathing.
The presumptive diagnosis of diphtheria is primarily based on the clinical symptoms (i.e., the pseudomembrane) and vaccination history, and is typically confirmed by identifying bacterial cultures obtained from throat swabs. The diphtheria toxin itself can be directly detected in vitro using polymerase chain reaction (PCR)-based, direct detection systems for the diphtheria tox gene, and immunological techniques like radial immunodiffusion or Elek’s immunodiffusion test.
Broad-spectrum antibiotics like penicillin and erythromycin tend to effectively control C. diphtheriae infections. Regrettably, they have no effect against preformed toxins. If toxin production has already occurred in the patient, antitoxins (preformed antibodies against the toxin) are administered. Although this is effective in neutralizing the toxin, the antitoxins may lead to serum sickness because they are produced in horses (see Hypersensitivities).
Widespread vaccination efforts have reduced the occurrence of diphtheria worldwide. There are currently four combination toxoid vaccines available that provide protection against diphtheria and other diseases: DTaP, Tdap, DT, and Td. In all cases, the letters “d,” “t,” and “p” stand for diphtheria, tetanus, and pertussis, respectively; the “a” stands for acellular. If capitalized, the letters indicate a full-strength dose; lowercase letters indicate reduced dosages. According to current recommendations, children should receive five doses of the DTaP vaccine in their youth and a Td booster every 10 years. Children with adverse reactions to the pertussis vaccine may be given the DT vaccine in place of the DTaP.
Exercise \(3\)
1. What effect does diphtheria toxin have?
2. What is the pseudomembrane composed of?
Bacterial Pneumonia
Pneumonia is a general term for infections of the lungs that lead to inflammation and accumulation of fluids and white blood cells in the alveoli. Pneumonia can be caused by bacteria, viruses, fungi, and other organisms, although the vast majority of pneumonias are bacterial in origin. Bacterial pneumonia is a prevalent, potentially serious infection; it caused more 50,000 deaths in the United States in 2014.3 As the alveoli fill with fluids and white blood cells (consolidation), air exchange becomes impaired and patients experience respiratory distress (Figure \(5\)). In addition, pneumonia can lead to pleurisy, an infection of the pleural membrane surrounding the lungs, which can make breathing very painful. Although many different bacteria can cause pneumonia under the right circumstances, three bacterial species cause most clinical cases: Streptococcus pneumoniae, H. influenzae, and Mycoplasma pneumoniae. In addition to these, we will also examine some of the less common causes of pneumonia.
Pneumococcal Pneumonia
The most common cause of community-acquired bacterial pneumonia is Streptococcus pneumoniae. This gram-positive, alpha hemolytic streptococcus is commonly found as part of the normal microbiota of the human respiratory tract. The cells tend to be somewhat lancet-shaped and typically appear as pairs (Figure \(6\)). The pneumococci initially colonize the bronchioles of the lungs. Eventually, the infection spreads to the alveoli, where the microbe’s polysaccharide capsule interferes with phagocytic clearance. Other virulence factors include autolysins like Lyt A, which degrade the microbial cell wall, resulting in cell lysis and the release of cytoplasmic virulence factors. One of these factors, pneumolysin O, is important in disease progression; this pore-forming protein damages host cells, promotes bacterial adherence, and enhances pro-inflammatory cytokine production. The resulting inflammatory response causes the alveoli to fill with exudate rich in neutrophils and red blood cells. As a consequence, infected individuals develop a productive cough with bloody sputum.
Pneumococci can be presumptively identified by their distinctive gram-positive, lancet-shaped cell morphology and diplococcal arrangement. In blood agar cultures, the organism demonstrates alpha hemolytic colonies that are autolytic after 24 to 48 hours. In addition, S. pneumoniae is extremely sensitive to optochin and colonies are rapidly destroyed by the addition of 10% solution of sodium deoxycholate. All clinical pneumococcal isolates are serotyped using the quellung reaction with typing antisera produced by the CDC. Positive quellung reactions are considered definitive identification of pneumococci.
Antibiotics remain the mainstay treatment for pneumococci. β-Lactams like penicillin are the first-line drugs, but resistance to β-lactams is a growing problem. When β-lactam resistance is a concern, macrolides and fluoroquinolones may be prescribed. However, S. pneumoniae resistance to macrolides and fluoroquinolones is increasing as well, limiting the therapeutic options for some infections. There are currently two pneumococcal vaccines available: pneumococcal conjugate vaccine (PCV13) and pneumococcal polysaccharide vaccine (PPSV23). These are generally given to the most vulnerable populations of individuals: children younger than 2 years and adults older than 65 years.
Haemophilus Pneumonia
Encapsulated strains of Haemophilus influenzae are known for causing meningitis, but nonencapsulated strains are important causes of pneumonia. This small, gram-negative coccobacillus is found in the pharynx of the majority of healthy children; however, Haemophilus pneumonia is primarily seen in the elderly. Like other pathogens that cause pneumonia, H. influenzae is spread by droplets and aerosols produced by coughing. A fastidious organism, H. influenzae will only grow on media with available factor X (hemin) and factor V (NAD), like chocolate agar (Figure \(7\)). Serotyping must be performed to confirm identity of H. influenzae isolates.
Infections of the alveoli by H. influenzae result in inflammation and accumulation of fluids. Increasing resistance to β-lactams, macrolides, and tetracyclines presents challenges for the treatment of Haemophilus pneumonia. Resistance to the fluoroquinolones is rare among isolates of H. influenzae but has been observed. As discussed for AOM, a vaccine directed against nonencapsulated H. influenzae, if developed, would provide protection against pneumonia caused by this pathogen.
Why Me?
Tracy is a 6-year old who developed a serious cough that would not seem to go away. After 2 weeks, her parents became concerned and took her to the pediatrician, who suspected a case of bacterial pneumonia. Tests confirmed that the cause was Haemophilus influenzae. Fortunately, Tracy responded well to antibiotic treatment and eventually made a full recovery.
Because there had been several other cases of bacterial pneumonia at Tracy’s elementary school, local health officials urged parents to have their children screened. Of the children who were screened, it was discovered that greater than 50% carried H. influenzae in their nasal cavities, yet all but two of them were asymptomatic.
Why is it that some individuals become seriously ill from bacterial infections that seem to have little or no effect on others? The pathogenicity of an organism—its ability to cause host damage—is not solely a property of the microorganism. Rather, it is the product of a complex relationship between the microbe’s virulence factors and the immune defenses of the individual. Preexisting conditions and environmental factors such as exposure to secondhand smoke can make some individuals more susceptible to infection by producing conditions favorable to microbial growth or compromising the immune system. In addition, individuals may have genetically determined immune factors that protect them—or not—from particular strains of pathogens. The interactions between these host factors and the pathogenicity factors produced by the microorganism ultimately determine the outcome of the infection. A clearer understanding of these interactions may allow for better identification of at-risk individuals and prophylactic interventions in the future.
Mycoplasma Pneumonia (Walking Pneumonia)
Primary atypical pneumonia is caused by Mycoplasma pneumoniae. This bacterium is not part of the respiratory tract’s normal microbiota and can cause epidemic disease outbreaks. Also known as walking pneumonia, mycoplasmapneumonia infections are common in crowded environments like college campuses and military bases. It is spread by aerosols formed when coughing or sneezing. The disease is often mild, with a low fever and persistent cough. These bacteria, which do not have cell walls, use a specialized attachment organelle to bind to ciliated cells. In the process, epithelial cells are damaged and the proper function of the cilia is hindered (Figure \(8\)).
Mycoplasma grow very slowly when cultured. Therefore, penicillin and thallium acetate are added to agar to prevent the overgrowth by faster-growing potential contaminants. Since M. pneumoniae does not have a cell wall, it is resistant to these substances. Without a cell wall, the microbial cells appear pleomorphic. M. pneumoniae infections tend to be self-limiting but may also respond well to macrolide antibiotic therapy. β-lactams, which target cell wall synthesis, are not indicated for treatment of infections with this pathogen.
Chlamydial Pneumonias and Psittacosis
Chlamydial pneumonia can be caused by three different species of bacteria: Chlamydophila pneumoniae (formerly known as Chlamydia pneumoniae), Chlamydophila psittaci (formerly known as Chlamydia psittaci), and Chlamydia trachomatis. All three are obligate intracellular pathogens and cause mild to severe pneumonia and bronchitis. Of the three, Chlamydophila pneumoniae is the most common and is transmitted via respiratory droplets or aerosols. C. psittaci causes psittacosis, a zoonotic disease that primarily affects domesticated birds such as parakeets, turkeys, and ducks, but can be transmitted from birds to humans. Psittacosis is a relatively rare infection and is typically found in people who work with birds. Chlamydia trachomatis, the causative agent of the sexually transmitted disease chlamydia, can cause pneumonia in infants when the infection is passed from mother to baby during birth.
Diagnosis of chlamydia by culturing tends to be difficult and slow. Because they are intracellular pathogens, they require multiple passages through tissue culture. Recently, a variety of PCR- and serologically based tests have been developed to enable easier identification of these pathogens. Tetracycline and macrolide antibiotics are typically prescribed for treatment.
Health Care-Associated Pneumonia
A variety of opportunistic bacteria that do not typically cause respiratory disease in healthy individuals are common causes of health care-associated pneumonia. These include Klebsiella pneumoniae, Staphylococcus aureus, and proteobacteria such as species of Escherichia, Proteus, and Serratia. Patients at risk include the elderly, those who have other preexisting lung conditions, and those who are immunocompromised. In addition, patients receiving supportive therapies such as intubation, antibiotics, and immunomodulatory drugs may also be at risk because these interventions disrupt the mucociliary escalator and other pulmonary defenses. Invasive medical devices such as catheters, medical implants, and ventilators can also introduce opportunistic pneumonia-causing pathogens into the body.4
Pneumonia caused by K. pneumoniae is characterized by lung necrosis and “currant jelly sputum,” so named because it consists of clumps of blood, mucus, and debris from the thick polysaccharide capsule produced by the bacterium. K. pneumoniae is often multidrug resistant. Aminoglycoside and cephalosporin are often prescribed but are not always effective. Klebsiella pneumonia is frequently fatal even when treated.
Pseudomonas Pneumonia
Pseudomonas aeruginosa is another opportunistic pathogen that can cause serious cases of bacterial pneumonia in patients with cystic fibrosis (CF) and hospitalized patients assisted with artificial ventilators. This bacterium is extremely antibiotic resistant and can produce a variety of exotoxins. Ventilator-associated pneumonia with P. aeruginosa is caused by contaminated equipment that causes the pathogen to be aspirated into the lungs. In patients with CF, a genetic defect in the cystic fibrosis transmembrane receptor (CFTR) leads to the accumulation of excess dried mucus in the lungs. This decreases the effectiveness of the defensins and inhibits the mucociliary escalator. P. aeruginosa is known to infect more than half of all patients with CF. It adapts to the conditions in the patient’s lungs and begins to produce alginate, a viscous exopolysaccharide that inhibits the mucociliary escalator. Lung damage from the chronic inflammatory response that ensues is the leading cause of mortality in patients with CF.5
Exercise \(4\)
1. What three pathogens are responsible for the most prevalent types of bacterial pneumonia?
2. Which cause of pneumonia is most likely to affect young people?
3. In what contexts does Pseudomonas aeruginosa cause pneumonia?
Clinical Focus: Part 2
John’s chest radiograph revealed an extensive consolidation in the right lung, and his sputum cultures revealed the presence of a gram-negative rod. His physician prescribed a course of the antibiotic clarithromycin. He also ordered the rapid influenza diagnostic tests (RIDTs) for type A and B influenza to rule out a possible underlying viral infection. Despite antibiotic therapy, John’s condition continued to deteriorate, so he was admitted to the hospital.
Exercise \(5\)
What are some possible causes of pneumonia that would not have responded to the prescribed antibiotic?
Tuberculosis
Tuberculosis (TB) is one of the deadliest infectious diseases in human history. Although tuberculosis infection rates in the United States are extremely low, the CDC estimates that about one-third of the world’s population is infected with Mycobacterium tuberculosis, the causal organism of TB, with 9.6 million new TB cases and 1.5 million deaths worldwide in 2014.6
M. tuberculosis is an acid-fast, high G + C, gram-positive, nonspore-forming rod. Its cell wall is rich in waxy mycolic acids, which make the cells impervious to polar molecules. It also causes these organisms to grow slowly. M. tuberculosis causes a chronic granulomatous disease that can infect any area of the body, although it is typically associated with the lungs. M. tuberculosis is spread by inhalation of respiratory droplets or aerosols from an infected person. The infectious dose of M. tuberculosis is only 10 cells.7
After inhalation, the bacteria enter the alveoli (Figure \(9\)). The cells are phagocytized by macrophages but can survive and multiply within these phagocytes because of the protection by the waxy mycolic acid in their cell walls. If not eliminated by macrophages, the infection can progress, causing an inflammatory response and an accumulation of neutrophils and macrophages in the area. Several weeks or months may pass before an immunological response is mounted by T cells and B cells. Eventually, the lesions in the alveoli become walled off, forming small round lesions called tubercles. Bacteria continue to be released into the center of the tubercles and the chronic immune response results in tissue damage and induction of apoptosis (programmed host-cell death) in a process called liquefaction. This creates a caseous center, or air pocket, where the aerobic M. tuberculosis can grow and multiply. Tubercles may eventually rupture and bacterial cells can invade pulmonary capillaries; from there, bacteria can spread through the bloodstream to other organs, a condition known as miliary tuberculosis. The rupture of tubercles also facilitates transmission of the bacteria to other individuals via droplet aerosols that exit the body in coughs. Because these droplets can be very small and stay aloft for a long time, special precautions are necessary when caring for patients with TB, such as the use of face masks and negative-pressure ventilation and filtering systems.
Eventually, most lesions heal to form calcified Ghon complexes. These structures are visible on chest radiographs and are a useful diagnostic feature. But even after the disease has apparently ended, viable bacteria remain sequestered in these locations. Release of these organisms at a later time can produce reactivation tuberculosis (or secondary TB). This is mainly observed in people with alcoholism, the elderly, or in otherwise immunocompromised individuals (Figure \(9\)).
Because TB is a chronic disease, chemotherapeutic treatments often continue for months or years. Multidrug resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains of M. tuberculosis are a growing clinical concern. These strains can arise due to misuse or mismanagement of antibiotic therapies. Therefore, it is imperative that proper multidrug protocols are used to treat these infections. Common antibiotics included in these mixtures are isoniazid, rifampin, ethambutol, and pyrazinamide.
A TB vaccine is available that is based on the so-called bacillus Calmette-Guérin (BCG) strain of M. bovis commonly found in cattle. In the United States, the BCG vaccine is only given to health-care workers and members of the military who are at risk of exposure to active cases of TB. It is used more broadly worldwide. Many individuals born in other countries have been vaccinated with BCG strain. BCG is used in many countries with a high prevalence of TB, to prevent childhood tuberculous meningitis and miliary disease.
The Mantoux tuberculin skin test (Figure \(10\)) is regularly used in the United States to screen for potential TB exposure (see Hypersensitivities). However, prior vaccinations with the BCG vaccine can cause false-positive results. Chest radiographs to detect Ghon complex formation are required, therefore, to confirm exposure.
Exercise \(6\)
1. What characteristic of Mycobacterium tuberculosis allows it to evade the immune response?
2. What happens to cause miliary tuberculosis?
3. Explain the limitations of the Mantoux tuberculin skin test.
Pertussis (Whooping Cough)
The causative agent of pertussis, commonly called whooping cough, is Bordetella pertussis, a gram-negative coccobacillus. The disease is characterized by mucus accumulation in the lungs that leads to a long period of severe coughing. Sometimes, following a bout of coughing, a sound resembling a “whoop” is produced as air is inhaled through the inflamed and restricted airway—hence the name whooping cough. Although adults can be infected, the symptoms of this disease are most pronounced in infants and children. Pertussis is highly communicable through droplet transmission, so the uncontrollable coughing produced is an efficient means of transmitting the disease in a susceptible population.
Following inhalation, B. pertussis specifically attaches to epithelial cells using an adhesin, filamentous hemagglutinin. The bacteria then grow at the site of infection and cause disease symptoms through the production of exotoxins. One of the main virulence factors of this organism is an A-B exotoxin called the pertussis toxin (PT). When PT enters the host cells, it increases the cyclic adenosine monophosphate (cAMP) levels and disrupts cellular signaling. PT is known to enhance inflammatory responses involving histamine and serotonin. In addition to PT, B. pertussis produces a tracheal cytotoxin that damages ciliated epithelial cells and results in accumulation of mucus in the lungs. The mucus can support the colonization and growth of other microbes and, as a consequence, secondary infections are common. Together, the effects of these factors produce the cough that characterizes this infection.
A pertussis infection can be divided into three distinct stages. The initial infection, termed the catarrhal stage, is relatively mild and unremarkable. The signs and symptoms may include nasal congestion, a runny nose, sneezing, and a low-grade fever. This, however, is the stage in which B. pertussis is most infectious. In the paroxysmal stage, mucus accumulation leads to uncontrollable coughing spasms that can last for several minutes and frequently induce vomiting. The paroxysmal stage can last for several weeks. A long convalescence stage follows the paroxysmal stage, during which time patients experience a chronic cough that can last for up to several months. In fact, the disease is sometimes called the 100-day cough.
In infants, coughing can be forceful enough to cause fractures to the ribs, and prolonged infections can lead to death. The CDC reported 20 pertussis-related deaths in 2012,9 but that number had declined to five by 2015.10
During the first 2 weeks of infection, laboratory diagnosis is best performed by culturing the organism directly from a nasopharyngeal (NP) specimen collected from the posterior nasopharynx. The NP specimen is streaked onto Bordet-Gengou medium. The specimens must be transported to the laboratory as quickly as possible, even if transport media are used. Transport times of longer than 24 hours reduce the viability of B. pertussis significantly.
Within the first month of infection, B. pertussis can be diagnosed using PCR techniques. During the later stages of infection, pertussis-specific antibodies can be immunologically detected using an enzyme-linked immunosorbent assay (ELISA).
Pertussis is generally a self-limiting disease. Antibiotic therapy with erythromycin or tetracycline is only effective at the very earliest stages of disease. Antibiotics given later in the infection, and prophylactically to uninfected individuals, reduce the rate of transmission. Active vaccination is a better approach to control this disease. The DPT vaccine was once in common use in the United States. In that vaccine, the P component consisted of killed whole-cell B. pertussis preparations. Because of some adverse effects, that preparation has now been superseded by the DTaP and Tdap vaccines. In both of these new vaccines, the “aP” component is a pertussis toxoid.
Widespread vaccination has greatly reduced the number of reported cases and prevented large epidemics of pertussis. Recently, however, pertussis has begun to reemerge as a childhood disease in some states because of declining vaccination rates and an increasing population of susceptible children.
Exercise \(7\)
1. What accounts for the mucus production in a pertussis infection?
2. What are the signs and symptoms associated with the three stages of pertussis?
3. Why is pertussis becoming more common in the United States?
Legionnaires Disease
An atypical pneumonia called Legionnaires disease (also known as legionellosis) is caused by an aerobic gram-negative bacillus, Legionella pneumophila. This bacterium infects free-living amoebae that inhabit moist environments, and infections typically occur from human-made reservoirs such as air-conditioning cooling towers, humidifiers, misting systems, and fountains. Aerosols from these reservoirs can lead to infections of susceptible individuals, especially those suffering from chronic heart or lung disease or other conditions that weaken the immune system.
When L. pneumophila bacteria enter the alveoli, they are phagocytized by resident macrophages. However, L. pneumophila uses a secretion system to insert proteins in the endosomal membrane of the macrophage; these proteins prevent lysosomal fusion, allowing L. pneumophila to continue to proliferate within the phagosome. The resulting respiratory disease can range from mild to severe pneumonia, depending on the status of the host’s immune defenses. Although this disease primarily affects the lungs, it can also cause fever, nausea, vomiting, confusion, and other neurological effects.
Diagnosis of Legionnaires disease is somewhat complicated. L. pneumophila is a fastidious bacterium and is difficult to culture. In addition, since the bacterial cells are not efficiently stained with the Gram stain, other staining techniques, such as the Warthin-Starry silver-precipitate procedure, must be used to visualize this pathogen. A rapid diagnostic test has been developed that detects the presence of Legionella antigen in a patient’s urine; results take less than 1 hour, and the test has high selectivity and specificity (greater than 90%). Unfortunately, the test only works for one serotype of L. pneumophila (type 1, the serotype responsible for most infections). Consequently, isolation and identification of L. pneumophila from sputum remains the defining test for diagnosis.
Once diagnosed, Legionnaire disease can be effectively treated with fluoroquinolone and macrolide antibiotics. However, the disease is sometimes fatal; about 10% of patients die of complications.11 There is currently no vaccine available.
Exercise \(8\)
• Why is Legionnaires disease associated with air-conditioning systems?
• How does Legionella pneumophila circumvent the immune system?
Q Fever
The zoonotic disease Q fever is caused by a rickettsia, Coxiella burnetii. The primary reservoirs for this bacterium are domesticated livestock such as cattle, sheep, and goats. The bacterium may be transmitted by ticks or through exposure to the urine, feces, milk, or amniotic fluid of an infected animal. In humans, the primary route of infection is through inhalation of contaminated farmyard aerosols. It is, therefore, largely an occupational disease of farmers. Humans are acutely sensitive to C. burnetii—the infective dose is estimated to be just a few cells.12 In addition, the organism is hardy and can survive in a dry environment for an extended time. Symptoms associated with acute Q fever include high fever, headache, coughing, pneumonia, and general malaise. In a small number of patients (less than 5%13), the condition may become chronic, often leading to endocarditis, which may be fatal.
Diagnosing rickettsial infection by cultivation in the laboratory is both difficult and hazardous because of the easy aerosolization of the bacteria, so PCR and ELISA are commonly used. Doxycycline is the first-line drug to treat acute Q fever. In chronic Q fever, doxycycline is often paired with hydroxychloroquine.
Bacterial Diseases of the Respiratory Tract
Numerous pathogens can cause infections of the respiratory tract. Many of these infections produce similar signs and symptoms, but appropriate treatment depends on accurate diagnosis through laboratory testing. The tables in Figure \(11\) and Figure \(12\) summarize the most important bacterial respiratory infections, with the latter focusing specifically on forms of bacterial pneumonia.
Key Concepts and Summary
• A wide variety of bacteria can cause respiratory diseases; most are treatable with antibiotics or preventable with vaccines.
• Streptococcus pyogenes causes strep throat, an infection of the pharynx that also causes high fever and can lead to scarlet fever, acute rheumatic fever, and acute glomerulonephritis.
• Acute otitis media is an infection of the middle ear that may be caused by several bacteria, including Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. The infection can block the eustachian tubes, leading to otitis media with effusion.
• Diphtheria, caused by Corynebacterium diphtheriae, is now a rare disease because of widespread vaccination. The bacteria produce exotoxins that kill cells in the pharynx, leading to the formation of a pseudomembrane; and damage other parts of the body.
• Bacterial pneumonia results from infections that cause inflammation and fluid accumulation in the alveoli. It is most commonly caused by S. pneumoniae or H. influenzae. The former is commonly multidrug resistant.
• Mycoplasma pneumonia results from infection by Mycoplasma pneumoniae; it can spread quickly, but the disease is mild and self-limiting.
• Chlamydial pneumonia can be caused by three pathogens that are obligate intracellular parasites. Chlamydophila pneumoniae is typically transmitted from an infected person, whereas C. psittaci is typically transmitted from an infected bird. Chlamydia trachomatis, may cause pneumonia in infants.
• Several other bacteria can cause pneumonia in immunocompromised individuals and those with cystic fibrosis.
• Tuberculosis is caused by Mycobacterium tuberculosis. Infection leads to the production of protective tubercles in the alveoli and calcified Ghon complexes that can harbor the bacteria for a long time. Antibiotic-resistant forms are common and treatment is typically long term.
• Pertussis is caused by Bordetella pertussis. Mucus accumulation in the lungs leads to prolonged severe coughing episodes (whooping cough) that facilitate transmission. Despite an available vaccine, outbreaks are still common.
• Legionnaires disease is caused by infection from environmental reservoirs of the Legionella pneumophila bacterium. The bacterium is endocytic within macrophages and infection can lead to pneumonia, particularly among immunocompromised individuals.
• Q fever is caused by Coxiella burnetii, whose primary hosts are domesticated mammals (zoonotic disease). It causes pneumonia primarily in farm workers and can lead to serious complications, such as endocarditis.
Footnotes
1. 1 WL Lean et al. “Rapid Diagnostic Tests for Group A Streptococcal Pharyngitis: A Meta-Analysis.” Pediatrics 134, no. 4 (2014):771–781.
2. 2 G. Worrall. “Acute Otitis Media.” Canadian Family Physician 53 no. 12 (2007):2147–2148.
3. 3 KD Kochanek et al. “Deaths: Final Data for 2014.” National Vital Statistics Reports 65 no 4 (2016).
4. 4 SM Koenig et al. “Ventilator-Associated Pneumonia: Diagnosis, Treatment, and Prevention.” Clinical Microbiology Reviews 19 no. 4 (2006):637–657.
5. 5 R. Sordé et al. “Management of Refractory Pseudomonas aeruginosa Infection in Cystic Fibrosis.” Infection and Drug Resistance 4 (2011):31–41.
6. 6 Centers for Disease Control and Prevention. “Tuberculosis (TB). Data and Statistics.” http://www.cdc.gov/tb/statistics/default.htm
7. 7 D. Saini et al. “Ultra-Low Dose of Mycobacterium tuberculosis Aerosol Creates Partial Infection in Mice.” Tuberculosis 92 no. 2 (2012):160–165.
8. 8 G. Kaplan et al. “Mycobacterium tuberculosis Growth at the Cavity Surface: A Microenvironment with Failed Immunity.” Infection and Immunity 71 no.12 (2003):7099–7108.
9. 9 Centers for Disease Control and Prevention. “2012 Final Pertussis Surveillance Report.” 2015. http://www.cdc.gov/pertussis/downloa...eport-2012.pdf. Accessed July 6, 2016.
10. 10 Centers for Disease Control and Prevention. “2015 Provisional Pertussis Surveillance Report.” 2016. http://www.cdc.gov/pertussis/downloa...rovisional.pdf. Accessed July 6, 2016.
11. 11 Centers for Disease Control and Prevention. “Legionella (Legionnaires’ Disease and Pontiac Fever: Diagnosis, Treatment, and Complications).” http://www.cdc.gov/legionella/about/diagnosis.html. Accessed Sept 14, 2016.
12. 12 WD Tigertt et al. “Airborne Q Fever.” Bacteriological Reviews 25 no. 3 (1961):285–293.
13. 13 Centers for Disease Control and Prevention. “Q fever. Symptoms, Diagnosis, and Treatment.” 2013. http://www.cdc.gov/qfever/symptoms/index.html. Accessed July 6, 2016. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/22%3A_Respiratory_System_Infections/22.02%3A_Bacterial_Infections_of_the_Respiratory_Tract.txt |
Learning Objectives
• Identify the most common viruses that can cause infections of the upper and lower respiratory tract
• Compare the major characteristics of specific viral diseases of the respiratory tract
Viruses are the most frequent cause of respiratory tract infections. Unlike the bacterial pathogens, we have few effective therapies to combat viral respiratory infections. Fortunately, many of these diseases are mild and self-limiting. A few respiratory infections manifest their primary symptoms at other locations in the body.
The Common Cold
The common cold is a generic term for a variety of mild viral infections of the nasal cavity. More than 200 different viruses are known to cause the common cold. The most common groups of cold viruses include rhinoviruses, coronaviruses, and adenoviruses. These infections are widely disseminated in the human population and are transmitted through direct contact and droplet transmission. Coughing and sneezing efficiently produce infectious aerosols, and rhinoviruses are known to persist on environmental surfaces for up to a week.1
Viral contact with the nasal mucosa or eyes can lead to infection. Rhinoviruses tend to replicate best between 33 °C (91.4 °F) and 35 °C (95 °F), somewhat below normal body temperature (37 °C [98.6 °F]). As a consequence, they tend to infect the cooler tissues of the nasal cavities. Colds are marked by an irritation of the mucosa that leads to an inflammatory response. This produces common signs and symptoms such as nasal excess nasal secretions (runny nose), congestion, sore throat, coughing, and sneezing. The absence of high fever is typically used to differentiate common colds from other viral infections, like influenza. Some colds may progress to cause otitis media, pharyngitis, or laryngitis, and patients may also experience headaches and body aches. The disease, however, is self-limiting and typically resolves within 1–2 weeks.
There are no effective antiviral treatments for the common cold and antibacterial drugs should not be prescribed unless secondary bacterial infections have been established. Many of the viruses that cause colds are related, so immunity develops throughout life. Given the number of viruses that cause colds, however, individuals are never likely to develop immunity to all causes of the common cold.
Exercise \(1\)
1. How are colds transmitted?
2. What is responsible for the symptoms of a cold?
Clinical Focus: Part 3
Since antibiotic treatment had proven ineffective, John’s doctor suspects that a viral or fungal pathogen may be the culprit behind John’s case of pneumonia. Another possibility is that John could have an antibiotic-resistant bacterial infection that will require a different antibiotic or combination of antibiotics to clear.
The RIDT tests both came back negative for type A and type B influenza. However, the diagnostic laboratory identified the sputum isolate as Legionella pneumophila. The doctor ordered tests of John’s urine and, on the second day after his admission, results of an enzyme immunoassay (EIA) were positive for the Legionella antigen. John’s doctor added levofloxacin to his antibiotic therapy and continued to monitor him. The doctor also began to ask John where he had been over the past 10 to 14 days.
Exercise \(2\)
1. Do negative RIDT results absolutely rule out influenza virus as the etiologic agent? Why or why not?
2. What is John’s prognosis?
Influenza
Commonly known as the flu, influenza is a common viral disease of the lower respiratory system caused by an orthomyxovirus. Influenza is pervasive worldwide and causes 3,000–50,000 deaths each year in the United States. The annual mortality rate can vary greatly depending on the virulence of the strain(s) responsible for seasonal epidemics. 2
Influenza infections are most typically characterized by fever, chills, and body aches. This is followed by symptoms similar to the common cold that may last a week or more. Table \(1\) compares the signs and symptoms of influenza and the common cold.
Table \(1\): Comparing the Common Cold and Influenza
Sign/Symptom Common Cold Influenza
Fever Low (37.2 °C [99 °F]) High (39 °C [102.2 °F])
Headache Common Common
Aches and pains Mild Severe
Fatigue Slight Severe
Nasal congestion Common Rare
Sneezing Common Rare
In general, influenza is self-limiting. However, serious cases can lead to pneumonia and other complications that can be fatal. Such cases are more common in the very young and the elderly; however, certain strains of influenza virus (like the 1918–1919 variant discussed later in this chapter) are more lethal to young adults than to the very young or old. Strains that affect young adults are believed to involve a cytokine storm—a positive feedback loop that forms between cytokine production and leukocytes. This cytokine storm produces an acute inflammatory response that leads to rapid fluid accumulation in the lungs, culminating in pulmonary failure. In such cases, the ability to mount a vigorous immune response is actually detrimental to the patient. The very young and very old are less susceptible to this effect because their immune systems are less robust.
A complication of influenza that occurs primarily in children and teenagers is Reye syndrome. This sequela causes swelling in the liver and brain, and may progress to neurological damage, coma, or death. Reye syndrome may follow other viral infections, like chickenpox, and has been associated with the use of aspirin. For this reason, the CDC and other agencies recommend that aspirin and products containing aspirin never be used to treat viral illnesses in children younger than age 19 years.3
The influenza virus is primarily transmitted by direct contact and inhalation of aerosols. The RNA genome of this virus exists as seven or eight segments, each coated with ribonucleoprotein and encoding one or two specific viral proteins. The influenza virus is surrounded by a lipid membrane envelope, and two of the main antigens of the influenza virus are the spike proteins hemagglutinin (H) and neuraminidase (N), as shown in Figure \(1\). These spike proteins play important roles in the viral infectious cycle.
Following inhalation, the influenza virus uses the hemagglutinin protein to bind to sialic acid receptors on host respiratory epithelial cells. This facilitates endocytosis of the viral particle. Once inside the host cell, the negative strand viral RNA is replicated by the viral RNA polymerase to form mRNA, which is translated by the host to produce viral proteins. Additional viral RNA molecules are transcribed to produce viral genomic RNA, which assemble with viral proteins to form mature virions. Release of the virions from the host cell is facilitated by viral neuraminidase, which cleaves sialic-acid receptors to allow progeny viruses to make a clean exit when budding from an infected cell.
There are three genetically related influenza viruses, called A, B, and C. The influenza A viruses have different subtypes based on the structure of their hemagglutinin and neuraminidase proteins. There are currently 18 known subtypes of hemagglutinin and 11 known subtypes of neuraminidase. Influenza viruses are serologically characterized by the type of H and N proteins that they possess. Of the nearly 200 different combinations of H and N, only a few, such as the H1N1 strain, are associated with human disease. The influenza viruses A, B, and C make up three of the five major groups of orthomyxoviruses. The differences between the three types of influenza are summarized in Table \(2\). The most virulent group is the influenza A viruses, which cause seasonal pandemics of influenza each year. Influenza A virus can infect a variety of animals, including pigs, horses, pigs, and even whales and dolphins. Influenza B virus is less virulent and is sometimes associated with epidemic outbreaks. Influenza C virus generally produces the mildest disease symptoms and is rarely connected with epidemics. Neither influenza B virus nor influenza C virus has significant animal reservoirs.
Table \(2\): The Three Major Groups of Influenza Viruses
Influenza A virus Influenza B virus Influenza C virus
Severity Severe Moderate Mild
Animal reservoir Yes No No
Genome segments 8 8 7
Population spread Epidemic and pandemic Epidemic Sporadic
Antigenic variation Shift/drift Drift Drift
Influenza virus infections elicit a strong immune response, particularly to the hemagglutinin protein, which would protect the individual if they encountered the same virus. Unfortunately, the antigenic properties of the virus change relatively rapidly, so new strains are evolving that immune systems previously challenged by influenza virus cannot recognize. When an influenza virus gains a new hemagglutinin or neuraminidase type, it is able to evade the host’s immune response and be successfully transmitted, often leading to an epidemic.
There are two mechanisms by which these evolutionary changes may occur. The mechanisms of antigen drift and antigenic shift for influenza virus have been described in Virulence Factors of Bacterial and Viral Pathogens. Of these two genetic processes, it is viruses produced by antigenic shift that have the potential to be extremely virulent because individuals previously infected by other strains are unlikely to produce any protective immune response against these novel variants.
The most lethal influenza pandemic in recorded history occurred from 1918 through 1919. Near the end of World War I, an antigenic shift involving the recombination of avian and human viruses is thought to have produced a new H1N1 virus. This strain rapidly spread worldwide and is commonly claimed to have killed as many as 40 million to 50 million people—more than double the number killed in the war. Although referred to as the Spanish flu, this disease is thought to have originated in the United States. Regardless of its source, the conditions of World War I greatly contributed to the spread of this disease. Crowding, poor sanitation, and rapid mobilization of large numbers of personnel and animals facilitated the dissemination of the new virus once it appeared.
Several of the most important influenza pandemics of modern times have been associated with antigenic shifts. A few of these are summarized in Table \(3\).
Table \(3\): Historical Influenza Outbreaks4 5 6
Years Common Name Serotype Estimated Number of Deaths
1918–1919 Spanish flu H1N1 20,000,000–40,000,000
1957–1958 Asian flu N2N2 1,000,000–2,000,000
1968–1969 Hong Kong flu H3N2 1,000,000–3,000,000
2009–2010 Swine flu H1N1/09 152,000–575,000
Laboratory diagnosis of influenza is typically performed using a variety of RIDTs. These tests are inoculated by point-of-care personnel and give results within 15–20 minutes. Unfortunately, these tests have variable sensitivity and commonly yield false-negative results. Other tests include hemagglutination of erythrocytes (due to hemagglutinin action) or complement fixation. Patient serum antibodies against influenza viruses can also be detected in blood samples. Because influenza is self-limiting disease, diagnosis through these more time-consuming and expensive methods is not typically used.
Three drugs that inhibit influenza neuraminidase activity are available: inhaled zanamivir, oral oseltamivir, and intravenous peramivir. If taken at the onset of symptoms, these drugs can shorten the course of the disease. These drugs are thought to impair the ability of the virus to efficiently exit infected host cells. A more effective means of controlling influenza outbreaks, though, is vaccination. Every year, new influenza vaccines are developed to be effective against the strains expected to be predominant. This is determined in February by a review of the dominant strains around the world from a network of reporting sites; their reports are used to generate a recommendation for the vaccine combination for the following winter in the northern hemisphere. In September, a similar recommendation is made for the winter in the southern hemisphere.7 These recommendations are used by vaccine manufacturers to formulate each year’s vaccine. In most cases, three or four viruses are selected—the two most prevalent influenza A strains and one or two influenza B strains. The chosen strains are typically cultivated in eggs and used to produce either an inactivated or a live attenuated vaccine (e.g., FluMist). For individuals 18 years or older with an allergy to egg products, a recombinant egg-free trivalent vaccine is available. Most of the influenza vaccines over the past decade have had an effectiveness of about 50%.8
Flu Pandemic
During the spring of 2013, a new strain of H7N9 influenza was reported in China. A total of 132 people were infected. Of those infected, 44 (33%) died. A genetic analysis of the virus suggested that this strain arose from the reassortment of three different influenza viruses: a domestic duck H7N3 virus, a wild bird H7N9 virus, and a domestic poultry H9N2 virus. The virus was detected in the Chinese domestic bird flocks and contact with this reservoir is thought to have been the primary source of infection. This strain of influenza was not able to spread from person to person. Therefore, the disease did not become a global problem. This case does, though, illustrate the potential threat that influenza still represents. If a strain like the H7N9 virus were to undergo another antigenic shift, it could become more communicable in the human population. With a mortality rate of 33%, such a pandemic would be disastrous. For this reason, organizations like the World Health Organization and the Centers for Disease Control and Prevention keep all known influenza outbreaks under constant surveillance.
Exercise \(3\)
1. Compare the severity of the three types of influenza viruses.
2. Why must new influenza vaccines be developed each year?
Viral Pneumonia
Viruses cause fewer cases of pneumonia than bacteria; however, several viruses can lead to pneumonia in children and the elderly. The most common sources of viral pneumonia are adenoviruses, influenza viruses, parainfluenza viruses, and respiratory syncytial viruses. The signs and symptoms produced by these viruses can range from mild cold-like symptoms to severe cases of pneumonia, depending on the virulence of the virus strain and the strength of the host defenses of the infected individual. Occasionally, infections can result in otitis media.
Respiratory syncytial virus (RSV) infections are fairly common in infants; most people have been infected by the age of 2 years. During infection, a viral surface protein causes host cells to fuse and form multinucleated giant cells called syncytia. There are no specific antiviral therapies or vaccines available for viral pneumonia. In adults, these infections are self-limiting, resemble the common cold, and tend to resolve uneventfully within 1 or 2 weeks. Infections in infants, however, can be life-threatening. RSV is highly contagious and can be spread through respiratory droplets from coughing and sneezing. RSV can also survive for a long time on environmental surfaces and, thus, be transmitted indirectly via fomites.
Exercise \(4\)
1. Who is most likely to contract viral pneumonia?
2. What is the recommended treatment for viral pneumonia?
SARS and MERS
Severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) are two acute respiratory infections caused by coronaviruses. In both cases, these are thought to be zoonotic infections. Bats and civet cats are thought to have been the reservoirs for SARS; camels seem to be the reservoir for MERS.
SARS originated in southern China in the winter of 2002 and rapidly spread to 37 countries. Within about 1 year, more than 8,000 people experienced influenza-like symptoms and nearly 800 people died. The rapid spread and severity of these infections caused grave concern at the time. However, the outbreak was controlled in 2003 and no further cases of SARS have been recorded since 2004.9 Signs and symptoms of SARS include high fever, headache, body aches, and cough, and most patients will develop pneumonia.
MERS was first reported in Saudi Arabia in 2013. Although some infected individuals will be asymptomatic or have mild cold-like symptoms, most will develop a high fever, aches, cough and a severe respiratory infection that can progress to pneumonia. As of 2015, over 1,300 people in 27 countries have been infected. About 500 people have died. There are no specific treatments for either MERS or SARS. In addition, no vaccines are currently available. Several recombinant vaccines, however, are being developed.
Exercise \(5\)
1. What is the cause of SARS?
2. What are the signs and symptoms of MERS?
Viral Respiratory Diseases Causing Skin Rashes
Measles, rubella (German measles), and chickenpox are three important viral diseases often associated with skin rashes. However, their symptoms are systemic, and because their portal of entry is the respiratory tract, they can be considered respiratory infections.
Measles (Rubeola)
The measles virus (MeV) causes the highly contagious disease measles, also known as rubeola, which is a major cause of childhood mortality worldwide. Although vaccination efforts have greatly reduced the incidence of measles in much of the world, epidemics are still common in unvaccinated populations in certain countries.10
The measles virus is a single-stranded, negative-strand RNA virus and, like the influenza virus, it possesses an envelope with spikes of embedded hemagglutinin. The infection is spread by direct contact with infectious secretions or inhalation of airborne droplets spread by breathing, coughing, or sneezing. Measles is initially characterized by a high fever, conjunctivitis, and a sore throat. The virus then moves systemically through the bloodstream and causes a characteristic rash. The measles rash initially forms on the face and later spreads to the extremities. The red, raised macular rash will eventually become confluent and can last for several days. At the same time, extremely high fevers (higher than 40.6 °C [105 °F]) can occur. Another diagnostic sign of measles infections is Koplik’s spots, white spots that form on the inner lining of inflamed cheek tissues (Figure \(2\)).
Although measles is usually self-limiting, it can lead to pneumonia, encephalitis, and death. In addition, the inhibition of immune system cells by the measles virus predisposes patients to secondary infections. In severe infections with highly virulent strains, measles fatality rates can be as high as 10% to 15%. There were more than 145,000 measles deaths (mostly young children) worldwide in 2013.11
The preliminary diagnosis of measles is typically based on the appearance of the rash and Koplik’s spots. Hemagglutination inhibition tests and serological tests may be used to confirm measles infections in low-prevalence settings.
There are no effective treatments for measles. Vaccination is widespread in developed countries as part of the measles, mumps, and rubella (MMR) vaccine. As a result, there are typically fewer than 200 cases of measles in the United States annually.12 When it is seen, it is often associated with children who have not been vaccinated.
Preventable Measles Outbreaks
In December 2014, a measles epidemic began at Disneyland in southern California. Within just 4 months, this outbreak affected 134 people in 24 states.13 Characterization of the virus suggests that an unidentified infected individual brought the disease to the United States from the Philippines, where a similar virus had sickened more than 58,000 people and killed 110.14 Measles is highly communicable, and its spread at Disneyland may have been facilitated by the low vaccination rate in some communities in California.15
Several factors could conceivably lead to a strong comeback of measles in the U.S. Measles is still an epidemic disease in many locations worldwide. Air travel enables infected individuals to rapidly translocate these infections globally. Compounding this problem, low vaccination rates in some local areas in the United States (such as in Amish communities) provide populations of susceptible hosts for the virus to establish itself. Finally, measles has been a low-prevalence infection in the U.S. for some time. As a consequence, physicians are not as likely to recognize the initial symptoms and make accurate diagnoses. Until vaccination rates become high enough to ensure herd immunity, measles is likely to be an ongoing problem in the United States.
Rubella (German Measles)
Rubella, or the German measles, is a relatively mild viral disease that produces a rash somewhat like that caused by the measles, even though the two diseases are unrelated. The rubella virus is an enveloped RNA virus that can be found in the respiratory tract. It is transmitted from person to person in aerosols produced by coughing or sneezing. Nearly half of all infected people remain asymptomatic. However, the virus is shed and spread by asymptomatic carriers. Like rubeola, rubella begins with a facial rash that spreads to the extremities (Figure \(3\)). However, the rash is less intense, shorter lived (2–3 days), not associated with Koplik’s spots, and the resulting fever is lower (101 °F [38.3 °C]).
Congenital rubella syndrome is the most severe clinical complication of the German measles. This occurs if a woman is infected with rubella during pregnancy. The rubella virus is teratogenic, meaning it can cause developmental defects if it crosses the placenta during pregnancy. There is a very high incidence of stillbirth, spontaneous abortion, or congenital birth defects if the mother is infected before 11 weeks of pregnancy and 35% if she is infected between weeks 13–16; after this time the incidence is low.16 For this reason, prenatal screening for rubella is commonly practiced in the United States. Postnatal infections are usually self-limiting and rarely cause severe complications.
Like measles, the preliminary diagnosis of rubella is based on the patient’s history, vaccination records, and the appearance of the rash. The diagnosis can be confirmed by hemagglutinin inhibition assays and a variety of other immunological techniques. There are no antiviral therapies for rubella, but an effective vaccine (MMR) is widely available. Vaccination efforts have essentially eliminated rubella in the United States; fewer than a dozen cases are reported in a typical year.
Chickenpox and Shingles
Chickenpox, also known as varicella, was once a common viral childhood disease. The causative agent of chickenpox, the varicella-zoster virus, is a member of the herpesvirus family. In children, the disease is mild and self-limiting, and is easily transmitted by direct contact or inhalation of material from the skin lesions. In adults, however, chickenpox infections can be much more severe and can lead to pneumonia and birth defects in the case of infected pregnant women. Reye syndrome, mentioned earlier in this chapter, is also a serious complication associated with chickenpox, generally in children.
Once infected, most individuals acquire a lifetime immunity to future chickenpox outbreaks. For this reason, parents once held “chickenpox parties” for their children. At these events, uninfected children were intentionally exposed to an infected individual so they would contract the disease earlier in life, when the incidence of complications is very low, rather than risk a more severe infection later.
After the initial viral exposure, chickenpox has an incubation period of about 2 weeks. The initial infection of the respiratory tract leads to viremia and eventually produces fever and chills. A pustular rash then develops on the face, progresses to the trunk, and then the extremities, although most form on the trunk (Figure \(4\)). Eventually, the lesions burst and form a crusty scab. Individuals with chickenpox are infectious from about 2 days before the outbreak of the rash until all the lesions have scabbed over.
Like other herpesviruses, the varicella-zoster virus can become dormant in nerve cells. While the pustular vesicles are developing, the virus moves along sensory nerves to the dorsal ganglia in the spinal cord. Once there, the varicella-zoster virus can remain latent for decades. These dormant viruses may be reactivated later in life by a variety of stimuli, including stress, aging, and immunosuppression. Once reactivated, the virus moves along sensory nerves to the skin of the face or trunk. This results in the production of the painful lesions in a condition known as shingles (Figure \(5\)). These symptoms generally last for 2–6 weeks, and may recur more than once. Postherpetic neuralgia, pain signals sent from damaged nerves long after the other symptoms have subsided, is also possible. In addition, the virus can spread to other organs in immunocompromised individuals. A person with shingles lesions can transmit the virus to a nonimmune contact, and the newly infected individual would develop chickenpox as the primary infection. Shingles cannot be transmitted from one person to another.
The primary diagnosis of chickenpox in children is mainly based on the presentation of a pustular rash of the trunk. Serological and PCR-based tests are available to confirm the initial diagnosis. Treatment for chickenpox infections in children is usually not required. In patients with shingles, acyclovir treatment can often reduce the severity and length of symptoms, and diminish the risk of postherpetic neuralgia. An effective vaccine is now available for chickenpox. A vaccine is also available for adults older than 60 years who were infected with chickenpox in their youth. This vaccine reduces the likelihood of a shingles outbreak by boosting the immune defenses that are keeping the latent infection in check and preventing reactivation.
Exercise \(6\)
1. Why does measles often lead to secondary infections?
2. What signs or symptoms would distinguish rubella and measles?
3. Why can chickenpox lead to shingles later in life?
Smallpox Stockpiles
Smallpox has probably killed more humans than any other infectious disease, with the possible exception of tuberculosis. This disease, caused by the variola major virus, is transmitted by inhalation of viral particles shed from lesions in the throat. The smallpox virus spreads systemically in the bloodstream and produces a pustular skin rash. Historical epidemics of smallpox had fatality rates of 50% or greater in susceptible populations. Concerted worldwide vaccination efforts eradicated smallpox from the general population in 1977. This was the first microbial disease in history to be eradicated, a feat made possible by the fact that the only reservoir for the smallpox virus is infected humans.
Although the virus is no longer present in the wild, laboratory samples of the virus still exist in the United States and Russia.17 The question is, why do these samples still exist? Some claim that these stocks should be maintained for research purposes. Should the smallpox virus ever reappear, they say, we would need access to such stocks for development of vaccines and treatments. Concerns about a re-emergence of the virus are not totally unfounded. Although there are no living reservoirs of the virus, there is always the possibility that smallpox could re-emerge from mummified human bodies or human remains preserved in permafrost. It is also possible that there are as-yet undiscovered samples of the virus in other locations around the world. An example of such "lost" samples was discovered in a drawer in a Food and Drug Administration lab in Maryland.18 If an outbreak from such a source were to occur, it could lead to uncontrolled epidemics, since the population is largely unvaccinated now.
Critics of this argument, including many research scientists and the World Health Organization, claim that there is no longer any rational argument for keeping the samples. They view the “re-emergence scenarios” as a thinly veiled pretense for harboring biological weapons. These scenarios, they say, are less probable than an intentional reintroduction of the virus from militarized stocks by humans. Furthermore, they point out that if we needed to research smallpox in the future, we could rebuild the virus from its DNA sequence.
What do you think? Are there legitimate arguments for maintaining stockpiles of smallpox, or should all forms of this deadly disease be eradicated?
Viral Infections of the Respiratory Tract
Many viruses are capable of entering and causing disease in the respiratory system, and a number are able to spread beyond the respiratory system to cause systemic infections. Most of these infections are highly contagious and, with a few exceptions, antimicrobial drugs are not effective for treatment. Although some of these infections are self-limiting, others can have serious or fatal complications. Effective vaccines have been developed for several of these diseases, as summarized in Figure \(6\).
Key Concepts and Summary
• Viruses cause respiratory tract infections more frequently than bacteria, and most viral infections lead to mild symptoms.
• The common cold can be caused by more than 200 viruses, typically rhinoviruses, coronaviruses, and adenoviruses, transmitted by direct contact, aerosols, or environmental surfaces.
• Due to its ability to rapidly mutate through antigenic drift and antigenic shift, influenza remains an important threat to human health. Two new influenza vaccines are developed annually.
• Several viral infections, including respiratory syncytial virus infections, which frequently occur in the very young, can begin with mild symptoms before progressing to viral pneumonia.
• SARS and MERS are acute respiratory infections caused by coronaviruses, and both appear to originate in animals. SARS has not been seen in the human population since 2004 but had a high mortality rate during its outbreak. MERS also has a high mortality rate and continues to appear in human populations.
• Measles, rubella, and chickenpox are highly contagious, systemic infections that gain entry through the respiratory system and cause rashes and fevers. Vaccines are available for all three. Measles is the most severe of the three and is responsible for significant mortality around the world. Chickenpox typically causes mild infections in children but the virus can reactivate to cause painful cases of shingles later in life.
Footnotes
1. 1 AG L’Huillier et al. “Survival of Rhinoviruses on Human Fingers.” Clinical Microbiology and Infection 21, no. 4 (2015):381–385.
2. 2 Centers for Disease Control and Prevention. “Estimating Seasonal Influenza-Associated Deaths in the United States: CDC Study Confirms Variability of Flu.” 2016. https://www.cdc.gov/flu/about/burden/preliminary-in-season-estimates.htm. Accessed July 6, 2016.
3. 3 ED Belay et al. “Reye’s Syndrome in the United States From 1981 Through 1997.” New England Journal of Medicine 340 no. 18 (1999):1377–1382.
4. 4 CE Mills et al. “Transmissibility of 1918 Pandemic Influenza.” Nature 432, no. 7019 (2004):904–906.
5. 5 E. Tognotti. “Influenza Pandemics: A Historical Retrospect.” Journal of Infection in Developing Countries 3, no. 5 (2009):331–334.
6. 6 FS Dawood et al. “Estimated Global Mortality Associated with the First 12 Months of 2009 Pandemic Influenza A H1N1 Virus Circulation: A Modelling Study.” The Lancet Infectious Diseases 12, no. 9 (2012):687–695.
7. 7 World Health Organization. “WHO Report on Global Surveillance of Epidemic-Prone Infectious Diseases.” 2000. http://www.who.int/csr/resources/pub.../Influenza.pdf. Accessed July 6, 2016.
8. 8 Centers of Disease Control and Prevention. “Vaccine Effectiveness - How Well Does the Flu Vaccine Work?” 2016. http://www.cdc.gov/flu/about/qa/vaccineeffect.htm. Accessed July 6, 2016.
9. 9 Y. Huang. “The SARS Epidemic and Its Aftermath in China: A Political Perspective.” In Learning from SARS: Preparing for the Next Disease Outbreak. Edited by S. Knobler et al. Washington, DC: National Academies Press; 2004. Available at: www.ncbi.nlm.nih.gov/books/NBK92479/
10. 10 Centers for Disease Control and Prevention. “Global Health - Measles, Rubella, and CRS, Eliminating Measles, Rubella & Congenital Rubella Syndrome (CRS) Worldwide.” 2015. http://www.cdc.gov/globalhealth/measles/. Accessed July 7, 2016.
11. 11 World Health Organization. “Measles Factsheet.” 2016. http://www.who.int/mediacentre/factsheets/fs286/en/. Accessed July 7, 2016.
12. 12 Centers for Disease Control and Prevention. “Measles Cases and Outbreaks.” 2016. http://www.cdc.gov/measles/cases-outbreaks.html. Accessed July 7, 2016.
13. 13 Ibid.
14. 14 World Health Organization. “Measles-Rubella Bulletin.” Manila, Philippines; Expanded Programme on Immunization Regional Office for the Western Pacific World Health Organization; 9 no. 1 (2015). http://www.wpro.who.int/immunization...vol9issue1.pdf
15. 15 M. Bloch et al. “Vaccination Rates for Every Kindergartener in California.” The New York Times February 6, 2015. http://www.nytimes.com/interactive/2...-map.html?_r=1. Accessed July 7, 2016.
16. 16 E. Miller et al. “Consequences of Confirmed Maternal Rubella at Successive Stages of Pregnancy.” The Lancet 320, no. 8302 (1982):781–784.
17. 17 Centers for Disease Control and Prevention. “CDC Media Statement on Newly Discovered Smallpox Specimens.” July 8, 2014. http://www.cdc.gov/media/releases/2014/s0708-nih.html. Accessed on July 7, 2016.
18. 18 Ibid. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/22%3A_Respiratory_System_Infections/22.03%3A_Viral_Infections_of_the_Respiratory_Tract.txt |
Learning Objectives
• Identify the most common fungi that can cause infections of the respiratory tract
• Compare the major characteristics of specific fungal diseases of the respiratory tract
Fungal pathogens are ubiquitous in the environment. Serological studies have demonstrated that most people have been exposed to fungal respiratory pathogens during their lives. Yet symptomatic infections by these microbes are rare in healthy individuals. This demonstrates the efficacy of the defenses of our respiratory system. In this section, we will examine some of the fungi that can cause respiratory infections.
Histoplasmosis
Histoplasmosis is a fungal disease of the respiratory system and most commonly occurs in the Mississippi Valley of the United States and in parts of Central and South America, Africa, Asia, and Australia. The causative agent, Histoplasma capsulatum, is a dimorphic fungus. This microbe grows as a filamentous mold in the environment but occurs as a budding yeast during human infections. The primary reservoir for this pathogen is soil, particularly in locations rich in bat or bird feces.
Histoplasmosis is acquired by inhaling microconidial spores in the air; this disease is not transmitted from human to human. The incidence of histoplasmosis exposure is high in endemic areas, with 60%–90% of the population having anti-Histoplasma antibodies, depending on location;1 however, relatively few individuals exposed to the fungus actually experience symptoms. Those most likely to be affected are the very young, the elderly, and immunocompromised people.
In many ways, the course of this disease is similar to that of tuberculosis. Following inhalation, the spores enter the lungs and are phagocytized by alveolar macrophages. The fungal cells then survive and multiply within these phagocytes (see Figure 5.3.2). Focal infections cause the formation of granulomatous lesions, which can lead to calcifications that resemble the Ghon complexes of tuberculosis, even in asymptomatic cases. Also like tuberculosis, histoplasmosis can become chronic and reactivation can occur, along with dissemination to other areas of the body (e.g., the liver or spleen).
Signs and symptoms of pulmonary histoplasmosis include fever, headache, and weakness with some chest discomfort. The initial diagnosis is often based on chest radiographs and cultures grown on fungal selective media like Sabouraud's dextrose agar. Direct fluorescence antibody staining and Giemsa staining can also be used to detect this pathogen. In addition, serological tests including a complement fixation assay and histoplasmin sensitivity can be used to confirm the diagnosis. In most cases, these infections are self-limiting and antifungal therapy is not required. However, in disseminated disease, the antifungal agents amphotericin B and ketoconazole are effective; itraconazole may be effective in immunocompromised patients, in whom the disease can be more serious.
Exercise \(1\)
1. In what environments is one more likely to be infected with histoplasmosis?
2. Identify at least two similarities between histoplasmosis and tuberculosis.
Coccidioidomycosis
Infection by the dimorphic fungus Coccidioides immitis causes coccidioidomycosis. Because the microbe is endemic to the San Joaquin Valley of California, the disease is sometimes referred to as Valley fever. A related species that causes similar infections is found in semi-arid and arid regions of the southwestern United States, Mexico, and Central and South America.2
Like histoplasmosis, coccidioidomycosis is acquired by inhaling fungal spores—in this case, arthrospores formed by hyphal fragmentation. Once in the body, the fungus differentiates into spherules that are filled with endospores. Most C. immitis infections are asymptomatic and self-limiting. However, the infection can be very serious for immunocompromised patients. The endospores may be transported in the blood, disseminating the infection and leading to the formation of granulomatous lesions on the face and nose (Figure \(1\)). In severe cases, other major organs can become infected, leading to serious complications such as fatal meningitis.
Coccidioidomycosis can be diagnosed by culturing clinical samples. C. immitis readily grows on laboratory fungal media, such as Sabouraud's dextrose agar, at 35 °C (95 °F). Culturing the fungus, however, is rather dangerous. C. immitis is one of the most infectious fungal pathogens known and is capable of causing laboratory-acquired infections. Indeed, until 2012, this organism was considered a “select agent” of bioterrorism and classified as a BSL-3 microbe. Serological tests for antibody production are more often used for diagnosis. Although mild cases generally do not require intervention, disseminated infections can be treated with intravenous antifungal drugs like amphotericin B.
Clinical Focus: Resolution
John’s negative RIDT tests do not rule out influenza, since false-negative results are common, but the Legionella infection still must be treated with antibiotic therapy and is the more serious condition. John's prognosis is good, provided the physician can find an antibiotic therapy to which the infection responds.
While John was undergoing treatment, three of the employees from the home improvement store also reported to the clinic with very similar symptoms. All three were older than 55 years and had Legionella antigen in their urine; L. pneumophila was also isolated from their sputum. A team from the health department was sent to the home improvement store to identify a probable source for these infections. Their investigation revealed that about 3 weeks earlier, the store's air conditioning system, which was located where the employees ate lunch, had been undergoing maintenance. L. pneumophila was isolated from the cooling coils of the air conditioning system and intracellular L. pneumophila was observed in amoebae in samples of condensed water from the cooling coils as well (Figure \(2\)). The amoebae provide protection for the Legionella bacteria and are known to enhance their pathogenicity.3
In the wake of the infections, the store ordered a comprehensive cleaning of the air conditioning system and implemented a regular maintenance program to prevent the growth of biofilms within the cooling tower. They also reviewed practices at their other facilities.
After a month of rest at home, John recovered from his infection enough to return to work, as did the other three employees of the store. However, John experienced lethargy and joint pain for more than a year after his treatment.
Blastomycosis
Blastomycosis is a rare disease caused by another dimorphic fungus, Blastomyces dermatitidis. Like Histoplasma and Coccidioides, Blastomyces uses the soil as a reservoir, and fungal spores can be inhaled from disturbed soil. The pulmonary form of blastomycosis generally causes mild flu-like symptoms and is self-limiting. It can, however, become disseminated in immunocompromised people, leading to chronic cutaneous disease with subcutaneous lesions on the face and hands (Figure \(3\)). These skin lesions eventually become crusty and discolored and can result in deforming scars. Systemic blastomycosis is rare, but if left untreated, it is always fatal.
Preliminary diagnosis of pulmonary blastomycosis can be made by observing the characteristic budding yeast forms in sputum samples. Commercially available urine antigen tests are now also available. Additional confirmatory tests include serological assays such as immunodiffusion tests or EIA. Most cases of blastomycosis respond well to amphotericin Bor ketoconazole treatments.
Mucormycosis
A variety of fungi in the order Mucorales cause mucormycosis, a rare fungal disease. These include bread molds, like Rhizopus and Mucor; the most commonly associated species is Rhizopus arrhizus (oryzae) (see Figure 5.3.4). These fungi can colonize many different tissues in immunocompromised patients, but often infect the skin, sinuses, or the lungs.
Although most people are regularly exposed to the causative agents of mucormycosis, infections in healthy individuals are rare. Exposure to spores from the environment typically occurs through inhalation, but the spores can also infect the skin through a wound or the gastrointestinal tract if ingested. Respiratory mucormycosis primarily affects immunocompromised individuals, such as patients with cancer or those who have had a transplant.4
After the spores are inhaled, the fungi grow by extending hyphae into the host’s tissues. Infections can occur in both the upper and lower respiratory tracts. Rhinocerebral mucormycosis is an infection of the sinuses and brain; symptoms include headache, fever, facial swelling, congestion, and tissue necrosis causing black lesions in the oral cavity. Pulmonary mucormycosis is an infection of the lungs; symptoms include fever, cough, chest pain, and shortness of breath. In severe cases, infections may become disseminated and involve the central nervous system, leading to coma and death.5
Diagnosing mucormycosis can be challenging. Currently, there are no serological or PCR-based tests available to identify these infections. Tissue biopsy specimens must be examined for the presence of the fungal pathogens. The causative agents, however, are often difficult to distinguish from other filamentous fungi. Infections are typically treated by the intravenous administration of amphotericin B, and superficial infections are removed by surgical debridement. Since the patients are often immunocompromised, viral and bacterial secondary infections commonly develop. Mortality rates vary depending on the site of the infection, the causative fungus, and other factors, but a recent study found an overall mortality rate of 54%.6
Exercise \(2\)
1. Compare the modes of transmission for coccidioidomycosis, blastomycosis, and mucormycosis.
2. In general, which are more serious: the pulmonary or disseminated forms of these infections?
Aspergillosis
Aspergillus is a common filamentous fungus found in soils and organic debris. Nearly everyone has been exposed to this mold, yet very few people become sick. In immunocompromised patients, however, Aspergillus may become established and cause aspergillosis. Inhalation of spores can lead to asthma-like allergic reactions. The symptoms commonly include shortness of breath, wheezing, coughing, runny nose, and headaches. Fungal balls, or aspergilloma, can form when hyphal colonies collect in the lungs (Figure \(4\)). The fungal hyphae can invade the host tissues, leading to pulmonary hemorrhage and a bloody cough. In severe cases, the disease may progress to a disseminated form that is often fatal. Death most often results from pneumonia or brain hemorrhages.
Laboratory diagnosis typically requires chest radiographs and a microscopic examination of tissue and respiratory fluid samples. Serological tests are available to identify Aspergillus antigens. In addition, a skin test can be performed to determine if the patient has been exposed to the fungus. This test is similar to the Mantoux tuberculin skin test used for tuberculosis. Aspergillosis is treated with intravenous antifungal agents, including itraconazole and voriconazole. Allergic symptoms can be managed with corticosteroids because these drugs suppress the immune system and reduce inflammation. However, in disseminated infections, corticosteroids must be discontinued to allow a protective immune response to occur.
Pneumocystis Pneumonia
A type of pneumonia called Pneumocystis pneumonia (PCP) is caused by Pneumocystis jirovecii. Once thought to be a protozoan, this organism was formerly named P. carinii but it has been reclassified as a fungus and renamed based on biochemical and genetic analyses. Pneumocystis is a leading cause of pneumonia in patients with acquired immunodeficiency syndrome (AIDS) and can be seen in other compromised patients and premature infants. Respiratory infection leads to fever, cough, and shortness of breath. Diagnosis of these infections can be difficult. The organism is typically identified by microscopic examination of tissue and fluid samples from the lungs (Figure \(5\)). A PCR-based test is available to detect P. jirovecii in asymptomatic patients with AIDS. The best treatment for these infections is the combination drug trimethoprim-sulfamethoxazole (TMP/SMZ). These sulfa drugs often have adverse effects, but the benefits outweigh these risks. Left untreated, PCP infections are often fatal.
Cryptococcosis
Infection by the encapsulated yeast Cryptococcus neoformans causes cryptococcosis. This fungus is ubiquitous in the soil and can be isolated from bird feces. Immunocompromised people are infected by inhaling basidiospores found in aerosols. The thick polysaccharide capsule surrounding these microbes enables them to avoid clearance by the alveolar macrophage. Initial symptoms of infection include fever, fatigue, and a dry cough. In immunocompromised patients, pulmonary infections often disseminate to the brain. The resulting meningitis produces headaches, sensitivity to light, and confusion. Left untreated, such infections are often fatal.
Cryptococcus infections are often diagnosed based on microscopic examination of lung tissues or cerebrospinal fluids. India ink preparations (Figure \(6\)) can be used to visualize the extensive capsules that surround the yeast cells. Serological tests are also available to confirm the diagnosis. Amphotericin B, in combination with flucytosine, is typically used for the initial treatment of pulmonary infections. Amphotericin B is a broad-spectrum antifungal drug that targets fungal cell membranes. It can also adversely impact host cells and produce side effects. For this reason, clinicians must carefully balance the risks and benefits of treatments in these patients. Because it is difficult to eradicate cryptococcal infections, patients usually need to take fluconazole for up to 6 months after treatment with amphotericin B and flucytosine to clear the fungus. Cryptococcal infections are more common in immunocompromised people, such as those with AIDS. These patients typically require life-long suppressive therapy to control this fungal infection.
Exercise \(3\)
1. What populations are most at risk for developing Pneumocystis pneumonia or cryptococcosis?
2. Why are these infections fatal if left untreated?
Fungal Diseases of the Respiratory Tract
Most respiratory mycoses are caused by fungi that inhabit the environment. Such infections are generally transmitted via inhalation of fungal spores and cannot be transmitted between humans. In addition, healthy people are generally not susceptible to infection even when exposed; the fungi are only virulent enough to establish infection in patients with HIV, AIDS, or another condition that compromises the immune defenses. Figure \(7\) summarizes the features of important respiratory mycoses.
Key Concepts and Summary
• Fungal pathogens rarely cause respiratory disease in healthy individuals, but inhalation of fungal spores can cause severe pneumonia and systemic infections in immunocompromised patients.
• Antifungal drugs like amphotericin B can control most fungal respiratory infections.
• Histoplasmosis is caused by a mold that grows in soil rich in bird or bat droppings. Few exposed individuals become sick, but vulnerable individuals are susceptible. The yeast-like infectious cells grow inside phagocytes.
• Coccidioidomycosis is also acquired from soil and, in some individuals, will cause lesions on the face. Extreme cases may infect other organs, causing death.
• Blastomycosis, a rare disease caused by a soil fungus, typically produces a mild lung infection but can become disseminated in the immunocompromised. Systemic cases are fatal if untreated.
• Mucormycosis is a rare disease, caused by fungi of the order Mucorales. It primarily affects immunocompromised people. Infection involves growth of the hyphae into infected tissues and can lead to death in some cases.
• Aspergillosis, caused by the common soil fungus Aspergillus, infects immunocompromised people. Hyphal balls may impede lung function and hyphal growth into tissues can cause damage. Disseminated forms can lead to death.
• Pneumocystis pneumonia is caused by the fungus P. jirovecii. The disease is found in patients with AIDS and other immunocompromised individuals. Sulfa drug treatments have side effects, but untreated cases may be fatal.
• Cryptococcosis is caused by Cryptococcus neoformans. Lung infections may move to the brain, causing meningitis, which can be fatal.
Footnotes
1. 1 NE Manos et al. “Geographic Variation in the Prevalence of Histoplasmin Sensitivity.” Dis Chest 29, no. 6 (1956):649–668.
2. 2 DR Hospenthal. “Coccioidomycosis.” Medscape. 2015. http://emedicine.medscape.com/article/215978-overview. Accessed July 7, 2016.
3. 3 HY Lau and NJ Ashbolt. “The Role of Biofilms and Protozoa in Legionella Pathogenesis: Implications for Drinking Water.” Journal of Applied Microbiology 107 no. 2 (2009):368–378.
4. 4 Centers for Disease Control and Prevention. “Fungal Diseases. Definition of Mucormycosis.” 2015 http://www.cdc.gov/fungal/diseases/m...efinition.html. Accessed July 7, 2016.
5. 5 Centers for Disease Control and Prevention. “Fungal Diseases. Symptoms of Mucormycosis.” 2015 http://www.cdc.gov/fungal/diseases/m.../symptoms.html. Accessed July 7, 2016.
6. 6 MM Roden et al. “Epidemiology and Outcome of Zygomycosis: A Review of 929 Reported Cases.” Clinical Infectious Diseases 41 no. 5 (2005):634–653. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/22%3A_Respiratory_System_Infections/22.04%3A_Respiratory_Mycoses.txt |
22.1: Anatomy and Normal Microbiota of the Respiratory Tract
The upper respiratory tract is colonized by an extensive and diverse normal microbiota, many of which are potential pathogens. Few microbial inhabitants have been found in the lower respiratory tract, and these may be transients. Members of the normal microbiota may cause opportunistic infections, using a variety of strategies to overcome the innate nonspecific defenses (including the mucociliary escalator) and adaptive specific defenses of the respiratory system.
Multiple Choice
Which of the following is not directly connected to the nasopharynx?
1. middle ear
2. oropharynx
3. lacrimal glands
4. nasal cavity
Answer
C
What type of cells produce the mucus for the mucous membranes?
1. goblet cells
2. macrophages
3. phagocytes
4. ciliated epithelial cells
Answer
A
Which of these correctly orders the structures through which air passes during inhalation?
1. pharynx → trachea → larynx → bronchi
2. pharynx → larynx → trachea → bronchi
3. larynx → pharynx → bronchi → trachea
4. larynx → pharynx → trachea → bronchi
Answer
B
The ___________ separates the upper and lower respiratory tract.
1. bronchi
2. larynx
3. epiglottis
4. palatine tonsil
Answer
C
Which microbial virulence factor is most important for attachment to host respiratory tissues?
1. adhesins
2. lipopolysaccharide
3. hyaluronidase
4. capsules
Answer
A
Fill in the Blank
Unattached microbes are moved from the lungs to the epiglottis by the _______ effect.
Answer
mucociliary escalator
Many bacterial pathogens produce _______ to evade phagocytosis.
Answer
capsules
The main type of antibody in the mucous membrane defenses is _______.
Answer
IgA
_______ results from an inflammation of the “voice box.”
Answer
Laryngitis
_______ phagocytize potential pathogens in the lower lung.
Answer
Alveolar macrophages
Short Answer
Explain why the lower respiratory tract is essentially sterile.
Explain why pneumonia is often a life-threatening disease.
Critical Thinking
Name each of the structures of the respiratory tract shown, and state whether each has a relatively large or small normal microbiota.
(credit: modification of work by National Cancer Institute)
Cystic fibrosis causes, among other things, excess mucus to be formed in the lungs. The mucus is very dry and caked, unlike the moist, more-fluid mucus of normal lungs. What effect do you think that has on the lung’s defenses?
Why do you think smokers are more likely to suffer from respiratory tract infections?
22.2: Bacterial Infections of the Respiratory Tract
The respiratory tract can be infected by a variety of bacteria, both gram positive and gram negative. Although the diseases that they cause may range from mild to severe, in most cases, the microbes remain localized within the respiratory system. Fortunately, most of these infections also respond well to antibiotic therapy.
Multiple Choice
Which of the following does not involve a bacterial exotoxin?
1. diphtheria
2. whooping cough
3. scarlet fever
4. Q fever
Answer
D
What disease is caused by Coxiella burnetii?
1. Q fever
2. tuberculosis
3. diphtheria
4. walking pneumonia
Answer
A
In which stage of pertussis is the characteristic whooping sound made?
1. convalescence
2. catarrhal
3. paroxysmal
4. prodromal
Answer
C
What is the causative agent of Q fever?
1. Coxiella burnetii
2. Chlamydophila psittaci
3. Mycoplasma pneumoniae
4. Streptococcus pyogenes
Answer
A
Which of these microbes causes “walking pneumonia”?
1. Klebsiella pneumoniae
2. Streptococcus pneumoniae
3. Mycoplasma pneumoniae
4. Chlamydophila pneumoniae
Answer
C
Fill in the Blank
Calcified lesions called _______ form in the lungs of patients with TB.
Answer
Ghon complexes
An inflammation of the middle ear is called _______.
Answer
otitis media
The _______ is used to serologically identify Streptococcus pneumoniae isolates.
Answer
quellung reaction
_______ is a zoonotic infection that can be contracted by people who handle birds.
Answer
Psittacosis
The main virulence factor involved in scarlet fever is the _______.
Answer
erythrogenic toxin
Short Answer
Name three bacteria that commonly cause pneumonia. Which is the most common cause?
How does smoking make an individual more susceptible to infections?
How does the diphtheria pathogen form a pseudomembrane?
Critical Thinking
Why might β-lactam antibiotics be ineffective against Mycoplasma pneumoniae infections?
Why is proper antibiotic therapy especially important for patients with tuberculosis?
22.3: Viral Infections of the Respiratory Tract
Viruses cause respiratory tract infections more frequently than bacteria, and most viral infections lead to mild symptoms. The common cold can be caused by more than 200 viruses, typically rhinoviruses, coronaviruses, and adenoviruses, transmitted by direct contact, aerosols, or environmental surfaces. Due to its ability to rapidly mutate through antigenic drift and antigenic shift, influenza remains an important threat to human health. Two new influenza vaccines are developed annually.
Multiple Choice
Which of the following viruses is not commonly associated with the common cold?
1. coronavirus
2. adenovirus
3. rhinovirus
4. varicella-zoster virus
Answer
D
Which of the following viral diseases has been eliminated from the general population worldwide?
1. smallpox
2. measles
3. German measles
4. influenza
Answer
A
What term refers to multinucleated cells that form when many host cells fuse together during infections?
1. Ghon elements
2. Reye syndrome
3. Koplik’s spots
4. syncytia
Answer
D
Which of the following diseases is not associated with coronavirus infections?
1. Middle East respiratory syndrome
2. German measles
3. the common cold
4. severe acute respiratory syndrome
Answer
B
Which of these viruses is responsible for causing shingles?
1. rubella virus
2. measles virus
3. varicella-zoster virus
4. variola major virus
Answer
C
Fill in the Blank
The _______ virus is responsible for causing German measles.
Answer
rubella
A(n) _______ is an uncontrolled positive feedback loop between cytokines and leucocytes.
Answer
cytokine storm
In cases of shingles, the antiviral drug _______ may be prescribed.
Answer
acyclovir
The slow accumulation of genetic changes to an influenza virus over time is referred to as _______.
Answer
antigenic drift
The _______ vaccine is effective in controlling both measles and rubella.
Answer
MMR
Short Answer
Since we all have experienced many colds in our lifetime, why are we not resistant to future infections?
Critical Thinking
What role does the common cold have in the rise of antibiotic-resistant strains of bacteria in the United States?
Why is it highly unlikely that influenza A virus will ever be eradicated, like the smallpox virus?
22.4: Respiratory Mycoses
Fungal pathogens are ubiquitous in the environment. Serological studies have demonstrated that most people have been exposed to fungal respiratory pathogens during their lives. Yet symptomatic infections by these microbes are rare in healthy individuals. This demonstrates the efficacy of the defenses of our respiratory system. In this section, we examine some of the fungi that can cause respiratory infections.
Multiple Choice
Which of these infections is also referred to as Valley fever?
1. histoplasmosis
2. coccidioidomycosis
3. blastomycosis
4. aspergillosis
Answer
B
Which of the following is not caused by a dimorphic fungus?
1. histoplasmosis
2. coccidioidomycosis
3. blastomycosis
4. aspergillosis
Answer
D
Which of the following is caused by infections by bread molds?
1. mucormycosis
2. coccidioidomycosis
3. cryptococcosis
4. Pneumocystis pneumonia
Answer
A
In the United States, most histoplasmosis cases occur
1. in the Pacific northwest.
2. in the desert southwest.
3. in the Mississippi river valley.
4. in Colorado river valley.
Answer
C
Which of the following infections can be diagnosed using a skin test similar to the tuberculin test?
1. histoplasmosis
2. cryptococcosis
3. blastomycosis
4. aspergillosis
Answer
D
Fill in the Blank
In coccidioidomycosis, _______ containing many endospores form in the lungs.
Answer
spherules
In cryptococcosis, the main fungal virulence factor is the _______, which helps the pathogen avoid phagocytosis.
Answer
capsule
In some mycoses, fungal balls called _______ form in the lungs
Answer
aspergillomas
Most US cases of coccidioidomycosis occur in _______.
Answer
the desert southwest
Coccidioidomycosis may develop when Coccidioides immitis _______ are inhaled.
Answer
arthrospores
Short Answer
Which pulmonary fungal infection is most likely to be confused with tuberculosis? How can we discriminate between these two types of infection?
Compare and contrast aspergillosis and mucormycosis.
Critical Thinking
Why are fungal pulmonary infections rarely transmissible from person to person? | textbooks/bio/Microbiology/Microbiology_(OpenStax)/22%3A_Respiratory_System_Infections/22.E%3A_Respiratory_System_Infections_%28Exercises%29.txt |
The urogenital system is a combination of the urinary tract and reproductive system. Because both systems are open to the external environment, they are prone to infections. Some infections are introduced from outside, whereas others result from imbalances in the microbiota of the urogenital tract.
Urinary tract infections (UTIs) are one the most common bacterial infections worldwide, affecting over 100 million people each year. During 2007 in the United States, doctor office visits for UTIs exceeded 10 million, and an additional 2–3 million emergency department visits were attributed to UTIs. Sexually transmitted infections (STIs) also primarily affect the urogenital system and are an important cause of patient morbidity. The Centers for Disease Control and Prevention (CDC) estimates that there are approximately 20 million new cases of reportable STIs annually in the United States, half of which occur in people aged 15–24 years old. When STIs spread to the reproductive organs, they can be associated with severe morbidity and loss of fertility.
Because males and females have different urogenital anatomy, urogenital infections may affect males and females differently. In this chapter, we will discuss the various microbes that cause urogenital disease and the factors that contribute to their pathogenicity.
• 23.1: Anatomy and Normal Microbiota of the Urogenital Tract
The urinary system is responsible for filtering the blood, excreting wastes, and helping to regulate electrolyte and water balance. The urinary system includes the kidneys, ureters, urinary bladder, and urethra; the bladder and urethra are the most common sites of infection. Common sites of infection in the male reproductive system include the urethra, as well as the testes, prostate and epididymis. The common sites of infection in females are the vulva, vagina, cervix, and fallopian tubes.
• 23.2: Bacterial Infections of the Urinary System
Urinary tract infections (UTIs) include infections of the urethra, bladder, and kidneys, and are common causes of urethritis, cystitis, pyelonephritis, and glomerulonephritis. Bacteria are the most common causes of UTIs, especially in the urethra and bladder. Bacterial cystitis is commonly caused by fecal bacteria such as E. coli. Pyelonephritis is a serious kidney infection that is often caused by bacteria that travel from infections elsewhere in the urinary tract.
• 23.3: Bacterial Infections of the Reproductive System
In addition to infections of the urinary tract, bacteria commonly infect the reproductive tract. As with the urinary tract, parts of the reproductive system closest to the external environment are the most likely sites of infection. Often, the same microbes are capable of causing urinary tract and reproductive tract infections. Bacterial vaginosis, Chlamydia, Gonorrhea, and Chancroid are diseases caused by bacteria.
• 23.4: Viral Infections of the Reproductive System
Genital herpes is usually caused by HSV-2 (although HSV-1 can also be responsible) and may cause the development of infectious, potentially recurrent vesicles. Neonatal herpes can occur in babies born to infected mothers and can cause symptoms that range from relatively mild (more common) to severe. Human papillomaviruses are the most common sexually transmitted viruses and include strains that cause genital warts as well as strains that cause cervical cancer.
• 23.5: Fungal Infections of the Reproductive System
Candida spp. are typically present in the normal microbiota in the body, including the skin, respiratory tract, gastrointestinal tract, and female urogenital system. Disruptions in the normal vaginal microbiota can lead to an overgrowth of Candida, causing vaginal candidiasis. Vaginal candidiasis can be treated with topical or oral fungicides. Prevention is difficult.
• 23.6: Protozoan Infections of the Reproductive System
Trichomoniasis is a common STI caused by Trichomonas vaginalis. T. vaginalis is common at low levels in the normal microbiota. Trichomoniasis is often asymptomatic. When symptoms develop, trichomoniasis causes urinary discomfort, irritation, itching, burning, discharge from the penis (in men), and vaginal discharge (in women). Trichomoniasis is treated with the antiflagellate drugs tinidazole and metronidazole.
• 23.E: Urogenital System Infections (Exercises)
Thumbnail: Candida blastospores (asexual spores that result from budding) and chlamydospores (resting spores produced through asexual reproduction) are visible in this micrograph. (credit: modification of work by Centers for Disease Control and Prevention).
23: Urogenital System Infections
Learning Objectives
• Compare the anatomy, function, and normal microbiota associated with the male and female urogenital systems
• Explain how microorganisms, in general, overcome the defenses of the urogenital system to cause infection
• Name, describe, and differentiate between general signs and symptoms associated with infections of the urogenital tract
Clinical Focus: Part 1
Nadia is a newly married 26-year-old graduate student in economics. Recently she has been experiencing an unusual vaginal discharge, as well as some itching and discomfort. Since she is due for her annual physical exam, she makes an appointment with her doctor hoping that her symptoms can be quickly treated. However, she worries that she may have some sort of sexually transmitted infection (STI). Although she is now in a monogamous relationship, she is not fully certain of her spouse’s sexual history and she is reluctant to ask him about it.
At her checkup, Nadia describes her symptoms to her primary care physician and, somewhat awkwardly, explains why she thinks she might have an STI. Nadia’s doctor reassures her that she regularly sees patients with similar concerns and encourages her to be fully transparent about her symptoms because some STIs can have serious complications if left untreated. After some further questioning, the doctor takes samples of Nadia’s blood, urine, and vaginal discharge to be sent to the lab for testing.
Exercise \(1\)
1. What are some possible causes of Nadia’s symptoms?
2. Why does the doctor take so many different samples?
The urinary system filters blood, excretes wastes, and maintains an appropriate electrolyte and water balance. The reproductive system is responsible for the production of gametes and participates in conception and, in females, development of offspring. Due to their proximity and overlap, these systems are often studied together and referred to as the urogenital system (or genitourinary system).
Anatomy of the Urinary Tract
The basic structures of the urinary tract are common in males and females. However, there are unique locations for these structures in females and males, and there is a significant amount of overlap between the urinary and genital structures in males. Figure \(1\) illustrates the urinary anatomy common to females and males.
The kidneys carry out the urinary system’s primary functions of filtering the blood and maintaining water and electrolyte balance. The kidneys are composed of millions of filtration units called nephrons. Each nephron is in intimate contact with blood through a specialized capillary bed called the glomerulus (plural glomeruli). Fluids, electrolytes, and molecules from the blood pass from the glomerulus into the nephron, creating the filtrate that becomes urine (Figure \(2\)). Urine that collects in each kidney empties through a ureter and drains to the urinary bladder, which stores urine. Urine is released from the bladder to the urethra, which transports it to be excreted from the body through the urinary meatus, the opening of the urethra.
Anatomy of the Reproductive System
The male reproductive system (Figure \(3\)) is located in close proximity to the urinary system, and the urethra is part of both systems. The testes are responsible for the production of sperm. The epididymis is a coiled tube that collects sperm from the testes and passes it on to the vas deferens. The epididymis is also the site of sperm maturation after they leave the testes. The seminal vesicles and prostate are accessory glands that produce fluid that supports sperm. During ejaculation, the vas deferens releases this mixture of fluid and sperm, called semen, into the urethra, which extends to the end of the penis.
The female reproductive system is located near the urinary system (Figure \(3\)). The external genitalia (vulva) in females open to the vagina, a muscular passageway that connects to the cervix. The cervix is the lower part of the uterus (the organ where a fertilized egg will implant and develop). The cervix is a common site of infection, especially for viruses that may lead to cervical cancer. The uterus leads to the fallopian tubes and eventually to the ovaries. Ovaries are the site of ova (egg) production, as well as the site of estrogen and progesterone production that are involved in maturation and maintenance of reproductive organs, preparation of the uterus for pregnancy, and regulation of the menstrual cycle.
Exercise \(2\)
1. What are the major structures of the urinary system, starting where urine is formed?
2. What structure in males is shared by the reproductive and the urinary systems?
Normal Microbiota of the Urogenital System
The normal microbiota of different body sites provides an important nonspecific defense against infectious diseases (see Physical Defenses), and the urogenital tract is no exception. In both men and women, however, the kidneys are sterile. Although urine does contain some antibacterial components, bacteria will grow in urine left out at room temperature. Therefore, it is primarily the flushing action that keeps the ureters and bladder free of microbes.
Below the bladder, the normal microbiota of the male urogenital system is found primarily within the distal urethra and includes bacterial species that are commonly associated with the skin microbiota. In women, the normal microbiota is found within the distal one third of the urethra and the vagina. The normal microbiota of the vagina becomes established shortly after birth and is a complex and dynamic population of bacteria that fluctuates in response to environmental changes. Members of the vaginal microbiota play an important role in the nonspecific defense against vaginal infections and sexually transmitted infections by occupying cellular binding sites and competing for nutrients. In addition, the production of lactic acid by members of the microbiota provides an acidic environment within the vagina that also serves as a defense against infections. For the majority of women, the lactic-acid–producing bacteria in the vagina are dominated by a variety of species of Lactobacillus. For women who lack sufficient lactobacilli in their vagina, lactic acid production comes primarily from other species of bacteria such as Leptotrichia spp., Megasphaera spp., and Atopobium vaginae. Lactobacillus spp. use glycogen from vaginal epithelial cells for metabolism and production of lactic acid. This process is tightly regulated by the hormone estrogen. Increased levels of estrogen correlate with increased levels of vaginal glycogen, increased production of lactic acid, and a lower vaginal pH. Therefore, decreases in estrogen during the menstrual cycle and with menopause are associated with decreased levels of vaginal glycogen and lactic acid, and a higher pH. In addition to producing lactic acid, Lactobacillus spp. also contribute to the defenses against infectious disease through their production of hydrogen peroxide and bacteriocins (antibacterial peptides).
Exercise \(3\)
What factors affect the microbiota of the female reproductive tract?
General Signs and Symptoms of Urogenital Infections
Infections of the urinary tract most commonly cause inflammation of the bladder (cystitis) or of the urethra (urethritis). Urethritis can be associated with cystitis, but can also be caused by sexually transmitted infections. Symptoms of urethritis in men include burning sensation while urinating, discharge from the penis, and blood in the semen or the urine. In women, urethritis is associated with painful and frequent urination, vaginal discharge, fever, chills, and abdominal pain. The symptoms of cystitis are similar to those of urethritis. When urethritis is caused by a sexually transmitted pathogen, additional symptoms involving the genitalia can occur. These can include painful vesicles (blisters), warts, and ulcers. Ureteritis, a rare infection of the ureter, can also occur with cystitis. These infections can be acute or chronic.
Pyelonephritis and glomerulonephritis are infections of the kidney that are potentially serious. Pyelonephritis is an infection of one or both of the kidneys and may develop from a lower urinary tract infection; the upper urinary tract, including the ureters, is often affected. Signs and symptoms of pyelonephritis include fever, chills, nausea, vomiting, lower back pain, and frequent painful urination. Pyelonephritis usually only becomes chronic in individuals who have malformations in or damage to the kidneys.
Glomerulonephritis is an inflammation of the glomeruli of the nephrons. Symptoms include excessive protein and blood in urine, increased blood pressure, and fluid retention leading to edema of face, hands, and feet. Glomerulonephritis may be an acute infection or it can become chronic.
Infections occurring within the reproductive structures of males include epididymitis, orchitis, and prostatitis. Bacterial infections may cause inflammation of the epididymis, called epididymitis. This inflammation causes pain in the scrotum, testicles, and groin; swelling, redness, and warm skin in these areas may also be observed. Inflammation of the testicle, called orchitis, is usually caused by a bacterial infection spreading from the epididymis, but it can also be a complication of mumps, a viral disease. The symptoms are similar to those of epididymitis, and it is not uncommon for them both to occur together, in which case the condition is called epididymo-orchitis. Inflammation of the prostate gland, called prostatitis, can result from a bacterial infection. The signs and symptoms of prostatitis include fever, chills, and pain in the bladder, testicles, and penis. Patients may also experience burning during urination, difficulty emptying the bladder, and painful ejaculation.
Because of its proximity to the exterior, the vagina is a common site for infections in women. The general term for any inflammation of the vagina is vaginitis. Vaginitis often develops as a result of an overgrowth of bacteria or fungi that normally reside in the vaginal microbiota, although it can also result from infections by transient pathogens. Bacterial infections of the vagina are called bacterial vaginosis, whereas fungal infections (typically involving Candida spp.) are called yeast infections. Dynamic changes affecting the normal microbiota, acid production, and pH variations can be involved in the initiation of the microbial overgrowth and the development of vaginitis. Although some individuals may have no symptoms, vaginosis and vaginitis can be associated with discharge, odor, itching, and burning.
Pelvic inflammatory disease (PID) is an infection of the female reproductive organs including the uterus, cervix, fallopian tubes, and ovaries. The two most common pathogens are the sexually transmitted bacterial pathogens Neisseria gonorrhoeae and Chlamydia trachomatis. Inflammation of the fallopian tubes, called salpingitis, is the most serious form of PID. Symptoms of PID can vary between women and include pain in the lower abdomen, vaginal discharge, fever, chills, nausea, diarrhea, vomiting, and painful urination.
Exercise \(4\)
1. What conditions can result from infections affecting the urinary system?
2. What are some common causes of vaginitis in women?
General Causes and Modes of Transmission of Urogenital Infections
Hormonal changes, particularly shifts in estrogen in women due to pregnancy or menopause, can increase susceptibility to urogenital infections. As discussed earlier, estrogen plays an important role in regulating the availability of glycogen and subsequent production of lactic acid by Lactobacillus species. Low levels of estrogen are associated with an increased vaginal pH and an increased risk of bacterial vaginosis and yeast infections. Estrogen also plays a role in maintaining the elasticity, strength, and thickness of the vaginal wall, and keeps the vaginal wall lubricated, reducing dryness. Low levels of estrogen are associated with thinning of the vaginal wall. This thinning increases the risk of tears and abrasions, which compromise the protective barrier and increase susceptibility to pathogens.
Another common cause of urogenital infections in females is fecal contamination that occurs because of the close proximity of the anus and the urethra. Escherichia coli, an important member of the digestive tract microbiota, is the most common cause of urinary tract infections (urethritis and cystitis) in women; it generally causes infection when it is introduced to the urethra in fecal matter. Good hygiene can reduce the risk of urinary tract infections by this route. In men, urinary tract infections are more commonly associated with other conditions, such as an enlarged prostate, kidney stones, or placement of a urinary catheter. All of these conditions impair the normal emptying of the bladder, which serves to flush out microbes capable of causing infection.
Infections that are transmitted between individuals through sexual contact are called sexually transmitted infections(STIs) or sexually transmitted diseases (STDs). (The CDC prefers the term STD, but WHO prefers STI,1 which encompasses infections that result in disease as well as those that are subclinical or asymptomatic.) STIs often affect the external genitalia and skin, where microbes are easily transferred through physical contact. Lymph nodes in the genital region may also become swollen as a result of infection. However, many STIs have systemic effects as well, causing symptoms that range from mild (e.g., general malaise) to severe (e.g., liver damage or serious immunosuppression).
Exercise \(5\)
1. What role does Lactobacillus play in the health of the female reproductive system?
2. Why do urinary tract infections have different causes in males and females?
Key Concepts and Summary
• The urinary system is responsible for filtering the blood, excreting wastes, and helping to regulate electrolyte and water balance.
• The urinary system includes the kidneys, ureters, urinary bladder, and urethra; the bladder and urethra are the most common sites of infection.
• Common sites of infection in the male reproductive system include the urethra, as well as the testes, prostate and epididymis.
• The most common sites of infection in the female reproductive system are the vulva, vagina, cervix, and fallopian tubes.
• Infections of the urogenital tract can occur through colonization from the external environment, alterations in microbiota due to hormonal or other physiological and environmental changes, fecal contamination, and sexual transmission (STIs).
Footnotes
1. 1 World Health Organization. “Guidelines for the Management of Sexually Transmitted Infections.” World Health Organization, 2003. http://www.who.int/hiv/pub/sti/en/ST...elines2003.pdf. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/23%3A_Urogenital_System_Infections/23.01%3A_Anatomy_and_Normal_Microbiota_of_the_Urogenital_Tract.txt |
Learning Objectives
• Identify the most common bacterial pathogens that can cause urinary tract infections
• Compare the major characteristics of specific bacterial diseases affecting the urinary tract
Urinary tract infections (UTIs) include infections of the urethra, bladder, and kidneys, and are common causes of urethritis, cystitis, pyelonephritis, and glomerulonephritis. Bacteria are the most common causes of UTIs, especially in the urethra and bladder.
Cystitis
Cystitis is most often caused by a bacterial infection of the bladder, but it can also occur as a reaction to certain treatments or irritants such as radiation treatment, hygiene sprays, or spermicides. Common symptoms of cystitis include dysuria (urination accompanied by burning, discomfort, or pain), pyuria (pus in the urine), hematuria (blood in the urine), and bladder pain.
In women, bladder infections are more common because the urethra is short and located in close proximity to the anus, which can result in infections of the urinary tract by fecal bacteria. Bladder infections are also more common in the elderly because the bladder may not empty fully, causing urine to pool; the elderly may also have weaker immune systems that make them more vulnerable to infection. Conditions such as prostatitis in men or kidney stones in both men and women can impact proper drainage of urine and increase risk of bladder infections. Catheterization can also increase the risk of bladder infection (see Case in Point: Cystitis in the Elderly).
Gram-negative bacteria such as Escherichia coli (most commonly), Proteus vulgaris, Pseudomonas aeruginosa, and Klebsiella pneumoniae cause most bladder infections. Gram-positive pathogens associated with cystitis include the coagulase-negative Staphylococcus saprophyticus, Enterococcus faecalis, and Streptococcus agalactiae. Routine manual urinalysis using a urine dipstick or test strip can be used for rapid screening of infection. These test strips (Figure \(1\)) are either held in a urine stream or dipped in a sample of urine to test for the presence of nitrites, leukocyte esterase, protein, or blood that can indicate an active bacterial infection. The presence of nitrite may indicate the presence of E. coli or K. pneumonia; these bacteria produce nitrate reductase, which converts nitrate to nitrite. The leukocyte esterase (LE) test detects the presence of neutrophils as an indication of active infection.
Low specificity, sensitivity, or both, associated with these rapid screening tests require that care be taken in interpretation of results and in their use in diagnosis of urinary tract infections. Therefore, positive LE or nitrite results are followed by a urine culture to confirm a bladder infection. Urine culture is generally accomplished using blood agar and MacConkey agar, and it is important to culture a clean catch of urine to minimize contamination with normal microbiota of the penis and vagina. A clean catch of urine is accomplished by first washing the labia and urethral opening of female patients or the penis of male patients. The patient then releases a small amount of urine into the toilet bowl before stopping the flow of urine. Finally, the patient resumes urination, this time filling the container used to collect the specimen.
Bacterial cystitis is commonly treated with fluoroquinolones, nitrofurantoin, cephalosporins, or a combination of trimethoprim and sulfamethoxazole. Pain medications may provide relief for patients with dysuria. Treatment is more difficult in elderly patients, who experience a higher rate of complications such as sepsis and kidney infections.
Case in Point: Cystitis in the Elderly
Robert, an 81-year-old widower with early onset Alzheimer’s, was recently moved to a nursing home because he was having difficulty living on his own. Within a few weeks of his arrival, he developed a fever and began to experience pain associated with urination. He also began having episodes of confusion and delirium. The doctor assigned to examine Robert read his file and noticed that Robert was treated for prostatitis several years earlier. When he asked Robert how often he had been urinating, Robert explained that he had been trying not to drink too much so that he didn’t have to walk to the restroom.
All of this evidence suggests that Robert likely has a urinary tract infection. Robert’s age means that his immune system has probably begun to weaken, and his previous prostate condition may be making it difficult for him to empty his bladder. In addition, Robert’s avoidance of fluids has led to dehydration and infrequent urination, which may have allowed an infection to establish itself in his urinary tract. The fever and dysuria are common signs of a UTI in patients of all ages, and UTIs in elderly patients are often accompanied by a notable decline in mental function.
Physical challenges often discourage elderly individuals from urinating as frequently as they would otherwise. In addition, neurological conditions that disproportionately affect the elderly (e.g., Alzheimer’s and Parkinson’s disease) may also reduce their ability to empty their bladders. Robert’s doctor noted that he was having difficulty navigating his new home and recommended that he be given more assistance and that his fluid intake be monitored. The doctor also took a urine sample and ordered a laboratory culture to confirm the identity of the causative agent.
Exercise \(1\)
1. Why is it important to identify the causative agent in a UTI?
2. Should the doctor prescribe a broad-spectrum or narrow-spectrum antibiotic to treat Robert’s UTI? Why?
Kidney Infections (Pyelonephritis and Glomerulonephritis)
Pyelonephritis, an inflammation of the kidney, can be caused by bacteria that have spread from other parts of the urinary tract (such as the bladder). In addition, pyelonephritis can develop from bacteria that travel through the bloodstream to the kidney. When the infection spreads from the lower urinary tract, the causative agents are typically fecal bacteria such as E. coli. Common signs and symptoms include back pain (due to the location of the kidneys), fever, and nausea or vomiting. Gross hematuria (visible blood in the urine) occurs in 30–40% of women but is rare in men.1 The infection can become serious, potentially leading to bacteremia and systemic effects that can become life-threatening. Scarring of the kidney can occur and persist after the infection has cleared, which may lead to dysfunction.
Diagnosis of pyelonephritis is made using microscopic examination of urine, culture of urine, testing for leukocyte esterase and nitrite levels, and examination of the urine for blood or protein. It is also important to use blood cultures to evaluate the spread of the pathogen into the bloodstream. Imaging of the kidneys may be performed in high-risk patients with diabetes or immunosuppression, the elderly, patients with previous renal damage, or to rule out an obstruction in the kidney. Pyelonephritis can be treated with either oral or intravenous antibiotics, including penicillins, cephalosporins, vancomycin, fluoroquinolones, carbapenems, and aminoglycosides.
Glomerulonephritis occurs when the glomeruli of the nephrons are damaged from inflammation. Whereas pyelonephritis is usually acute, glomerulonephritis may be acute or chronic. The most well-characterized mechanism of glomerulonephritis is the post-streptococcal sequelae associated with Streptococcus pyogenes throat and skin infections. Although S. pyogenes does not directly infect the glomeruli of the kidney, immune complexes that form in blood between S. pyogenes antigens and antibodies lodge in the capillary endothelial cell junctions of the glomeruli and trigger a damaging inflammatory response. Glomerulonephritis can also occur in patients with bacterial endocarditis(infection and inflammation of heart tissue); however, it is currently unknown whether glomerulonephritis associated with endocarditis is also immune-mediated.
Leptospirosis
Leptospira are generally harmless spirochetes that are commonly found in the soil. However, some pathogenic species can cause an infection called leptospirosis in the kidneys and other organs (Figure \(2\)). Leptospirosis can produce fever, headache, chills, vomiting, diarrhea, and rash with severe muscular pain. If the disease continues to progress, infection of the kidney, meninges, or liver may occur and may lead to organ failure or meningitis. When the kidney and liver become seriously infected, it is called Weil’s disease. Pulmonary hemorrhagic syndrome can also develop in the lungs, and jaundice may occur.
Leptospira spp. are found widely in animals such as dogs, horses, cattle, pigs, and rodents, and are excreted in their urine. Humans generally become infected by coming in contact with contaminated soil or water, often while swimming or during flooding; infection can also occur through contact with body fluids containing the bacteria. The bacteria may enter the body through mucous membranes, skin injuries, or by ingestion. The mechanism of pathogenicity is not well understood.
Leptospirosis is extremely rare in the United States, although it is endemic in Hawaii; 50% of all cases in the United States come from Hawaii.2 It is more common in tropical than in temperate climates, and individuals who work with animals or animal products are most at risk. The bacteria can also be cultivated in specialized media, with growth observed in broth in a few days to four weeks; however, diagnosis of leptospirosis is generally made using faster methods, such as detection of antibodies to Leptospira spp. in patient samples using serologic testing. Polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), slide agglutination, and indirect immunofluorescence tests may all be used for diagnosis. Treatment for leptospirosis involves broad-spectrum antibiotics such as penicillin and doxycycline. For more serious cases of leptospirosis, antibiotics may be given intravenously.
Exercise \(2\)
• What is the most common cause of a kidney infection?
• What are the most common symptoms of a kidney infection?
Nongonococcal Urethritis (NGU)
There are two main categories of bacterial urethritis: gonorrheal and nongonococcal. Gonorrheal urethritis is caused by Neisseria gonorrhoeae and is associated with gonorrhea, a common STI. This cause of urethritis will be discussed in Bacterial Infections of the Reproductive System. The term nongonococcal urethritis (NGU) refers to inflammation of the urethra that is unrelated to N. gonorrhoeae. In women, NGU is often asymptomatic. In men, NGU is typically a mild disease, but can lead to purulent discharge and dysuria. Because the symptoms are often mild or nonexistent, most infected individuals do not know that they are infected, yet they are carriers of the disease. Asymptomatic patients also have no reason to seek treatment, and although not common, untreated NGU can spread to the reproductive organs, causing pelvic inflammatory disease and salpingitis in women and epididymitis and prostatitis in men. Important bacterial pathogens that cause nongonococcal urethritis include Chlamydia trachomatis, Mycoplasma genitalium, Ureaplasma urealyticum, and Mycoplasma hominis.
C. trachomatis is a difficult-to-stain, gram-negative bacterium with an ovoid shape. An intracellular pathogen, C. trachomatis causes the most frequently reported STI in the United States, chlamydia. Although most persons infected with C. trachomatis are asymptomatic, some patients can present with NGU. C. trachomatis can also cause non-urogenital infections such as the ocular disease trachoma (see Bacterial Infections of the Skin and Eyes). The life cycle of C. trachomatis is illustrated in Figure 4.2.2.
C. trachomatis has multiple possible virulence factors that are currently being studied to evaluate their roles in causing disease. These include polymorphic outer-membrane autotransporter proteins, stress response proteins, and type III secretion effectors. The type III secretion effectors have been identified in gram-negative pathogens, including C. trachomatis. This virulence factor is an assembly of more than 20 proteins that form what is called an injectisome for the transfer of other effector proteins that target the infected host cells. The outer-membrane autotransporter proteins are also an effective mechanism of delivering virulence factors involved in colonization, disease progression, and immune system evasion.
Other species associated with NGU include Mycoplasma genitalium, Ureaplasma urealyticum, and Mycoplasma hominis. These bacteria are commonly found in the normal microbiota of healthy individuals, who may acquire them during birth or through sexual contact, but they can sometimes cause infections leading to urethritis (in males and females) or vaginitis and cervicitis (in females).
M. genitalium is a more common cause of urethritis in most settings than N. gonorrhoeae, although it is less common than C. trachomatis. It is responsible for approximately 30% of recurrent or persistent infections, 20–25% of nonchlamydial NGU cases, and 15%–20% of NGU cases. M. genitalium attaches to epithelial cells and has substantial antigenic variation that helps it evade host immune responses. It has lipid-associated membrane proteins that are involved in causing inflammation.
Several possible virulence factors have been implicated in the pathogenesis of U. urealyticum (Figure \(3\)). These include the ureaplasma proteins phospholipase A, phospholipase C, multiple banded antigen (MBA), urease, and immunoglobulin α protease. The phospholipases are virulence factors that damage the cytoplasmic membrane of target cells. The immunoglobulin α protease is an important defense against antibodies. It can generate hydrogen peroxide, which may adversely affect host cell membranes through the production of reactive oxygen species.
Treatments differ for gonorrheal and nongonococcal urethritis. However, N. gonorrhoeae and C. trachomatis are often simultaneously present, which is an important consideration for treatment. NGU is most commonly treated using tetracyclines (such as doxycycline) and azithromycin; erythromycin is an alternative option. Tetracyclines and fluoroquinolones are most commonly used to treat U. urealyticum, but resistance to tetracyclines is becoming an increasing problem.3 While tetracyclines have been the treatment of choice for M. hominis, increasing resistance means that other options must be used. Clindamycin and fluoroquinolones are alternatives. M. genitalium is generally susceptible to doxycycline, azithromycin, and moxifloxacin. Like other mycoplasma, M. genitalium does not have a cell wall and therefore β-lactams (including penicillins and cephalosporins) are not effective treatments.
Exercise \(3\)
1. What are the three most common causes of urethritis?
2. What three members of the normal microbiota can cause urethritis?
Bacterial Infections of the Urinary Tract
Urinary tract infections can cause inflammation of the urethra (urethritis), bladder (cystitis), and kidneys (pyelonephritis), and can sometimes spread to other body systems through the bloodstream. Figure \(4\) captures the most important features of various types of UTIs.
Key Concepts and Summary
• Bacterial cystitis is commonly caused by fecal bacteria such as E. coli.
• Pyelonephritis is a serious kidney infection that is often caused by bacteria that travel from infections elsewhere in the urinary tract and may cause systemic complications.
• Leptospirosis is a bacterial infection of the kidney that can be transmitted by exposure to infected animal urine, especially in contaminated water. It is more common in tropical than in temperate climates.
• Nongonococcal urethritis (NGU) is commonly caused by C. trachomatis, M. genitalium, Ureaplasma urealyticum, and M. hominis.
• Diagnosis and treatment for bacterial urinary tract infections varies. Urinalysis (e.g., for leukocyte esterase levels, nitrite levels, microscopic evaluation, and culture of urine) is an important component in most cases. Broad-spectrum antibiotics are typically used.
Footnotes
1. 1 Tibor Fulop. “Acute Pyelonephritis” Medscape, 2015. http://emedicine.medscape.com/article/245559-overview.
2. 2 Centers for Disease Control and Prevention. “Leptospirosis.” 2015. http://www.cdc.gov/leptospirosis/health_care_workers.
3. 3 Ken B Waites. “Ureaplasma Infection Medication.” Medscape, 2015. emedicine.medscape.com/articl...470-medication. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/23%3A_Urogenital_System_Infections/23.02%3A_Bacterial_Infections_of_the_Urinary_System.txt |
Learning Objectives
• Identify the most common bacterial pathogens that can cause infections of the reproductive system
• Compare the major characteristics of specific bacterial diseases affecting the reproductive system
In addition to infections of the urinary tract, bacteria commonly infect the reproductive tract. As with the urinary tract, parts of the reproductive system closest to the external environment are the most likely sites of infection. Often, the same microbes are capable of causing urinary tract and reproductive tract infections.
Bacterial Vaginitis and Vaginosis
Inflammation of the vagina is called vaginitis, often caused by a bacterial infection. It is also possible to have an imbalance in the normal vaginal microbiota without inflammation called bacterial vaginosis (BV). Vaginosis may be asymptomatic or may cause mild symptoms such as a thin, white-to-yellow, homogeneous vaginal discharge, burning, odor, and itching. The major causative agent is Gardnerella vaginalis, a gram-variable to gram-negative pleomorphic bacterium. Other causative agents include anaerobic species such as members of the genera Bacteroides and Fusobacterium. Additionally, ureaplasma and mycoplasma may be involved. The disease is usually self-limiting, although antibiotic treatment is recommended if symptoms develop.
G. vaginalis appears to be more virulent than other vaginal bacterial species potentially associated with BV. Like Lactobacillus spp., G. vaginalis is part of the normal vaginal microbiota, but when the population of Lactobacillus spp. decreases and the vaginal pH increases, G. vaginalis flourishes, causing vaginosis by attaching to vaginal epithelial cells and forming a thick protective biofilm. G. vaginalis also produces a cytotoxin called vaginolysin that lyses vaginal epithelial cells and red blood cells.Since G. vaginalis can also be isolated from healthy women, the “gold standard” for the diagnosis of BV is direct examination of vaginal secretions and not the culture of G. vaginalis. Diagnosis of bacterial vaginosis from vaginal secretions can be accurately made in three ways. The first is to use a DNA probe. The second method is to assay for sialidase activity (sialidase is an enzyme produced by G. vaginalis and other bacteria associated with vaginosis, including Bacteroides spp., Prevotella spp., and Mobiluncus spp.). The third method is to assess gram-stained vaginal smears for microscopic morphology and relative numbers and types of bacteria, squamous epithelial cells, and leukocytes. By examining slides prepared from vaginal swabs, it is possible to distinguish lactobacilli (long, gram-positive rods) from other gram-negative species responsible for BV. A shift in predominance from gram-positive bacilli to gram-negative coccobacilli can indicate BV. Additionally, the slide may contain so-called clue cells, which are epithelial cells that appear to have a granular or stippled appearance due to bacterial cells attached to their surface (Figure \(1\)).
Presumptive diagnosis of bacterial vaginosis can involve an assessment of clinical symptoms and evaluation of vaginal fluids using Amsel’s diagnostic criteria which include 3 out of 4 of the following characteristics:
1. white to yellow discharge;
2. a fishy odor, most noticeable when 10% KOH is added;
3. pH greater than 4.5;
4. the presence of clue cells.
Treatment is often unnecessary because the infection often clears on its own. However, in some cases, antibiotics such as topical or oral clindamycin or metronidazole may be prescribed. Alternative treatments include oral tinidazole or clindamycin ovules (vaginal suppositories).
Exercise \(1\)
1. Explain the difference between vaginosis and vaginitis.
2. What organisms are responsible for vaginosis and what organisms typically hold it at bay?
Clinical Focus: Part 2
There is no catch-all test for STIs, so several tests, in addition to a physical exam, are necessary to diagnose an infection. Nadia tries to relax in the exam room while she waits for the doctor to return, but she is nervous about the results.
When the doctor finally returns, she has some unexpected news: Nadia is pregnant. Surprised and excited, Nadia wants to know if the pregnancy explains her unusual symptoms. The doctor explains that the irritation that Nadia is experiencing is vaginitis, which can be caused by several types of microorganisms. One possibility is bacterial vaginosis, which develops when there is an imbalance in the bacteria in the vagina, as often occurs during pregnancy. Vaginosis can increase the risk of preterm birth and low birth weight, and a few studies have also shown that it can cause second-trimester miscarriage; however, the condition can be treated. To check for it, the doctor has asked the lab to perform a Gram stain on Nadia’s sample.
Exercise \(2\)
• What result would you expect from the Gram stain if Nadia has bacterial vaginosis?
• What is the relationship between pregnancy, estrogen levels, and development of bacterial vaginosis?
Gonorrhea
Also known as the clap, gonorrhea is a common sexually transmitted disease of the reproductive system that is especially prevalent in individuals between the ages of 15 and 24. It is caused by Neisseria gonorrhoeae, often called gonococcus or GC, which have fimbriae that allow the cells to attach to epithelial cells. It also has a type of lipopolysaccharide endotoxin called lipooligosaccharide as part of the outer membrane structure that enhances its pathogenicity. In addition to causing urethritis, N. gonorrhoeae can infect other body tissues such as the skin, meninges, pharynx, and conjunctiva.
Many infected individuals (both men and women) are asymptomatic carriers of gonorrhea. When symptoms do occur, they manifest differently in males and females. Males may develop pain and burning during urination and discharge from the penis that may be yellow, green, or white (Figure \(2\)). Less commonly, the testicles may become swollen or tender. Over time, these symptoms can increase and spread. In some cases, chronic infection develops. The disease can also develop in the rectum, causing symptoms such as discharge, soreness, bleeding, itching, and pain (especially in association with bowel movements).
Women may develop pelvic pain, discharge from the vagina, intermenstrual bleeding (i.e., bleeding not associated with normal menstruation), and pain or irritation associated with urination. As with men, the infection can become chronic. In women, however, chronic infection can cause increases in menstrual flow. Rectal infection can also occur, with the symptoms previously described for men. Infections that spread to the endometrium and fallopian tubes can cause pelvic inflammatory disease (PID), characterized by pain in the lower abdominal region, dysuria, vaginal discharge, and fever. PID can also lead to infertility through scarring and blockage of the fallopian tubes (salpingitis); it may also increase the risk of a life-threatening ectopic pregnancy, which occurs when a fertilized egg begins developing somewhere other than the uterus (e.g., in the fallopian tube or ovary).
When a gonorrhea infection disseminates throughout the body, serious complications can develop. The infection may spread through the blood (bacteremia) and affect organs throughout the body, including the heart (gonorrheal endocarditis), joints (gonorrheal arthritis), and meninges encasing the brain (meningitis).
Urethritis caused by N. gonorrhoeae can be difficult to treat due to antibiotic resistance (see Micro Connections below). Some strains have developed resistance to the fluoroquinolones, so cephalosporins are often a first choice for treatment. Because co-infection with C. trachomatis is common, the CDC recommends treating with a combination regimen of ceftriaxone and azithromycin. Treatment of sexual partners is also recommended to avoid reinfection and spread of infection to others.1
Exercise \(3\)
1. What are some of the serious consequences of a gonorrhea infection?
2. What organism commonly coinfects with N. gonorrhoeae?
Antibiotic Resistance in Neisseria
Antibiotic resistance in many pathogens is steadily increasing, causing serious concern throughout the public health community. Increased resistance has been especially notable in some species, such as Neisseria gonorrhoeae. The CDC monitors the spread of antibiotic resistance in N. gonorrhoeae, which it classifies as an urgent threat, and makes recommendations for treatment. So far, N. gonorrhoeae has shown resistance to cefixime (a cephalosporin), ceftriaxone (another cephalosporin), azithromycin, and tetracycline. Resistance to tetracycline is the most common, and was seen in 188,600 cases of gonorrhea in 2011 (out of a total 820,000 cases). In 2011, some 246,000 cases of gonorrhea involved strains of N. gonorrhoeae that were resistant to at least one antibiotic.2 These resistance genes are spread by plasmids, and a single bacterium may be resistant to multiple antibiotics. The CDC currently recommends treatment with two medications, ceftriaxone and azithromycin, to attempt to slow the spread of resistance. If resistance to cephalosporins increases, it will be extremely difficult to control the spread of N. gonorrhoeae.
Chlamydia
Chlamydia trachomatis is the causative agent of the STI chlamydia (Figure \(3\)). While many Chlamydia infections are asymptomatic, chlamydia is a major cause of nongonococcal urethritis (NGU) and may also cause epididymitis and orchitis in men. In women, chlamydia infections can cause urethritis, salpingitis, and PID. In addition, chlamydial infections may be associated with an increased risk of cervical cancer.
Because chlamydia is widespread, often asymptomatic, and has the potential to cause substantial complications, routine screening is recommended for sexually active women who are under age 25, at high risk (i.e., not in a monogamous relationship), or beginning prenatal care.
Certain serovars of C. trachomatis can cause an infection of the lymphatic system in the groin known as lymphogranuloma venereum. This condition is commonly found in tropical regions and can also co-occur in conjunction with human immunodeficiency virus (HIV) infection. After the microbes invade the lymphatic system, buboes (large lymph nodes, see Figure \(3\)) form and can burst, releasing pus through the skin. The male genitals can become greatly enlarged and in women the rectum may become narrow.
Urogenital infections caused by C. trachomatis can be treated using azithromycin or doxycycline (the recommended regimen from the CDC). Erythromycin, levofloxacin, and ofloxacin are alternatives.
Exercise \(4\)
Compare the signs and symptoms of chlamydia infection in men and women.
Syphilis
Syphilis is spread through direct physical (generally sexual) contact, and is caused by the gram-negative spirochete Treponema pallidum. T. pallidum has a relatively simple genome and lacks lipopolysaccharide endotoxin characteristic of gram-negative bacteria. However, it does contain lipoproteins that trigger an immune response in the host, causing tissue damage that may enhance the pathogen’s ability to disseminate while evading the host immune system.
After entering the body, T. pallidum moves rapidly into the bloodstream and other tissues. If not treated effectively, syphilis progresses through three distinct stages: primary, secondary, and tertiary. Primary syphilis appears as a single lesion on the cervix, penis, or anus within 10 to 90 days of transmission. Such lesions contain many T. pallidum cells and are highly infectious. The lesion, called a hard chancre, is initially hard and painless, but it soon develops into an ulcerated sore (Figure \(4\)). Localized lymph node swelling may occur as well. In some cases, these symptoms may be relatively mild, and the lesion may heal on its own within two to six weeks. Because the lesions are painless and often occur in hidden locations (e.g., the cervix or anus), infected individuals sometimes do not notice them.
The secondary stage generally develops once the primary chancre has healed or begun to heal. Secondary syphilis is characterized by a rash that affects the skin and mucous membranes of the mouth, vagina, or anus. The rash often begins on the palms or the soles of the feet and spreads to the trunk and the limbs (Figure \(4\)). The rash may take many forms, such as macular or papular. On mucous membranes, it may manifest as mucus patches or white, wartlike lesions called condylomata lata. The rash may be accompanied by malaise, fever, and swelling of lymph nodes. Individuals are highly contagious in the secondary stage, which lasts two to six weeks and is recurrent in about 25% of cases.
After the secondary phase, syphilis can enter a latent phase, in which there are no symptoms but microbial levels remain high. Blood tests can still detect the disease during latency. The latent phase can persist for years.
Tertiary syphilis, which may occur 10 to 20 years after infection, produces the most severe symptoms and can be fatal. Granulomatous lesions called gummas may develop in a variety of locations, including mucous membranes, bones, and internal organs (Figure \(4\)). Gummas can be large and destructive, potentially causing massive tissue damage. The most deadly lesions are those of the cardiovascular system (cardiovascular syphilis) and the central nervous system (neurosyphilis). Cardiovascular syphilis can result in a fatal aortic aneurysm (rupture of the aorta) or coronary stenosis (a blockage of the coronary artery). Damage to the central nervous system can cause dementia, personality changes, seizures, general paralysis, speech impairment, loss of vision and hearing, and loss of bowel and bladder control.
The recommended methods for diagnosing early syphilis are darkfield or brightfield (silver stain) microscopy of tissue or exudate from lesions to detect T. pallidum (Figure \(5\)). If these methods are not available, two types of serologic tests (treponemal and nontreponemal) can be used for a presumptive diagnosis once the spirochete has spread in the body. Nontreponemal serologic tests include the Venereal Disease Research Laboratory (VDRL) and rapid plasma reagin(RPR) tests. These are similar screening tests that detect nonspecific antibodies (those for lipid antigens produced during infection) rather than those produced against the spirochete. Treponemal serologic tests measure antibodies directed against T. pallidum antigens using particle agglutination (T. pallidum passive particle agglutination or TP-PA), immunofluorescence (the fluorescent T. pallidum antibody absorption or FTA-ABS), various enzyme reactions (enzyme immunoassays or EIAs) and chemiluminescence immunoassays (CIA). Confirmatory testing, rather than screening, must be done using treponemal rather than nontreponemal tests because only the former tests for antibodies to spirochete antigens. Both treponemal and nontreponemal tests should be used (as opposed to just one) since both tests have limitations than can result in false positives or false negatives.
Neurosyphilis cannot be diagnosed using a single test. With or without clinical signs, it is generally necessary to assess a variety of factors, including reactive serologic test results, cerebrospinal fluid cell count abnormalities, cerebrospinal fluid protein abnormalities, or reactive VDRL-CSF (the VDRL test of cerebrospinal fluid). The VDRL-CSF is highly specific, but not sufficiently sensitive for conclusive diagnosis.
The recommended treatment for syphilis is parenteral penicillin G (especially long-acting benzathine penicillin, although the exact choice depends on the stage of disease). Other options include tetracycline and doxycycline.
Congenital Syphilis
Congenital syphilis is passed by mother to fetus when untreated primary or secondary syphilis is present. In many cases, infection may lead to miscarriage or stillbirth. Children born with congenital syphilis show symptoms of secondary syphilis and may develop mucus patches that deform the nose. In infants, gummas can cause significant tissue damage to organs and teeth. Many other complications may develop, such as osteochondritis, anemia, blindness, bone deformations, neurosyphilis, and cardiovascular lesions. Because congenital syphilis poses such a risk to the fetus, expectant mothers are screened for syphilis infection during the first trimester of pregnancy as part of the TORCH panel of prenatal tests.
Exercise \(5\)
1. What aspect of tertiary syphilis can lead to death?
2. How do treponemal serologic tests detect an infection?
Chancroid
The sexually transmitted infection chancroid is caused by the gram-negative rod Haemophilus ducreyi. It is characterized by soft chancres (Figure \(6\)) on the genitals or other areas associated with sexual contact, such as the mouth and anus. Unlike the hard chancres associated with syphilis, soft chancres develop into painful, open sores that may bleed or produce fluid that is highly contagious. In addition to causing chancres, the bacteria can invade the lymph nodes, potentially leading to pus discharge through the skin from lymph nodes in the groin. Like other genital lesions, soft chancres are of particular concern because they compromise the protective barriers of the skin or mucous membranes, making individuals more susceptible to HIV and other sexually transmitted diseases.
Several virulence factors have been associated with H. ducreyi, including lipooligosaccharides, protective outer membrane proteins, antiphagocytic proteins, secretory proteins, and collagen-specific adhesin NcaA. The collagen-specific adhesion NcaA plays an important role in initial cellular attachment and colonization. Outer membrane proteins DsrA and DltA have been shown to provide protection from serum-mediated killing by antibodies and complement.
H. ducreyi is difficult to culture; thus, diagnosis is generally based on clinical observation of genital ulcers and tests that rule out other diseases with similar ulcers, such as syphilis and genital herpes. PCR tests for H. ducreyi have been developed in some laboratories, but as of 2015 none had been cleared by the US Food and Drug Administration (FDA).3 Recommended treatments for chancroid include antibiotics such as azithromycin, ciprofloxacin, erythromycin and ceftriaxone. Resistance to ciprofloxacin and erythromycin has been reported.4
Exercise \(6\)
1. What is the key difference between chancroid lesions and those associated with syphilis?
2. Why is it difficult to definitively diagnose chancroid?
Bacterial Reproductive Tract Infections
Many bacterial infections affecting the reproductive system are transmitted through sexual contact, but some can be transmitted by other means. In the United States, gonorrhea and chlamydia are common illnesses with incidences of about 350,000 and 1.44 million, respectively, in 2014. Syphilis is a rarer disease with an incidence of 20,000 in 2014. Chancroid is exceedingly rare in the United States with only six cases in 2014 and a median of 10 cases per year for the years 2010–2014.5 Figure \(7\) summarizes bacterial infections of the reproductive tract.
Key Concepts and Summary
• Bacterial vaginosis is caused by an imbalance in the vaginal microbiota, with a decrease in lactobacilli and an increase in vaginal pH. G. vaginalis is the most common cause of bacterial vaginosis, which is associated with vaginal discharge, odor, burning, and itching.
• Gonorrhea is caused by N. gonorrhoeae, which can cause infection of the reproductive and urinary tracts and is associated with symptoms of urethritis. If left untreated, it can progress to epididymitis, salpingitis, and pelvic inflammatory disease and enter the bloodstream to infect other sites in the body.
• Chlamydia is the most commonly reported STI and is caused by C. trachomatis. Most infections are asymptomatic, and infections that are not treated can spread to involve the epididymis of men and cause salpingitis and pelvic inflammatory disease in women.
• Syphilis is caused by T. pallidum and has three stages, primary, secondary, and tertiary. Primary syphilis is associated with a painless hard chancre lesion on genitalia. Secondary syphilis is associated with skin and mucous membrane lesions. Tertiary syphilis is the most serious and life-threatening, and can involve serious nervous system damage.
• Chancroid is an infection of the reproductive tract caused by H. ducreyi that results in the development of characteristic soft chancres.
Footnotes
1. 1 Centers for Disease Control and Prevention. “2015 Sexually Transmitted Diseases Treatment Guidelines: Gonococcal Infections,” 2015. http://www.cdc.gov/std/tg2015/gonorrhea.htm.
2. 2 Centers for Disease Control and Prevention. “Antibiotic Resistance Threats in the United States, 2013,” 2013. http://www.cdc.gov/drugresistance/pd...s-2013-508.pdf.
3. 3 Centers for Disease Control and Prevention. “2015 Sexually Transmitted Diseases Treatment Guidelines: Chancroid,” 2015. http://www.cdc.gov/std/tg2015/chancroid.htm.
4. 4 Ibid.
5. 5 Centers for Disease Control and Prevention. “2014 Sexually Transmitted Disease Surveillance,” 2015. http://www.cdc.gov/std/stats14/default.htm. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/23%3A_Urogenital_System_Infections/23.03%3A_Bacterial_Infections_of_the_Reproductive_System.txt |
Learning Objectives
• Identify the most common viruses that cause infections of the reproductive system
• Compare the major characteristics of specific viral diseases affecting the reproductive system
Several viruses can cause serious problems for the human reproductive system. Most of these viral infections are incurable, increasing the risk of persistent sexual transmission. In addition, such viral infections are very common in the United States. For example, human papillomavirus (HPV) is the most common STI in the country, with an estimated prevalence of 79.1 million infections in 2008; herpes simplex virus 2 (HSV-2) is the next most prevalent STI at 24.1 million infections.1 In this section, we will examine these and other major viral infections of the reproductive system.
Genital Herpes
Genital herpes is a common condition caused by the herpes simplex virus (Figure \(1\)), an enveloped, double-stranded DNA virus that is classified into two distinct types. Herpes simplex virus has several virulence factors, including infected cell protein (ICP) 34.5, which helps in replication and inhibits the maturation of dendritic cells as a mechanism of avoiding elimination by the immune system. In addition, surface glycoproteins on the viral envelope promote the coating of herpes simplex virus with antibodies and complement factors, allowing the virus to appear as “self” and prevent immune system activation and elimination.
There are two herpes simplex virus types. While herpes simplex virus type 1 (HSV-1) is generally associated with oral lesions like cold sores or fever blisters (see Viral Infections of the Skin and Eyes), herpes simplex virus type 2 (HSV-2)is usually associated with genital herpes. However, both viruses can infect either location as well as other parts of the body. Oral-genital contact can spread either virus from the mouth to the genital region or vice versa.
Many infected individuals do not develop symptoms, and thus do not realize that they carry the virus. However, in some infected individuals, fever, chills, malaise, swollen lymph nodes, and pain precede the development of fluid-filled vesicles that may be irritating and uncomfortable. When these vesicles burst, they release infectious fluid and allow transmission of HSV. In addition, open herpes lesions can increase the risk of spreading or acquiring HIV.
In men, the herpes lesions typically develop on the penis and may be accompanied by a watery discharge. In women, the vesicles develop most commonly on the vulva, but may also develop on the vagina or cervix (Figure \(2\)). The symptoms are typically mild, although the lesions may be irritating or accompanied by urinary discomfort. Use of condoms may not always be an effective means of preventing transmission of genital herpes since the lesions can occur on areas other than the genitals.
Herpes simplex viruses can cause recurrent infections because the virus can become latent and then be reactivated. This occurs more commonly with HSV-2 than with HSV-1.2 The virus moves down peripheral nerves, typically sensory neurons, to ganglia in the spine (either the trigeminal ganglion or the lumbar-sacral ganglia) and becomes latent. Reactivation can later occur, causing the formation of new vesicles. HSV-2 most effectively reactivates from the lumbar-sacral ganglia. Not everyone infected with HSV-2 experiences reactivations, which are typically associated with stressful conditions, and the frequency of reactivation varies throughout life and among individuals. Between outbreaks or when there are no obvious vesicles, the virus can still be transmitted.
Virologic and serologic techniques are used for diagnosis. The virus may be cultured from lesions. The immunostaining methods that are used to detect virus from cultures generally require less expertise than methods based on cytopathic effect (CPE), as well as being a less expensive option. However, PCR or other DNA amplification methods may be preferred because they provide the most rapid results without waiting for culture amplification. PCR is also best for detecting systemic infections. Serologic techniques are also useful in some circumstances, such as when symptoms persist but PCR testing is negative.
While there is no cure or vaccine for HSV-2 infections, antiviral medications are available that manage the infection by keeping the virus in its dormant or latent phase, reducing signs and symptoms. If the medication is discontinued, then the condition returns to its original severity. The recommended medications, which may be taken at the start of an outbreak or daily as a method of prophylaxis, are acyclovir, famciclovir, and valacyclovir.
Neonatal Herpes
Herpes infections in newborns, referred to as neonatal herpes, are generally transmitted from the mother to the neonate during childbirth, when the child is exposed to pathogens in the birth canal. Infections can occur regardless of whether lesions are present in the birth canal. In most cases, the infection of the newborn is limited to skin, mucous membranes, and eyes, and outcomes are good. However, sometimes the virus becomes disseminated and spreads to the central nervous system, resulting in motor function deficits or death.
In some cases, infections can occur before birth when the virus crosses the placenta. This can cause serious complications in fetal development and may result in spontaneous abortion or severe disabilities if the fetus survives. The condition is most serious when the mother is infected with HSV for the first time during pregnancy. Thus, expectant mothers are screened for HSV infection during the first trimester of pregnancy as part of the TORCH panel of prenatal tests (see How Pathogens Cause Disease). Systemic acyclovir treatment is recommended to treat newborns with neonatal herpes.
Exercise \(1\)
1. Why are latent herpes virus infections still of clinical concern?
2. How is neonatal herpes contracted?
Human Papillomas
Warts of all types are caused by a variety of strains of human papillomavirus (HPV) (see Viral Infections of the Skin and Eyes). Condylomata acuminata, more commonly called genital warts or venereal warts (Figure \(3\)), are an extremely prevalent STI caused by certain strains of HPV. Condylomata are irregular, soft, pink growths that are found on external genitalia or the anus.
HPV is a small, non-enveloped virus with a circular double-stranded DNA genome. Researchers have identified over 200 different strains (called types) of HPV, with approximately 40 causing STIs. While some types of HPV cause genital warts, HPV infection is often asymptomatic and self-limiting. However, genital HPV infection often co-occurs with other STIs like syphilis or gonorrhea. Additionally, some forms of HPV (not the same ones associated with genital warts) are associated with cervical cancers. At least 14 oncogenic (cancer-causing) HPV types are known to have a causal association with cervical cancers. Examples of oncogenic HPV are types 16 and 18, which are associated with 70% of cervical cancers.3 Oncogenic HPV types can also cause oropharyngeal cancer, anal cancer, vaginal cancer, vulvar cancer, and penile cancer. Most of these cancers are caused by HPV type 16. HPV virulence factors include proteins (E6 and E7) that are capable of inactivating tumor suppressor proteins, leading to uncontrolled cell division and the development of cancer.
HPV cannot be cultured, so molecular tests are the primary method used to detect HPV. While routine HPV screening is not recommended for men, it is included in guidelines for women. An initial screening for HPV at age 30, conducted at the same time as a Pap test, is recommended. If the tests are negative, then further HPV testing is recommended every five years. More frequent testing may be needed in some cases. The protocols used to collect, transport, and store samples vary based on both the type of HPV testing and the purpose of the testing. This should be determined in individual cases in consultation with the laboratory that will perform the testing.
Because HPV testing is often conducted concurrently with Pap testing, the most common approach uses a single sample collection within one vial for both. This approach uses liquid-based cytology (LBC). The samples are then used for Pap smear cytology as well as HPV testing and genotyping. HPV can be recognized in Pap smears by the presence of cells called koilocytes (called koilocytosis or koilocytotic atypia). Koilocytes have a hyperchromatic atypical nucleus that stains darkly and a high ratio of nuclear material to cytoplasm. There is a distinct clear appearance around the nucleus called a perinuclear halo (Figure \(4\)).
Most HPV infections resolve spontaneously; however, various therapies are used to treat and remove warts. Topical medications such as imiquimod (which stimulates the production of interferon), podofilox, or sinecatechins, may be effective. Warts can also be removed using cryotherapy or surgery, but these approaches are less effective for genital warts than for other types of warts. Electrocauterization and carbon dioxide laser therapy are also used for wart removal.
Regular Pap testing can detect abnormal cells that might progress to cervical cancer, followed by biopsy and appropriate treatment. Vaccines for some of the high risk HPV types are now available. Gardasil vaccine includes types 6, 11, 16 and 18 (types 6 and 11 are associated with 90% of genital wart infections and types 16 and 18 are associated with 70% of cervical cancers). Gardasil 9 vaccinates against the previous four types and an additional five high-risk types (31, 33, 45, 52, and 58). Cervarix vaccine includes just HPV types 16 and 18. Vaccination is the most effective way to prevent infection with oncogenic HPV, but it is important to note that not all oncogenic HPV types are covered by the available vaccines. It is recommended for both boys and girls prior to sexual activity (usually between the ages of nine and fifteen).
Exercise \(2\)
1. What is diagnostic of an HPV infection in a Pap smear?
2. What is the motivation for HPV vaccination?
Secret STIs
Few people who have an STI (or think they may have one) are eager to share that information publicly. In fact, many patients are even uncomfortable discussing the symptoms privately with their doctors. Unfortunately, the social stigma associated with STIs makes it harder for infected individuals to seek the treatment they need and creates the false perception that STIs are rare. In reality, STIs are quite common, but it is difficult to determine exactly how common.
A recent study on the effects of HPV vaccination found a baseline HPV prevalence of 26.8% for women between the ages of 14 and 59. Among women aged 20–24, the prevalence was 44.8%; in other words, almost half of the women in this age bracket had a current infection.4 According to the CDC, HSV-2 infection was estimated to have a prevalence of 15.5% in younger individuals (14–49 years of age) in 2007–2010, down from 20.3% in the same age group in 1988–1994. However, the CDC estimates that 87.4% of infected individuals in this age group have not been diagnosed by a physician.5
Another complicating factor is that many STIs can be asymptomatic or have long periods of latency. For example, the CDC estimates that among women ages 14–49 in the United States, about 2.3 million (3.1%) are infected with the sexually transmitted protozoan Trichomonas (see Protozoan Infections of the Urogenital System); however, in a study of infected women, 85% of those diagnosed with the infection were asymptomatic.6
Even when patients are treated for symptomatic STIs, it can be difficult to obtain accurate data on the number of cases. Whereas STIs like chlamydia, gonorrhea, and syphilis are notifiable diseases—meaning each diagnosis must be reported by healthcare providers to the CDC—other STIs are not notifiable (e.g., genital herpes, genital warts, and trichomoniasis). Between the social taboos, the inconsistency of symptoms, and the lack of mandatory reporting, it can be difficult to estimate the true prevalence of STIs—but it is safe to say they are much more prevalent than most people think.
Viral Reproductive Tract Infections
Figure \(5\) summarizes the most important features of viral diseases affecting the human reproductive tract.
Key Concepts and Summary
• Genital herpes is usually caused by HSV-2 (although HSV-1 can also be responsible) and may cause the development of infectious, potentially recurrent vesicles.
• Neonatal herpes can occur in babies born to infected mothers and can cause symptoms that range from relatively mild (more common) to severe.
• Human papillomaviruses are the most common sexually transmitted viruses and include strains that cause genital warts as well as strains that cause cervical cancer.
Footnotes
1. 1 Catherine Lindsey Satterwhite, Elizabeth Torrone, Elissa Meites, Eileen F. Dunne, Reena Mahajan, M. Cheryl Bañez Ocfemia, John Su, Fujie Xu, and Hillard Weinstock. “Sexually Transmitted Infections Among US Women and Men: Prevalence and Incidence Estimates, 2008.” Sexually Transmitted Diseases 40, no. 3 (2013): 187–193.
2. 2 Centers for Disease Control and Prevention. “2015 Sexually Transmitted Disease Treatment Guidelines: Genital Herpes,” 2015. http://www.cdc.gov/std/tg2015/herpes.htm.
3. 3 Lauren Thaxton and Alan G. Waxman. “Cervical Cancer Prevention: Immunization and Screening 2015.” Medical Clinics of North America 99, no. 3 (2015): 469–477.
4. 4 Eileen F. Dunne, Elizabeth R. Unger, Maya Sternberg, Geraldine McQuillan, David C. Swan, Sonya S. Patel, and Lauri E. Markowitz. “Prevalence of HPV Infection Among Females in the United States.” Journal of the American Medical Association 297, no. 8 (2007): 813–819.
5. 5 Centers for Disease Control and Prevention. “Genital Herpes - CDC Fact Sheet,” 2015. www.cdc.gov/std/herpes/stdfac...s-detailed.htm.
6. 6 Centers for Disease Control and Prevention. “Trichomoniasis Statistics,” 2015. http://www.cdc.gov/std/trichomonas/stats.htm. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/23%3A_Urogenital_System_Infections/23.04%3A_Viral_Infections_of_the_Reproductive_System.txt |
Learning Objectives
• Summarize the important characteristics of vaginal candidiasis
Only one major fungal pathogen affects the urogenital system. Candida is a genus of fungi capable of existing in a yeast form or as a multicellular fungus. Candida spp. are commonly found in the normal, healthy microbiota of the skin, gastrointestinal tract, respiratory system, and female urogenital tract (Figure \(1\)). They can be pathogenic due to their ability to adhere to and invade host cells, form biofilms, secrete hydrolases (e.g., proteases, phospholipases, and lipases) that assist in their spread through tissues, and change their phenotypes to protect themselves from the immune system. However, they typically only cause disease in the female reproductive tract under conditions that compromise the host’s defenses. While there are at least 20 Candida species of clinical importance, C. albicans is the species most commonly responsible for fungal vaginitis.
As discussed earlier, lactobacilli in the vagina inhibit the growth of other organisms, including bacteria and Candida, but disruptions can allow Candida to increase in numbers. Typical disruptions include antibiotic therapy, illness (especially diabetes), pregnancy, and the presence of transient microbes. Immunosuppression can also play a role, and the severe immunosuppression associated with HIV infection often allows Candida to thrive. This can cause genital or vaginal candidiasis, a condition characterized by vaginitis and commonly known as a yeast infection. When a yeast infection develops, inflammation occurs along with symptoms of pruritus (itching), a thick white or yellow discharge, and odor.
Other forms of candidiasis include cutaneous candidiasis (see Mycoses of the Skin) and oral thrush (see Microbial Diseases of the Mouth and Oral Cavity). Although Candida spp. are found in the normal microbiota, Candida spp. may also be transmitted between individuals. Sexual contact is a common mode of transmission, although candidiasis is not considered an STI.
Diagnosis of vaginal candidiasis can be made using microscopic evaluation of vaginal secretions to determine whether there is an excess of Candida. Culturing approaches are less useful because Candida is part of the normal microbiota and will regularly appear. It is also easy to contaminate samples with Candida because it is so common, so care must be taken to handle clinical material appropriately. Samples can be refrigerated if there is a delay in handling. Candida is a dimorphic fungus, so it does not only exist in a yeast form; cultivation can be used to identify chlamydospores and pseudohyphae, which develop from germ tubes (Figure \(2\)). The presence of the germ tube can be used in a diagnostic test in which cultured yeast cells are combined with rabbit serum and observed after a few hours for the presence of germ tubes. Molecular tests are also available if needed. The Affirm VPII Microbial Identification Test, for instance, tests simultaneously for the vaginal microbes C. albicans, G. vaginalis (see Bacterial Infections of the Urinary System), and Trichomonas vaginalis (see Protozoan Infections of the Urogenital System).
Topical antifungal medications for vaginal candidiasis include butoconazole, miconazole, clotrimazole, tioconazole, and nystatin. Oral treatment with fluconazole can be used. There are often no clear precipitating factors for infection, so prevention is difficult.
Exercise \(1\)
1. What factors can lead to candidiasis?
2. How is candidiasis typically diagnosed?
Clinical Focus: Part 3
The Gram stain of Nadia’s vaginal smear showed that the concentration of lactobacilli relative to other species in Nadia’s vaginal sample was abnormally low. However, there were no clue cells visible, which suggests that the infection is not bacterial vaginosis. But a wet-mount slide showed an overgrowth of yeast cells, suggesting that the problem is candidiasis, or a yeast infection (Figure \(3\)). This, Nadia’s doctor assures her, is good news. Candidiasis is common during pregnancy and easily treatable.
Exercise \(2\)
Knowing that the problem is candidiasis, what treatments might the doctor suggest?
Key Concepts and Summary
• Candida spp. are typically present in the normal microbiota in the body, including the skin, respiratory tract, gastrointestinal tract, and female urogenital system.
• Disruptions in the normal vaginal microbiota can lead to an overgrowth of Candida, causing vaginal candidiasis.
• Vaginal candidiasis can be treated with topical or oral fungicides. Prevention is difficult. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/23%3A_Urogenital_System_Infections/23.05%3A_Fungal_Infections_of_the_Reproductive_System.txt |
Learning Objectives
• Identify the most common protozoan pathogen that causes infections of the reproductive system
• Summarize the important characteristics of trichomoniasis
Only one major protozoan species causes infections in the urogenital system. Trichomoniasis, or “trich,” is the most common nonviral STI and is caused by a flagellated protozoan Trichomonas vaginalis. T. vaginalis has an undulating membrane and, generally, an amoeboid shape when attached to cells in the vagina. In culture, it has an oval shape.
T. vaginalis is commonly found in the normal microbiota of the vagina. As with other vaginal pathogens, it can cause vaginitis when there is disruption to the normal microbiota. It is found only as a trophozoite and does not form cysts. T. vaginalis can adhere to cells using adhesins such as lipoglycans; it also has other cell-surface virulence factors, including tetraspanins that are involved in cell adhesion, motility, and tissue invasion. In addition, T. vaginalis is capable of phagocytosing other microbes of the normal microbiota, contributing to the development of an imbalance that is favorable to infection.
Both men and women can develop trichomoniasis. Men are generally asymptomatic, and although women are more likely to develop symptoms, they are often asymptomatic as well. When symptoms do occur, they are characteristic of urethritis. Men experience itching, irritation, discharge from the penis, and burning after urination or ejaculation. Women experience dysuria; itching, burning, redness, and soreness of the genitalia; and vaginal discharge. The infection may also spread to the cervix. Infection increases the risk of transmitting or acquiring HIV and is associated with pregnancy complications such as preterm birth.
Microscopic evaluation of wet mounts is an inexpensive and convenient method of diagnosis, but the sensitivity of this method is low (Figure \(1\)). Nucleic acid amplification testing (NAAT) is preferred due to its high sensitivity. Using wet mounts and then NAAT for those who initially test negative is one option to improve sensitivity. Samples may be obtained for NAAT using urine, vaginal, or endocervical specimens for women and with urine and urethral swabs for men. It is also possible to use other methods such as the OSOM Trichomonas Rapid Test (an immunochromatographic test that detects antigen) and a DNA probe test for multiple species associated with vaginitis (the Affirm VPII Microbial Identification Test discussed in section 23.5).1 T. vaginalis is sometimes detected on a Pap test, but this is not considered diagnostic due to high rates of false positives and negatives. The recommended treatment for trichomoniasis is oral metronidazole or tinidazole. Sexual partners should be treated as well.
Exercise \(1\)
What are the symptoms of trichomoniasis?
STIs and Privacy
For many STIs, it is common to contact and treat sexual partners of the patient. This is especially important when a new illness has appeared, as when HIV became more prevalent in the 1980s. But to contact sexual partners, it is necessary to obtain their personal information from the patient. This raises difficult questions. In some cases, providing the information may be embarrassing or difficult for the patient, even though withholding such information could put their sexual partner(s) at risk.
Legal considerations further complicate such situations. The Health Insurance Portability and Accountability Act (HIPPA), passed into law in 1996, sets the standards for the protection of patient information. It requires businesses that use health information, such as insurance companies and healthcare providers, to maintain strict confidentiality of patient records. Contacting a patient’s sexual partners may therefore violate the patient’s privacy rights if the patient’s diagnosis is revealed as a result.
From an ethical standpoint, which is more important: the patient’s privacy rights or the sexual partner’s right to know that they may be at risk of a sexually transmitted disease? Does the answer depend on the severity of the disease or are the rules universal? Suppose the physician knows the identity of the sexual partner but the patient does not want that individual to be contacted. Would it be a violation of HIPPA rules to contact the individual without the patient’s consent?
Questions related to patient privacy become even more complicated when dealing with patients who are minors. Adolescents may be reluctant to discuss their sexual behavior or health with a health professional, especially if they believe that healthcare professionals will tell their parents. This leaves many teens at risk of having an untreated infection or of lacking the information to protect themselves and their partners. On the other hand, parents may feel that they have a right to know what is going on with their child. How should physicians handle this? Should parents always be told even if the adolescent wants confidentiality? Does this affect how the physician should handle notifying a sexual partner?
Clinical Focus: Resolution
Vaginal candidiasis is generally treated using topical antifungal medications such as butoconazole, miconazole, clotrimazole, ticonozole, nystatin, or oral fluconazole. However, it is important to be careful in selecting a treatment for use during pregnancy. Nadia’s doctor recommended treatment with topical clotrimazole. This drug is classified as a category B drug by the FDA for use in pregnancy, and there appears to be no evidence of harm, at least in the second or third trimesters of pregnancy. Based on Nadia’s particular situation, her doctor thought that it was suitable for very short-term use even though she was still in the first trimester. After a seven-day course of treatment, Nadia’s yeast infection cleared. She continued with a normal pregnancy and delivered a healthy baby eight months later.
Higher levels of hormones during pregnancy can shift the typical microbiota composition and balance in the vagina, leading to high rates of infections such as candidiasis or vaginosis. Topical treatment has an 80–90% success rate, with only a small number of cases resulting in recurrent or persistent infections. Longer term or intermittent treatment is usually effective in these cases.
Fungal and Protozoan Reproductive Tract Infections
Figure \(2\) summarizes the most important features of candidiasis and trichomoniasis.
Link to Learning
Take an online quiz for a review of sexually transmitted infections.
Key Concepts and Summary
• Trichomoniasis is a common STI caused by Trichomonas vaginalis.
• T. vaginalis is common at low levels in the normal microbiota.
• Trichomoniasis is often asymptomatic. When symptoms develop, trichomoniasis causes urinary discomfort, irritation, itching, burning, discharge from the penis (in men), and vaginal discharge (in women).
• Trichomoniasis is treated with the antiflagellate drugs tinidazole and metronidazole.
Footnotes
1. 1 Association of Public Health Laboratories. “Advances in Laboratory Detection of Trichomonas vaginalis,” 2013. http://www.aphl.org/AboutAPHL/public...-vaginalis.pdf. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/23%3A_Urogenital_System_Infections/23.06%3A_Protozoan_Infections_of_the_Reproductive_System.txt |
23.1: Anatomy and Normal Microbiota of the Urogenital Tract
The urinary system is responsible for filtering the blood, excreting wastes, and helping to regulate electrolyte and water balance. The urinary system includes the kidneys, ureters, urinary bladder, and urethra; the bladder and urethra are the most common sites of infection. Common sites of infection in the male reproductive system include the urethra, as well as the testes, prostate and epididymis. The common sites of infection in females are the vulva, vagina, cervix, and fallopian tubes.
Multiple Choice
When it first leaves the kidney, urine flows through
1. the urinary bladder.
2. the urethra.
3. the ureter.
4. the glomeruli.
Answer
C
What part of the male urogenital tract is shared by the urinary and reproductive systems?
1. the prostate gland
2. the seminal vesicles
3. the vas deferens
4. the urethra
Answer
D
Fill in the Blank
The genus of bacteria found in the vagina that is important in maintaining a healthy environment, including an acidic pH, is _____.
Answer
Lactobacillus
Short Answer
When the microbial balance of the vagina is disrupted, leading to overgrowth of resident bacteria without necessarily causing inflammation, the condition is called _____.
Answer
vaginosis
Explain the difference between a sexually transmitted infection and a sexually transmitted disease.
Answer
An STI is a broader term, including colonization by organisms that may not necessarily cause disease.
In the figure shown here, where would cystitis occur?
Critical Thinking
Epidemiological data show that the use of antibiotics is often followed by cases of vaginosis or vaginitis in women. Can you explain this finding?
23.2: Bacterial Infections of the Urinary System
Urinary tract infections (UTIs) include infections of the urethra, bladder, and kidneys, and are common causes of urethritis, cystitis, pyelonephritis, and glomerulonephritis. Bacteria are the most common causes of UTIs, especially in the urethra and bladder. Bacterial cystitis is commonly caused by fecal bacteria such as E. coli. Pyelonephritis is a serious kidney infection that is often caused by bacteria that travel from infections elsewhere in the urinary tract.
Multiple Choice
Which species is not associated with NGU?
1. Neisseria gonorrhoeae
2. Mycoplasma hominis
3. Chlamydia trachomatis
4. Mycoplasma genitalium
Answer
A
A strain of bacteria associated with a bladder infection shows gram-negative rods. What species is most likely to be the causative agent?
1. Mycoplasma hominis
2. Escherichia coli
3. Neisseria gonorrhoeae
4. Chlamydia trachomatis
Answer
B
Fill in the Blank
Pyelonephritis is a potentially severe infection of the _____.
Answer
kidneys
What is pyuria?
Critical Thinking
What are some factors that would increase an individual’s risk of contracting leptospirosis?
23.3: Bacterial Infections of the Reproductive System
In addition to infections of the urinary tract, bacteria commonly infect the reproductive tract. As with the urinary tract, parts of the reproductive system closest to the external environment are the most likely sites of infection. Often, the same microbes are capable of causing urinary tract and reproductive tract infections. Bacterial vaginosis, Chlamydia, Gonorrhea, and Chancroid are diseases caused by bacteria.
Multiple Choice
Treponemal and non-treponemal serological testing can be used to test for
1. vaginosis.
2. chlamydia.
3. syphilis.
4. gonorrhea.
Answer
C
Lymphogranuloma venereum is caused by serovars of
1. Neisseria gonorrhoeae.
2. Chlamydia trachomatis.
3. Treponema pallidum.
4. Haemophilis ducreyi.
Answer
B
The latent stage of syphilis, which may last for years, can occur between
1. the secondary and tertiary stages.
2. the primary and secondary stages.
3. initial infection and the primary stage.
4. any of the three stages.
Answer
A
Based on its shape, which microbe is this?
(credit: modification of work by Centers for Disease Control and Prevention)
1. Neisseria gonorrhoeae
2. Chlamydia trachomatis
3. Treponema pallidum
4. Haemophilis ducreyi
Answer
C
Fill in the Blank
Soft chancres on the genitals are characteristic of the sexually transmitted disease known as _____.
Answer
chancroid
Short Answer
Compare gonococcal and nongonoccocal urethritis with respect to their symptoms and the pathogens that cause each disease.
Critical Thinking
Chlamydia is often asymptomatic. Why might it be important for an individual to know if he or she were infected?
Why does the CDC recommend a two-drug treatment regimen to cover both C. trachomatis and N. gonorrhoeae if testing to distinguish between the two is not available? Additionally, how does the two-drug treatment regimen address antibiotic resistance?
23.4: Viral Infections of the Reproductive System
Genital herpes is usually caused by HSV-2 (although HSV-1 can also be responsible) and may cause the development of infectious, potentially recurrent vesicles. Neonatal herpes can occur in babies born to infected mothers and can cause symptoms that range from relatively mild (more common) to severe. Human papillomaviruses are the most common sexually transmitted viruses and include strains that cause genital warts as well as strains that cause cervical cancer.
Multiple Choice
Genital herpes is most commonly caused by
1. herpes simplex virus 1.
2. varicella-zoster virus.
3. herpes simplex virus 2.
4. cytomegalovirus.
Answer
C
Koilocytes are characteristic of
1. cells infected with human papillomavirus
2. cells infected with herpes simplex virus 2
3. cells infected with all forms of herpesviruses
4. cervical cancer cells
Answer
A
Fill in the Blank
Condylomata are _____.
Answer
warts
Short Answer
Is it true that human papillomaviruses can always be detected by the presence of genital warts?
How is neonatal herpes transmitted?
Critical Thinking
Recently, studies have shown a reduction in the prevalence of some strains of HPV in younger women. What might be the reason for this?
23.5: Fungal Infections of the Reproductive System
Candida spp. are typically present in the normal microbiota in the body, including the skin, respiratory tract, gastrointestinal tract, and female urogenital system. Disruptions in the normal vaginal microbiota can lead to an overgrowth of Candida, causing vaginal candidiasis. Vaginal candidiasis can be treated with topical or oral fungicides. Prevention is difficult.
Multiple Choice
Which oral medication is recommended as an initial topical treatment for genital yeast infections?
1. penicillin
2. acyclovir
3. fluconazole
4. miconazole
Answer
D
Fill in the Blank
The most common Candida species associated with yeast infections is _____.
Answer
C. albicans
23.6: Protozoan Infections of the Reproductive System
Trichomoniasis is a common STI caused by Trichomonas vaginalis. T. vaginalis is common at low levels in the normal microbiota. Trichomoniasis is often asymptomatic. When symptoms develop, trichomoniasis causes urinary discomfort, irritation, itching, burning, discharge from the penis (in men), and vaginal discharge (in women). Trichomoniasis is treated with the antiflagellate drugs tinidazole and metronidazole.
Multiple Choice
What is the only common infection of the reproductive tract caused by a protozoan?
1. gonorrhea
2. chlamydia
3. trichomoniasis
4. candidiasis
Answer
C
Which test is preferred for detecting T. vaginalis because of its high sensitivity?
1. NAAT
2. wet mounts
3. Pap tests
4. all of the above are equally good
Answer
A
Fill in the Blank
Trichomoniasis is caused by _____.
Answer
Trichomonas vaginalis
Short Answer
Name three organisms (a bacterium, a fungus, and a protozoan) that are associated with vaginitis. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/23%3A_Urogenital_System_Infections/23.E%3A_Urogenital_System_Infections_%28Exercises%29.txt |
Gastrointestinal (GI) diseases are so common that, unfortunately, most people have had first-hand experience with the unpleasant symptoms, such as diarrhea, vomiting, and abdominal discomfort. The causes of gastrointestinal illness can vary widely, but such diseases can be grouped into two categories: those caused by infection (the growth of a pathogen in the GI tract) or intoxication (the presence of a microbial toxin in the GI tract).
Foodborne pathogens like Escherichia coli O157:H7 are among the most common sources of gastrointestinal disease. Contaminated food and water have always posed a health risk for humans, but in today’s global economy, outbreaks can occur on a much larger scale. E. coli O157:H7 is a potentially deadly strain of E. coli with a history of contaminating meat and produce that are not properly processed. The source of an E. coli O157:H7 outbreak can be difficult to trace, especially if the contaminated food is processed in a foreign country. Once the source is identified, authorities may issue recalls of the contaminated food products, but by then there are typically numerous cases of food poisoning, some of them fatal.
• 24.1: Anatomy and Normal Microbiota of the Digestive System
The human digestive system, or the gastrointestinal (GI) tract, begins with the mouth and ends with the anus. The parts of the mouth include the teeth, the gums, the tongue, the oral vestibule (the space between the gums, lips, and teeth), and the oral cavity proper (the space behind the teeth and gums). Other parts of the GI tract are the pharynx, esophagus, stomach, small intestine, large intestine, rectum, and anus.
• 24.2: Microbial Diseases of the Mouth and Oral Cavity
Despite the presence of saliva and the mechanical forces of chewing and eating, some microbes thrive in the mouth. These microbes can cause damage to the teeth and can cause infections that have the potential to spread beyond the mouth and sometimes throughout the body.
• 24.3: Bacterial Infections of the Gastrointestinal Tract
Major causes of gastrointestinal illness include Salmonella spp., Staphylococcus spp., Helicobacter pylori, Clostridium perfringens, Clostridium difficile, Bacillus cereus, and Yersinia bacteria. C. difficile is an important cause of hospital acquired infection. Vibrio cholerae causes cholera, which can be a severe diarrheal illness. Different strains of E. coli, including ETEC, EPEC, EIEC, and EHEC, cause different illnesses with varying degrees of severity.
• 24.4: Viral Infections of the Gastrointestinal Tract
Common viral causes of gastroenteritis include rotaviruses, noroviruses, and astroviruses. Hepatitis may be caused by several unrelated viruses: hepatitis viruses A, B, C, D, and E. The hepatitis viruses differ in their modes of transmission, treatment, and potential for chronic infection.
• 24.5: Protozoan Infections of the Gastrointestinal Tract
Like other microbes, protozoa are abundant in natural microbiota but can also be associated with significant illness. Gastrointestinal diseases caused by protozoa are generally associated with exposure to contaminated food and water, meaning that those without access to good sanitation are at greatest risk. Even in developed countries, infections can occur and these microbes have sometimes caused significant outbreaks from contamination of public water supplies.
• 24.6: Helminthic Infections of the Gastrointestinal Tract
Helminths are widespread intestinal parasites. These parasites can be divided into three common groups: round-bodied worms also described as nematodes, flat-bodied worms that are segmented (also described as cestodes), and flat-bodied worms that are non-segmented (also described as trematodes). The nematodes include roundworms, pinworms, hookworms, and whipworms. Many of these parasites are so well adapted to the human host that there is little obvious disease.
• 24.E: Digestive System Infections (Exercises)
Thumbnail: This is an adult Taenia saginata tapeworm. Humans become infected by ingesting raw or undercooked infected meat. In the human intestine, the cysticercus develops over 2 mo. into an adult tapeworm, which can survive for years, attaching to, and residing in the small intestine. (Public Domain; UC CDC)
24: Digestive System Infections
Learning Objectives
• Describe the major anatomical features of the human digestive system
• Describe the normal microbiota of various regions in the human digestive system
• Explain how microorganisms overcome the defenses of the digestive tract to cause infection or intoxication
• Describe general signs and symptoms associated with infections of the digestive system
Clinical Focus: Part 1
After a morning of playing outside, four-year-old Carli ran inside for lunch. After taking a bite of her fried egg, she pushed it away and whined, “It’s too slimy, Mommy. I don’t want any more.” But her mother, in no mood for games, curtly replied that if she wanted to go back outside she had better finish her lunch. Reluctantly, Carli complied, trying hard not to gag as she choked down the runny egg.
That night, Carli woke up feeling nauseated. She cried for her parents and then began to vomit. Her parents tried to comfort her, but she continued to vomit all night and began to have diarrhea and run a fever. By the morning, her parents were very worried. They rushed her to the emergency room.
Exercise \(1\)
What could have caused Carli’s signs and symptoms?
The human digestive system, or the gastrointestinal (GI) tract, begins with the mouth and ends with the anus. The parts of the mouth include the teeth, the gums, the tongue, the oral vestibule (the space between the gums, lips, and teeth), and the oral cavity proper (the space behind the teeth and gums). Other parts of the GI tract are the pharynx, esophagus, stomach, small intestine, large intestine, rectum, and anus (Figure \(1\)). Accessory digestive organs include the salivary glands, liver, gallbladder, spleen, and pancreas.
The digestive system contains normal microbiota, including archaea, bacteria, fungi, protists, and even viruses. Because this microbiota is important for normal functioning of the digestive system, alterations to the microbiota by antibiotics or diet can be harmful. Additionally, the introduction of pathogens to the GI tract can cause infections and diseases. In this section, we will review the microbiota found in a healthy digestive tract and the general signs and symptoms associated with oral and GI infections.
Anatomy and Normal Microbiota of the Oral Cavity
Food enters the digestive tract through the mouth, where mechanical digestion (by chewing) and chemical digestion (by enzymes in saliva) begin. Within the mouth are the tongue, teeth, and salivary glands, including the parotid, sublingual, and submandibular glands (Figure \(2\)). The salivary glands produce saliva, which lubricates food and contains digestive enzymes.
The structure of a tooth (Figure \(3\)) begins with the visible outer surface, called the crown, which has to be extremely hard to withstand the force of biting and chewing. The crown is covered with enamel, which is the hardest material in the body. Underneath the crown, a layer of relatively hard dentin extends into the root of the tooth around the innermost pulp cavity, which includes the pulp chamber at the top of the tooth and pulp canal, or root canal, located in the root. The pulp that fills the pulp cavity is rich in blood vessels, lymphatic vessels, connective tissue, and nerves. The root of the tooth and some of the crown are covered with cementum, which works with the periodontal ligament to anchor the tooth in place in the jaw bone. The soft tissues surrounding the teeth and bones are called gums, or gingiva. The gingival space or gingival crevice is located between the gums and teeth.
Microbes such as bacteria and archaea are abundant in the mouth and coat all of the surfaces of the oral cavity. However, different structures, such as the teeth or cheeks, host unique communities of both aerobic and anaerobic microbes. Some factors appear to work against making the mouth hospitable to certain microbes. For example, chewing allows microbes to mix better with saliva so they can be swallowed or spit out more easily. Saliva also contains enzymes, including lysozyme, which can damage microbial cells. Recall that lysozyme is part of the first line of defense in the innate immune system and cleaves the β-(1,4) glycosidic linkages between N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) in bacterial peptidoglycan (see Chemical Defenses). Additionally, fluids containing immunoglobulins and phagocytic cells are produced in the gingival spaces. Despite all of these chemical and mechanical activities, the mouth supports a large microbial community.
Exercise \(2\)
What factors make the mouth inhospitable for certain microbes?
Anatomy and Normal Microbiota of the GI Tract
As food leaves the oral cavity, it travels through the pharynx, or the back of the throat, and moves into the esophagus, which carries the food from the pharynx to the stomach without adding any additional digestive enzymes. The stomach produces mucus to protect its lining, as well as digestive enzymes and acid to break down food. Partially digested food then leaves the stomach through the pyloric sphincter, reaching the first part of the small intestine called the duodenum. Pancreatic juice, which includes enzymes and bicarbonate ions, is released into the small intestine to neutralize the acidic material from the stomach and to assist in digestion. Bile, produced by the liver but stored in the gallbladder, is also released into the small intestine to emulsify fats so that they can travel in the watery environment of the small intestine. Digestion continues in the small intestine, where the majority of nutrients contained in the food are absorbed. Simple columnar epithelial cells called enterocytes line the lumen surface of the small intestinal folds called villi. Each enterocyte has smaller microvilli (cytoplasmic membrane extensions) on the cellular apical surface that increase the surface area to allow more absorption of nutrients to occur (Figure \(4\)).
Digested food leaves the small intestine and moves into the large intestine, or colon, where there is a more diverse microbiota. Near this junction, there is a small pouch in the large intestine called the cecum, which attaches to the appendix. Further digestion occurs throughout the colon and water is reabsorbed, then waste is excreted through the rectum, the last section of the colon, and out of the body through the anus (Figure \(1\)).
The environment of most of the GI tract is harsh, which serves two purposes: digestion and immunity. The stomach is an extremely acidic environment (pH 1.5–3.5) due to the gastric juices that break down food and kill many ingested microbes; this helps prevent infection from pathogens. The environment in the small intestine is less harsh and is able to support microbial communities. Microorganisms present in the small intestine can include lactobacilli, diptherioids and the fungus Candida. On the other hand, the large intestine (colon) contains a diverse and abundant microbiota that is important for normal function. These microbes include Bacteriodetes (especially the genera Bacteroides and Prevotella) and Firmicutes (especially members of the genus Clostridium). Methanogenic archaea and some fungi are also present, among many other species of bacteria. These microbes all aid in digestion and contribute to the production of feces, the waste excreted from the digestive tract, and flatus, the gas produced from microbial fermentation of undigested food. They can also produce valuable nutrients. For example, lactic acid bacteria such as bifidobacteria can synthesize vitamins, such as vitamin B12, folate, and riboflavin, that humans cannot synthesize themselves. E. coli found in the intestine can also break down food and help the body produce vitamin K, which is important for blood coagulation.
The GI tract has several other methods of reducing the risk of infection by pathogens. Small aggregates of underlying lymphoid tissue in the ileum, called Peyer’s patches (Figure \(4\)), detect pathogens in the intestines via microfold (M) cells, which transfer antigens from the lumen of the intestine to the lymphocytes on Peyer’s patches to induce an immune response. The Peyer’s patches then secrete IgA and other pathogen-specific antibodies into the intestinal lumen to help keep intestinal microbes at safe levels. Goblet cells, which are modified simple columnar epithelial cells, also line the GI tract (Figure \(5\)). Goblet cells secrete a gel-forming mucin, which is the major component of mucus. The production of a protective layer of mucus helps reduce the risk of pathogens reaching deeper tissues.
The constant movement of materials through the gastrointestinal tract also helps to move transient pathogens out of the body. In fact, feces are composed of approximately 25% microbes, 25% sloughed epithelial cells, 25% mucus, and 25% digested or undigested food. Finally, the normal microbiota provides an additional barrier to infection via a variety of mechanisms. For example, these organisms outcompete potential pathogens for space and nutrients within the intestine. This is known as competitive exclusion. Members of the microbiota may also secrete protein toxins known as bacteriocins that are able to bind to specific receptors on the surface of susceptible bacteria.
Exercise \(3\)
Compare and contrast the microbiota of the small and large intestines.
General Signs and Symptoms of Oral and GI Disease
Despite numerous defense mechanisms that protect against infection, all parts of the digestive tract can become sites of infection or intoxication. The term food poisoning is sometimes used as a catch-all for GI infections and intoxications, but not all forms of GI disease originate with foodborne pathogens or toxins.
In the mouth, fermentation by anaerobic microbes produces acids that damage the teeth and gums. This can lead to tooth decay, cavities, and periodontal disease, a condition characterized by chronic inflammation and erosion of the gums. Additionally, some pathogens can cause infections of the mucosa, glands, and other structures in the mouth, resulting in inflammation, sores, cankers, and other lesions. An open sore in the mouth or GI tract is typically called an ulcer.
Infections and intoxications of the lower GI tract often produce symptoms such as nausea, vomiting, diarrhea, aches, and fever. In some cases, vomiting and diarrhea may cause severe dehydration and other complications that can become serious or fatal. Various clinical terms are used to describe gastrointestinal symptoms. For example, gastritis is an inflammation of the stomach lining that results in swelling and enteritis refers to inflammation of the intestinal mucosa. When the inflammation involves both the stomach lining and the intestinal lining, the condition is called gastroenteritis. Inflammation of the liver is called hepatitis. Inflammation of the colon, called colitis, commonly occurs in cases of food intoxication. Because an inflamed colon does not reabsorb water as effectively as it normally does, stools become watery, causing diarrhea. Damage to the epithelial cells of the colon can also cause bleeding and excess mucus to appear in watery stools, a condition called dysentery.
Exercise \(4\)
List possible causes and signs and symptoms of food poisoning.
Key Concepts and Summary
• The digestive tract, consisting of the oral cavity, pharynx, esophagus, stomach, small intestine, and large intestine, has a normal microbiota that is important for health.
• The constant movement of materials through the gastrointestinal canal, the protective layer of mucus, the normal microbiota, and the harsh chemical environment in the stomach and small intestine help to prevent colonization by pathogens.
• Infections or microbial toxins in the oral cavity can cause tooth decay, periodontal disease, and various types of ulcers.
• Infections and intoxications of the gastrointestinal tract can cause general symptoms such as nausea, vomiting, diarrhea, and fever. Localized inflammation of the GI tract can result in gastritis, enteritis, gastroenteritis, hepatitis, or colitis, and damage to epithelial cells of the colon can lead to dysentery.
• Foodborne illness refers to infections or intoxications that originate with pathogens or toxins ingested in contaminated food or water. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/24%3A_Digestive_System_Infections/24.01%3A_Anatomy_and_Normal_Microbiota_of_the_Digestive_System.txt |
Learning Objectives
• Explain the role of microbial activity in diseases of the mouth and oral cavity
• Compare the major characteristics of specific oral diseases and infections
Despite the presence of saliva and the mechanical forces of chewing and eating, some microbes thrive in the mouth. These microbes can cause damage to the teeth and can cause infections that have the potential to spread beyond the mouth and sometimes throughout the body.
Dental Caries
Cavities of the teeth, known clinically as dental caries, are microbial lesions that cause damage to the teeth. Over time, the lesion can grow through the outer enamel layer to infect the underlying dentin or even the innermost pulp. If dental caries are not treated, the infection can become an abscess that spreads to the deeper tissues of the teeth, near the roots, or to the bloodstream.
Tooth decay results from the metabolic activity of microbes that live on the teeth. A layer of proteins and carbohydrates forms when clean teeth come into contact with saliva. Microbes are attracted to this food source and form a biofilmcalled plaque. The most important cariogenic species in these biofilms is Streptococcus mutans. When sucrose, a disaccharide sugar from food, is broken down by bacteria in the mouth, glucose and fructose are produced. The glucose is used to make dextran, which is part of the extracellular matrix of the biofilm. Fructose is fermented, producing organic acids such as lactic acid. These acids dissolve the minerals of the tooth, including enamel, even though it is the hardest material in the body. The acids work even more quickly on exposed dentin (Figure \(1\)). Over time, the plaque biofilm can become thick and eventually calcify. When a heavy plaque deposit becomes hardened in this way, it is called tartar or dental calculus (Figure \(2\)). These substantial plaque biofilms can include a variety of bacterial species, including Streptococcus and Actinomyces species.
Some tooth decay is visible from the outside, but it is not always possible to see all decay or the extent of the decay. X-ray imaging is used to produce radiographs that can be studied to look for deeper decay and damage to the root or bone (Figure \(2\)). If not detected, the decay can reach the pulp or even spread to the bloodstream. Painful abscesses can develop.
To prevent tooth decay, prophylactic treatment and good hygiene are important. Regular tooth brushing and flossing physically removes microbes and combats microbial growth and biofilm formation. Toothpaste contains fluoride, which becomes incorporated into the hydroxyapatite of tooth enamel, protecting it against acidity caused by fermentation of mouth microbiota. Fluoride is also bacteriostatic, thus slowing enamel degradation. Antiseptic mouthwashes commonly contain plant-derived phenolics like thymol and eucalyptol and/or heavy metals like zinc chloride (see Using Chemicals to Control Microorganisms). Phenolics tend to be stable and persistent on surfaces, and they act through denaturing proteins and disrupting membranes.
Regular dental cleanings allow for the detection of decay at early stages and the removal of tartar. They may also help to draw attention to other concerns, such as damage to the enamel from acidic drinks. Reducing sugar consumption may help prevent damage that results from the microbial fermentation of sugars. Additionally, sugarless candies or gum with sugar alcohols (such as xylitol) can reduce the production of acids because these are fermented to nonacidic compounds (although excess consumption may lead to gastrointestinal distress). Fluoride treatment or ingesting fluoridated water strengthens the minerals in teeth and reduces the incidence of dental caries.
If caries develop, prompt treatment prevents worsening. Smaller areas of decay can be drilled to remove affected tissue and then filled. If the pulp is affected, then a root canal may be needed to completely remove the infected tissues to avoid continued spread of the infection, which could lead to painful abscesses.
Exercise \(1\)
1. Name some ways that microbes contribute to tooth decay.
2. What is the most important cariogenic species of bacteria?
Periodontal Disease
In addition to damage to the teeth themselves, the surrounding structures can be affected by microbes. Periodontal disease is the result of infections that lead to inflammation and tissue damage in the structures surrounding the teeth. The progression from mild to severe periodontal disease is generally reversible and preventable with good oral hygiene.
Inflammation of the gums that can lead to irritation and bleeding is called gingivitis. When plaque accumulates on the teeth, bacteria colonize the gingival space. As this space becomes increasingly blocked, the environment becomes anaerobic. This allows a wide variety of microbes to colonize, including Porphyromonas, Streptococcus, and Actinomyces. The bacterial products, which include lipopolysaccharide (LPS), proteases, lipoteichoic acids, and others, cause inflammation and gum damage (Figure \(3\)). It is possible that methanogenic archaeans (including Methanobrevibacter oralis and other Methanobrevibacter species) also contribute to disease progression as some species have been identified in patients with periodontal disease, but this has proven difficult to study.123 Gingivitis is diagnosed by visual inspection, including measuring pockets in the gums, and X-rays, and is usually treated using good dental hygiene and professional dental cleaning, with antibiotics reserved for severe cases.
Over time, chronic gingivitis can develop into the more serious condition of periodontitis (Figure \(4\)). When this happens, the gums recede and expose parts of the tooth below the crown. This newly exposed area is relatively unprotected, so bacteria can grow on it and spread underneath the enamel of the crown and cause cavities. Bacteria in the gingival space can also erode the cementum, which helps to hold the teeth in place. If not treated, erosion of cementum can lead to the movement or loss of teeth. The bones of the jaw can even erode if the infection spreads. This condition can be associated with bleeding and halitosis (bad breath). Cleaning and appropriate dental hygiene may be sufficient to treat periodontitis. However, in cases of severe periodontitis, an antibiotic may be given. Antibiotics may be given in pill form or applied directly to the gum (local treatment). Antibiotics given can include tetracycline, doxycycline, macrolides or β-lactams. Because periodontitis can be caused by a mix of microbes, a combination of antibiotics may be given.
Trench Mouth
When certain bacteria, such as Prevotella intermedia, Fusobacterium species, and Treponema vicentii, are involved and periodontal disease progresses, acute necrotizing ulcerative gingivitis or trench mouth, also called Vincent's disease, can develop. This is severe periodontitis characterized by erosion of the gums, ulcers, substantial pain with chewing, and halitosis (Figure \(5\)) that can be diagnosed by visual examination and X-rays. In countries with good medical and dental care, it is most common in individuals with weakened immune systems, such as patients with AIDS. In addition to cleaning and pain medication, patients may be prescribed antibiotics such as amoxicillin, amoxicillin clavulanate, clindamycin, or doxycycline.
Exercise \(2\)
How does gingivitis progress to periodontitis?
Healthy Mouth, Healthy Body
Good oral health promotes good overall health, and the reverse is also true. Poor oral health can lead to difficulty eating, which can cause malnutrition. Painful or loose teeth can also cause a person to avoid certain foods or eat less. Malnutrition due to dental problems is of greatest concern for the elderly, for whom it can worsen other health conditions and contribute to mortality. Individuals who have serious illnesses, especially AIDS, are also at increased risk of malnutrition from dental problems.
Additionally, poor oral health can contribute to the development of disease. Increased bacterial growth in the mouth can cause inflammation and infection in other parts of the body. For example, Streptococcus in the mouth, the main contributor to biofilms on teeth, tartar, and dental caries, can spread throughout the body when there is damage to the tissues inside the mouth, as can happen during dental work. S. mutans produces a surface adhesin known as P1, which binds to salivary agglutinin on the surface of the tooth. P1 can also bind to extracellular matrix proteins including fibronectin and collagen. When Streptococcus enters the bloodstream as a result of tooth brushing or dental cleaning, it causes inflammation that can lead to the accumulation of plaque in the arteries and contribute to the development of atherosclerosis, a condition associated with cardiovascular disease, heart attack, and stroke. In some cases, bacteria that spread through the blood vessels can lodge in the heart and cause endocarditis (an example of a focal infection).
Oral Infections
As noted earlier, normal oral microbiota can cause dental and periodontal infections. However, there are number of other infections that can manifest in the oral cavity when other microbes are present.
Herpetic Gingivostomatitis
As described in Viral Infections of the Skin and Eyes, infections by herpes simplex virus type 1 (HSV-1) frequently manifest as oral herpes, also called acute herpes labialis and characterized by cold sores on the lips, mouth, or gums. HSV-1 can also cause acute herpetic gingivostomatitis, a condition that results in ulcers of the mucous membranes inside the mouth (Figure \(6\)). Herpetic gingivostomatitis is normally self-limiting except in immunocompromised patients. Like oral herpes, the infection is generally diagnosed through clinical examination, but cultures or biopsies may be obtained if other signs or symptoms suggest the possibility of a different causative agent. If treatment is needed, mouthwashes or antiviral medications such as acyclovir, famciclovir, or valacyclovir may be used.
Oral Thrush
The yeast Candida is part of the normal human microbiota, but overgrowths, especially of Candida albicans, can lead to infections in several parts of the body. When Candida infection develops in the oral cavity, it is called oral thrush. Oral thrush is most common in infants because they do not yet have well developed immune systems and have not acquired the robust normal microbiota that keeps Candida in check in adults. Oral thrush is also common in immunodeficient patients and is a common infection in patients with AIDS.
Oral thrush is characterized by the appearance of white patches and pseudomembranes in the mouth (Figure \(7\)) and can be associated with bleeding. The infection may be treated topically with nystatin or clotrimazole oral suspensions, although systemic treatment is sometimes needed. In serious cases, systemic azoles such as fluconazole or itraconazole (for strains resistant to fluconazole), may be used. Amphotericin B can also be used if the infection is severe or if the Candida species is azole-resistant.
Mumps
The viral disease mumps is an infection of the parotid glands, the largest of the three pairs of salivary glands (Figure 24.1.2). The causative agent is mumps virus (MuV), a paramyxovirus with an envelope that has hemagglutinin and neuraminidase spikes. A fusion protein located on the surface of the envelope helps to fuse the viral envelope to the host cell plasma membrane.
Mumps virus is transmitted through respiratory droplets or through contact with contaminated saliva, making it quite contagious so that it can lead easily to epidemics. It causes fever, muscle pain, headache, pain with chewing, loss of appetite, fatigue, and weakness. There is swelling of the salivary glands and associated pain (Figure \(8\)). The virus can enter the bloodstream (viremia), allowing it to spread to the organs and the central nervous system. The infection ranges from subclinical cases to cases with serious complications, such as encephalitis, meningitis, and deafness. Inflammation of the pancreas, testes, ovaries, and breasts may also occur and cause permanent damage to those organs; despite these complications, a mumps infection rarely cause sterility.
Mumps can be recognized based on clinical signs and symptoms, and a diagnosis can be confirmed with laboratory testing. The virus can be identified using culture or molecular techniques such as RT-PCR. Serologic tests are also available, especially enzyme immunoassays that detect antibodies. There is no specific treatment for mumps, so supportive therapies are used. The most effective way to avoid infection is through vaccination. Although mumps used to be a common childhood disease, it is now rare in the United States due to vaccination with the measles, mumps, and rubella (MMR) vaccine.
Exercise \(3\)
Compare and contrast the signs and symptoms of herpetic gingivostomatitis, oral thrush, and mumps.
Oral Infections
Infections of the mouth and oral cavity can be caused by a variety of pathogens, including bacteria, viruses, and fungi. Many of these infections only affect the mouth, but some can spread and become systemic infections. Figure \(9\) summarizes the main characteristics of common oral infections.
Key Concepts and Summary
• Dental caries, tartar, and gingivitis are caused by overgrowth of oral bacteria, usually Streptococcus and Actinomyces species, as a result of insufficient dental hygiene.
• Gingivitis can worsen, allowing Porphyromonas, Streptococcus, and Actinomyces species to spread and cause periodontitis. When Prevotella intermedia, Fusobacterium species, and Treponema vicentii are involved, it can lead to acute necrotizing ulcerative gingivitis.
• The herpes simplex virus type 1 can cause lesions of the mouth and throat called herpetic gingivostomatitis.
• Other infections of the mouth include oral thrush, a fungal infection caused by overgrowth of Candida yeast, and mumps, a viral infection of the salivary glands caused by the mumps virus, a paramyxovirus.
Footnotes
1. 1 Hans-Peter Horz and Georg Conrads. “Methanogenic Archaea and Oral Infections—Ways to Unravel the Black Box.” Journal of Oral Microbiology 3(2011). doi: 10.3402/jom.v3i0.5940.
2. 2 Hiroshi Maeda, Kimito Hirai, Junji Mineshiba, Tadashi Yamamoto, Susumu Kokeguchi, and Shogo Takashiba. “Medical Microbiological Approach to Archaea in Oral Infectious Diseases.” Japanese Dental Science Review 49: 2, p. 72–78.
3. 3 Paul W. Lepp, Mary M. Brinig, Cleber C. Ouverney, Katherine Palm, Gary C. Armitage, and David A. Relman. “Methanogenic Archaea and Human Periodontal Disease.” Proceedings of the National Academy of Sciences of the United States of America 101 (2003): 16, pp. 6176–6181. doi: 10.1073/pnas.0308766101. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/24%3A_Digestive_System_Infections/24.02%3A_Microbial_Diseases_of_the_Mouth_and_Oral_Cavity.txt |
Learning Objectives
• Identify the most common bacteria that can cause infections of the GI tract
• Compare the major characteristics of specific bacterial diseases affecting the GI tract
A wide range of gastrointestinal diseases are caused by bacterial contamination of food. Recall that foodborne diseasecan arise from either infection or intoxication. In both cases, bacterial toxins are typically responsible for producing disease signs and symptoms. The distinction lies in where the toxins are produced. In an infection, the microbial agent is ingested, colonizes the gut, and then produces toxins that damage host cells. In an intoxication, bacteria produce toxins in the food before it is ingested. In either case, the toxins cause damage to the cells lining the gastrointestinal tract, typically the colon. This leads to the common signs and symptoms of diarrhea or watery stool and abdominal cramps, or the more severe dysentery. Symptoms of foodborne diseases also often include nausea and vomiting, which are mechanisms the body uses to expel the toxic materials.
Most bacterial gastrointestinal illness is short-lived and self-limiting; however, loss of fluids due to severe diarrheal illness can lead to dehydration that can, in some cases, be fatal without proper treatment. Oral rehydration therapy with electrolyte solutions is an essential aspect of treatment for most patients with GI disease, especially in children and infants.
Staphylococcal Food Poisoning
Staphylococcal food poisoning is one form of food intoxication. When Staphylococcus aureus grows in food, it may produce enterotoxins that, when ingested, can cause symptoms such as nausea, diarrhea, cramping, and vomiting within one to six hours. In some severe cases, it may cause headache, dehydration, and changes in blood pressure and heart rate. Signs and symptoms resolve within 24 to 48 hours. S. aureus is often associated with a variety of raw or undercooked and cooked foods including meat (e.g., canned meat, ham, and sausages) and dairy products (e.g., cheeses, milk, and butter). It is also commonly found on hands and can be transmitted to prepared foods through poor hygiene, including poor handwashing and the use of contaminated food preparation surfaces, such as cutting boards. The greatest risk is for food left at a temperature below 60 °C (140 °F), which allows the bacteria to grow. Cooked foods should generally be reheated to at least 60 °C (140 °F) for safety and most raw meats should be cooked to even higher internal temperatures (Figure \(1\)).
There are at least 21 Staphylococcal enterotoxins and Staphylococcal enterotoxin-like toxins that can cause food intoxication. The enterotoxins are proteins that are resistant to low pH, allowing them to pass through the stomach. They are heat stable and are not destroyed by boiling at 100 °C. Even though the bacterium itself may be killed, the enterotoxins alone can cause vomiting and diarrhea, although the mechanisms are not fully understood. At least some of the symptoms may be caused by the enterotoxin functioning as a superantigen and provoking a strong immune response by activating T cell proliferation.
The rapid onset of signs and symptoms helps to diagnose this foodborne illness. Because the bacterium does not need to be present for the toxin to cause symptoms, diagnosis is confirmed by identifying the toxin in a food sample or in biological specimens (feces or vomitus) from the patient. Serological techniques, including ELISA, can also be used to identify the toxin in food samples.
The condition generally resolves relatively quickly, within 24 hours, without treatment. In some cases, supportive treatment in a hospital may be needed.
Exercise \(1\)
How can S. aureus cause food intoxication?
Shigellosis (Bacillary Dysentery)
When gastrointestinal illness is associated with the rod-shaped, gram-negative bacterium Shigella, it is called bacillary dysentery, or shigellosis. Infections can be caused by S. dysenteriae, S. flexneri, S. boydii, and/or S. sonnei that colonize the GI tract. Shigellosis can be spread from hand to mouth or through contaminated food and water. Most commonly, it is transmitted through the fecal-oral route.
Shigella bacteria invade intestinal epithelial cells. When taken into a phagosome, they can escape and then live within the cytoplasm of the cell or move to adjacent cells. As the organisms multiply, the epithelium and structures with M cells of the Peyer’s patches in the intestine may become ulcerated and cause loss of fluid. Stomach cramps, fever, and watery diarrhea that may also contain pus, mucus, and/or blood often develop. More severe cases may result in ulceration of the mucosa, dehydration, and rectal bleeding. Additionally, patients may later develop hemolytic uremic syndrome (HUS), a serious condition in which damaged blood cells build up in the kidneys and may cause kidney failure, or reactive arthritis, a condition in which arthritis develops in multiple joints following infection. Patients may also develop chronic post-infection irritable bowel syndrome (IBS).
S. dysenteriae type 1 is able to produce Shiga toxin, which targets the endothelial cells of small blood vessels in the small and large intestine by binding to a glycosphingolipid. Once inside the endothelial cells, the toxin targets the large ribosomal subunit, thus affecting protein synthesis of these cells. Hemorrhaging and lesions in the colon can result. The toxin can target the kidney’s glomerulus, the blood vessels where filtration of blood in the kidney begins, thus resulting in HUS.
Stool samples, which should be processed promptly, are analyzed using serological or molecular techniques. One common method is to perform immunoassays for S. dysenteriae. (Other methods that can be used to identify Shigella include API test strips, Enterotube systems, or PCR testing. The presence of white blood cells and blood in fecal samples occurs in about 70% of patients1 (Figure \(2\)). Severe cases may require antibiotics such as ciprofloxacin and azithromycin, but these must be carefully prescribed because resistance is increasingly common.
Exercise \(2\)
Compare and contrast Shigella infections and intoxications.
Salmonellosis
Salmonella gastroenteritis, also called salmonellosis, is caused by the rod-shaped, gram-negative bacterium Salmonella. Two species, S. enterica and S. bongori, cause disease in humans, but S. enterica is the most common. The most common serotypes of S. enterica are Enteritidis and Typhi. We will discuss typhoid fever caused by serotypes Typhi and Paratyphi A separately. Here, we will focus on salmonellosis caused by other serotypes.
Salmonella is a part of the normal intestinal microbiota of many individuals. However, salmonellosis is caused by exogenous agents, and infection can occur depending on the serotype, size of the inoculum, and overall health of the host. Infection is caused by ingestion of contaminated food, handling of eggshells, or exposure to certain animals. Salmonella is part of poultry’s microbiota, so exposure to raw eggs and raw poultry can increase the risk of infection. Handwashing and cooking foods thoroughly greatly reduce the risk of transmission. Salmonella bacteria can survive freezing for extended periods but cannot survive high temperatures.
Once the bacteria are ingested, they multiply within the intestines and penetrate the epithelial mucosal cells via M cells where they continue to grow (Figure \(3\)). They trigger inflammatory processes and the hypersecretion of fluids. Once inside the body, they can persist inside the phagosomes of macrophages. Salmonella can cross the epithelial cell membrane and enter the bloodstream and lymphatic system. Some strains of Salmonella also produce an enterotoxin that can cause an intoxication.
Infected individuals develop fever, nausea, abdominal cramps, vomiting, headache, and diarrhea. These signs and symptoms generally last a few days to a week. According to the Centers for Disease Control and Prevention (CDC), there are 1,000,000 cases annually, with 380 deaths each year.2 However, because the disease is usually self-limiting, many cases are not reported to doctors and the overall incidence may be underreported. Diagnosis involves culture followed by serotyping and DNA fingerprinting if needed. Positive results are reported to the CDC. When an unusual serotype is detected, samples are sent to the CDC for further analysis. Serotyping is important for determining treatment. Oral rehydration therapy is commonly used. Antibiotics are only recommended for serious cases. When antibiotics are needed, as in immunocompromised patients, fluoroquinolones, third-generation cephalosporins, and ampicillin are recommended. Antibiotic resistance is a serious concern.
Typhoid Fever
Certain serotypes of S. enterica, primarily serotype Typhi (S. typhi) but also Paratyphi, cause a more severe type of salmonellosis called typhoid fever. This serious illness, which has an untreated mortality rate of 10%, causes high fever, body aches, headache, nausea, lethargy, and a possible rash.
Some individuals carry S. typhi without presenting signs or symptoms (known as asymptomatic carriers) and continually shed them through their feces. These carriers often have the bacteria in the gallbladder or intestinal epithelium. Individuals consuming food or water contaminated with these feces can become infected.
S. typhi penetrate the intestinal mucosa, grow within the macrophages, and are transported through the body, most notably to the liver and gallbladder. Eventually, the macrophages lyse, releasing S. typhi into the bloodstream and lymphatic system. Mortality can result from ulceration and perforation of the intestine. A wide range of complications, such as pneumonia and jaundice, can occur with disseminated disease.
S. typhi have Salmonella pathogenicity islands (SPIs) that contain the genes for many of their virulence factors. Two examples of important typhoid toxins are the Vi antigen, which encodes for capsule production, and chimeric A2B5 toxin, which causes many of the signs and symptoms of the acute phase of typhoid fever.
Clinical examination and culture are used to make the diagnosis. The bacteria can be cultured from feces, urine, blood, or bone marrow. Serology, including ELISA, is used to identify the most pathogenic strains, but confirmation with DNA testing or culture is needed. A PCR test can also be used, but is not widely available.
The recommended antibiotic treatment involves fluoroquinolones, ceftriaxone, and azithromycin. Individuals must be extremely careful to avoid infecting others during treatment. Typhoid fever can be prevented through vaccination for individuals traveling to parts of the world where it is common.
Exercise \(3\)
Why is serotyping particularly important in Salmonella infections and typhoid fever?
Typhoid Mary
Mary Mallon was an Irish immigrant who worked as a cook in New York in the early 20th century. Over seven years, from 1900 to 1907, Mallon worked for a number of different households, unknowingly spreading illness to the people who lived in each one. In 1906, one family hired George Soper, an expert in typhoid fever epidemics, to determine the cause of the illnesses in their household. Eventually, Soper tracked Mallon down and directly linked 22 cases of typhoid fever to her. He discovered that Mallon was a carrier for typhoid but was immune to it herself. Although active carriers had been recognized before, this was the first time that an asymptomatic carrier of infection had been identified.
Because she herself had never been ill, Mallon found it difficult to believe she could be the source of the illness. She fled from Soper and the authorities because she did not want to be quarantined or forced to give up her profession, which was relatively well paid for someone with her background. However, Mallon was eventually caught and kept in an isolation facility in the Bronx, where she remained until 1910, when the New York health department released her under the condition that she never again work with food. Unfortunately, Mallon did not comply, and she soon began working as a cook again. After new cases began to appear that resulted in the death of two individuals, the authorities tracked her down again and returned her to isolation, where she remained for 23 more years until her death in 1938. Epidemiologists were able to trace 51 cases of typhoid fever and three deaths directly to Mallon, who is unflatteringly remembered as “Typhoid Mary.”
The Typhoid Mary case has direct correlations in the health-care industry. Consider Kaci Hickox, an American nurse who treated Ebola patients in West Africa during the 2014 epidemic. After returning to the United States, Hickox was quarantined against her will for three days and later found not to have Ebola. Hickox vehemently opposed the quarantine. In an editorial published in the British newspaper The Guardian,3 Hickox argued that quarantining asymptomatic health-care workers who had not tested positive for a disease would not only prevent such individuals from practicing their profession, but discourage others from volunteering to work in disease-ridden areas where health-care workers are desperately needed.
What is the responsibility of an individual like Mary Mallon to change her behavior to protect others? What happens when an individual believes that she is not a risk, but others believe that she is? How would you react if you were in Mallon’s shoes and were placed in a quarantine you did not believe was necessary, at the expense of your own freedom and possibly your career? Would it matter if you were definitely infected or not?
E. coli Infections
The gram-negative rod Escherichia coli is a common member of the normal microbiota of the colon. Although the vast majority of E. coli strains are helpful commensal bacteria, some can be pathogenic and may cause dangerous diarrheal disease. The pathogenic strains have additional virulence factors such as type 1 fimbriae that promote colonization of the colon or may produce toxins (see Virulence Factors of Bacterial and Viral Pathogens). These virulence factors are acquired through horizontal gene transfer.
Extraintestinal disease can result if the bacteria spread from the gastrointestinal tract. Although these bacteria can be spread from person to person, they are often acquired through contaminated food or water. There are six recognized pathogenic groups of E. coli, but we will focus here on the four that are most commonly transmitted through food and water.
Enterotoxigenic E. coli (ETEC), also known as traveler’s diarrhea, causes diarrheal illness and is common in less developed countries. In Mexico, ETEC infection is called Montezuma’s Revenge. Following ingestion of contaminated food or water, infected individuals develop a watery diarrhea, abdominal cramps, malaise (a feeling of being unwell), and a low fever. ETEC produces a heat-stable enterotoxin similar to cholera toxin, and adhesins called colonization factors that help the bacteria to attach to the intestinal wall. Some strains of ETEC also produce heat-labile toxins. The disease is usually relatively mild and self-limiting. Diagnosis involves culturing and PCR. If needed, antibiotic treatment with fluoroquinolones, doxycycline, rifaximin, and trimethoprim-sulfamethoxazole (TMP/SMZ) may shorten infection duration. However, antibiotic resistance is a problem.
Enteroinvasive E. coli (EIEC) is very similar to shigellosis, including its pathogenesis of intracellular invasion into intestinal epithelial tissue. This bacterium carries a large plasmid that is involved in epithelial cell penetration. The illness is usually self-limiting, with symptoms including watery diarrhea, chills, cramps, malaise, fever, and dysentery. Culturing and PCR testing can be used for diagnosis. Antibiotic treatment is not recommended, so supportive therapy is used if needed.
Enteropathogenic E. coli (EPEC) can cause a potentially fatal diarrhea, especially in infants and those in less developed countries. Fever, vomiting, and diarrhea can lead to severe dehydration. These E. coli inject a protein (Tir) that attaches to the surface of the intestinal epithelial cells and triggers rearrangement of host cell actin from microvilli to pedestals. Tir also happens to be the receptor for Intimin, a surface protein produced by EPEC, thereby allowing E. coli to “sit” on the pedestal. The genes necessary for this pedestal formation are encoded on the locus for enterocyte effacement (LEE) pathogenicity island. As with ETEC, diagnosis involves culturing and PCR. Treatment is similar to that for ETEC.
The most dangerous strains are enterohemorrhagic E. coli (EHEC), which are the strains capable of causing epidemics. In particular, the strain O157:H7 has been responsible for several recent outbreaks. Recall that the O and H refer to surface antigens that contribute to pathogenicity and trigger a host immune response (“O” refers to the O-side chain of the lipopolysaccharide and the “H” refers to the flagella). Similar to EPEC, EHEC also forms pedestals. EHEC also produces a Shiga-like toxin. Because the genome of this bacterium has been sequenced, it is known that the Shiga toxin genes were most likely acquired through transduction (horizontal gene transfer). The Shiga toxin genes originated from Shigella dysenteriae. Prophage from a bacteriophage that previously infected Shigella integrated into the chromosome of E. coli. The Shiga-like toxin is often called verotoxin.
EHEC can cause disease ranging from relatively mild to life-threatening. Symptoms include bloody diarrhea with severe cramping, but no fever. Although it is often self-limiting, it can lead to hemorrhagic colitis and profuse bleeding. One possible complication is HUS. Diagnosis involves culture, often using MacConkey with sorbitol agar to differentiate between E. coli O157:H7, which does not ferment sorbitol, and other less virulent strains of E. coli that can ferment sorbitol.
Serological typing or PCR testing also can be used, as well as genetic testing for Shiga toxin. To distinguish EPEC from EHEC, because they both form pedestals on intestinal epithelial cells, it is necessary to test for genes encoding for both the Shiga-like toxin and for the LEE. Both EPEC and EHEC have LEE, but EPEC lacks the gene for Shiga toxin. Antibiotic therapy is not recommended and may worsen HUS because of the toxins released when the bacteria are killed, so supportive therapies must be used. Table \(1\) summarizes the characteristics of the four most common pathogenic groups.
Table \(1\): Some Pathogenic Groups of E. coli
Group Virulence Factors and Genes Signs and Symptoms Diagnostic Tests Treatment
Enterotoxigenic E. coli (ETEC) Heat stable enterotoxin similar to cholera toxin Relatively mild, watery diarrhea Culturing, PCR Self-limiting; if needed, fluoroquinolones, doxycycline, rifaximin, TMP/SMZ; antibiotic resistance is a problem
Enteroinvasive E. coli (EIEC) Inv (invasive plasmid) genes Relatively mild, watery diarrhea; dysentery or inflammatory colitis may occur Culturing, PCR; testing for inv gene; additional assays to distinguish from Shigella Supportive therapy only; antibiotics not recommended
Enteropathogenic E. coli (EPEC) Locus of enterocyte effacement (LEE) pathogenicity island Severe fever, vomiting, nonbloody diarrhea, dehydration; potentially fatal Culturing, PCR; detection of LEE lacking Shiga-like toxin genes Self-limiting; if needed, fluoroquinolones, doxycycline, rifaximin (TMP/SMZ); antibiotic resistance is a problem
Enterohemorrhagic E. coli (EHEC) Verotoxin May be mild or very severe; bloody diarrhea; may result in HUS Culturing; plate on MacConkey agar with sorbitol agar as it does not ferment sorbitol; PCR detection of LEE containing Shiga-like toxin genes Antibiotics are not recommended due to the risk of HUS
Exercise \(4\)
Compare and contrast the virulence factors and signs and symptoms of infections with the four main E. coli groups.
Cholera and Other Vibrios
The gastrointestinal disease cholera is a serious infection often associated with poor sanitation, especially following natural disasters, because it is spread through contaminated water and food that has not been heated to temperatures high enough to kill the bacteria. It is caused by Vibrio cholerae serotype O1, a gram-negative, flagellated bacterium in the shape of a curved rod (vibrio). According to the CDC, cholera causes an estimated 3 to 5 million cases and 100,000 deaths each year.4
Because V. cholerae is killed by stomach acid, relatively large doses are needed for a few microbial cells to survive to reach the intestines and cause infection. The motile cells travel through the mucous layer of the intestines, where they attach to epithelial cells and release cholera enterotoxin. The toxin is an A-B toxin with activity through adenylate cyclase (see Virulence Factors of Bacterial and Viral Pathogens). Within the intestinal cell, cyclic AMP (cAMP) levels increase, which activates a chloride channel and results in the release of ions into the intestinal lumen. This increase in osmotic pressure in the lumen leads to water also entering the lumen. As the water and electrolytes leave the body, it causes rapid dehydration and electrolyte imbalance. Diarrhea is so profuse that it is often called “rice water stool,” and patients are placed on cots with a hole in them to monitor the fluid loss (Figure \(4\)).
Cholera is diagnosed by taking a stool sample and culturing for Vibrio. The bacteria are oxidase positive and show non-lactose fermentation on MacConkey agar. Gram-negative lactose fermenters will produce red colonies while non-fermenters will produce white/colorless colonies. Gram-positive bacteria will not grow on MacConkey. Lactose fermentation is commonly used for pathogen identification because the normal microbiota generally ferments lactose while pathogens do not. V. cholerae may also be cultured on thiosulfate citrate bile salts sucrose (TCBS) agar, a selective and differential media for Vibrio spp., which produce a distinct yellow colony.
Cholera may be self-limiting and treatment involves rehydration and electrolyte replenishment. Although antibiotics are not typically needed, they can be used for severe or disseminated disease. Tetracyclines are recommended, but doxycycline, erythromycin, orfloxacin, ciprofloxacin, and TMP/SMZ may be used. Recent evidence suggests that azithromycin is also a good first-line antibiotic. Good sanitation—including appropriate sewage treatment, clean supplies for cooking, and purified drinking water—is important to prevent infection (Figure \(4\))
V. cholera is not the only Vibrio species that can cause disease. V. parahemolyticus is associated with consumption of contaminated seafood and causes gastrointestinal illness with signs and symptoms such as watery diarrhea, nausea, fever, chills, and abdominal cramps. The bacteria produce a heat-stable hemolysin, leading to dysentery and possible disseminated disease. It also sometimes causes wound infections. V. parahemolyticus is diagnosed using cultures from blood, stool, or a wound. As with V. cholera, selective medium (especially TCBS agar) works well. Tetracycline and ciprofloxacin can be used to treat severe cases, but antibiotics generally are not needed.
Vibrio vulnificus is found in warm seawater and, unlike V. cholerae, is not associated with poor sanitary conditions. The bacteria can be found in raw seafood, and ingestion causes gastrointestinal illness. It can also be acquired by individuals with open skin wounds who are exposed to water with high concentrations of the pathogen. In some cases, the infection spreads to the bloodstream and causes septicemia. Skin infection can lead to edema, ecchymosis (discoloration of skin due to bleeding), and abscesses. Patients with underlying disease have a high fatality rate of about 50%. It is of particular concern for individuals with chronic liver disease or who are otherwise immunodeficient because a healthy immune system can often prevent infection from developing. V. vulnificus is diagnosed by culturing for the pathogen from stool samples, blood samples, or skin abscesses. Adult patients are treated with doxycycline combined with a third generation cephalosporin or with fluoroquinolones, and children are treated with TMP/SMZ.
Two other vibrios, Aeromonas hydrophila and Plesiomonas shigelloides, are also associated with marine environments and raw seafood; they can also cause gastroenteritis. Like V. vulnificus, A. hydrophila is more often associated with infections in wounds, generally those acquired in water. In some cases, it can also cause septicemia. Other species of Aeromonas can cause illness. P. shigelloides is sometimes associated with more serious systemic infections if ingested in contaminated food or water. Culture can be used to diagnose A. hydrophila and P. shigelloides infections, for which antibiotic therapy is generally not needed. When necessary, tetracycline and ciprofloxacin, among other antibiotics, may be used for treatment of A. hydrophila, and fluoroquinolones and trimethoprim are the effective treatments for P. shigelloides.
Exercise \(5\)
How does V. cholera infection cause rapid dehydration?
Campylobacter jejuni Gastroenteritis
Campylobacter is a genus of gram-negative, spiral or curved bacteria. They may have one or two flagella. Campylobacter jejuni gastroenteritis, a form of campylobacteriosis, is a widespread illness that is caused by Campylobacter jejuni. The primary route of transmission is through poultry that becomes contaminated during slaughter. Handling of the raw chicken in turn contaminates cooking surfaces, utensils, and other foods. Unpasteurized milk or contaminated water are also potential vehicles of transmission. In most cases, the illness is self-limiting and includes fever, diarrhea, cramps, vomiting, and sometimes dysentery. More serious signs and symptoms, such as bacteremia, meningitis, pancreatitis, cholecystitis, and hepatitis, sometimes occur. It has also been associated with autoimmune conditions such as Guillain-Barré syndrome, a neurological disease that occurs after some infections and results in temporary paralysis. HUS following infection can also occur. The virulence in many strains is the result of hemolysin production and the presence of Campylobacter cytolethal distending toxin (CDT), a powerful deoxyribonuclease (DNase) that irreversibly damages host cell DNA.
Diagnosis involves culture under special conditions, such as elevated temperature, low oxygen tension, and often medium supplemented with antimicrobial agents. These bacteria should be cultured on selective medium (such as Campy CV, charcoal selective medium, or cefaperazone charcoal deoxycholate agar) and incubated under microaerophilic conditions for at least 72 hours at 42 °C. Antibiotic treatment is not usually needed, but erythromycin or ciprofloxacin may be used.
Peptic Ulcers
The gram-negative bacterium Helicobacter pylori is able to tolerate the acidic environment of the human stomach and has been shown to be a major cause of peptic ulcers, which are ulcers of the stomach or duodenum. The bacterium is also associated with increased risk of stomach cancer (Figure \(5\)). According to the CDC, approximately two-thirds of the population is infected with H. pylori, but less than 20% have a risk of developing ulcers or stomach cancer. H. pylori is found in approximately 80% of stomach ulcers and in over 90% of duodenal ulcers.5
H. pylori colonizes epithelial cells in the stomach using pili for adhesion. These bacteria produce urease, which stimulates an immune response and creates ammonia that neutralizes stomach acids to provide a more hospitable microenvironment. The infection damages the cells of the stomach lining, including those that normally produce the protective mucus that serves as a barrier between the tissue and stomach acid. As a result, inflammation (gastritis) occurs and ulcers may slowly develop. Ulcer formation can also be caused by toxin activity. It has been reported that 50% of clinical isolates of H. pylori have detectable levels of exotoxin activity in vitro.6 This toxin, VacA, induces vacuole formation in host cells. VacA has no primary sequence homology with other bacterial toxins, and in a mouse model, there is a correlation between the presence of the toxin gene, the activity of the toxin, and gastric epithelial tissue damage.
Signs and symptoms include nausea, lack of appetite, bloating, burping, and weight loss. Bleeding ulcers may produce dark stools. If no treatment is provided, the ulcers can become deeper, more tissues can be involved, and stomach perforation can occur. Because perforation allows digestive enzymes and acid to leak into the body, it is a very serious condition.
To diagnose H. pylori infection, multiple methods are available. In a breath test, the patient swallows radiolabeled urea. If H. pylori is present, the bacteria will produce urease to break down the urea. This reaction produces radiolabeled carbon dioxide that can be detected in the patient’s breath. Blood testing can also be used to detect antibodies to H. pylori. The bacteria themselves can be detected using either a stool test or a stomach wall biopsy.
Antibiotics can be used to treat the infection. However, unique to H. pylori, the recommendation from the US Food and Drug Administration is to use a triple therapy. The current protocols are 10 days of treatment with omeprazole, amoxicillin, and clarithromycin (OAC); 14 days of treatment with bismuth subsalicylate, metronidazole, and tetracycline (BMT); or 10 or 14 days of treatment with lansoprazole, amoxicillin, and clarithromycin (LAC). Omeprazole, bismuth subsalicylate, and lansoprazole are not antibiotics but are instead used to decrease acid levels because H. pylori prefers acidic environments.
Although treatment is often valuable, there are also risks to H. pylori eradication. Infection with H. pylori may actually protect against some cancers, such as esophageal adenocarcinoma and gastroesophageal reflux disease.78
Exercise \(6\)
How does H. pylori cause peptic ulcers?
Clostridium perfringens Gastroenteritis
Clostridium perfringens gastroenteritis is a generally mild foodborne disease that is associated with undercooked meats and other foods. C. perfringens is a gram-positive, rod-shaped, endospore-forming anaerobic bacterium that is tolerant of high and low temperatures. At high temperatures, the bacteria can form endospores that will germinate rapidly in foods or within the intestine. Food poisoning by type A strains is common. This strain always produces an enterotoxin, sometimes also present in other strains, that causes the clinical symptoms of cramps and diarrhea. A more severe form of the illness, called pig-bel or enteritis necroticans, causes hemorrhaging, pain, vomiting, and bloating. Gangrene of the intestines may result. This form has a high mortality rate but is rare in the United States.
Diagnosis involves detecting the C. perfringens toxin in stool samples using either molecular biology techniques (PCR detection of the toxin gene) or immunology techniques (ELISA). The bacteria itself may also be detected in foods or in fecal samples. Treatment includes rehydration therapy, electrolyte replacement, and intravenous fluids. Antibiotics are not recommended because they can damage the balance of the microbiota in the gut, and there are concerns about antibiotic resistance. The illness can be prevented through proper handling and cooking of foods, including prompt refrigeration at sufficiently low temperatures and cooking food to a sufficiently high temperature.
Clostridium difficile
Clostridium difficile is a gram-positive rod that can be a commensal bacterium as part of the normal microbiota of healthy individuals. When the normal microbiota is disrupted by long-term antibiotic use, it can allow the overgrowth of this bacterium, resulting in antibiotic-associated diarrhea caused by C. difficile. Antibiotic-associated diarrhea can also be considered a nosocomial disease. Patients at the greatest risk of C. difficile infection are those who are immunocompromised, have been in health-care settings for extended periods, are older, have recently taken antibiotics, have had gastrointestinal procedures done, or use proton pump inhibitors, which reduce stomach acidity and allow proliferation of C. difficile. Because this species can form endospores, it can survive for extended periods of time in the environment under harsh conditions and is a considerable concern in health-care settings.
This bacterium produces two toxins, Clostridium difficile toxin A (TcdA) and Clostridium difficile toxin B (TcdB). These toxins inactivate small GTP-binding proteins, resulting in actin condensation and cell rounding, followed by cell death. Infections begin with focal necrosis, then ulceration with exudate, and can progress to pseudomembranous colitis, which involves inflammation of the colon and the development of a pseudomembrane of fibrin containing dead epithelial cells and leukocytes (Figure \(6\)). Watery diarrhea, dehydration, fever, loss of appetite, and abdominal pain can result. Perforation of the colon can occur, leading to septicemia, shock, and death. C. difficile is also associated with necrotizing enterocolitis in premature babies and neutropenic enterocolitis associated with cancer therapies.
Diagnosis is made by considering the patient history (such as exposure to antibiotics), clinical presentation, imaging, endoscopy, lab tests, and other available data. Detecting the toxin in stool samples is used to confirm diagnosis. Although culture is preferred, it is rarely practical in clinical practice because the bacterium is an obligate anaerobe. Nucleic acid amplification tests, including PCR, are considered preferable to ELISA testing for molecular analysis.
The first step of conventional treatment is to stop antibiotic use, and then to provide supportive therapy with electrolyte replacement and fluids. Metronidazole is the preferred treatment if the C. difficile diagnosis has been confirmed. Vancomycin can also be used, but it should be reserved for patients for whom metronidazole was ineffective or who meet other criteria (e.g., under 10 years of age, pregnant, or allergic to metronidazole).
A newer approach to treatment, known as a fecal transplant, focuses on restoring the microbiota of the gut in order to combat the infection. In this procedure, a healthy individual donates a stool sample, which is mixed with saline and transplanted to the recipient via colonoscopy, endoscopy, sigmoidoscopy, or enema. It has been reported that this procedure has greater than 90% success in resolving C. difficile infections.9
Exercise \(7\)
How does antibiotic use lead to C. difficile infections?
Foodborne Illness Due to Bacillus cereus
Bacillus cereus, commonly found in soil, is a gram-positive endospore-forming bacterium that can sometimes cause foodborne illness. B. cereus endospores can survive cooking and produce enterotoxins in food after it has been heated; illnesses often occur after eating rice and other prepared foods left at room temperature for too long. The signs and symptoms appear within a few hours of ingestion and include nausea, pain, and abdominal cramps. B. cereus produces two toxins: one causing diarrhea, and the other causing vomiting. More severe signs and symptoms can sometimes develop.
Diagnosis can be accomplished by isolating bacteria from stool samples or vomitus and uneaten infected food. Treatment involves rehydration and supportive therapy. Antibiotics are not typically needed, as the illness is usually relatively mild and is due to toxin activity.
Foodborne Illness Due to Yersinia
The genus Yersinia is best known for Yersinia pestis, a gram-negative rod that causes the plague. However, Y. enterocolitica and Y. pseudotuberculosis can cause gastroenteritis. The infection is generally transmitted through the fecal-oral route, with ingestion of food or water that has been contaminated by feces. Intoxication can also result because of the activity of its endotoxin and exotoxins (enterotoxin and cytotoxin necrotizing factor). The illness is normally relatively mild and self-limiting. However, severe diarrhea and dysentery can develop in infants. In adults, the infection can spread and cause complications such as reactive arthritis, thyroid disorders, endocarditis, glomerulonephritis, eye inflammation, and/or erythema nodosum. Bacteremia may develop in rare cases.
Diagnosis is generally made by detecting the bacteria in stool samples. Samples may also be obtained from other tissues or body fluids. Treatment is usually supportive, including rehydration, without antibiotics. If bacteremia or other systemic disease is present, then antibiotics such as fluoroquinolones, aminoglycosides, doxycycline, and trimethoprim-sulfamethoxazole may be used. Recovery can take up to two weeks.
Exercise \(8\)
Compare and contrast foodborne illnesses due to B. cereus and Yersinia.
Bacterial Infections of the Gastrointestinal Tract
Bacterial infections of the gastrointestinal tract generally occur when bacteria or bacterial toxins are ingested in contaminated food or water. Toxins and other virulence factors can produce gastrointestinal inflammation and general symptoms such as diarrhea and vomiting. Bacterial GI infections can vary widely in terms of severity and treatment. Some can be treated with antibiotics, but in other cases antibiotics may be ineffective in combating toxins or even counterproductive if they compromise the GI microbiota. Figure \(7\) and Figure \(8\) the key features of common bacterial GI infections.
Clinical Focus: Part 2
At the hospital, Carli’s doctor began to think about possible causes of her severe gastrointestinal distress. One possibility was food poisoning, but no one else in her family was sick. The doctor asked about what Carli had eaten the previous day; her mother mentioned that she’d had eggs for lunch, and that they may have been a little undercooked. The doctor took a sample of Carli’s stool and sent it for laboratory testing as part of her workup. She suspected that Carli could have a case of bacterial or viral gastroenteritis, but she needed to know the cause in order to prescribe an appropriate treatment.
In the laboratory, technicians microscopically identified gram-negative bacilli in Carli’s stool sample. They also established a pure culture of the bacteria and analyzed it for antigens. This testing showed that the causative agent was Salmonella.
Exercise \(9\)
What should the doctor do now to treat Carli?
Key Concepts and Summary
• Major causes of gastrointestinal illness include Salmonella spp., Staphylococcus spp., Helicobacter pylori, Clostridium perfringens, Clostridium difficile, Bacillus cereus, and Yersinia bacteria.
• C. difficile is an important cause of hospital acquired infection.
• Vibrio cholerae causes cholera, which can be a severe diarrheal illness.
• Different strains of E. coli, including ETEC, EPEC, EIEC, and EHEC, cause different illnesses with varying degrees of severity.
• H. pylori is associated with peptic ulcers.
• Salmonella enterica serotypes can cause typhoid fever, a more severe illness than salmonellosis.
• Rehydration and other supportive therapies are often used as general treatments.
• Careful antibiotic use is required to reduce the risk of causing C. difficile infections and when treating antibiotic-resistant infections.
Footnotes
1. 1 Jaya Sureshbabu. “Shigella Infection Workup.” Medscape. Updated Jun 28, 2016. http://emedicine.medscape.com/article/968773-workup.
2. 2 Centers for Disease Control and Prevention. Salmonella. Updated August 25, 2016. https://www.cdc.gov/salmonella.
3. 3 Kaci Hickox. “Stop Calling Me the ‘Ebola Nurse.’” The Guardian. November 17, 2014. www.theguardian.com/commentis...se-kaci-hickox.
4. 4 Centers for Disease Control and Prevention. Cholera—Vibrio cholerae Infection. Updated November 6, 2014. http://www.cdc.gov/cholera/general. Accessed Sept 14, 2016.
5. 5 Centers for Disease Control and Prevention. “Helicobacter pylori: Fact Sheet for Health Care Providers.” Updated July 1998. www.cdc.gov/ulcer/files/hpfacts.pdf.
6. 6 T. L. Cover. “The Vacuolating Cytotoxin of Helicobacter pylori.” Molecular Microbiology 20 (1996) 2: pp. 241–246. http://www.ncbi.nlm.nih.gov/pubmed/8733223.
7. 7 Martin J. Blaser. “Disappearing Microbiota: Helicobacter pylori Protection against Esophageal Adenocarcinoma.” Cancer Prevention Research 1 (2008) 5: pp. 308–311. http://cancerpreventionresearch.aacr....full.pdf+html.
8. 8 Ivan F. N. Hung and Benjamin C. Y. Wong. “Assessing the Risks and Benefits of Treating Helicobacter pylori Infection.” Therapeutic Advances in Gastroenterology 2 (2009) 3: pp, 141–147. doi: 10.1177/1756283X08100279.
9. 9 Faith Rohlke and Neil Stollman. “Fecal Microbiota Transplantation in Relapsing Clostridium difficile Infection,” Therapeutic Advances in Gastroenterology 5 (2012) 6: 403–420. doi: 10.1177/1756283X12453637. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/24%3A_Digestive_System_Infections/24.03%3A_Bacterial_Infections_of_the_Gastrointestinal_Tract.txt |
Learning Objectives
• Identify the most common viruses that can cause infections of the GI tract
• Compare the major characteristics of specific viral diseases affecting the GI tract and liver
In the developing world, acute viral gastroenteritis is devastating and a leading cause of death for children.1 Worldwide, diarrhea is the second leading cause of mortality for children under age five, and 70% of childhood gastroenteritis is viral.2 As discussed, there are a number of bacteria responsible for diarrhea, but viruses can also cause diarrhea. E. coli and rotavirus are the most common causative agents in the developing world. In this section, we will discuss rotaviruses and other, less common viruses that can also cause gastrointestinal illnesses.
Gastroenteritis Caused by Rotaviruses
Rotaviruses are double-stranded RNA viruses in the family Reoviridae. They are responsible for common diarrheal illness, although prevention through vaccination is becoming more common. The virus is primarily spread by the fecal-oral route (Figure \(1\)).
These viruses are widespread in children, especially in day-care centers. The CDC estimates that 95% of children in the United States have had at least one rotavirus infection by the time they reach age five.3 Due to the memory of the body’s immune system, adults who come into contact with rotavirus will not contract the infection or, if they do, are asymptomatic. The elderly, however, are vulnerable to rotavirus infection due to weakening of the immune system with age, so infections can spread through nursing homes and similar facilities. In these cases, the infection may be transmitted from a family member who may have subclinical or clinical disease. The virus can also be transmitted from contaminated surfaces, on which it can survive for some time.
Infected individuals exhibit fever, vomiting, and diarrhea. The virus can survive in the stomach following a meal, but is normally found in the small intestines, particularly the epithelial cells on the villi. Infection can cause food intolerance, especially with respect to lactose. The illness generally appears after an incubation period of about two days and lasts for approximately one week (three to eight days). Without supportive treatment, the illness can cause severe fluid loss, dehydration, and even death. Even with milder illness, repeated infections can potentially lead to malnutrition, especially in developing countries, where rotavirus infection is common due to poor sanitation and lack of access to clean drinking water. Patients (especially children) who are malnourished after an episode of diarrhea are more susceptible to future diarrheal illness, increasing their risk of death from rotavirus infection.
The most common clinical tool for diagnosis is enzyme immunoassay, which detects the virus from fecal samples. Latex agglutination assays are also used. Additionally, the virus can be detected using electron microscopy and RT-PCR.
Treatment is supportive with oral rehydration therapy. Preventive vaccination is also available. In the United States, rotavirus vaccines are part of the standard vaccine schedule and administration follows the guidelines of the World Health Organization (WHO). The WHO recommends that all infants worldwide receive the rotavirus vaccine, the first dose between six and 15 weeks of age and the second before 32 weeks.4
Gastroenteritis Caused by Noroviruses
Noroviruses, commonly identified as Norwalk viruses, are caliciviruses. Several strains can cause gastroenteritis. There are millions of cases a year, predominately in infants, young children, and the elderly. These viruses are easily transmitted and highly contagious. They are known for causing widespread infections in groups of people in confined spaces, such as on cruise ships. The viruses can be transmitted through direct contact, through touching contaminated surfaces, and through contaminated food. Because the virus is not killed by disinfectants used at standard concentrations for killing bacteria, the risk of transmission remains high, even after cleaning.
The signs and symptoms of norovirus infection are similar to those for rotavirus, with watery diarrhea, mild cramps, and fever. Additionally, these viruses sometimes cause projectile vomiting. The illness is usually relatively mild, develops 12 to 48 hours after exposure, and clears within a couple of days without treatment. However, dehydration may occur.
Norovirus can be detected using PCR or enzyme immunoassay (EIA) testing. RT-qPCR is the preferred approach as EIA is insufficiently sensitive. If EIA is used for rapid testing, diagnosis should be confirmed using PCR. No medications are available, but the illness is usually self-limiting. Rehydration therapy and electrolyte replacement may be used. Good hygiene, hand washing, and careful food preparation reduce the risk of infection.
Gastroenteritis Caused by Astroviruses
Astroviruses are single-stranded RNA viruses (family Astroviridae) that can cause severe gastroenteritis, especially in infants and children. Signs and symptoms include diarrhea, nausea, vomiting, fever, abdominal pain, headache, and malaise. The viruses are transmitted through the fecal-oral route (contaminated food or water). For diagnosis, stool samples are analyzed. Testing may involve enzyme immunoassays and immune electron microscopy. Treatment involves supportive rehydration and electrolyte replacement if needed.
Exercise \(1\)
Why are rotaviruses, noroviruses, and astroviruses more common in children?
Viral Infections of the Gastrointestinal Tract
A number of viruses can cause gastroenteritis, characterized by inflammation of the GI tract and other signs and symptoms with a range of severities. As with bacterial GI infections, some cases can be relatively mild and self-limiting, while others can become serious and require intensive treatment. Antimicrobial drugs are generally not used to treat viral gastroenteritis; generally, these illnesses can be treated effectively with rehydration therapy to replace fluids lost in bouts of diarrhea and vomiting. Because most viral causes of gastroenteritis are quite contagious, the best preventive measures involve avoiding and/or isolating infected individuals and limiting transmission through good hygiene and sanitation.
Hepatitis
Hepatitis is a general term meaning inflammation of the liver, which can have a variety of causes. In some cases, the cause is viral infection. There are five main hepatitis viruses that are clinically significant: hepatitisviruses A (HAV), B (HBV), C (HCV), D, (HDV) and E (HEV) (Figure \(3\)). Note that other viruses, such as Epstein-Barr virus (EBV), yellow fever, and cytomegalovirus (CMV) can also cause hepatitis and are discussed in Viral Infections of the Circulatory and Lymphatic Systems.
Although the five hepatitis viruses differ, they can cause some similar signs and symptoms because they all have an affinity for hepatocytes (liver cells). HAV and HEV can be contracted through ingestion while HBV, HCV, and HDV are transmitted by parenteral contact. It is possible for individuals to become long term or chronic carriers of hepatitis viruses.
The virus enters the blood (viremia), spreading to the spleen, the kidneys, and the liver. During viral replication, the virus infects hepatocytes. The inflammation is caused by the hepatocytes replicating and releasing more hepatitis virus. Signs and symptoms include malaise, anorexia, loss of appetite, dark urine, pain in the upper right quadrant of the abdomen, vomiting, nausea, diarrhea, joint pain, and gray stool. Additionally, when the liver is diseased or injured, it is unable to break down hemoglobin effectively, and bilirubin can build up in the body, giving the skin and mucous membranes a yellowish color, a condition called jaundice (Figure \(4\)). In severe cases, death from liver necrosis may occur.
Despite having many similarities, each of the hepatitis viruses has its own unique characteristics. HAV is generally transmitted through the fecal-oral route, close personal contact, or exposure to contaminated water or food. Hepatitis A can develop after an incubation period of 15 to 50 days (the mean is 30). It is normally mild or even asymptomatic and is usually self-limiting within weeks to months. A more severe form, fulminant hepatitis, rarely occurs but has a high fatality rate of 70–80%. Vaccination is available and is recommended especially for children (between ages one and two), those traveling to countries with higher risk, those with liver disease and certain other conditions, and drug users.
Although HBV is associated with similar signs and symptoms, transmission and outcomes differ. This virus has a mean incubation period of 120 days and is generally associated with exposure to infectious blood or body fluids such as semen or saliva. Exposure can occur through skin puncture, across the placenta, or through mucosal contact, but it is not spread through casual contact such as hugging, hand holding, sneezing, or coughing, or even through breastfeeding or kissing. Risk of infection is greatest for those who use intravenous drugs or who have sexual contact with an infected individual. Health-care workers are also at risk from needle sticks and other injuries when treating infected patients. The infection can become chronic and may progress to cirrhosis or liver failure. It is also associated with liver cancer. Chronic infections are associated with the highest mortality rates and are more common in infants. Approximately 90% of infected infants become chronic carriers, compared with only 6–10% of infected adults.5 Vaccination is available and is recommended for children as part of the standard vaccination schedule (one dose at birth and the second by 18 months of age) and for adults at greater risk (e.g., those with certain diseases, intravenous drug users, and those who have sex with multiple partners). Health-care agencies are required to offer the HBV vaccine to all workers who have occupational exposure to blood and/or other infectious materials.
HCV is often undiagnosed and therefore may be more widespread than is documented. It has a mean incubation period of 45 days and is transmitted through contact with infected blood. Although some cases are asymptomatic and/or resolve spontaneously, 75%–85% of infected individuals become chronic carriers. Nearly all cases result from parenteral transmission often associated with IV drug use or transfusions. The risk is greatest for individuals with past or current history of intravenous drug use or who have had sexual contact with infected individuals. It has also been spread through contaminated blood products and can even be transmitted through contaminated personal products such as toothbrushes and razors. New medications have recently been developed that show great effectiveness in treating HCV and that are tailored to the specific genotype causing the infection.
HDV is uncommon in the United States and only occurs in individuals who are already infected with HBV, which it requires for replication. Therefore, vaccination against HBV is also protective against HDV infection. HDV is transmitted through contact with infected blood.
HEV infections are also rare in the United States but many individuals have a positive antibody titer for HEV. The virus is most commonly spread by the fecal-oral route through food and/or water contamination, or person-to-person contact, depending on the genotype of the virus, which varies by location. There are four genotypes that differ somewhat in their mode of transmission, distribution, and other factors (for example, two are zoonotic and two are not, and only one causes chronic infection). Genotypes three and four are only transmitted through food, while genotypes one and two are also transmitted through water and fecal-oral routes. Genotype one is the only type transmitted person-to-person and is the most common cause of HEV outbreaks. Consumption of undercooked meat, especially deer or pork, and shellfish can lead to infection. Genotypes three and four are zoonoses, so they can be transmitted from infected animals that are consumed. Pregnant women are at particular risk. This disease is usually self-limiting within two weeks and does not appear to cause chronic infection.
General laboratory testing for hepatitis begins with blood testing to examine liver function (Figure \(5\)). When the liver is not functioning normally, the blood will contain elevated levels of alkaline phosphatase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), direct bilirubin, total bilirubin, serum albumin, serum total protein, and calculated globulin, albumin/globulin (A/G) ratio. Some of these are included in a complete metabolic panel (CMP), which may first suggest a possible liver problem and indicate the need for more comprehensive testing. A hepatitis virus serological test panel can be used to detect antibodies for hepatitis viruses A, B, C, and sometimes D. Additionally, other immunological and genomic tests are available.
Specific treatments other than supportive therapy, rest, and fluids are often not available for hepatitis virus infection, except for HCV, which is often self-limited. Immunoglobulins can be used prophylactically following possible exposure. Medications are also used, including interferon alpha 2b and antivirals (e.g., lamivudine, entecavir, adefovir, and telbivudine) for chronic infections. Hepatitis C can be treated with interferon (as monotherapy or combined with other treatments), protease inhibitors, and other antivirals (e.g., the polymerase inhibitor sofosbuvir). Combination treatments are commonly used. Antiviral and immunosuppressive medications may be used for chronic cases of HEV. In severe cases, liver transplants may be necessary. Additionally, vaccines are available to prevent infection with HAV and HBV. The HAV vaccine is also protective against HEV. The HBV vaccine is also protective against HDV. There is no vaccine against HCV.
Link to Learning
Learn more information about hepatitis virus infections.
Exercise \(2\)
Why do the five different hepatitis viruses all cause similar signs and symptoms?
Preventing HBV Transmission in Health-Care Settings
Hepatitis B was once a leading on-the-job hazard for health-care workers. Many health-care workers over the years have become infected, some developing cirrhosis and liver cancer. In 1982, the CDC recommended that health-care workers be vaccinated against HBV, and rates of infection have declined since then. Even though vaccination is now common, it is not always effective and not all individuals are vaccinated. Therefore, there is still a small risk for infection, especially for health-care workers working with individuals who have chronic infections, such as drug addicts, and for those with higher risk of needle sticks, such as phlebotomists. Dentists are also at risk.
Health-care workers need to take appropriate precautions to prevent infection by HBV and other illnesses. Blood is the greatest risk, but other body fluids can also transmit infection. Damaged skin, as occurs with eczema or psoriasis, can also allow transmission. Avoiding contact with body fluids, especially blood, by wearing gloves and face protection and using disposable syringes and needles reduce the risk of infection. Washing exposed skin with soap and water is recommended. Antiseptics may also be used, but may not help. Post-exposure treatment, including treatment with hepatitis B immunoglobulin (HBIG) and vaccination, may be used in the event of exposure to the virus from an infected patient. Detailed protocols are available for managing these situations. The virus can remain infective for up to seven days when on surfaces, even if no blood or other fluids are visible, so it is important to consider the best choices for disinfecting and sterilizing equipment that could potentially transmit the virus. The CDC recommends a solution of 10% bleach to disinfect surfaces.6 Finally, testing blood products is important to reduce the risk of transmission during transfusions and similar procedures.
Viral Hepatitis
Hepatitis involves inflammation of the liver that typically manifests with signs and symptoms such as jaundice, nausea, vomiting, joint pain, gray stool, and loss of appetite. However, the severity and duration of the disease can vary greatly depending on the causative agent. Some infections may be completely asymptomatic, whereas others may be life threatening. The five different viruses capable of causing hepatitis are compared in Figure \(5\). For the sake of comparison, this table presents only the unique aspects of each form of viral hepatitis, not the commonalities.
Key Concepts and Summary
• Common viral causes of gastroenteritis include rotaviruses, noroviruses, and astroviruses.
• Hepatitis may be caused by several unrelated viruses: hepatitis viruses A, B, C, D, and E.
• The hepatitis viruses differ in their modes of transmission, treatment, and potential for chronic infection.
Footnotes
1. 1 Caleb K. King, Roger Glass, Joseph S. Bresee, Christopher Duggan. “Managing Acute Gastroenteritis Among Children: Oral Rehydration, Maintenance, and Nutritional Therapy.” MMWR 52 (2003) RR16: pp. 1–16. http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5216a1.htm.
2. 2 Elizabeth Jane Elliott. “Acute Gastroenteritis in Children.” British Medical Journal 334 (2007) 7583: 35–40, doi: 10.1136/bmj.39036.406169.80; S. Ramani and G. Kang. “Viruses Causing Diarrhoea in the Developing World.” Current Opinions in Infectious Diseases 22 (2009) 5: pp. 477–482. doi: 10.1097/QCO.0b013e328330662f; Michael Vincent F Tablang. “Viral Gastroenteritis.” Medscape. http://emedicine.medscape.com/article/176515-overview.
3. 3 Centers for Disease Control and Prevention. “Rotavirus,” The Pink Book. Updated September 8, 2015. http://www.cdc.gov/vaccines/pubs/pinkbook/rota.html.
4. 4 World Health Organization. “Rotavirus.” Immunization, Vaccines, and Biologicals. Updated April 21, 2010. www.who.int/immunization/topics/rotavirus/en/.
5. 5 Centers for Disease Control and Prevention. “The ABCs of Hepatitis.” Updated 2016. http://www.cdc.gov/hepatitis/resourc...s/abctable.pdf.
6. 6 Centers for Disease Control and Prevention. “Hepatitis B FAQs for Health Professionals.” Updated August 4, 2016. http://www.cdc.gov/hepatitis/HBV/HBVfaq.htm. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/24%3A_Digestive_System_Infections/24.04%3A_Viral_Infections_of_the_Gastrointestinal_Tract.txt |
Learning Objectives
• Identify the most common protozoans that can cause infections of the GI tract
• Compare the major characteristics of specific protozoan diseases affecting the GI tract
Like other microbes, protozoa are abundant in natural microbiota but can also be associated with significant illness. Gastrointestinal diseases caused by protozoa are generally associated with exposure to contaminated food and water, meaning that those without access to good sanitation are at greatest risk. Even in developed countries, infections can occur and these microbes have sometimes caused significant outbreaks from contamination of public water supplies.
Giardiasis
Also called backpacker’s diarrhea or beaver fever, giardiasis is a common disease in the United States caused by the flagellated protist Giardia lamblia, also known as Giardia intestinalis or Giardia duodenalis (Figure 1.3.5). To establish infection, G. lamblia uses a large adhesive disk to attach to the intestinal mucosa. The disk is comprised of microtubules. During adhesion, the flagella of G. lamblia move in a manner that draws fluid out from under the disk, resulting in an area of lower pressure that promotes its adhesion to the intestinal epithelial cells. Due to its attachment, Giardia also blocks absorption of nutrients, including fats.
Transmission occurs through contaminated food or water or directly from person to person. Children in day-care centers are at risk due to their tendency to put items into their mouths that may be contaminated. Large outbreaks may occur if a public water supply becomes contaminated. Giardia have a resistant cyst stage in their life cycle that is able to survive cold temperatures and the chlorination treatment typically used for drinking water in municipal reservoirs. As a result, municipal water must be filtered to trap and remove these cysts. Once consumed by the host, Giardia develops into the active tropozoite.
Infected individuals may be asymptomatic or have gastrointestinal signs and symptoms, sometimes accompanied by weight loss. Common symptoms, which appear one to three weeks after exposure, include diarrhea, nausea, stomach cramps, gas, greasy stool (because fat absorption is being blocked), and possible dehydration. The parasite remains in the colon and does not cause systemic infection. Signs and symptoms generally clear within two to six weeks. Chronic infections may develop and are often resistant to treatment. These are associated with weight loss, episodic diarrhea, and malabsorption syndrome due to the blocked nutrient absorption.
Diagnosis may be made using observation under the microscope. A stool ova and parasite (O&P) exam involves direct examination of a stool sample for the presence of cysts and trophozoites; it can be used to distinguish common parasitic intestinal infections. ELISA and other immunoassay tests, including commercial direct fluorescence antibody kits, are also used. The most common treatments use metronidazole as the first-line choice, followed by tinidazole. If the infection becomes chronic, the parasites may become resistant to medications.
Cryptosporidiosis
Another protozoan intestinal illness is cryptosporidiosis, which is usually caused by Cryptosporidium parvum or C. hominis. (Figure \(1\)) These pathogens are commonly found in animals and can be spread in feces from mice, birds, and farm animals. Contaminated water and food are most commonly responsible for transmission. The protozoan can also be transmitted through human contact with infected animals or their feces.
In the United States, outbreaks of cryptosporidiosis generally occur through contamination of the public water supply or contaminated water at water parks, swimming pools, and day-care centers. The risk is greatest in areas with poor sanitation, making the disease more common in developing countries.
Signs and symptoms include watery diarrhea, nausea, vomiting, cramps, fever, dehydration, and weight loss. The illness is generally self-limiting within a month. However, immunocompromised patients, such as those with HIV/AIDS, are at particular risk of severe illness or death.
Diagnosis involves direct examination of stool samples, often over multiple days. As with giardiasis, a stool O&P exam may be helpful. Acid fast staining is often used. Enzyme immunoassays and molecular analysis (PCR) are available.
The first line of treatment is typically oral rehydration therapy. Medications are sometimes used to treat the diarrhea. The broad-range anti-parasitic drug nitazoxanide can be used to treat cryptosporidiosis. Other anti-parasitic drugs that can be used include azithromycin and paromomycin.
Amoebiasis (Amebiasis)
The protozoan parasite Entamoeba histolytica causes amoebiasis, which is known as amoebic dysentery in severe cases. E. histolytica is generally transmitted through water or food that has fecal contamination. The disease is most widespread in the developing world and is one of the leading causes of mortality from parasitic disease worldwide. Disease can be caused by as few as 10 cysts being transmitted.
Signs and symptoms range from nonexistent to mild diarrhea to severe amoebic dysentery. Severe infection causes the abdomen to become distended and may be associated with fever. The parasite may live in the colon without causing signs or symptoms or may invade the mucosa to cause colitis. In some cases, the disease spreads to the spleen, brain, genitourinary tract, or lungs. In particular, it may spread to the liver and cause an abscess. When a liver abscess develops, fever, nausea, liver tenderness, weight loss, and pain in the right abdominal quadrant may occur. Chronic infection may occur and is associated with intermittent diarrhea, mucus, pain, flatulence, and weight loss.
Direct examination of fecal specimens may be used for diagnosis. As with cryptosporidiosis, samples are often examined on multiple days. A stool O&P exam of fecal or biopsy specimens may be helpful. Immunoassay, serology, biopsy, molecular, and antibody detection tests are available. Enzyme immunoassay may not distinguish current from past illness. Magnetic resonance imaging (MRI) can be used to detect any liver abscesses. The first line of treatment is metronidazole or tinidazole, followed by diloxanide furoate, iodoquinol, or paromomycin to eliminate the cysts that remain.
Cyclosporiasis
The intestinal disease cyclosporiasis is caused by the protozoan Cyclospora cayetanensis. It is endemic to tropical and subtropical regions and therefore uncommon in the United States, although there have been outbreaks associated with contaminated produce imported from regions where the protozoan is more common. This protist is transmitted through contaminated food and water and reaches the lining of the small intestine, where it causes infection. Signs and symptoms begin within seven to ten days after ingestion. Based on limited data, it appears to be seasonal in ways that differ regionally and that are poorly understood.
Some individuals do not develop signs or symptoms. Those who do may exhibit explosive and watery diarrhea, fever, nausea, vomiting, cramps, loss of appetite, fatigue, and bloating. These symptoms may last for months without treatment. Trimethoprim-sulfamethoxazole is the recommended treatment. Microscopic examination is used for diagnosis. A stool O&P examination may be helpful. The oocysts have a distinctive blue halo when viewed using ultraviolet fluorescence microscopy (Figure \(2\)).
Exercise \(1\)
Which protozoan GI infections are common in the United States?
Protozoan Gastrointestinal Infections
Protozoan GI infections are generally transmitted through contaminated food or water, triggering diarrhea and vomiting that can lead to dehydration. Rehydration therapy is an important aspect of treatment, but most protozoan GI infections can also be treated with drugs that target protozoans.
Key Concepts and Summary
• Giardiasis, cryptosporidiosis, amoebiasis, and cyclosporiasis are intestinal infections caused by protozoans.
• Protozoan intestinal infections are commonly transmitted through contaminated food and water.
• Treatment varies depending on the causative agent, so proper diagnosis is important.
• Microscopic examination of stool or biopsy specimens is often used in diagnosis, in combination with other approaches.
Footnotes
1. 1 Centers for Disease Control and Prevention. “Cyclosporiasis FAQs for Health Professionals.” Updated June 13, 2014. http://www.cdc.gov/parasites/cyclosp...s/hp-faqs.html. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/24%3A_Digestive_System_Infections/24.05%3A_Protozoan_Infections_of_the_Gastrointestinal_Tract.txt |
Learning Objectives
• Identify the most common helminths that cause infections of the GI tract
• Compare the major characteristics of specific helminthic diseases affecting GI tract
Helminths are widespread intestinal parasites. These parasites can be divided into three common groups: round-bodied worms also described as nematodes, flat-bodied worms that are segmented (also described as cestodes), and flat-bodied worms that are non-segmented (also described as trematodes). The nematodes include roundworms, pinworms, hookworms, and whipworms. Cestodes include beef, pork, and fish tapeworms. Trematodes are collectively called flukes and more uniquely identified with the body site where the adult flukes are located. Although infection can have serious consequences, many of these parasites are so well adapted to the human host that there is little obvious disease.
Ascariasis
Infections caused by the large nematode roundworm Ascaris lumbricoides, a soil-transmitted helminth, are called ascariasis. Over 800 million to 1 billion people are estimated to be infected worldwide.1 Infections are most common in warmer climates and at warmer times of year. At present, infections are uncommon in the United States. The eggs of the worms are transmitted through contaminated food and water. This may happen if food is grown in contaminated soil, including when manure is used as fertilizer.
When an individual consumes embryonated eggs (those with a developing embryo), the eggs travel to the intestine and the larvae are able to hatch. Ascaris is able to produce proteases that allow for penetration and degradation of host tissue. The juvenile worms can then enter the circulatory system and migrate to the lungs where they enter the alveoli (air sacs). From here they crawl to the pharynx and then follow the gut lumen to return to the small intestine, where they mature into adult roundworms. Females in the host will produce and release eggs that leave the host via feces. In some cases, the worms can block ducts such as those of the pancreas or gallbladder.
The infection is commonly asymptomatic. When signs and symptoms are present, they include shortness of breath, cough, nausea, diarrhea, blood in the stool, abdominal pain, weight loss, and fatigue. The roundworms may be visible in the stool. In severe cases, children with substantial infections may experience intestinal blockage.
The eggs can be identified by microscopic examination of the stool (Figure \(1\)). In some cases, the worms themselves may be identified if coughed up or excreted in stool. They can also sometimes be identified by X-rays, ultrasounds, or MRIs.
Ascariasis is self-limiting, but can last one to two years because the worms can inhibit the body’s inflammatory response through glycan gimmickry (see Virulence Factors of Eukaryotic Pathogens). The first line of treatment is mebendazole or albendazole. In some severe cases, surgery may be required.
Exercise \(1\)
Describe the route by which A. lumbricoides reaches the host’s intestines as an adult worm.
Hookworm
Two species of nematode worms are associated with hookworm infection. Both species are found in the Americas, Africa, and Asia. Necator americanus is found predominantly in the United States and Australia. Another species, Ancylostoma doudenale, is found in southern Europe, North Africa, the Middle East, and Asia. The eggs of these species develop into larvae in soil contaminated by dog or cat feces. These larvae can penetrate the skin. After traveling through the venous circulation, they reach the lungs. When they are coughed up, they are then swallowed and can enter the intestine and develop into mature adults. At this stage, they attach to the wall of the intestine, where they feed on blood and can potentially cause anemia. Signs and symptoms include cough, an itchy rash, loss of appetite, abdominal pain, and diarrhea. In children, hookworms can affect physical and cognitive growth.
Some hookworm species, such as Ancylostoma braziliense that is commonly found in animals such as cats and dogs, can penetrate human skin and migrate, causing cutaneous larva migrans, a skin disease caused by the larvae of hookworms. As they move across the skin, in the subcutaneous tissue, pruritic tracks appear (Figure \(2\)).
The infection is diagnosed using microscopic examination of the stool, allowing for observation of eggs in the feces. Medications such as albendazole, mebendazole, and pyrantel pamoate are used as needed to treat systemic infection. In addition to systemic medication for symptoms associated with cutaneous larva migrans, topical thiabendazole is applied to the affected areas.
Strongyloidiasis
Strongyloidiasis is generally caused by Strongyloides stercoralis, a soil-transmitted helminth with both free-living and parasitic forms. In the parasitic form, the larvae of these nematodes generally penetrate the body through the skin, especially through bare feet, although transmission through organ transplantation or at facilities like day-care centers can also occur. When excreted in the stool, larvae can become free-living adults rather than developing into the parasitic form. These free-living worms reproduce, laying eggs that hatch into larvae that can develop into the parasitic form. In the parasitic life cycle, infective larvae enter the skin, generally through the feet. The larvae reach the circulatory system, which allows them to travel to the alveolar spaces of the lungs. They are transported to the pharynx where, like many other helminths, the infected patient coughs them up and swallows them again so that they return to the intestine. Once they reach the intestine, females live in the epithelium and produce eggs that develop asexually, unlike the free-living forms, which use sexual reproduction. The larvae may be excreted in the stool or can reinfect the host by entering the tissue of the intestines and skin around the anus, which can lead to chronic infections.
The condition is generally asymptomatic, although severe symptoms can develop after treatment with corticosteroids for asthma or chronic obstructive pulmonary disease, or following other forms of immunosuppression. When the immune system is suppressed, the rate of autoinfection increases, and huge amounts of larvae migrate to organs throughout the body.
Signs and symptoms are generally nonspecific. The condition can cause a rash at the site of skin entry, cough (dry or with blood), fever, nausea, difficulty breathing, bloating, pain, heartburn, and, rarely, arthritis, or cardiac or kidney complications. Disseminated strongyloidiasis or hyperinfection is a life-threatening form of the disease that can occur, usually following immunosuppression such as that caused by glucocorticoid treatment (most commonly), with other immunosuppressive medications, with HIV infection, or with malnutrition.
As with other helminths, direct examination of the stool is important in diagnosis. Ideally, this should be continued over seven days. Serological testing, including antigen testing, is also available. These can be limited by cross-reactions with other similar parasites and by the inability to distinguish current from resolved infection. Ivermectin is the preferred treatment, with albendazole as a secondary option.
Exercise \(2\)
How does an acute infection of S. stercoralis become chronic?
Pinworms (Enterobiasis)
Enterobius vermicularis, commonly called pinworms, are tiny (2–13 mm) nematodes that cause enterobiasis. Of all helminthic infections, enterobiasis is the most common in the United States, affecting as many as one-third of American children.2 Although the signs and symptoms are generally mild, patients may experience abdominal pain and insomnia from itching of the perianal region, which frequently occurs at night when worms leave the anus to lay eggs. The itching contributes to transmission, as the disease is transmitted through the fecal-oral route. When an infected individual scratches the anal area, eggs may get under the fingernails and later be deposited near the individual’s mouth, causing reinfection, or on fomites, where they can be transferred to new hosts. After being ingested, the larvae hatch within the small intestine and then take up residence in the colon and develop into adults. From the colon, the female adult exits the body at night to lay eggs (Figure \(3\)).
Infection is diagnosed in any of three ways. First, because the worms emerge at night to lay eggs, it is possible to inspect the perianal region for worms while an individual is asleep. An alternative is to use transparent tape to remove eggs from the area around the anus first thing in the morning for three days to yield eggs for microscopic examination. Finally, it may be possible to detect eggs through examination of samples from under the fingernails, where eggs may lodge due to scratching. Once diagnosis has been made, mebendazole, albendazole, and pyrantel pamoate are effective for treatment.
Trichuriasis
The nematode whipworm Trichuris trichiura is a parasite that is transmitted by ingestion from soil-contaminated hands or food and causes trichuriasis. Infection is most common in warm environments, especially when there is poor sanitation and greater risk of fecal contamination of soil, or when food is grown in soil using manure as a fertilizer. The signs and symptoms may be minimal or nonexistent. When a substantial infection develops, signs and symptoms include painful, frequent diarrhea that may contain mucus and blood. It is possible for the infection to cause rectal prolapse, a condition in which a portion of the rectum becomes detached from the inside of the body and protrudes from the anus (Figure \(4\)). Severely infected children may experience reduced growth and their cognitive development may be affected.
When fertilized eggs are ingested, they travel to the intestine and the larvae emerge, taking up residence in the walls of the colon and cecum. They attach themselves with part of their bodies embedded in the mucosa. The larvae mature and live in the cecum and ascending colon. After 60 to 70 days, females begin to lay 3000 to 20,000 eggs per day.
Diagnosis involves examination of the feces for the presence of eggs. It may be necessary to use concentration techniques and to collect specimens on multiple days. Following diagnosis, the infection may be treated with mebendazole, albendazole, or ivermectin.
Trichinosis
Trichinosis (trichenellosis) develops following consumption of food that contains Trichinella spiralis (most commonly) or other Trichinella species. These microscopic nematode worms are most commonly transmitted in meat, especially pork, that has not been cooked thoroughly. T. spiralis larvae in meat emerge from cysts when exposed to acid and pepsin in the stomach. They develop into mature adults within the large intestine. The larvae produced in the large intestine are able to migrate into the muscles mechanically via the stylet of the parasite, forming cysts. Muscle proteins are reduced in abundance or undetectable in cells that contain Trichinella (nurse cells). Animals that ingest the cysts from other animals can later develop infection (Figure \(5\)).
Although infection may be asymptomatic, symptomatic infections begin within a day or two of consuming the nematodes. Abdominal symptoms arise first and can include diarrhea, constipation, and abdominal pain. Other possible symptoms include headache, light sensitivity, muscle pain, fever, cough, chills, and conjunctivitis. More severe symptoms affecting motor coordination, breathing, and the heart sometimes occur. It may take months for the symptoms to resolve, and the condition is occasionally fatal. Mild cases may be mistaken for influenza or similar conditions.
Infection is diagnosed using clinical history, muscle biopsy to look for larvae, and serological testing, including immunoassays. Enzyme immunoassay is the most common test. It is difficult to effectively treat larvae that have formed cysts in the muscle, although medications may help. It is best to begin treatment as soon as possible because medications such as mebendazole and albendazole are effective in killing only the adult worms in the intestine. Steroids may be used to reduce inflammation if larvae are in the muscles.
Exercise \(3\)
Compare and contrast the transmissions of pinworms and whipworms.
Tapeworms (Taeniasis)
Taeniasis is a tapeworm infection, generally caused by pork (Taenia solium), beef (Taenia saginata), and Asian (Taenia asiatica) tapeworms found in undercooked meat. Consumption of raw or undercooked fish, including contaminated sushi, can also result in infection from the fish tapeworm (Diphyllobothrium latum). Tapeworms are flatworms (cestodes) with multiple body segments and a head called a scolex that attaches to the intestinal wall. Tapeworms can become quite large, reaching 4 to 8 meters long (Figure \(6\)). Figure 5.2.5 illustrates the life cycle of a tapeworm.
Tapeworms attached to the intestinal wall produce eggs that are excreted in feces. After ingestion by animals, the eggs hatch and the larvae emerge. They may take up residence in the intestine, but can sometimes move to other tissues, especially muscle or brain tissue. When T. solium larvae form cysts in tissue, the condition is called cysticercosis. This occurs through ingestion of eggs via the fecal-oral route, not through consumption of undercooked meat. It can develop in the muscles, eye (ophthalmic cysticercosis), or brain (neurocysticercosis).
Infections may be asymptomatic or they may cause mild gastrointestinal symptoms such as epigastric discomfort, nausea, diarrhea, flatulence, or hunger pains. It is also common to find visible tapeworm segments passed in the stool. In cases of cysticercosis, symptoms differ depending upon where the cysts become established. Neurocysticercosis can have severe, life-threatening consequences and is associated with headaches and seizures because of the presence of the tapeworm larvae encysted in the brain. Cysts in muscles may be asymptomatic, or they may be painful.
To diagnose these conditions, microscopic analysis of stool samples from three separate days is generally recommended. Eggs or body segments, called proglottids, may be visible in these samples. Molecular methods have been developed but are not yet widely available. Imaging, such as CT and MRI, may be used to detect cysts. Praziquantel or niclosamide are used for treatment.
What’s in Your Sushi Roll?
As foods that contain raw fish, such as sushi and sashimi, continue to increase in popularity throughout the world, so does the risk of parasitic infections carried by raw or undercooked fish. Diphyllobothrium species, known as fish tapeworms, is one of the main culprits. Evidence suggests that undercooked salmon caused an increase in Diphyllobothrium infections in British Columbia in the 1970s and early 1980s. In the years since, the number of reported cases in the United States and Canada has been low, but it is likely that cases are underreported because the causative agent is not easily recognized.3
Another illness transmitted in undercooked fish is herring worm disease, or anisakiasis, in which nematodes attach to the epithelium of the esophagus, stomach, or small intestine. Cases have increased around the world as raw fish consumption has increased.4
Although the message may be unpopular with sushi lovers, fish should be frozen or cooked before eating. The extremely low and high temperatures associated with freezing and cooking kill worms and larvae contained in the meat, thereby preventing infection. Ingesting fresh, raw sushi may make for a delightful meal, but it also entails some risk.
Hydatid Disease
Another cestode, Echinococcus granulosus, causes a serious infection known as hydatid disease (cystic echinococcosis). E. granulosus is found in dogs (the definitive host), as well as several intermediate hosts (sheep, pigs, goats, cattle). The cestodes are transmitted through eggs in the feces from infected animals, which can be an occupational hazard for individuals who work in agriculture.
Once ingested, E. granulosus eggs hatch in the small intestine and release the larvae. The larvae invade the intestinal wall to gain access to the circulatory system. They form hydatid cysts in internal organs, especially in the lungs and liver, that grow slowly and are often undetected until they become large. If the cysts burst, a severe allergic reaction (anaphylaxis) may occur.
Cysts present in the liver can cause enlargement of the liver, nausea, vomiting, right epigastric pain, pain in the right upper quadrant, and possible allergic signs and symptoms. Cysts in the lungs can lead to alveolar disease. Abdominal pain, weight loss, pain, and malaise may occur, and inflammatory processes develop.
E. granulosus can be detected through imaging (ultrasonography, CT, MRI) that shows the cysts. Serologic tests, including ELISA and indirect hemagglutinin tests, are used. Cystic disease is most effectively treated with surgery to remove cysts, but other treatments are also available, including chemotherapy with anti-helminthic drugs (albendazoleor mebendazole).
Exercise \(4\)
Describe the risks of the cysts associated with taeniasis and hydatid disease.
Flukes
Flukes are flatworms that have a leaflike appearance. They are a type of trematode worm, and multiple species are associated with disease in humans. The most common are liver flukes and intestinal flukes (Figure \(7\)).
Liver Flukes
The liver flukes are several species of trematodes that cause disease by interfering with the bile duct. Fascioliasis is caused by Fasciola hepatica and Fasciola gigantica in contaminated raw or undercooked aquatic plants (e.g., watercress). In Fasciola infection, adult flukes develop in the bile duct and release eggs into the feces. Clonochiasis is caused by Clonorchis sinensis in contaminated freshwater fish. Other flukes, such as Opisthorchis viverrini (found in fish) and Opisthorchis felineus (found in freshwater snails), also cause infections. Liver flukes spend part of their life cycle in freshwater snails, which serve as an intermediate host. Humans are typically infected after eating aquatic plants contaminated by the infective larvae after they have left the snail. Once they reach the human intestine, they migrate back to the bile duct, where they mature. The life cycle is similar for the other infectious liver flukes (see Figure 5.2.4).
When Fasciola flukes cause acute infection, signs and symptoms include nausea, vomiting, abdominal pain, rash, fever, malaise, and breathing difficulties. If the infection becomes chronic, with adult flukes living in the bile duct, then cholangitis, cirrhosis, pancreatitis, cholecystitis, and gallstones may develop. Symptoms are similar for infections by other liver flukes. Cholangiocarcinoma can occur from C. sinensis infection. The Opisthorchis species can also be associated with cancer development.
Diagnosis is accomplished using patient history and examination of samples from feces or other samples (such as vomitus). Because the eggs may appear similar, immunoassay techniques are available that can help distinguish species. The preferred treatment for fascioliasis is triclabendazole. C. sinensis and Opisthorchis spp. infections are treated with praziquantel or albendazole.
Intestinal Flukes
The intestinal flukes are trematodes that develop in the intestines. Many, such as Fasciolopsis buski, which causes fasciolopsiasis, are closely related to liver flukes. Intestinal flukes are ingested from contaminated aquatic plants that have not been properly cooked. When the cysts are consumed, the larvae emerge in the duodenum and develop into adults while attached to the intestinal epithelium. The eggs are released in stool.
Intestinal fluke infection is often asymptomatic, but some cases may involve mild diarrhea and abdominal pain. More severe symptoms such as vomiting, nausea, allergic reactions, and anemia can sometimes occur, and high parasite loads may sometimes lead to intestinal obstructions.
Diagnosis is the same as with liver flukes: examination of feces or other samples and immunoassay. Praziquantel is used to treat infections caused by intestinal flukes.
Exercise \(5\)
How are flukes transmitted?
Helminthic Gastrointestinal Infections
Numerous helminths are capable of colonizing the GI tract. Many such infections are asymptomatic, but others may cause signs and symptoms ranging from mild GI stress to severe systemic infection. Helminths have complex and unique life cycles that dictate their specific modes of transmission. Most helminthic infections can be treated with medications.
Clinical Focus: Resolution
Carli’s doctor explained that she had bacterial gastroenteritis caused by Salmonella bacteria. The source of these bacteria was likely the undercooked egg. Had the egg been fully cooked, the high temperature would have been sufficient to kill any Salmonella in or on the egg. In this case, enough bacteria survived to cause an infection once the egg was eaten.
Carli’s signs and symptoms continued to worsen. Her fever became higher, her vomiting and diarrhea continued, and she began to become dehydrated. She felt thirsty all the time and had continual abdominal cramps. Carli’s doctor treated her with intravenous fluids to help with her dehydration, but did not prescribe antibiotics. Carli’s parents were confused because they thought a bacterial infection should always be treated with antibiotics.
The doctor explained that the worst medical problem for Carli was dehydration. Except in the most vulnerable and sick patients, such as those with HIV/AIDS, antibiotics do not reduce recovery time or improve outcomes in Salmonella infections. In fact, antibiotics can actually delay the natural excretion of bacteria from the body. Rehydration therapy replenishes lost fluids, diminishing the effects of dehydration and improving the patient’s condition while the infection resolves.
After two days of rehydration therapy, Carli’s signs and symptoms began to fade. She was still somewhat thirsty, but the amount of urine she passed became larger and the color lighter. She stopped vomiting. Her fever was gone, and so was the diarrhea. At that point, stool analysis found very few Salmonella bacteria. In one week, Carli was discharged as fully recovered.
Key Concepts and Summary
• Helminths often cause intestinal infections after transmission to humans through exposure to contaminated soil, water, or food. Signs and symptoms are often mild, but severe complications may develop in some cases.
• Ascaris lumbricoides eggs are transmitted through contaminated food or water and hatch in the intestine. Juvenile larvae travel to the lungs and then to the pharynx, where they are swallowed and returned to the intestines to mature. These nematode roundworms cause ascariasis.
• Necator americanus and Ancylostoma doudenale cause hookworm infection when larvae penetrate the skin from soil contaminated by dog or cat feces. They travel to the lungs and are then swallowed to mature in the intestines.
• Strongyloides stercoralis are transmitted from soil through the skin to the lungs and then to the intestine where they cause strongyloidiasis.
• Enterobius vermicularis are nematode pinworms transmitted by the fecal-oral route. After ingestion, they travel to the colon where they cause enterobiasis.
• Trichuris trichiura can be transmitted through soil or fecal contamination and cause trichuriasis. After ingestion, the eggs travel to the intestine where the larvae emerge and mature, attaching to the walls of the colon and cecum.
• Trichinella spp. is transmitted through undercooked meat. Larvae in the meat emerge from cysts and mature in the large intestine. They can migrate to the muscles and form new cysts, causing trichinosis.
• Taenia spp. and Diphyllobothrium latum are tapeworms transmitted through undercooked food or the fecal-oral route. Taenia infections cause taeniasis. Tapeworms use their scolex to attach to the intestinal wall. Larvae may also move to muscle or brain tissue.
• Echinococcus granulosus is a cestode transmitted through eggs in the feces of infected animals, especially dogs. After ingestion, eggs hatch in the small intestine, and the larvae invade the intestinal wall and travel through the circulatory system to form dangerous cysts in internal organs, causing hydatid disease.
• Flukes are transmitted through aquatic plants or fish. Liver flukes cause disease by interfering with the bile duct. Intestinal flukes develop in the intestines, where they attach to the intestinal epithelium.
Footnotes
1. 1 Centers for Disease Control and Prevention. “Parasites–Ascariasis.” Updated May 24, 2016. http://www.cdc.gov/parasites/ascariasis/index.html.
2. 2 “Roundworms.” University of Maryland Medical Center Medical Reference Guide. Last reviewed December 9, 2014. https://umm.edu/health/medical/altme...ion/roundworms.
3. 3 Nancy Craig. “Fish Tapeworm and Sushi.” Canadian Family Physician 58 (2012) 6: pp. 654–658. www.ncbi.nlm.nih.gov/pmc/articles/PMC3374688/.
4. 4 Centers for Disease Control and Prevention. “Anisakiasis FAQs.” Updated November 12, 2012. http://www.cdc.gov/parasites/anisakiasis/faqs.html. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/24%3A_Digestive_System_Infections/24.06%3A_Helminthic_Infections_of_the_Gastrointestinal_Tract.txt |
24.1: Anatomy and Normal Microbiota of the Digestive System
The human digestive system, or the gastrointestinal (GI) tract, begins with the mouth and ends with the anus. The parts of the mouth include the teeth, the gums, the tongue, the oral vestibule (the space between the gums, lips, and teeth), and the oral cavity proper (the space behind the teeth and gums). Other parts of the GI tract are the pharynx, esophagus, stomach, small intestine, large intestine, rectum, and anus.
Multiple Choice
Which of the following is NOT a way the normal microbiota of the intestine helps to prevent infection?
1. It produces acids that lower the pH of the stomach.
2. It speeds up the process by which microbes are flushed from the digestive tract.
3. It consumes food and occupies space, outcompeting potential pathogens.
4. It generates large quantities of oxygen that kill anaerobic pathogens.
Answer
D
What types of microbes live in the intestines?
1. Diverse species of bacteria, archaea, and fungi, especially Bacteroides and Firmicutes bacteria
2. A narrow range of bacteria, especially Firmicutes
3. A narrow range of bacteria and fungi, especially Bacteroides
4. Archaea and fungi only
Answer
A
Fill in the Blank
The part of the gastrointestinal tract with the largest natural microbiota is the _________.
Answer
Large intestine or colon
Short Answer
How does the diarrhea caused by dysentery differ from other types of diarrhea?
24.2: Microbial Diseases of the Mouth and Oral Cavity
Despite the presence of saliva and the mechanical forces of chewing and eating, some microbes thrive in the mouth. These microbes can cause damage to the teeth and can cause infections that have the potential to spread beyond the mouth and sometimes throughout the body.
Multiple Choice
What pathogen is the most important contributor to biofilms in plaque?
1. Staphylococcus aureus
2. Streptococcus mutans
3. Escherichia coli
4. Clostridium difficile
Answer
B
What type of organism causes thrush?
1. a bacterium
2. a virus
3. a fungus
4. a protozoan
Answer
C
In mumps, what glands swell to produce the disease’s characteristic appearance?
1. the sublingual glands
2. the gastric glands
3. the parotid glands
4. the submandibular glands
Answer
C
Which of the following is true of HSV-1?
1. It causes oral thrush in immunocompromised patients.
2. Infection is generally self-limiting.
3. It is a bacterium.
4. It is usually treated with amoxicillin.
Answer
B
Fill in the Blank
When plaque becomes heavy and hardened, it is called dental calculus or _________.
Answer
tartar
Short Answer
Why do sugary foods promote dental caries?
24.3: Bacterial Infections of the Gastrointestinal Tract
Major causes of gastrointestinal illness include Salmonella spp., Staphylococcus spp., Helicobacter pylori, Clostridium perfringens, Clostridium difficile, Bacillus cereus, and Yersinia bacteria. C. difficile is an important cause of hospital acquired infection. Vibrio cholerae causes cholera, which can be a severe diarrheal illness. Different strains of E. coli, including ETEC, EPEC, EIEC, and EHEC, cause different illnesses with varying degrees of severity.
Multiple Choice
Which type of E. coli infection can be severe with life-threatening consequences such as hemolytic uremic syndrome?
1. ETEC
2. EPEC
3. EHEC
4. EIEC
Answer
C
Which species of Shigella has a type that produces Shiga toxin?
1. S. boydii
2. S. flexneri
3. S. dysenteriae
4. S. sonnei
Answer
C
Which type of bacterium produces an A-B toxin?
1. Salmonella
2. Vibrio cholera
3. ETEC
4. Shigella dysenteriae
Answer
B
Fill in the Blank
Antibiotic associated pseudomembranous colitis is caused by _________.
Answer
Clostridium difficile
Critical Thinking
Why does use of antibiotics and/or proton pump inhibitors contribute to the development of C. difficile infections?
Why did scientists initially think it was unlikely that a bacterium caused peptic ulcers?
Does it makes a difference in treatment to know if a particular illness is caused by a bacterium (an infection) or a toxin (an intoxication)?
24.4: Viral Infections of the Gastrointestinal Tract
Common viral causes of gastroenteritis include rotaviruses, noroviruses, and astroviruses. Hepatitis may be caused by several unrelated viruses: hepatitis viruses A, B, C, D, and E. The hepatitis viruses differ in their modes of transmission, treatment, and potential for chronic infection.
Multiple Choice
Which form of hepatitisvirus can only infect an individual who is already infected with another hepatitisvirus?
1. HDV
2. HAV
3. HBV
4. HEV
Answer
A
Which cause of viral gastroenteritis commonly causes projectile vomiting?
1. hepatitisvirus
2. Astroviruses
3. Rotavirus
4. Noroviruses
Answer
D
Fill in the Blank
Jaundice results from a buildup of _________.
Answer
bilirubin
Short Answer
Which forms of viral hepatitis are transmitted through the fecal-oral route?
Critical Thinking
Based on what you know about HBV, what are some ways that its transmission could be reduced in a health-care setting?
24.5: Protozoan Infections of the Gastrointestinal Tract
Like other microbes, protozoa are abundant in natural microbiota but can also be associated with significant illness. Gastrointestinal diseases caused by protozoa are generally associated with exposure to contaminated food and water, meaning that those without access to good sanitation are at greatest risk. Even in developed countries, infections can occur and these microbes have sometimes caused significant outbreaks from contamination of public water supplies.
Multiple Choice
Which protozoan is associated with the ability to cause severe dysentery?
1. Giardia lamblia
2. Cryptosporidium hominis
3. Cyclospora cayetanesis
4. Entamoeba histolytica
Answer
D
Which protozoan has a unique appearance, with a blue halo, when viewed using ultraviolet fluorescence microscopy?
1. Giardia lamblia
2. Cryptosporidium hominis
3. Cyclospora cayetanesis
4. Entamoeba histolytica
Answer
C
The micrograph shows protozoans attached to the intestinal wall of a gerbil. Based on what you know about protozoan intestinal parasites, what is it?
(credit: Dr. Stan Erlandsen, Centers for Disease Control and Prevention)
1. Giardia lamblia
2. Cryptosporidium hominis
3. Cyclospora cayetanesis
4. Entamoeba histolytica
Fill in the Blank
Chronic _________ infections cause the unique sign of disease of greasy stool and are often resistant to treatment.
Answer
giardia
Short Answer
What is an O&P exam?
24.6: Helminthic Infections of the Gastrointestinal Tract
Helminths are widespread intestinal parasites. These parasites can be divided into three common groups: round-bodied worms also described as nematodes, flat-bodied worms that are segmented (also described as cestodes), and flat-bodied worms that are non-segmented (also described as trematodes). The nematodes include roundworms, pinworms, hookworms, and whipworms. Many of these parasites are so well adapted to the human host that there is little obvious disease.
Multiple Choice
What is another name for Trichuris trichiura?
1. pinworm
2. whipworm
3. hookworm
4. ascariasis
Answer
B
Which type of helminth infection can be diagnosed using tape?
1. pinworm
2. whipworm
3. hookworm
4. tapeworm
Answer
A
Fill in the Blank
Liver flukes are often found in the _________ duct.
Answer
bile
Short Answer
Why does the coughing up of worms play an important part in the life cycle of some helminths, such as the roundworm Ascaris lumbricoides?
Critical Thinking
Cases of strongyloidiasis are often more severe in patients who are using corticosteroids to treat another disorder. Explain why this might occur. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/24%3A_Digestive_System_Infections/24.E%3A_Digestive_System_Infections_%28Exercises%29.txt |
Yellow fever was once common in the southeastern US, with annual outbreaks of more than 25,000 infections in New Orleans in the mid-1800s.1 In the early 20th century, efforts to eradicate the virus that causes yellow fever were successful thanks to vaccination programs and effective control (mainly through the insecticide dichlorodiphenyltrichloroethane [DDT]) of Aedes aegypti, the mosquito that serves as a vector. Today, the virus has been largely eradicated in North America.
Elsewhere, efforts to contain yellow fever have been less successful. Despite mass vaccination campaigns in some regions, the risk for yellow fever epidemics is rising in dense urban cities in Africa and South America.2 In an increasingly globalized society, yellow fever could easily make a comeback in North America, where A. aegypti is still present. If these mosquitoes were exposed to infected individuals, new outbreaks would be possible.
Like yellow fever, many of the circulatory and lymphatic diseases discussed in this chapter are emerging or re-emerging worldwide. Despite medical advances, diseases like malaria, Ebola, and others could become endemic in the US given the right circumstances.
• 25.1: Anatomy of the Circulatory and Lymphatic Systems
The circulatory and lymphatic systems are networks of vessels and a pump that transport blood and lymph, respectively, throughout the body. When these systems are infected with a microorganism, the network of vessels can facilitate the rapid dissemination of the microorganism to other regions of the body, sometimes with serious results. In this section, we examine some of the key anatomical features of the circulatory and lymphatic systems, as well as general signs and symptoms of infection.
• 25.2: Bacterial Infections of the Circulatory and Lymphatic Systems
Bacterial infections of the circulatory system are almost universally serious. Left untreated, most have high mortality rates. Bacterial pathogens usually require a breach in the immune defenses to colonize the circulatory system. Most often, this involves a wound or the bite of an arthropod vector, but it can also occur in hospital settings and result in nosocomial infections.
• 25.3: Viral Infections of the Circulatory and Lymphatic Systems
Viral pathogens of the circulatory system vary tremendously both in their virulence and distribution worldwide. Some of these pathogens are practically global in their distribution. Fortunately, the most ubiquitous viruses tend to produce the mildest forms of disease. In the majority of cases, those infected remain asymptomatic. On the other hand, other viruses are associated with life-threatening diseases that have impacted human history.
• 25.4: Parasitic Infections of the Circulatory and Lymphatic Systems
Some protozoa and parasitic flukes are also capable of causing infections of the human circulatory system. Although these infections are rare in the US, they continue to cause widespread suffering in the developing world today. Malaria, toxoplasmosis, babesiosis, Chagas disease, leishmaniasis, and schistosomiasis are discussed in this section.
• 25.E: Circulatory and Lymphatic System Infections (Exercises)
Footnotes
1. 1 Centers for Disease Control and Prevention. “The History of Yellow Fever.” http://www.cdc.gov/travel-training/local/HistoryEpidemiologyandVaccination/page27568.html
2. 2 C.L. Gardner, K.D. Ryman. “Yellow Fever: A Reemerging Threat.” Clinical Laboratory Medicine 30 no. 1 (2010):237–260.
Thumbnail: This "classic" bull's-eye rash is also called erythema migrans. A rash caused by Lyme does not always look like this and approximately 25% of those infected with Lyme disease may have no rash. (Public Domain; CDC/ James Gathany).
25: Circulatory and Lymphatic System Infections
Learning Objectives
• Describe the major anatomical features of the circulatory and lymphatic systems
• Explain why the circulatory and lymphatic systems lack normal microbiota
• Explain how microorganisms overcome defenses of the circulatory and lymphatic systems to cause infection
• Describe general signs and symptoms of disease associated with infections of the circulatory and lymphatic systems
Clinical Focus: Part 1
Barbara is a 43-year-old patient who has been diagnosed with metastatic inflammatory breast cancer. To facilitate her ongoing chemotherapy, her physician implanted a port attached to a central venous catheter. At a recent checkup, she reported feeling restless and complained that the site of the catheter had become uncomfortable. After removing the dressing, the physician observed that the surgical site appeared red and was warm to the touch, suggesting a localized infection. Barbara was also running a fever of 38.2 °C (100.8 °F). Her physician treated the affected area with a topical antiseptic and applied a fresh dressing. She also prescribed a course of the antibiotic oxacillin.
Exercise \(1\)
1. Based on this information, what factors likely contributed to Barbara’s condition?
2. What is the most likely source of the microbes involved?
The circulatory and lymphatic systems are networks of vessels and a pump that transport blood and lymph, respectively, throughout the body. When these systems are infected with a microorganism, the network of vessels can facilitate the rapid dissemination of the microorganism to other regions of the body, sometimes with serious results. In this section, we will examine some of the key anatomical features of the circulatory and lymphatic systems, as well as general signs and symptoms of infection.
The Circulatory System
The circulatory (or cardiovascular) system is a closed network of organs and vessels that moves blood around the body (Figure \(1\)). The primary purposes of the circulatory system are to deliver nutrients, immune factors, and oxygen to tissues and to carry away waste products for elimination. The heart is a four-chambered pump that propels the blood throughout the body. Deoxygenated blood enters the right atrium through the superior vena cava and the inferior vena cava after returning from the body. The blood next passes through the tricuspid valve to enter the right ventricle. When the heart contracts, the blood from the right ventricle is pumped through the pulmonary arteries to the lungs. There, the blood is oxygenated at the alveoli and returns to the heart through the pulmonary veins. The oxygenated blood is received at the left atrium and proceeds through the mitral valve to the left ventricle. When the heart contracts, the oxygenated blood is pumped throughout the body via a series of thick-walled vessels called arteries. The first and largest artery is called the aorta. The arteries sequentially branch and decrease in size (and are called arterioles) until they end in a network of smaller vessels called capillaries. The capillary beds are located in the interstitial spaces within tissues and release nutrients, immune factors, and oxygen to those tissues. The capillaries connect to a series of vessels called venules, which increase in size to form the veins. The veins join together into larger vessels as they transfer blood back to the heart. The largest veins, the superior and inferior vena cava, return the blood to the right atrium.
Other organs play important roles in the circulatory system as well. The kidneys filter the blood, removing waste products and eliminating them in the urine. The liver also filters the blood and removes damaged or defective red blood cells. The spleen filters and stores blood, removes damaged red blood cells, and is a reservoir for immune factors. All of these filtering structures serve as sites for entrapment of microorganisms and help maintain an environment free of microorganisms in the blood.
The Lymphatic System
The lymphatic system is also a network of vessels that run throughout the body (Figure \(2\)). However, these vessels do not form a full circulating system and are not pressurized by the heart. Rather, the lymphatic system is an open system with the fluid moving in one direction from the extremities toward two drainage points into veins just above the heart. Lymphatic fluids move more slowly than blood because they are not pressurized. Small lymph capillaries interact with blood capillaries in the interstitial spaces in tissues. Fluids from the tissues enter the lymph capillaries and are drained away (Figure \(3\)). These fluids, termed lymph, also contain large numbers of white blood cells.
The lymphatic system contains two types of lymphoid tissues. The primary lymphoid tissue includes bone marrow and the thymus. Bone marrow contains the hematopoietic stem cells (HSC) that differentiate and mature into the various types of blood cells and lymphocytes (see Figure 17.3.1). The secondary lymphoid tissues include the spleen, lymph nodes, and several areas of diffuse lymphoid tissues underlying epithelial membranes. The spleen, an encapsulated structure, filters blood and captures pathogens and antigens that pass into it (Figure \(4\)). The spleen contains specialized macrophages and dendritic cells that are crucial for antigen presentation, a mechanism critical for activation of T lymphocytes and B lymphocytes (see Major Histocompatibility Complexes and Antigen-Presenting Cells). Lymph nodes are bean-shaped organs situated throughout the body. These structures contain areas called germinal centers that are rich in B and T lymphocytes. The lymph nodes also contain macrophages and dendritic cells for antigen presentation. Lymph from nearby tissues enters the lymph node through afferent lymphatic vessels and encounters these lymphocytes as it passes through; the lymph exits the lymph node through the efferent lymphatic vessels (Figure \(4\)).
Link to Learning
The lymphatic system filters fluids that have accumulated in tissues before they are returned to the blood. A brief overview of this process is provided at this website.
Exercise \(2\)
What is the main function of the lymphatic system?
Infections of the Circulatory System
Under normal circumstances, the circulatory system and the blood should be sterile; the circulatory system has no normal microbiota. Because the system is closed, there are no easy portals of entry into the circulatory system for microbes. Those that are able to breach the body’s physical barriers and enter the bloodstream encounter a host of circulating immune defenses, such as antibodies, complement proteins, phagocytes, and other immune cells. Microbes often gain access to the circulatory system through a break in the skin (e.g., wounds, needles, intravenous catheters, insect bites) or spread to the circulatory system from infections in other body sites. For example, microorganisms causing pneumonia or renal infection may enter the local circulation of the lung or kidney and spread from there throughout the circulatory network.
If microbes in the bloodstream are not quickly eliminated, they can spread rapidly throughout the body, leading to serious, even life-threatening infections. Various terms are used to describe conditions involving microbes in the circulatory system. The term bacteremia refers to bacteria in the blood. If bacteria are reproducing in the blood as they spread, this condition is called septicemia. The presence of viruses in the blood is called viremia. Microbial toxins can also be spread through the circulatory system, causing a condition termed toxemia.
Microbes and microbial toxins in the blood can trigger an inflammatory response so severe that the inflammation damages host tissues and organs more than the infection itself. This counterproductive immune response is called systemic inflammatory response syndrome (SIRS), and it can lead to the life-threatening condition known as sepsis. Sepsis is characterized by the production of excess cytokines that leads to classic signs of inflammation such as fever, vasodilation, and edema (see Inflammation and Fever). In a patient with sepsis, the inflammatory response becomes dysregulated and disproportionate to the threat of infection. Critical organs such as the heart, lungs, liver, and kidneys become dysfunctional, resulting in increased heart and respiratory rates, and disorientation. If not treated promptly and effectively, patients with sepsis can go into shock and die.
Certain infections can cause inflammation in the heart and blood vessels. Inflammation of the endocardium, the inner lining of the heart, is called endocarditis and can result in damage to the heart valves severe enough to require surgical replacement. Inflammation of the pericardium, the sac surrounding the heart, is called pericarditis. The term myocarditis refers to the inflammation of the heart’s muscle tissue. Pericarditis and myocarditis can cause fluid to accumulate around the heart, resulting in congestive heart failure. Inflammation of blood vessels is called vasculitis. Although somewhat rare, vasculitis can cause blood vessels to become damaged and rupture; as blood is released, small red or purple spots called petechiae appear on the skin. If the damage of tissues or blood vessels is severe, it can result in reduced blood flow to the surrounding tissues. This condition is called ischemia, and it can be very serious. In severe cases, the affected tissues can die and become necrotic; these situations may require surgical debridement or amputation.
Exercise \(3\)
1. Why does the circulatory system have no normal microbiota?
2. Explain why the presence of microbes in the circulatory system can lead to serious consequences.
Infections of the Lymphatic System
Like the circulatory system, the lymphatic system does not have a normal microbiota, and the large numbers of immune cells typically eliminate transient microbes before they can establish an infection. Only microbes with an array of virulence factors are able to overcome these defenses and establish infection in the lymphatic system. However, when a localized infection begins to spread, the lymphatic system is often the first place the invading microbes can be detected.
Infections in the lymphatic system also trigger an inflammatory response. Inflammation of lymphatic vessels, called lymphangitis, can produce visible red streaks under the skin. Inflammation in the lymph nodes can cause them to swell. A swollen lymph node is referred to as a bubo, and the condition is referred to as lymphadenitis.
Key Concepts and Summary
• The circulatory system moves blood throughout the body and has no normal microbiota.
• The lymphatic system moves fluids from the interstitial spaces of tissues toward the circulatory system and filters the lymph. It also has no normal microbiota.
• The circulatory and lymphatic systems are home to many components of the host immune defenses.
• Infections of the circulatory system may occur after a break in the skin barrier or they may enter the bloodstream at the site of a localized infection. Pathogens or toxins in the bloodstream can spread rapidly throughout the body and can provoke systemic and sometimes fatal inflammatory responses such as SIRS, sepsis, and endocarditis.
• Infections of the lymphatic system can cause lymphangitis and lymphadenitis. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/25%3A_Circulatory_and_Lymphatic_System_Infections/25.01%3A_Anatomy_of_the_Circulatory_and_Lymphatic_Systems.txt |
Learning Objectives
• Identify and compare bacteria that most commonly cause infections of the circulatory and lymphatic systems
• Compare the major characteristics of specific bacterial diseases affecting the circulatory and lymphatic systems
Bacteria can enter the circulatory and lymphatic systems through acute infections or breaches of the skin barrier or mucosa. Breaches may occur through fairly common occurrences, such as insect bites or small wounds. Even the act of tooth brushing, which can cause small ruptures in the gums, may introduce bacteria into the circulatory system. In most cases, the bacteremia that results from such common exposures is transient and remains below the threshold of detection. In severe cases, bacteremia can lead to septicemia with dangerous complications such as toxemia, sepsis, and septic shock. In these situations, it is often the immune response to the infection that results in the clinical signs and symptoms rather than the microbes themselves.
Bacterial Sepsis, Septic and Toxic Shock
At low concentrations, pro-inflammatory cytokines such as interleukin 1 (IL-1) and tumor necrosis factor-α (TNF-α) play important roles in the host’s immune defenses. When they circulate systemically in larger amounts, however, the resulting immune response can be life threatening. IL-1 induces vasodilation (widening of blood vessels) and reduces the tight junctions between vascular endothelial cells, leading to widespread edema. As fluids move out of circulation into tissues, blood pressure begins to drop. If left unchecked, the blood pressure can fall below the level necessary to maintain proper kidney and respiratory functions, a condition known as septic shock. In addition, the excessive release of cytokines during the inflammatory response can lead to the formation of blood clots. The loss of blood pressure and occurrence of blood clots can result in multiple organ failure and death.
Bacteria are the most common pathogens associated with the development of sepsis, and septic shock.1 The most common infection associated with sepsis is bacterial pneumonia (see Bacterial Infections of the Respiratory Tract), accounting for about half of all cases, followed by intra-abdominal infections (Bacterial Infections of the Gastrointestinal Tract) and urinary tract infections (Bacterial Infections of the Urinary System).2 Infections associated with superficial wounds, animal bites, and indwelling catheters may also lead to sepsis and septic shock.
These initially minor, localized infections can be caused by a wide range of different bacteria, including Staphylococcus, Streptococcus, Pseudomonas, Pasteurella, Acinetobacter, and members of the Enterobacteriaceae. However, if left untreated, infections by these gram-positive and gram-negative pathogens can potentially progress to sepsis, shock, and death.
Toxic Shock Syndrome and Streptococcal Toxic Shock-Like Syndrome
Toxemia associated with infections caused by Staphylococcus aureus can cause staphylococcal toxic shock syndrome (TSS). Some strains of S. aureus produce a superantigen called toxic shock syndrome toxin-1 (TSST-1). TSS may occur as a complication of other localized or systemic infections such as pneumonia, osteomyelitis, sinusitis, and skin wounds (surgical, traumatic, or burns). Those at highest risk for staphylococcal TSS are women with preexisting S. aureus colonization of the vagina who leave tampons, contraceptive sponges, diaphragms, or other devices in the vagina for longer than the recommended time.
Staphylococcal TSS is characterized by sudden onset of vomiting, diarrhea, myalgia, body temperature higher than 38.9 °C (102.0 °F), and rapid-onset hypotension with a systolic blood pressure less than 90 mm Hg for adults; a diffuse erythematous rash that leads to peeling and shedding skin 1 to 2 weeks after onset; and additional involvement of three or more organ systems.3 The mortality rate associated with staphylococcal TSS is less than 3% of cases.
Diagnosis of staphylococcal TSS is based on clinical signs, symptoms, serologic tests to confirm bacterial species, and the detection of toxin production from staphylococcal isolates. Cultures of skin and blood are often negative; less than 5% are positive in cases of staphylococcal TSS. Treatment for staphylococcal TSS includes decontamination, debridement, vasopressors to elevate blood pressure, and antibiotic therapy with clindamycin plus vancomycin or daptomycin pending susceptibility results.
A syndrome with signs and symptoms similar to staphylococcal TSS can be caused by Streptococcus pyogenes. This condition, called streptococcal toxic shock-like syndrome (STSS), is characterized by more severe pathophysiology than staphylococcal TSS,4 with about 50% of patients developing S. pyogenes bacteremia and necrotizing fasciitis. In contrast to staphylococcal TSS, STSS is more likely to cause acute respiratory distress syndrome (ARDS), a rapidly progressive disease characterized by fluid accumulation in the lungs that inhibits breathing and causes hypoxemia (low oxygen levels in the blood). STSS is associated with a higher mortality rate (20%–60%), even with aggressive therapy. STSS usually develops in patients with a streptococcal soft-tissue infection such as bacterial cellulitis, necrotizing fasciitis, pyomyositis (pus formation in muscle caused by infection), a recent influenza A infection, or chickenpox.
Exercise \(1\)
How can large amounts of pro-inflammatory cytokines lead to septic shock?
Clinical Focus: Part 2
Despite oxacillin therapy, Barbara’s condition continued to worsen over the next several days. Her fever increased to 40.1 °C (104.2 °F) and she began to experience chills, rapid breathing, and confusion. Her doctor suspected bacteremia by a drug-resistant bacterium and admitted Barbara to the hospital. Cultures of the surgical site and blood revealed Staphylococcus aureus. Antibiotic susceptibility testing confirmed that the isolate was methicillin-resistant S. aureus (MRSA). In response, Barbara’s doctor changed her antibiotic therapy to vancomycin and arranged to have the port and venous catheter removed.
Exercise \(2\)
1. Why did Barbara’s infection not respond to oxacillin therapy?
2. Why did the physician have the port and catheter removed?
3. Based on the signs and symptoms described, what are some possible diagnoses for Barbara’s condition?
Puerperal Sepsis
A type of sepsis called puerperal sepsis, also known as puerperal infection, puerperal fever, or childbed fever, is a nosocomial infection associated with the period of puerperium—the time following childbirth during which the mother’s reproductive system returns to a nonpregnant state. Such infections may originate in the genital tract, breast, urinary tract, or a surgical wound. Initially the infection may be limited to the uterus or other local site of infection, but it can quickly spread, resulting in peritonitis, septicemia, and death. Before the 19th century work of Ignaz Semmelweis and the widespread acceptance of germ theory (see Modern Foundations of Cell Theory), puerperal sepsis was a major cause of mortality among new mothers in the first few days following childbirth.
Puerperal sepsis is often associated with Streptococcus pyogenes, but numerous other bacteria can also be responsible. Examples include gram-positive bacterial (e.g. Streptococcus spp., Staphylococcus spp., and Enterococcus spp.), gram-negative bacteria (e.g. Chlamydia spp., Escherichia coli, Klebsiella spp., and Proteus spp.), as well as anaerobes such as Peptostreptococcus spp., Bacteroides spp., and Clostridium spp. In cases caused by S. pyogenes, the bacteria attach to host tissues using M protein and produce a carbohydrate capsule to avoid phagocytosis. S. pyogenes also produces a variety of exotoxins, like streptococcal pyrogenic exotoxins A and B, that are associated with virulence and may function as superantigens.
Diagnosis of puerperal fever is based on the timing and extent of fever and isolation, and identification of the etiologic agent in blood, wound, or urine specimens. Because there are numerous possible causes, antimicrobial susceptibility testing must be used to determine the best antibiotic for treatment. Nosocomial incidence of puerperal fever can be greatly reduced through the use of antiseptics during delivery and strict adherence to handwashing protocols by doctors, midwives, and nurses.
Infectious Arthritis
Also called septic arthritis, infectious arthritis can be either an acute or a chronic condition. Infectious arthritis is characterized by inflammation of joint tissues and is most often caused by bacterial pathogens. Most cases of acute infectious arthritis are secondary to bacteremia, with a rapid onset of moderate to severe joint pain and swelling that limits the motion of the affected joint. In adults and young children, the infective pathogen is most often introduced directly through injury, such as a wound or a surgical site, and brought to the joint through the circulatory system. Acute infections may also occur after joint replacement surgery. Acute infectious arthritis often occurs in patients with an immune system impaired by other viral and bacterial infections. S. aureus is the most common cause of acute septic arthritis in the general population of adults and young children. Neisseria gonorrhoeae is an important cause of acute infectious arthritis in sexually active individuals.
Chronic infectious arthritis is responsible for 5% of all infectious arthritis cases and is more likely to occur in patients with other illnesses or conditions. Patients at risk include those who have an HIV infection, a bacterial or fungal infection, prosthetic joints, rheumatoid arthritis (RA), or who are undergoing immunosuppressive chemotherapy. Onset is often in a single joint; there may be little or no pain, aching pain that may be mild, gradual swelling, mild warmth, and minimal or no redness of the joint area.
Diagnosis of infectious arthritis requires the aspiration of a small quantity of synovial fluid from the afflicted joint. Direct microscopic evaluation, culture, antimicrobial susceptibility testing, and polymerase chain reaction (PCR) analyses of the synovial fluid are used to identify the potential pathogen. Typical treatment includes administration of appropriate antimicrobial drugs based on antimicrobial susceptibility testing. For nondrug-resistant bacterial strains, β-lactams such as oxacillin and cefazolin are often prescribed for staphylococcal infections. Third-generation cephalosporins (e.g., ceftriaxone) are used for increasingly prevalent β-lactam-resistant Neisseria infections. Infections by Mycobacterium spp. or fungi are treated with appropriate long-term antimicrobial therapy. Even with treatment, the prognosis is often poor for those infected. About 40% of patients with nongonnococcal infectious arthritis will suffer permanent joint damage and mortality rates range from 5% to 20%.5 Mortality rates are higher among the elderly.6
Osteomyelitis
Osteomyelitis is an inflammation of bone tissues most commonly caused by infection. These infections can either be acute or chronic and can involve a variety of different bacteria. The most common causative agent of osteomyelitis is S. aureus. However, M. tuberculosis, Pseudomonas aeruginosa, Streptococcus pyogenes, S. agalactiae, species in the Enterobacteriaceae, and other microorganisms can also cause osteomyelitis, depending on which bones are involved. In adults, bacteria usually gain direct access to the bone tissues through trauma or a surgical procedure involving prosthetic joints. In children, the bacteria are often introduced from the bloodstream, possibly spreading from focal infections. The long bones, such as the femur, are more commonly affected in children because of the more extensive vascularization of bones in the young.7
The signs and symptoms of osteomyelitis include fever, localized pain, swelling due to edema, and ulcers in soft tissues near the site of infection. The resulting inflammation can lead to tissue damage and bone loss. In addition, the infection may spread to joints, resulting in infectious arthritis, or disseminate into the blood, resulting in sepsis and thrombosis(formation of blood clots). Like septic arthritis, osteomyelitis is usually diagnosed using a combination of radiography, imaging, and identification of bacteria from blood cultures, or from bone cultures if blood cultures are negative. Parenteral antibiotic therapy is typically used to treat osteomyelitis. Because of the number of different possible etiologic agents, however, a variety of drugs might be used. Broad-spectrum antibacterial drugs such as nafcillin, oxacillin, or cephalosporin are typically prescribed for acute osteomyelitis, and ampicillin and piperacillin/tazobactam for chronic osteomyelitis. In cases of antibiotic resistance, vancomycin treatment is sometimes required to control the infection. In serious cases, surgery to remove the site of infection may be required. Other forms of treatment include hyperbaric oxygen therapy (see Using Physical Methods to Control Microorganisms) and implantation of antibiotic beads or pumps.
Exercise \(3\)
What bacterium the most common cause of both septic arthritis and osteomyelitis?
Rheumatic Fever
Infections with S. pyogenes have a variety of manifestations and complications generally called sequelae. As mentioned, the bacterium can cause suppurative infections like puerperal fever. However, this microbe can also cause nonsuppurative sequelae in the form of acute rheumatic fever (ARF), which can lead to rheumatic heart disease, thus impacting the circulatory system. Rheumatic fever occurs primarily in children a minimum of 2–3 weeks after an episode of untreated or inadequately treated pharyngitis (see Bacterial Infections of the Respiratory Tract). At one time, rheumatic fever was a major killer of children in the US; today, however, it is rare in the US because of early diagnosis and treatment of streptococcal pharyngitis with antibiotics. In parts of the world where diagnosis and treatment are not readily available, acute rheumatic fever and rheumatic heart disease are still major causes of mortality in children.8
Rheumatic fever is characterized by a variety of diagnostic signs and symptoms caused by nonsuppurative, immune-mediated damage resulting from a cross-reaction between patient antibodies to bacterial surface proteins and similar proteins found on cardiac, neuronal, and synovial tissues. Damage to the nervous tissue or joints, which leads to joint pain and swelling, is reversible. However, damage to heart valves can be irreversible and is worsened by repeated episodes of acute rheumatic fever, particularly during the first 3–5 years after the first rheumatic fever attack. The inflammation of the heart valves caused by cross-reacting antibodies leads to scarring and stiffness of the valve leaflets. This, in turn, produces a characteristic heart murmur. Patients who have previously developed rheumatic fever and who subsequently develop recurrent pharyngitis due to S. pyogenes are at high risk for a recurrent attacks of rheumatic fever.
The American Heart Association recommends9 a treatment regimen consisting of benzathine benzylpenicillin every 3 or 4 weeks, depending on the patient’s risk for reinfection. Additional prophylactic antibiotic treatment may be recommended depending on the age of the patient and risk for reinfection.
Bacterial Endocarditis and Pericarditis
The endocardium is a tissue layer that lines the muscles and valves of the heart. This tissue can become infected by a variety of bacteria, including gram-positive cocci such as Staphylococcus aureus, viridans streptococci, and Enterococcus faecalis, and the gram-negative so-called HACEK bacilli: Haemophilus spp., Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae. The resulting inflammation is called endocarditis, which can be described as either acute or subacute. Causative agents typically enter the bloodstream during accidental or intentional breaches in the normal barrier defenses (e.g., dental procedures, body piercings, catheterization, wounds). Individuals with preexisting heart damage, prosthetic valves and other cardiac devices, and those with a history of rheumatic fever have a higher risk for endocarditis. This disease can rapidly destroy the heart valves and, if untreated, lead to death in just a few days.
In subacute bacterial endocarditis, heart valve damage occurs slowly over a period of months. During this time, blood clots form in the heart, and these protect the bacteria from phagocytes. These patches of tissue-associated bacteria are called vegetations. The resulting damage to the heart, in part resulting from the immune response causing fibrosis of heart valves, can necessitate heart valve replacement (Figure \(1\)). Outward signs of subacute endocarditis may include a fever.
Diagnosis of infective endocarditis is determined using the combination of blood cultures, echocardiogram, and clinical symptoms. In both acute and subacute endocarditis, treatment typically involves relatively high doses of intravenous antibiotics as determined by antimicrobial susceptibility testing. Acute endocarditis is often treated with a combination of ampicillin, nafcillin, and gentamicin for synergistic coverage of Staphylococcus spp. and Streptococcus spp. Prosthetic-valve endocarditis is often treated with a combination of vancomycin, rifampin, and gentamicin. Rifampin is necessary to treat individuals with infection of prosthetic valves or other foreign bodies because rifampin can penetrate the biofilm of most of the pathogens that infect these devices.
Staphylcoccus spp. and Streptococcus spp. can also infect and cause inflammation in the tissues surrounding the heart, a condition called acute pericarditis. Pericarditis is marked by chest pain, difficulty breathing, and a dry cough. In most cases, pericarditis is self-limiting and clinical intervention is not necessary. Diagnosis is made with the aid of a chest radiograph, electrocardiogram, echocardiogram, aspirate of pericardial fluid, or biopsy of pericardium. Antibacterial medications may be prescribed for infections associated with pericarditis; however, pericarditis can also be caused other pathogens, including viruses (e.g., echovirus, influenza virus), fungi (e.g., Histoplasma spp., Coccidioides spp.), and eukaryotic parasites (e.g., Toxoplasma spp.).
Exercise \(4\)
Compare acute and subacute bacterial endocarditis.
Gas Gangrene
Traumatic injuries or certain medical conditions, such as diabetes, can cause damage to blood vessels that interrupts blood flow to a region of the body. When blood flow is interrupted, tissues begin to die, creating an anaerobic environment in which anaerobic bacteria can thrive. This condition is called ischemia. Endospores of the anaerobic bacterium Clostridium perfringens (along with a number of other Clostridium spp. from the gut) can readily germinate in ischemic tissues and colonize the anaerobic tissues.
The resulting infection, called gas gangrene, is characterized by rapidly spreading myonecrosis (death of muscle tissue). The patient experiences a sudden onset of excruciating pain at the infection site and the rapid development of a foul-smelling wound containing gas bubbles and a thin, yellowish discharge tinged with a small amount of blood. As the infection progresses, edema and cutaneous blisters containing bluish-purple fluid form. The infected tissue becomes liquefied and begins sloughing off. The margin between necrotic and healthy tissue often advances several inches per hour even with antibiotic therapy. Septic shock and organ failure frequently accompany gas gangrene; when patients develop sepsis, the mortality rate is greater than 50%.
α-Toxin and theta (θ) toxin are the major virulence factors of C. perfringens implicated in gas gangrene. α-Toxin is a lipase responsible for breaking down cell membranes; it also causes the formation of thrombi (blood clots) in blood vessels, contributing to the spread of ischemia. θ-Toxin forms pores in the patient’s cell membranes, causing cell lysis. The gas associated with gas gangrene is produced by Clostridium’s fermentation of butyric acid, which produces hydrogen and carbon dioxide that are released as the bacteria multiply, forming pockets of gas in tissues (Figure \(2\)).
Gas gangrene is initially diagnosed based on the presence of the clinical signs and symptoms described earlier in this section. Diagnosis can be confirmed through Gram stain and anaerobic cultivation of wound exudate (drainage) and tissue samples on blood agar. Treatment typically involves surgical debridement of any necrotic tissue; advanced cases may require amputation. Surgeons may also use vacuum-assisted closure (VAC), a surgical technique in which vacuum-assisted drainage is used to remove blood or serous fluid from a wound or surgical site to speed recovery. The most common antibiotic treatments include penicillin G and clindamycin. Some cases are also treated with hyperbaric oxygen therapy because Clostridium spp. are incapable of surviving in oxygen-rich environments.
Tularemia
Infection with the gram-negative bacterium Francisella tularensis causes tularemia (or rabbit fever), a zoonotic infection in humans. F. tularensis is a facultative intracellular parasite that primarily causes illness in rabbits, although a wide variety of domesticated animals are also susceptible to infection. Humans can be infected through ingestion of contaminated meat or, more typically, handling of infected animal tissues (e.g., skinning an infected rabbit). Tularemia can also be transmitted by the bites of infected arthropods, including the dog tick (Dermacentor variabilis), the lone star tick (Amblyomma americanum), the wood tick (Dermacentor andersoni), and deer flies (Chrysops spp.). Although the disease is not directly communicable between humans, exposure to aerosols of F. tularensis can result in life-threatening infections. F. tularensis is highly contagious, with an infectious dose of as few as 10 bacterial cells. In addition, pulmonary infections have a 30%–60% fatality rate if untreated.10 For these reasons, F. tularensis is currently classified and must be handled as a biosafety level-3 (BSL-3) organism and as a potential biological warfare agent.
Following introduction through a break in the skin, the bacteria initially move to the lymph nodes, where they are ingested by phagocytes. After escaping from the phagosome, the bacteria grow and multiply intracellularly in the cytoplasm of phagocytes. They can later become disseminated through the blood to other organs such as the liver, lungs, and spleen, where they produce masses of tissue called granulomas (Figure \(3\)). After an incubation period of about 3 days, skin lesions develop at the site of infection. Other signs and symptoms include fever, chills, headache, and swollen and painful lymph nodes.
A direct diagnosis of tularemia is challenging because it is so contagious. Once a presumptive diagnosis of tularemia is made, special handling is required to collect and process patients’ specimens to prevent the infection of health-care workers. Specimens suspected of containing F. tularensis can only be handled by BSL-2 or BSL-3 laboratories registered with the Federal Select Agent Program, and individuals handling the specimen must wear protective equipment and use a class II biological safety cabinet.
Tularemia is relatively rare in the US, and its signs and symptoms are similar to a variety of other infections that may need to be ruled out before a diagnosis can be made. Direct fluorescent-antibody (DFA) microscopic examination using antibodies specific for F. tularensis can rapidly confirm the presence of this pathogen. Culturing this microbe is difficult because of its requirement for the amino acid cysteine, which must be supplied as an extra nutrient in culturing media. Serological tests are available to detect an immune response against the bacterial pathogen. In patients with suspected infection, acute- and convalescent-phase serum samples are required to confirm an active infection. PCR-based tests can also be used for clinical identification of direct specimens from body fluids or tissues as well as cultured specimens. In most cases, diagnosis is based on clinical findings and likely incidents of exposure to the bacterium. The antibiotics streptomycin, gentamycin, doxycycline, and ciprofloxacin are effective in treating tularemia.
Brucellosis
Species in the genus Brucella are gram-negative facultative intracellular pathogens that appear as coccobacilli. Several species cause zoonotic infections in animals and humans, four of which have significant human pathogenicity: B. abortus from cattle and buffalo, B. canis from dogs, B. suis from swine, and B. melitensis from goats, sheep, and camels. Infections by these pathogens are called brucellosis, also known as undulant fever, “Mediterranean fever,” or “Malta fever.” Vaccination of animals has made brucellosis a rare disease in the US, but it is still common in the Mediterranean, south and central Asia, Central and South America, and the Caribbean. Human infections are primarily associated with the ingestion of meat or unpasteurized dairy products from infected animals. Infection can also occur through inhalation of bacteria in aerosols when handling animal products, or through direct contact with skin wounds. In the US, most cases of brucellosis are found in individuals with extensive exposure to potentially infected animals (e.g., slaughterhouse workers, veterinarians).
Two important virulence factors produced by Brucella spp. are urease, which allows ingested bacteria to avoid destruction by stomach acid, and lipopolysaccharide (LPS), which allows the bacteria to survive within phagocytes. After gaining entry to tissues, the bacteria are phagocytized by host neutrophils and macrophages. The bacteria then escape from the phagosome and grow within the cytoplasm of the cell. Bacteria phagocytized by macrophages are disseminated throughout the body. This results in the formation of granulomas within many body sites, including bone, liver, spleen, lung, genitourinary tract, brain, heart, eye, and skin. Acute infections can result in undulant (relapsing) fever, but untreated infections develop into chronic disease that usually manifests as acute febrile illness (fever of 40–41 °C [104–105.8 °F]) with recurring flu-like signs and symptoms.
Brucella is only reliably found in the blood during the acute fever stage; it is difficult to diagnose by cultivation. In addition, Brucella is considered a BSL-3 pathogen and is hazardous to handle in the clinical laboratory without protective clothing and at least a class II biological safety cabinet. Agglutination tests are most often used for serodiagnosis. In addition, enzyme-linked immunosorbent assays (ELISAs) are available to determine exposure to the organism. The antibiotics doxycycline or ciprofloxacin are typically prescribed in combination with rifampin; gentamicin, streptomycin, and trimethoprim-sulfamethoxazole (TMP-SMZ) are also effective against Brucella infections and can be used if needed.
Exercise \(5\)
Compare the pathogenesis of tularemia and brucellosis.
Cat-Scratch Disease
The zoonosis cat-scratch disease (CSD) (or cat-scratch fever) is a bacterial infection that can be introduced to the lymph nodes when a human is bitten or scratched by a cat. It is caused by the facultative intracellular gram-negative bacterium Bartonella henselae. Cats can become infected from flea feces containing B. henselae that they ingest while grooming. Humans become infected when flea feces or cat saliva (from claws or licking) containing B. henselae are introduced at the site of a bite or scratch. Once introduced into a wound, B. henselae infects red blood cells.
B. henselae invasion of red blood cells is facilitated by adhesins associated with outer membrane proteins and a secretion system that mediates transport of virulence factors into the host cell. Evidence of infection is indicated if a small nodule with pus forms in the location of the scratch 1 to 3 weeks after the initial injury. The bacteria then migrate to the nearest lymph nodes, where they cause swelling and pain. Signs and symptoms may also include fever, chills, and fatigue. Most infections are mild and tend to be self-limiting. However, immunocompromised patients may develop bacillary angiomatosis (BA), characterized by the proliferation of blood vessels, resulting in the formation of tumor-like masses in the skin and internal organs; or bacillary peliosis (BP), characterized by multiple cyst-like, blood-filled cavities in the liver and spleen. Most cases of CSD can be prevented by keeping cats free of fleas and promptly cleaning a cat scratch with soap and warm water.
The diagnosis of CSD is difficult because the bacterium does not grow readily in the laboratory. When necessary, immunofluorescence, serological tests, PCR, and gene sequencing can be performed to identify the bacterial species. Given the limited nature of these infections, antibiotics are not normally prescribed. For immunocompromised patients, rifampin, azithromycin, ciprofloxacin, gentamicin (intramuscularly), or TMP-SMZ are generally the most effective options.
Rat-Bite Fever
The zoonotic infection rat-bite fever can be caused by two different gram-negative bacteria: Streptobacillus moniliformis, which is more common in North America, and Spirillum minor, which is more common in Asia. Because of modern sanitation efforts, rat bites are rare in the US. However, contact with fomites, food, or water contaminated by rat feces or body fluids can also cause infections. Signs and symptoms of rat-bite fever include fever, vomiting, myalgia (muscle pain), arthralgia (joint pain), and a maculopapular rash on the hands and feet. An ulcer may also form at the site of a bite, along with some swelling of nearby lymph nodes. In most cases, the infection is self-limiting. Little is known about the virulence factors that contribute to these signs and symptoms of disease.
Cell culture, MALDI-TOF mass spectrometry, PCR, or ELISA can be used in the identification of Streptobacillus moniliformis. The diagnosis Spirillum minor may be confirmed by direct microscopic observation of the pathogens in blood using Giemsa or Wright stains, or darkfield microscopy. Serological tests can be used to detect a host immune response to the pathogens after about 10 days. The most commonly used antibiotics to treat these infections are penicillin or doxycycline.
Plague
The gram-negative bacillus Yersinia pestis causes the zoonotic infection plague. This bacterium causes acute febrile disease in animals, usually rodents or other small mammals, and humans. The disease is associated with a high mortality rate if left untreated. Historically, Y. pestis has been responsible for several devastating pandemics, resulting in millions of deaths (see Micro Connections: The History of the Plague). There are three forms of plague: bubonic plague (the most common form, accounting for about 80% of cases), pneumonic plague, and septicemic plague. These forms are differentiated by the mode of transmission and the initial site of infection. Figure \(4\) illustrates these various modes of transmission and infection between animals and humans.
In bubonic plague, Y. pestis is transferred by the bite of infected fleas. Since most flea bites occur on the legs and ankles, Y. pestis is often introduced into the tissues and blood circulation in the lower extremities. After a 2- to 6-day incubation period, patients experience an abrupt onset fever (39.5–41 °C [103.1–105.8 °F]), headache, hypotension, and chills. The pathogen localizes in lymph nodes, where it causes inflammation, swelling, and hemorrhaging that results in purple buboes (Figure \(5\)). Buboes often form in lymph nodes of the groin first because these are the nodes associated with the lower limbs; eventually, through circulation in the blood and lymph, lymph nodes throughout the body become infected and form buboes. The average mortality rate for bubonic plague is about 55% if untreated and about 10% with antibiotic treatment.
Septicemic plague occurs when Y. pestis is directly introduced into the bloodstream through a cut or wound and circulates through the body. The incubation period for septicemic plague is 1 to 3 days, after which patients develop fever, chills, extreme weakness, abdominal pain, and shock. Disseminated intravascular coagulation (DIC) can also occur, resulting in the formation of thrombi that obstruct blood vessels and promote ischemia and necrosis in surrounding tissues (Figure \(5\)). Necrosis occurs most commonly in extremities such as fingers and toes, which become blackened. Septicemic plague can quickly lead to death, with a mortality rate near 100% when it is untreated. Even with antibiotic treatment, the mortality rate is about 50%.
Pneumonic plague occurs when Y. pestis causes an infection of the lungs. This can occur through inhalation of aerosolized droplets from an infected individual or when the infection spreads to the lungs from elsewhere in the body in patients with bubonic or septicemic plague. After an incubation period of 1 to 3 days, signs and symptoms include fever, headache, weakness, and a rapidly developing pneumonia with shortness of breath, chest pain, and cough producing bloody or watery mucus. The pneumonia may result in rapid respiratory failure and shock. Pneumonic plague is the only form of plague that can be spread from person to person by infectious aerosol droplet. If untreated, the mortality rate is near 100%; with antibiotic treatment, the mortality rate is about 50%.
The high mortality rate for the plague is, in part, a consequence of it being unusually well equipped with virulence factors. To date, there are at least 15 different major virulence factors that have been identified from Y. pestis and, of these, eight are involved with adherence to host cells. In addition, the F1 component of the Y. pestis capsule is a virulence factor that allows the bacterium to avoid phagocytosis. F1 is produced in large quantities during mammalian infection and is the most immunogenic component.11 Successful use of virulence factors allows the bacilli to disseminate from the area of the bite to regional lymph nodes and eventually the entire blood and lymphatic systems.
Culturing and direct microscopic examination of a sample of fluid from a bubo, blood, or sputum is the best way to identify Y. pestis and confirm a presumptive diagnosis of plague. Specimens may be stained using either a Gram, Giemsa, Wright, or Wayson's staining technique (Figure \(6\)). The bacteria show a characteristic bipolar staining pattern, resembling safety pins, that facilitates presumptive identification. Direct fluorescent antibody tests (rapid test of outer-membrane antigens) and serological tests like ELISA can be used to confirm the diagnosis. The confirmatory method for identifying Y. pestis isolates in the US is bacteriophage lysis.
Prompt antibiotic therapy can resolve most cases of bubonic plague, but septicemic and pneumonic plague are more difficult to treat because of their shorter incubation stages. Survival often depends on an early and accurate diagnosis and an appropriate choice of antibiotic therapy. In the US, the most common antibiotics used to treat patients with plague are gentamicin, fluoroquinolones, streptomycin, levofloxacin, ciprofloxacin, and doxycycline.
Exercise \(6\)
Compare bubonic plague, septicemic plague, and pneumonic plague.
Micro Connections: The History of the Plague
The first recorded pandemic of plague, the Justinian plague, occurred in the sixth century CE. It is thought to have originated in central Africa and spread to the Mediterranean through trade routes. At its peak, more than 5,000 people died per day in Constantinople alone. Ultimately, one-third of that city’s population succumbed to plague.12 The impact of this outbreak probably contributed to the later fall of Emperor Justinian.
The second major pandemic, dubbed the Black Death, occurred during the 14th century. This time, the infections are thought to have originated somewhere in Asia before being transported to Europe by trade, soldiers, and war refugees. This outbreak killed an estimated one-quarter of the population of Europe (25 million, primarily in major cities). In addition, at least another 25 million are thought to have been killed in Asia and Africa.13 This second pandemic, associated with strain Yersinia pestis biovar Medievalis, cycled for another 300 years in Europe and Great Britain, and was called the Great Plague in the 1660s.
The most recent pandemic occurred in the 1890s with Yersinia pestis biovar Orientalis. This outbreak originated in the Yunnan province of China and spread worldwide through trade. It is at this time that plague made its way to the US. The etiologic agent of plague was discovered by Alexandre Yersin (1863–1943) during this outbreak as well. The overall number of deaths was lower than in prior outbreaks, perhaps because of improved sanitation and medical support.14 Most of the deaths attributed to this final pandemic occurred in India.
Link to Learning
Visit this link to see an article describing how similar the genome of the Black Death bacterium is to today’s strains of bubonic plague.
Zoonotic Febrile Diseases
A wide variety of zoonotic febrile diseases (diseases that cause fever) are caused by pathogenic bacteria that require arthropod vectors. These pathogens are either obligate intracellular species of Anaplasma, Bartonella, Ehrlichia, Orientia, and Rickettsia, or spirochetes in the genus Borrelia. Isolation and identification of pathogens in this group are best performed in BSL-3 laboratories because of the low infective dose associated with the diseases.
Anaplasmosis
The zoonotic tickborne disease human granulocytic anaplasmosis (HGA) is caused by the obligate intracellular pathogen Anaplasma phagocytophilum. HGA is endemic primarily in the central and northeastern US and in countries in Europe and Asia.
HGA is usually a mild febrile disease that causes flu-like symptoms in immunocompetent patients; however, symptoms are severe enough to require hospitalization in at least 50% of infections and, of those patients, less than 1% will die of HGA.15 Small mammals such as white-footed mice, chipmunks, and voles have been identified as reservoirs of A. phagocytophilum, which is transmitted by the bite of an Ixodes tick. Five major virulence factors16 have been reported in Anaplasma; three are adherence factors and two are factors that allow the pathogen to avoid the human immune response. Diagnostic approaches include locating intracellular microcolonies of Anaplasma through microscopic examination of neutrophils or eosinophils stained with Giemsa or Wright stain, PCR for detection of A. phagocytophilum, and serological tests to detect antibody titers against the pathogens. The primary antibiotic used for treatment is doxycycline.
Ehrlichiosis
Human monocytotropic ehrlichiosis (HME) is a zoonotic tickborne disease caused by the BSL-2, obligate intracellular pathogen Ehrlichia chaffeensis. Currently, the geographic distribution of HME is primarily the eastern half of the US, with a few cases reported in the West, which corresponds with the known geographic distribution of the primary vector, the lone star tick (Amblyomma americanum). Symptoms of HME are similar to the flu-like symptoms observed in anaplasmosis, but a rash is more common, with 60% of children and less than 30% of adults developing petechial, macula, and maculopapular rashes.17 Virulence factors allow E. chaffeensis to adhere to and infect monocytes, forming intracellular microcolonies in monocytes that are diagnostic for the HME. Diagnosis of HME can be confirmed with PCR and serologic tests. The first-line treatment for adults and children of all ages with HME is doxycycline.
Epidemic Typhus
The disease epidemic typhus is caused by Rickettsia prowazekii and is transmitted by body lice, Pediculus humanus. Flying squirrels are animal reservoirs of R. prowazekii in North America and can also be sources of lice capable of transmitting the pathogen. Epidemic typhus is characterized by a high fever and body aches that last for about 2 weeks. A rash develops on the abdomen and chest and radiates to the extremities. Severe cases can result in death from shock or damage to heart and brain tissues. Infected humans are an important reservoir for this bacterium because R. prowazekii is the only Rickettsia that can establish a chronic carrier state in humans.
Epidemic typhus has played an important role in human history, causing large outbreaks with high mortality rates during times of war or adversity. During World War I, epidemic typhus killed more than 3 million people on the Eastern front.18With the advent of effective insecticides and improved personal hygiene, epidemic typhus is now quite rare in the US. In the developing world, however, epidemics can lead to mortality rates of up to 40% in the absence of treatment.19 In recent years, most outbreaks have taken place in Burundi, Ethiopia, and Rwanda. For example, an outbreak in Burundi refugee camps in 1997 resulted in 45,000 illnesses in a population of about 760,000 people.20
A rapid diagnosis is difficult because of the similarity of the primary symptoms with those of many other diseases. Molecular and immunohistochemical diagnostic tests are the most useful methods for establishing a diagnosis during the acute stage of illness when therapeutic decisions are critical. PCR to detect distinctive genes from R. prowazekii can be used to confirm the diagnosis of epidemic typhus, along with immunofluorescent staining of tissue biopsy specimens. Serology is usually used to identify rickettsial infections. However, adequate antibody titers take up to 10 days to develop. Antibiotic therapy is typically begun before the diagnosis is complete. The most common drugs used to treat patients with epidemic typhus are doxycycline or chloramphenicol.
Murine (Endemic) Typhus
Murine typhus (also known as endemic typhus) is caused by Rickettsia typhi and is transmitted by the bite of the rat flea, Xenopsylla cheopis, with infected rats as the main reservoir. Clinical signs and symptoms of murine typhusinclude a rash and chills accompanied by headache and fever that last about 12 days. Some patients also exhibit a cough and pneumonia-like symptoms. Severe illness can develop in immunocompromised patients, with seizures, coma, and renal and respiratory failure.
Clinical diagnosis of murine typhus can be confirmed from a biopsy specimen from the rash. Diagnostic tests include indirect immunofluorescent antibody (IFA) staining, PCR for R. typhi, and acute and convalescent serologic testing. Primary treatment is doxycycline, with chloramphenicol as the second choice.
Rocky Mountain Spotted Fever
The disease Rocky Mountain spotted fever (RMSF) is caused by Rickettsia rickettsii and is transmitted by the bite of a hard-bodied tick such as the American dog tick (Dermacentor variabilis), Rocky Mountain wood tick (D. andersoni), or brown dog tick (Rhipicephalus sanguineus).
This disease is endemic in North and South America and its incidence is coincident with the arthropod vector range. Despite its name, most cases in the US do not occur in the Rocky Mountain region but in the Southeast; North Carolina, Oklahoma, Arkansas, Tennessee, and Missouri account for greater than 60% of all cases.21 The map in Figure \(7\) shows the distribution of prevalence in the US in 2010.
Signs and symptoms of RMSF include a high fever, headache, body aches, nausea, and vomiting. A petechial rash(similar in appearance to measles) begins on the hands and wrists, and spreads to the trunk, face, and extremities (Figure \(8\)). If untreated, RMSF is a serious illness that can be fatal in the first 8 days even in otherwise healthy patients. Ideally, treatment should begin before petechiae develop, because this is a sign of progression to severe disease; however, the rash usually does not appear until day 6 or later after onset of symptoms and only occurs in 35%–60% of patients with the infection. Increased vascular permeability associated with petechiae formation can result in fatality rates of 3% or greater, even in the presence of clinical support. Most deaths are due to hypotension and cardiac arrest or from ischemia following blood coagulation.
Diagnosis can be challenging because the disease mimics several other diseases that are more prevalent. The diagnosis of RMSF is made based on symptoms, fluorescent antibody staining of a biopsy specimen from the rash, PCR for Rickettsia rickettsii, and acute and convalescent serologic testing. Primary treatment is doxycycline, with chloramphenicol as the second choice.
Lyme Disease
Lyme disease is caused by the spirochete Borrelia burgdorferi that is transmitted by the bite of a hard-bodied, black-legged Ixodes tick. I. scapularis is the biological vector transmitting B. burgdorferi in the eastern and north-central US and I. pacificus transmits B. burgdorferi in the western US (Figure \(10\)). Different species of Ixodes ticks are responsible for B. burgdorferi transmission in Asia and Europe. In the US, Lyme disease is the most commonly reported vectorborne illness. In 2014, it was the fifth most common Nationally Notifiable disease.22
Ixodes ticks have complex life cycles and deer, mice, and even birds can act as reservoirs. Over 2 years, the ticks pass through four developmental stages and require a blood meal from a host at each stage. In the spring, tick eggs hatch into six-legged larvae. These larvae do not carry B. burgdorferi initially. They may acquire the spirochete when they take their first blood meal (typically from a mouse). The larvae then overwinter and molt into eight-legged nymphs in the following spring. Nymphs take blood meals primarily from small rodents, but may also feed on humans, burrowing into the skin. The feeding period can last several days to a week, and it typically takes 24 hours for an infected nymph to transmit enough B. burgdorferi to cause infection in a human host. Nymphs ultimately mature into male and female adult ticks, which tend to feed on larger animals like deer or, occasionally, humans. The adults then mate and produce eggs to continue the cycle (Figure \(9\)).
The symptoms of Lyme disease follow three stages: early localized, early disseminated, and late stage. During the early-localized stage, approximately 70%–80%23 of cases may be characterized by a bull's-eye rash, called erythema migrans, at the site of the initial tick bite. The rash forms 3 to 30 days after the tick bite (7 days is the average) and may also be warm to the touch (Figure \(10\)).24 This diagnostic sign is often overlooked if the tick bite occurs on the scalp or another less visible location. Other early symptoms include flu-like symptoms such as malaise, headache, fever, and muscle stiffness. If the patient goes untreated, the second early-disseminated stage of the disease occurs days to weeks later. The symptoms at this stage may include severe headache, neck stiffness, facial paralysis, arthritis, and carditis. The late-stage manifestations of the disease may occur years after exposure. Chronic inflammation causes damage that can eventually cause severe arthritis, meningitis, encephalitis, and altered mental states. The disease may be fatal if untreated.
A presumptive diagnosis of Lyme disease can be made based solely on the presence of a bull’s-eye rash at the site of infection, if it is present, in addition to other associated symptoms (Figure \(10\)). In addition, indirect immunofluorescent antibody (IFA) labeling can be used to visualize bacteria from blood or skin biopsy specimens. Serological tests like ELISA can also be used to detect serum antibodies produced in response to infection. During the early stage of infection (about 30 days), antibacterial drugs such as amoxicillin and doxycycline are effective. In the later stages, penicillin G, chloramphenicol, or ceftriaxone can be given intravenously.
Relapsing Fever
Borrelia spp. also can cause relapsing fever. Two of the most common species are B. recurrentis, which causes epidemics of louseborne relapsing fever, and B. hermsii, which causes tickborne relapsing fevers. These Borrelia species are transmitted by the body louse Pediculus humanus and the soft-bodied tick Ornithodoros hermsi, respectively. Lice acquire the spirochetes from human reservoirs, whereas ticks acquire them from rodent reservoirs. Spirochetes infect humans when Borrelia in the vector’s saliva or excreta enter the skin rapidly as the vector bites.
In both louse- and tickborne relapsing fevers, bacteremia usually occurs after the initial exposure, leading to a sudden high fever (39–43 °C [102.2–109.4 °F) typically accompanied by headache and muscle aches. After about 3 days, these symptoms typically subside, only to return again after about a week. After another 3 days, the symptoms subside again but return a week later, and this cycle may repeat several times unless it is disrupted by antibiotic treatment. Immune evasion through bacterial antigenic variation is responsible for the cyclical nature of the symptoms in these diseases.
The diagnosis of relapsing fever can be made by observation of spirochetes in blood, using darkfield microscopy (Figure \(11\)). For louseborne relapsing fever, doxycycline or erythromycin are the first-line antibiotics. For tickborne relapsing fever, tetracycline or erythromycin are the first-line antibiotics.
Trench Fever
The louseborne disease trench fever was first characterized as a specific disease during World War I, when approximately 1 million soldiers were infected. Today, it is primarily limited to areas of the developing world where poor sanitation and hygiene lead to infestations of lice (e.g., overpopulated urban areas and refugee camps). Trench fever is caused by the gram-negative bacterium Bartonella quintana, which is transmitted when feces from infected body lice, Pediculus humanus var corporis, are rubbed into the louse bite, abraded skin, or the conjunctiva. The symptoms typically follow a 5-day course marked by a high fever, body aches, conjunctivitis, ocular pain, severe headaches, and severe bone pain in the shins, neck, and back. Diagnosis can be made using blood cultures; serological tests like ELISA can be used to detect antibody titers to the pathogen and PCR can also be used. The first-line antibiotics are doxycycline, macrolide antibiotics, and ceftriaxone.
Exercise \(7\)
1. What is the vector associated with epidemic typhus?
2. Describe the life cycle of the deer tick and how it spreads Lyme disease.
Tick Tips
Many of the diseases covered in this chapter involve arthropod vectors. Of these, ticks are probably the most commonly encountered in the US. Adult ticks have eight legs and two body segments, the cephalothorax and the head (Figure \(12\)). They typically range from 2 mm to 4 mm in length, and feed on the blood of the host by attaching themselves to the skin.
Unattached ticks should be removed and eliminated as soon as they are discovered. When removing a tick that has already attached itself, keep the following guidelines in mind to reduce the chances of exposure to pathogens:
• Use blunt tweezers to gently pull near the site of attachment until the tick releases its hold on the skin.
• Avoid crushing the tick's body and do not handle the tick with bare fingers. This could release bacterial pathogens and actually increase your exposure. The tick can be killed by drowning in water or alcohol, or frozen if it may be needed later for identification and analysis.
• Disinfect the area thoroughly by swabbing with an antiseptic such as isopropanol.
• Monitor the site of the bite for rashes or other signs of infection.
Many ill-advised home remedies for tick removal have become popular in recent years, propagated by social media and pseudojournalism. Health professionals should discourage patients from resorting to any of the following methods, which are NOT recommended:
• using chemicals (e.g., petroleum jelly or fingernail polish) to dislodge an attached tick, because it can cause the tick to release fluid, which can increase the chance of infection
• using hot objects (matches or cigarette butts) to dislodge an attached tick
• squeezing the tick's body with fingers or tweezers
Bacterial Infections of the Circulatory and Lymphatic Systems
Although the circulatory system is a closed system, bacteria can enter the bloodstream through several routes. Wounds, animal bites, or other breaks in the skin and mucous membranes can result in the rapid dissemination of bacterial pathogens throughout the body. Localized infections may also spread to the bloodstream, causing serious and often fatal systemic infections. Figure \(13\) and Figure \(14\) summarize the major characteristics of bacterial infections of the circulatory and lymphatic systems.
Key Concepts and Summary
• Bacterial infections of the circulatory system are almost universally serious. Left untreated, most have high mortality rates.
• Bacterial pathogens usually require a breach in the immune defenses to colonize the circulatory system. Most often, this involves a wound or the bite of an arthropod vector, but it can also occur in hospital settings and result in nosocomial infections.
• Sepsis from both gram-negative and gram-positive bacteria, puerperal fever, rheumatic fever, endocarditis, gas gangrene, osteomyelitis, and toxic shock syndrome are typically a result of injury or introduction of bacteria by medical or surgical intervention.
• Tularemia, brucellosis, cat-scratch fever, rat-bite fever, and bubonic plague are zoonotic diseases transmitted by biological vectors
• Ehrlichiosis, anaplasmosis, endemic and murine typhus, Rocky Mountain spotted fever, Lyme disease, relapsing fever, and trench fever are transmitted by arthropod vectors.
• Because their symptoms are so similar to those of other diseases, many bacterial infections of the circulatory system are difficult to diagnose.
• Standard antibiotic therapies are effective for the treatment of most bacterial infections of the circulatory system, unless the bacterium is resistant, in which case synergistic treatment may be required.
• The systemic immune response to a bacteremia, which involves the release of excessive amounts of cytokines, can sometimes be more damaging to the host than the infection itself.
Footnotes
1. 1 S.P. LaRosa. “Sepsis.” 2010. http://www.clevelandclinicmeded.com/...isease/sepsis/.
2. 2 D.C. Angus, T. Van der Poll. “Severe Sepsis and Septic Shock.” New England Journal of Medicine 369, no. 9 (2013):840–851.
3. 3 Centers for Disease Control and Prevention. “Toxic Shock Syndrome (Other Than Streptococcal) (TSS) 2011 Case Definition.” https://wwwn.cdc.gov/nndss/condition...finition/2011/. Accessed July 25, 2016.
4. 4 Centers for Disease Control and Prevention. “Streptococcal Toxic Shock Syndrome (STSS) (Streptococcus pyogenes) 2010 Case Definition.” https://wwwn.cdc.gov/nndss/condition...finition/2010/. Accessed July 25, 2016.
5. 5 M.E. Shirtliff, Mader JT. “Acute Septic Arthritis.” Clinical Microbiology Reviews 15 no. 4 (2002):527–544.
6. 6 J.R. Maneiro et al. “Predictors of Treatment Failure and Mortality in Native Septic Arthritis.” Clinical Rheumatology 34, no. 11 (2015):1961–1967.
7. 7 M. Vazquez. “Osteomyelitis in Children.” Current Opinion in Pediatrics 14, no. 1 (2002):112–115.
8. 8 A. Beaudoin et al. “Acute Rheumatic Fever and Rheumatic Heart Disease Among Children—American Samoa, 2011–2012.” Morbidity and Mortality Weekly Report 64 no. 20 (2015):555–558.
9. 9 M.A. Gerber et al. “Prevention of Rheumatic Fever and Diagnosis and Treatment of Acute Streptococcal Pharyngitis: A Scientific Statement From the American Heart Association Rheumatic Fever, Endocarditis, and Kawasaki Disease Committee of the Council on Cardiovascular Disease in the Young, the Interdisciplinary Council on Functional Genomics and Translational Biology, and the Interdisciplinary Council on Quality of Care and Outcomes Research: Endorsed by the American Academy of Pediatrics.” Circulation 119, no. 11 (2009):1541–1551.
10. 10 World Health Organization. “WHO Guidelines on Tularaemia.” 2007. http://www.cdc.gov/tularemia/resources/whotularemiamanual.pdf. Accessed July 26, 2016.
11. 11 MOH Key Laboratory of Systems Biology of Pathogens. “Virulence Factors of Pathogenic Bacteria, Yersinia.” http://www.mgc.ac.cn/cgi-bin/VFs/gen...Genus=Yersinia. Accessed September 9, 2016.
12. 12 Rosen, William. Justinian’s Flea: Plague, Empire, and the Birth of Europe. Viking Adult; pg 3; ISBN 978-0-670-03855-8.
13. 13 Benedictow, Ole J. 2004. The Black Death 1346-1353: The Complete History. Woodbridge: Boydell Press.
14. 14 Centers for Disease Control and Prevention. “Plague: History.” http://www.cdc.gov/plague/history/. Accessed September 15, 2016.
15. 15 J.S. Bakken et al. “Diagnosis and Management of Tickborne Rickettsial Diseases: Rocky Mountain Spotted Fever, Ehrlichioses, and Anaplasmosis–United States. A Practical Guide for Physicians and Other Health Care and Public Health Professionals.” MMWR Recommendations and Reports 55 no. RR04 (2006):1–27.
16. 16 MOH Key Laboratory of Systems Biology of Pathogens, “Virulence Factors of Pathogenic Bacteria, Anaplasma” 2016. http://www.mgc.ac.cn/cgi-bin/VFs/jsif/main.cgi. Accessed July, 26, 2016.
17. 17 Centers for Disease Control and Prevention. “Ehrlichiosis, Symptoms, Diagnosis, and Treatment.” 2016. https://www.cdc.gov/ehrlichiosis/symptoms/index.html. Accessed July 29, 2016.
18. 18 Drali, R., Brouqui, P. and Raoult, D. “Typhus in World War I.” Microbiology Today 41 (2014) 2:58–61.
19. 19 Centers for Disease Control and Prevention. CDC Health Information for International Travel 2014: The Yellow Book. Oxford University Press, 2013. http://wwwnc.cdc.gov/travel/yellowbook/2016/infectious-diseases-related-to-travel/rickettsial-spotted-typhus-fevers-related-infections-anaplasmosis-ehrlichiosis. Accessed July 26, 2016.
20. 20 World Health Organization. “Typhus.” 1997. www.who.int/mediacentre/factsheets/fs162/en/. Accessed July 26, 2016.
21. 21 Centers for Disease Control and Prevention. “Rocky Mountain Spotted Fever (RMSF): Statistics and Epidemiology.” http://www.cdc.gov/rmsf/stats/index.html. Accessed Sept 16, 2016.
22. 22 Centers for Disease Control and Prevention. “Lyme Disease. Data and Statistics.” 2015. http://www.cdc.gov/lyme/stats/index.html. Accessed July 26, 2016.
23. 23 Centers for Disease Control and Prevention. “Signs and Symptoms of Untreated Lyme Disease.” 2015. http://www.cdc.gov/lyme/signs_symptoms/index.html. Accessed July 27, 2016.
24. 24 Centers for Disease Control and Prevention. “Ticks. Symptoms of Tickborne Illness.” 2015. http://www.cdc.gov/ticks/symptoms.html. Accessed July 27, 2016. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/25%3A_Circulatory_and_Lymphatic_System_Infections/25.02%3A_Bacterial_Infections_of_the_Circulatory_and_Lymphatic_Systems.txt |
Learning Objectives
• Identify common viral pathogens that cause infections of the circulatory and lymphatic systems
• Compare the major characteristics of specific viral diseases affecting the circulatory and lymphatic systems
Viral pathogens of the circulatory system vary tremendously both in their virulence and distribution worldwide. Some of these pathogens are practically global in their distribution. Fortunately, the most ubiquitous viruses tend to produce the mildest forms of disease. In the majority of cases, those infected remain asymptomatic. On the other hand, other viruses are associated with life-threatening diseases that have impacted human history.
Infectious Mononucleosis and Burkitt Lymphoma
Human herpesvirus 4, also known as Epstein-Barr virus (EBV), has been associated with a variety of human diseases, such as mononucleosis and Burkitt lymphoma. Exposure to the human herpesvirus 4 (HHV-4) is widespread and nearly all people have been exposed at some time in their childhood, as evidenced by serological tests on populations. The virus primarily resides within B lymphocytes and, like all herpes viruses, can remain dormant in a latent state for a long time.
When uninfected young adults are exposed to EBV, they may experience infectious mononucleosis. The virus is mainly spread through contact with body fluids (e.g., saliva, blood, and semen). The main symptoms include pharyngitis, fever, fatigue, and lymph node swelling. Abdominal pain may also occur as a result of spleen and liver enlargement in the second or third week of infection. The disease typically is self-limiting after about a month. The main symptom, extreme fatigue, can continue for several months, however. Complications in immunocompetent patients are rare but can include jaundice, anemia, and possible rupture of the spleen caused by enlargement.
In patients with malaria or HIV, Epstein-Barr virus can lead to a fast-growing malignant cancer known as Burkitt lymphoma (Figure \(1\)). This condition is a form of non-Hodgkin lymphoma that produces solid tumors chiefly consisting of aberrant B cells. Burkitt lymphoma is more common in Africa, where prevalence of HIV and malaria is high, and it more frequently afflicts children. Repeated episodes of viremia caused by reactivation of the virus are common in immunocompromised individuals. In some patients with AIDS, EBV may induce the formation of malignant B-cell lymphomas or oral hairy leukoplakia. Immunodeficiency-associated Burkitt lymphoma primarily occurs in patients with HIV. HIV infection, similar to malaria, leads to polyclonal B-cell activation and permits poorly controlled proliferation of EBV+ B cells, leading to the formation of lymphomas.
Infectious mononucleosis is typically diagnosed based on the initial clinical symptoms and a test for antibodies to EBV-associated antigens. Because the disease is self-limiting, antiviral treatments are rare for mononucleosis. Cases of Burkitt lymphoma are diagnosed from a biopsy specimen from a lymph node or tissue from a suspected tumor. Staging of the cancer includes computed tomography (CT) scans of the chest, abdomen, pelvis, and cytologic and histologic evaluation of biopsy specimens. Because the tumors grow so rapidly, staging studies must be expedited and treatment must be initiated promptly. An intensive alternating regimen of cyclophosphamide, vincristine, doxorubicin, methotrexate, ifosfamide, etoposide, and cytarabine (CODOX-M/IVAC) plus rituximab results in a cure rate greater than 90% for children and adults.
Cytomegalovirus Infections
Also known as cytomegalovirus (CMV), human herpesvirus 5 (HHV-5) is a virus with high infection rates in the human population. It is currently estimated that 50% of people in the US have been infected by the time they reach adulthood.1CMV is the major cause of non-Epstein-Barr infectious mononucleosis in the general human population. It is also an important pathogen in immunocompromised hosts, including patients with AIDS, neonates, and transplant recipients. However, the vast majority of CMV infections are asymptomatic. In adults, if symptoms do occur, they typically include fever, fatigue, swollen glands, and pharyngitis.
CMV can be transmitted between individuals through contact with body fluids such as saliva or urine. Common modes of transmission include sexual contact, nursing, blood transfusions, and organ transplants. In addition, pregnant women with active infections frequently pass this virus to their fetus, resulting in congenital CMV infections, which occur in approximately one in every 150 infants in US.2 Infants can also be infected during passage through the birth canal or through breast milk and saliva from the mother.
Perinatal infections tend to be milder but can occasionally cause lung, spleen, or liver damage. Serious symptoms in newborns include growth retardation, jaundice, deafness, blindness, and mental retardation if the virus crosses the placenta during the embryonic state when the body systems are developing in utero. However, a majority (approximately 80%) of infected infants will never have symptoms or experience long-term problems.3 Diagnosis of CMV infection during pregnancy is usually achieved by serology; CMV is the “C” in prenatal TORCH screening.
Many patients receiving blood transfusions and nearly all those receiving kidney transplants ultimately become infected with CMV. Approximately 60% of transplant recipients will have CMV infection and more than 20% will develop symptomatic disease.4 These infections may result from CMV-contaminated tissues but also may be a consequence of immunosuppression required for transplantation causing reactivation of prior CMV infections. The resulting viremia can lead to fever and leukopenia, a decrease in the number of white blood cells in the bloodstream. Serious consequences may include liver damage, transplant rejection, and death. For similar reasons, many patients with AIDS develop active CMV infections that can manifest as encephalitis or progressive retinitis leading to blindness.5
Diagnosis of a localized CMV infection can be achieved through direct microscopic evaluation of tissue specimens stained with routine stains (e.g., Wright-Giemsa, hematoxylin and eosin, Papanicolaou) and immunohistochemical stains. Cells infected by CMV produce characteristic inclusions with an "owl's eye" appearance; this sign is less sensitive than molecular methods like PCR but more predictive of localized disease (Figure \(2\)). For more severe CMV infection, tests such as enzyme immunoassay (EIA), indirect immunofluorescence antibody (IFA) tests, and PCR, which are based on detection of CMV antigen or DNA, have a higher sensitivity and can determine viral load. Cultivation of the virus from saliva or urine is still the method for detecting CMV in newborn babies up to 3 weeks old. Ganciclovir, valganciclovir, foscarnet, and cidofovir are the first-line antiviral drugs for serious CMV infections.
Exercise \(1\)
Compare the diseases caused by HHV-4 and HHV-5.
Arthropod-Borne Viral Diseases
There are a number of arthropod-borne viruses, or arboviruses, that can cause human disease. Among these are several important hemorrhagic fevers transmitted by mosquitoes. We will discuss three that pose serious threats: yellow fever, chikungunya fever, and dengue fever.
Yellow Fever
Yellow fever was once common in the US and caused several serious outbreaks between 1700 and 1900.6 Through vector control efforts, however, this disease has been eliminated in the US. Currently, yellow fever occurs primarily in tropical and subtropical areas in South America and Africa. It is caused by the yellow fever virus of the genus Flavivirus (named for the Latin word flavus meaning yellow), which is transmitted to humans by mosquito vectors. Sylvatic yellow fever occurs in tropical jungle regions of Africa and Central and South America, where the virus can be transmitted from infected monkeys to humans by the mosquitoes Aedes africanus or Haemagogus spp. In urban areas, the Aedes aegypti mosquito is mostly responsible for transmitting the virus between humans.
Most individuals infected with yellow fever virus have no illness or only mild disease. Onset of milder symptoms is sudden, with dizziness, fever of 39–40 °C (102–104 °F), chills, headache, and myalgias. As symptoms worsen, the face becomes flushed, and nausea, vomiting, constipation, severe fatigue, restlessness, and irritability are common. Mild disease may resolve after 1 to 3 days. However, approximately 15% of cases progress to develop moderate to severe yellow fever disease.7
In moderate or severe disease, the fever falls suddenly 2 to 5 days after onset, but recurs several hours or days later. Symptoms of jaundice, petechial rash, mucosal hemorrhages, oliguria (scant urine), epigastric tenderness with bloody vomit, confusion, and apathy also often occur for approximately 7 days of moderate to severe disease. After more than a week, patients may have a rapid recovery and no sequelae.
In its most severe form, called malignant yellow fever, symptoms include delirium, bleeding, seizures, shock, coma, and multiple organ failure; in some cases, death occurs. Patients with malignant yellow fever also become severely immunocompromised, and even those in recovery may become susceptible to bacterial superinfections and pneumonia. Of the 15% of patients who develop moderate or severe disease, up to half may die.
Diagnosis of yellow fever is often based on clinical signs and symptoms and, if applicable, the patient’s travel history, but infection can be confirmed by culture, serologic tests, and PCR. There are no effective treatments for patients with yellow fever. Whenever possible, patients with yellow fever should be hospitalized for close observation and given supportive care. Prevention is the best method of controlling yellow fever. Use of mosquito netting, window screens, insect repellents, and insecticides are all effective methods of reducing exposure to mosquito vectors. An effective vaccine is also available, but in the US, it is only administered to those traveling to areas with endemic yellow fever. In West Africa, the World Health Organization (WHO) launched a Yellow Fever Initiative in 2006 and, since that time, significant progress has been made in combating yellow fever. More than 105 million people have been vaccinated, and no outbreaks of yellow fever were reported in West Africa in 2015.
Yellow Fever: Altering the Course of History
Yellow fever originated in Africa and is still most prevalent there today. This disease is thought to have been translocated to the Americas by the slave trade in the 16th century.8 Since that time, yellow fever has been associated with many severe outbreaks, some of which had important impacts upon historic events.
Yellow fever virus was once an important cause of disease in the US. In the summer of 1793, there was a serious outbreak in Philadelphia (then the US capitol). It is estimated that 5,000 people (10% of the city’s population) died. All of the government officials, including George Washington, fled the city in the face of this epidemic. The disease only abated when autumn frosts killed the mosquito vector population.
In 1802, Napoleon Bonaparte sent an army of 40,000 to Hispaniola to suppress a slave revolution. This was seen by many as a part of a plan to use the Louisiana Territory as a granary as he reestablished France as a global power. Yellow fever, however, decimated his army and they were forced to withdraw. Abandoning his aspirations in the New World, Napoleon sold the Louisiana Territory to the US for \$15 million in 1803.
The most famous historic event associated with yellow fever is probably the construction of the Panama Canal. The French began work on the canal in the early 1880s. However, engineering problems, malaria, and yellow fever forced them to abandon the project. The US took over the task in 1904 and opened the canal a decade later. During those 10 years, yellow fever was a constant adversary. In the first few years of work, greater than 80% of the American workers in Panama were hospitalized with yellow fever. It was the work of Carlos Finlay and Walter Reed that turned the tide. Taken together, their work demonstrated that the disease was transmitted by mosquitoes. Vector control measures succeeded in reducing both yellow fever and malaria rates and contributed to the ultimate success of the project.
Dengue Fever
The disease dengue fever, also known as breakbone fever, is caused by four serotypes of dengue virus called dengue 1–4. These are Flavivirus species that are transmitted to humans by A. aegypti or A. albopictus mosquitoes. The disease is distributed worldwide but is predominantly located in tropical regions. The WHO estimates that 50 million to 100 million infections occur yearly, including 500,000 dengue hemorrhagic fever (DHF) cases and 22,000 deaths, most among children.9 Dengue fever is primarily a self-limiting disease characterized by abrupt onset of high fever up to 40 °C (104 °F), intense headaches, rash, slight nose or gum bleeding, and extreme muscle, joint, and bone pain, causing patients to feel as if their bones are breaking, which is the reason this disease is also referred to as breakbone fever. As the body temperature returns to normal, in some patients, signs of dengue hemorrhagic fever may develop that include drowsiness, irritability, severe abdominal pain, severe nose or gum bleeding, persistent vomiting, vomiting blood, and black tarry stools, as the disease progresses to DHF or dengue shock syndrome (DSS). Patients who develop DHF experience circulatory system failure caused by increased blood vessel permeability. Patients with dengue fever can also develop DSS from vascular collapse because of the severe drop in blood pressure. Patients who develop DHF or DSS are at greater risk for death without prompt appropriate supportive treatment. About 30% of patients with severe hemorrhagic disease with poor supportive treatment die, but mortality can be less than 1% with experienced support.10
Diagnostic tests for dengue fever include serologic testing, ELISA, and reverse transcriptase-polymerase chain reaction (RT-PCR) of blood. There are no specific treatments for dengue fever, nor is there a vaccine. Instead, supportive clinical care is provided to treat the symptoms of the disease. The best way to limit the impact of this viral pathogen is vector control.
Chikungunya Fever
The arboviral disease chikungunya fever is caused by chikungunya virus (CHIKV), which is transmitted to humans by A. aegypti and A. albopictus mosquitoes. Until 2013, the disease had not been reported outside of Africa, Asia, and a few European countries; however, CHIKV has now spread to mosquito populations in North and South America. Chikungunya fever is characterized by high fever, joint pain, rash, and blisters, with joint pain persisting for several months. These infections are typically self-limiting and rarely fatal.
The diagnostic approach for chikungunya fever is similar to that for dengue fever. Viruses can be cultured directly from patient serum during early infections. IFA, EIA, ELISA, PCR, and RT-PCR are available to detect CHIKV antigens and patient antibody response to the infection. There are no specific treatments for this disease except to manage symptoms with fluids, analgesics, and bed rest. As with most arboviruses, the best strategy for combating the disease is vector control.
Link to Learning
Use this interactive map to explore the global distribution of dengue.
Exercise \(2\)
1. Name three arboviral diseases and explain why they are so named.
2. What is the best method for controlling outbreaks of arboviral diseases?
Ebola Virus Disease
The Ebola virus disease (EVD) is a highly contagious disease caused by species of Ebolavirus, a BSL-4 filovirus (Figure \(3\)). Transmission to humans occurs through direct contact with body fluids (e.g., blood, saliva, sweat, urine, feces, or vomit), and indirect contact by contaminated fomites. Infected patients can easily transmit Ebola virus to others if appropriate containment and use of personal protective equipment is not available or used. Handling and working with patients with EVD is extremely hazardous to the general population and health-care workers. In almost every EVD outbreak there have been Ebola infections among health-care workers. This ease of Ebola virus transmission was recently demonstrated in the Ebola epidemic in Guinea, Liberia, and Sierra Leone in 2014, in which more than 28,000 people in 10 countries were infected and more than 11,000 died.11
After infection, the initial symptoms of Ebola are unremarkable: fever, severe headache, myalgia, cough, chest pain, and pharyngitis. As the disease progresses, patients experience abdominal pain, diarrhea, and vomiting. Hemorrhaging begins after about 3 days, with bleeding occurring in the gastrointestinal tract, skin, and many other sites. This often leads to delirium, stupor, and coma, accompanied by shock, multiple organ failure, and death. The mortality rates of EVD often range from 50% to 90%.
The initial diagnosis of Ebola is difficult because the early symptoms are so similar to those of many other illnesses. It is possible to directly detect the virus from patient samples within a few days after symptoms begin, using antigen-capture ELISA, immunoglobulin M (IgM) ELISA, PCR, and virus isolation. There are currently no effective, approved treatments for Ebola other than supportive care and proper isolation techniques to contain its spread.
Exercise \(3\)
How is Ebola transmitted?
Hantavirus
The genus Hantavirus consists of at least four serogroups with nine viruses causing two major clinical (sometimes overlapping) syndromes: hantavirus pulmonary syndrome (HPS) in North America and hemorrhagic fever with renal syndrome (HFRS) in other continents. Hantaviruses are found throughout the world in wild rodents that shed the virus in their urine and feces. Transmission occurs between rodents and to humans through inhalation of aerosols of the rodent urine and feces. Hantaviruses associated with outbreaks in the US and Canada are transmitted by the deer mouse, white-footed mouse, or cotton rat.
HPS begins as a nonspecific flu-like illness with headache, fever, myalgia, nausea, vomiting, diarrhea, and abdominal pain. Patients rapidly develop pulmonary edema and hypotension resulting in pneumonia, shock, and death, with a mortality rate of up to 50%.12 This virus can also cause HFRS, which has not been reported in the US. The initial symptoms of this condition include high fever, headache, chills, nausea, inflammation or redness of the eyes, or a rash. Later symptoms are hemorrhaging, hypotension, kidney failure, shock, and death. The mortality rate of HFRS can be as high as 15%.13
ELISA, Western blot, rapid immunoblot strip assay (RIBA), and RT-PCR detect host antibodies or viral proteins produced during infection. Immunohistological staining may also be used to detect the presence of viral antigens. There are no clinical treatments other than general supportive care available for HPS infections. Patients with HFRS can be treated with ribavirin.14
Exercise \(4\)
Compare the two Hantavirus diseases discussed in this section.
Human Immunodeficiency Virus
Human T-lymphotropic viruses (HTLV), also called human immunodeficiency viruses (HIV) are retroviruses that are the causative agent of acquired immune deficiency syndrome (AIDS). There are two main variants of human immunodeficiency virus (HIV). HIV-1 (Figure \(4\)) occurs in human populations worldwide, whereas HIV-2 is concentrated in West Africa. Currently, the most affected region in the world is sub-Saharan Africa, with an estimated 25.6 million people living with HIV in 2015.15 Sub-Saharan Africa also accounts for two-thirds of the global total of new HIV infections (Figure \(5\)).16
HIV is spread through direct contact with body fluids. Casual contact and insect vectors are not sufficient for disease transmission; common modes of transmission include sexual contact and sharing of needles by intravenous (IV) drug users. It generally takes many years before the effects of an HIV infection are detected. HIV infections are not dormant during this period: virions are continually produced, and the immune system continually attempts to clear the viral infection, while the virus persistently infects additional CD4 T cells. Over time, the CD4 T-cell population is devastated, ultimately leading to AIDS.
When people are infected with HIV, their disease progresses through three stages based on CD4 T-cell counts and the presence of clinical symptoms (Figure \(6\)).
• Stage 1: Acute HIV infection. Two to 4 weeks after infection with HIV, patients may experience a flu-like illness, which can last for a few weeks. Patients with acute HIV infection have more than 500 cells/μL CD4 T cells and a large amount of virus in their blood. Patients are very contagious during this stage. To confirm acute infection, either a fourth-generation antibody-antigen test or a nucleic acid test (NAT) must be performed.
• Stage 2: Clinical latency. During this period, HIV enters a period of dormancy. Patients have between 200 and 499 cells/μL CD4 T cells; HIV is still active but reproduces at low levels, and patients may not experience any symptoms of illness. For patients who are not taking medicine to treat HIV, this period can last a decade or longer. For patients receiving antiretroviral therapy, the stage may last for several decades, and those with low levels of the virus in their blood are much less likely to transmit HIV than those who are not virally suppressed. Near the end of the latent stage, the patient’s viral load starts to increase and the CD4 T-cell count begins to decrease, leading to the development of symptoms and increased susceptibility to opportunistic infections.
• Stage 3: Acquired immunodeficiency syndrome (AIDS). Patients are diagnosed with AIDS when their CD4 T-cell count drops below 200 cells/μL or when they develop certain opportunistic illnesses. During this stage, the immune system becomes severely damaged by HIV. Common symptoms of AIDS include chills, fever, sweats, swollen lymph glands, weakness, and weight loss; in addition, patients often develop rare cancers such as Kaposi’s sarcoma and opportunistic infections such as Pneumocystis pneumonia, tuberculosis, cryptosporidiosis, and toxoplasmosis. This is a fatal progression that, in the terminal stages, includes wasting syndrome and dementia complex. Patients with AIDS have a high viral load and are highly infectious; they typically survive about 3 years without treatment.
The initial diagnosis of HIV is performed using a serological test for antibody production against the pathogen. Positive test results are confirmed by Western blot or PCR tests. It can take weeks or months for the body to produce antibodies in response to an infection. There are fourth-generation tests that detect HIV antibodies and HIV antigens that are present even before the body begins producing antibodies. Nucleic acid tests (NATs) are a third type of test that is relatively expensive and uncommon; NAT can detect HIV in blood and determine the viral load.
As a consequence of provirus formation, it is currently not possible to eliminate HIV from an infected patient’s body. Elimination by specific antibodies is ineffective because the virus mutates rapidly—a result of the error-prone reverse transcriptase and the inability to correct errors. Antiviral treatments, however, can greatly extend life expectancy. To combat the problem of drug resistance, combinations of antiretroviral drugs called antiretroviral therapy (ART), sometimes called highly active ART or combined ART, are used. There are several different targets for antiviral drug action (and a growing list of drugs for each of these targets). One class of drugs inhibits HIV entry; other classes inhibit reverse transcriptase by blocking viral RNA-dependent and DNA-dependent DNA polymerase activity; and still others inhibit one of the three HIV enzymes needed to replicate inside human cells.
Exercise \(5\)
Why is it not yet possible to cure HIV infections?
HIV, AIDS, and Education
When the first outbreaks of AIDS in the US occurred in the early 1980s, very little was known about the disease or its origins. Erroneously, the disease quickly became stigmatized as one associated with what became identified as at-risk behaviors such as sexual promiscuity, homosexuality, and IV drug use, even though mounting evidence indicated the disease was also contracted through transfusion of blood and blood products or by fetuses of infected mothers. In the mid-1980s, scientists elucidated the identity of the virus, its mode of transmission, and mechanisms of pathogenesis. Campaigns were undertaken to educate the public about how HIV spreads to stem infection rates and encourage behavioral changes that reduced the risk for infection. Approaches to this campaign, however, emphasized very different strategies. Some groups favored educational programs that emphasized sexual abstinence, monogamy, heterosexuality, and “just say no to drugs.” Other groups placed an emphasis on “safe sex” in sex education programs and advocated social services programs that passed out free condoms to anyone, including sexually active minors, and provided needle exchange programs for IV drug users.
These are clear examples of the intersection between disease and cultural values. As a future health professional, what is your responsibility in terms of educating patients about behaviors that put them at risk for HIV or other diseases while possibly setting your own personal opinions aside? You will no doubt encounter patients whose cultural and moral values differ from your own. Is it ethical for you to promote your own moral agenda to your patients? How can you advocate for practical disease prevention while still respecting the personal views of your patients?
Viral Diseases of the Circulatory and Lymphatic Systems
Many viruses are able to cause systemic, difficult-to-treat infections because of their ability to replicate within the host. Some of the more common viruses that affect the circulatory system are summarized in Figure \(7\).
Key Concepts and Summary
• Human herpesviruses such Epstein-Barr virus (HHV-4) and cytomegalovirus (HHV-5) are widely distributed. The former is associated with infectious mononucleosis and Burkitt lymphoma, and the latter can cause serious congenital infections as well as serious disease in immunocompromised adults.
• Arboviral diseases such as yellow fever, dengue fever, and chikungunya fever are characterized by high fevers and vascular damage that can often be fatal. Ebola virus disease is a highly contagious and often fatal infection spread through contact with bodily fluids.
• Although there is a vaccine available for yellow fever, treatments for patients with yellow fever, dengue, chikungunya fever, and Ebola virus disease are limited to supportive therapies.
• Patients infected with human immunodeficiency virus (HIV) progress through three stages of disease, culminating in AIDS. Antiretroviral therapy (ART) uses various combinations of drugs to suppress viral loads, extending the period of latency and reducing the likelihood of transmission.
• Vector control and animal reservoir control remain the best defenses against most viruses that cause diseases of the circulatory system.
Footnotes
1. 1 Centers for Disease Control and Prevention. “Cytomegalovirus (CMV) and Congenital CMV Infection: About CMV.” 2016. www.cdc.gov/cmv/transmission.html. Accessed July 28, 2016.
2. 2 Centers for Disease Control and Prevention. “Cytomegalovirus (CMV) and Congenital CMV Infection: Babies Born with CMV (Congenital CMV Infection).” 2016. http://www.cdc.gov/cmv/congenital-infection.html. Accessed July 28, 2016.
3. 3 ibid.
4. 4 E. Cordero et al. “Cytomegalovirus Disease in Kidney Transplant Recipients: Incidence, Clinical Profile, and Risk Factors.” Transplantation Proceedings 44 no. 3 (2012):694–700.
5. 5 L.M. Mofenson et al. “Guidelines for the Prevention and Treatment of Opportunistic Infections Among HIV-Exposed and HIV-Infected Children: Recommendations From CDC, the National Institutes of Health, the HIV Medicine Association of the Infectious Diseases Society of America, the Pediatric Infectious Diseases Society, and the American Academy of Pediatrics.” MMWR Recommendations and Reports 58 no. RR-11 (2009):1–166.
6. 6 Centers for Disease Control and Prevention. “History Timeline Transcript.” www.cdc.gov/travel-training/l...Transcript.pdf. Accessed July 28, 2016.
7. 7 Centers for Disease Control and Prevention. “Yellow Fever, Symptoms and Treatment.” 2015 http://www.cdc.gov/yellowfever/symptoms/index.html. Accessed July 28, 2016.
8. 8 J.T. Cathey, J.S. Marr. “Yellow fever, Asia and the East African Slave Trade.” Transactions of the Royal Society of Tropical Medicine and Hygiene 108, no. 5 (2014):252–257.
9. 9 Centers for Disease Control and Prevention. “Dengue, Epidemiology.” 2014. http://www.cdc.gov/dengue/epidemiology/index.html. Accessed July 28, 2016.
10. 10 C.R. Pringle “Dengue.” MSD Manual: Consumer Version. www.msdmanuals.com/home/infe...ections/dengue. 2016. Accessed Sept 15, 2016.
11. 11 HealthMap. “2014 Ebola Outbreaks.” http://www.healthmap.org/ebola/#timeline. Accessed July 28, 2016.
12. 12 World Health Organization. “Hantavirus Diseases.” 2016. www.who.int/ith/diseases/hantavirus/en/. Accessed July 28, 2016.
13. 13 ibid.
14. 14 Centers for Disease Control and Prevention. “Hantavirus: Treatment.” 2012. www.cdc.gov/hantavirus/techni...treatment.html. Accessed July 28, 2016.
15. 15 World Health Organization. “HIV/AIDS: Fact Sheet.” 2016. http://www.who.int/mediacentre/factsheets/fs360/en/. Accessed July 28, 2016.
16. 16 ibid. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/25%3A_Circulatory_and_Lymphatic_System_Infections/25.03%3A_Viral_Infections_of_the_Circulatory_and_Lymphatic_Systems.txt |
Learning Objectives
• Identify common parasites that cause infections of the circulatory and lymphatic systems
• Compare the major characteristics of specific parasitic diseases affecting the circulatory and lymphatic systems
Some protozoa and parasitic flukes are also capable of causing infections of the human circulatory system. Although these infections are rare in the US, they continue to cause widespread suffering in the developing world today. Fungal infections of the circulatory system are very rare. Therefore, they are not discussed in this chapter.
Malaria
Despite more than a century of intense research and clinical advancements, malaria remains one of the most important infectious diseases in the world today. Its widespread distribution places more than half of the world’s population in jeopardy. In 2015, the WHO estimated there were about 214 million cases of malaria worldwide, resulting in about 438,000 deaths; about 88% of cases and 91% of deaths occurred in Africa.1 Although malaria is not currently a major threat in the US, the possibility of its reintroduction is a concern. Malaria is caused by several protozoan parasites in the genus Plasmodium: P. falciparum, P. knowlesi, P. malariae, P. ovale, and P. vivax. Plasmodium primarily infect red blood cells and are transmitted through the bite of Anopheles mosquitoes.
Currently, P. falciparum is the most common and most lethal cause of malaria, often called falciparum malaria. Falciparum malaria is widespread in highly populated regions of Africa and Asia, putting many people at risk for the most severe form of the disease.
The classic signs and symptoms of malaria are cycles of extreme fever and chills. The sudden, violent symptoms of malaria start with malaise, abrupt chills, and fever (39–41° C [102.2–105.8 °F]), rapid and faint pulse, polyuria, headache, myalgia, nausea, and vomiting. After 2 to 6 hours of these symptoms, the fever falls, and profuse sweating occurs for 2 to 3 hours, followed by extreme fatigue. These symptoms are a result of Plasmodium emerging from red blood cells synchronously, leading to simultaneous rupture of a large number of red blood cells, resulting in damage to the spleen, liver, lymph nodes, and bone marrow. The organ damage resulting from hemolysis causes patients to develop sludge blood (i.e., blood in which the red blood cells agglutinate into clumps) that can lead to lack of oxygen, necrosis of blood vessels, organ failure, and death.
In established infections, malarial cycles of fever and chills typically occur every 2 days in the disease described as tertian malaria, which is caused by P. vivax and P. ovale. The cycles occur every 3 days in the disease described as quartan malaria, which is caused by P. malariae. These intervals may vary among cases.
Plasmodium has a complex life cycle that includes several developmental stages alternately produced in mosquitoes and humans (Figure \(1\)). When an infected mosquito takes a blood meal, sporozoites in the mosquito salivary gland are injected into the host’s blood. These parasites circulate to the liver, where they develop into schizonts. The schizonts then undergo schizogony, resulting in the release of many merozoites at once. The merozoites move to the bloodstream and infect red blood cells. Inside red blood cells, merozoites develop into trophozoites that produce more merozoites. The synchronous release of merozoites from red blood cells in the evening leads to the symptoms of malaria.
In addition, some trophozoites alternatively develop into male and female gametocytes. The gametocytes are taken up when the mosquito takes a blood meal from an infected individual. Sexual sporogony occurs in the gut of the mosquito. The gametocytes fuse to form zygotes in the insect gut. The zygotes become motile and elongate into an ookinete. This form penetrates the midgut wall and develops into an oocyst. Finally, the oocyst releases new sporozoites that migrate to the mosquito salivary glands to complete the life cycle.
Diagnosis of malaria is by microscopic observation of developmental forms of Plasmodium in blood smears and rapid EIA assays that detect Plasmodium antigens or enzymes (Figure \(2\)). Drugs such as chloroquine, atovaquone , artemether, and lumefantrine may be prescribed for both acute and prophylactic therapy, although some Plasmodium spp. have shown resistance to antimalarial drugs. Use of insecticides and insecticide-treated bed nets can limit the spread of malaria. Despite efforts to develop a vaccine for malaria, none is currently available.
Link to Learning
The Nothing But Nets campaign, an initiative of the United Nations Foundation, has partnered with the Bill and Melinda Gates Foundation to make mosquito bed nets available in developing countries in Africa. Visit their website to learn more about their efforts to prevent malaria.
Exercise \(1\)
Why is malaria one of the most important infectious diseases?
Toxoplasmosis
The disease toxoplasmosis is caused by the protozoan Toxoplasma gondii. T. gondii is found in a wide variety of birds and mammals,2 and human infections are common. The Centers for Disease Control and Prevention (CDC) estimates that 22.5% of the population 12 years and older has been infected with T. gondii; but immunocompetent individuals are typically asymptomatic, however.3 Domestic cats are the only known definitive hosts for the sexual stages of T. gondii and, thus, are the main reservoirs of infection. Infected cats shed T. gondii oocysts in their feces, and these oocysts typically spread to humans through contact with fecal matter on cats’ bodies, in litter boxes, or in garden beds where outdoor cats defecate.
T. gondii has a complex life cycle that involves multiple hosts. The T. gondii life cycle begins when unsporulated oocysts are shed in the cat’s feces. These oocysts take 1–5 days to sporulate in the environment and become infective. Intermediate hosts in nature include birds and rodents, which become infected after ingesting soil, water, or plant material contaminated with the infective oocysts. Once ingested, the oocysts transform into tachyzoites that localize in the bird or rodent neural and muscle tissue, where they develop into tissue cysts. Cats may become infected after consuming birds and rodents harboring tissue cysts. Cats and other animals may also become infected directly by ingestion of sporulated oocysts in the environment. Interestingly, Toxoplasma infection appears to be able to modify the host’s behavior. Mice infected by Toxoplasma lose their fear of cat pheromones. As a result, they become easier prey for cats, facilitating the transmission of the parasite to the cat definitive host4 (Figure \(3\)).
Toxoplasma infections in humans are extremely common, but most infected people are asymptomatic or have subclinical symptoms. Some studies suggest that the parasite may be able to influence the personality and psychomotor performance of infected humans, similar to the way it modifies behavior in other mammals.5 When symptoms do occur, they tend to be mild and similar to those of mononucleosis. However, asymptomatic toxoplasmosis can become problematic in certain situations. Cysts can lodge in a variety of human tissues and lie dormant for years. Reactivation of these quiescent infections can occur in immunocompromised patients following transplantation, cancer therapy, or the development of an immune disorder such as AIDS. In patients with AIDS who have toxoplasmosis, the immune system cannot combat the growth of T. gondii in body tissues; as a result, these cysts can cause encephalitis, retinitis, pneumonitis, cognitive disorders, and seizures that can eventually be fatal.
Toxoplasmosis can also pose a risk during pregnancy because tachyzoites can cross the placenta and cause serious infections in the developing fetus. The extent of fetal damage resulting from toxoplasmosis depends on the severity of maternal disease, the damage to the placenta, the gestational age of the fetus when infected, and the virulence of the organism. Congenital toxoplasmosis often leads to fetal loss or premature birth and can result in damage to the central nervous system, manifesting as mental retardation, deafness, or blindness. Consequently, pregnant women are advised by the CDC to take particular care in preparing meat, gardening, and caring for pet cats.6 Diagnosis of toxoplasmosis infection during pregnancy is usually achieved by serology including TORCH testing (the “T” in TORCH stands for toxoplasmosis). Diagnosis of congenital infections can also be achieved by detecting T. gondii DNA in amniotic fluid, using molecular methods such as PCR.
In adults, diagnosis of toxoplasmosis can include observation of tissue cysts in tissue specimens. Tissue cysts may be observed in Giemsa- or Wright-stained biopsy specimens, and CT, magnetic resonance imaging, and lumbar puncture can also be used to confirm infection (Figure \(4\)).
Preventing infection is the best first-line defense against toxoplasmosis. Preventive measures include washing hands thoroughly after handling raw meat, soil, or cat litter, and avoiding consumption of vegetables possibly contaminated with cat feces. All meat should be cooked to an internal temperature of 73.9–76.7 °C (165–170 °F).
Most immunocompetent patients do not require clinical intervention for Toxoplasma infections. However, neonates, pregnant women, and immunocompromised patients can be treated with pyrimethamine and sulfadiazine—except during the first trimester of pregnancy, because these drugs can cause birth defects. Spiramycin has been used safely to reduce transmission in pregnant women with primary infection during the first trimester because it does not cross the placenta.
Exercise \(2\)
How does T. gondii infect humans?
Babesiosis
Babesiosis is a rare zoonotic infectious disease caused by Babesia spp. These parasitic protozoans infect various wild and domestic animals and can be transmitted to humans by black-legged Ixodes ticks. In humans, Babesia infect red blood cells and replicate inside the cell until it ruptures. The Babesia released from the ruptured red blood cell continue the growth cycle by invading other red blood cells. Patients may be asymptomatic, but those who do have symptoms often initially experience malaise, fatigue, chills, fever, headache, myalgia, and arthralgia. In rare cases, particularly in asplenic (absence of the spleen) patients, the elderly, and patients with AIDS, babesiosis may resemble falciparum malaria, with high fever, hemolytic anemia, hemoglobinuria (hemoglobin or blood in urine), jaundice, and renal failure, and the infection can be fatal. Previously acquired asymptomatic Babesia infection may become symptomatic if a splenectomy is performed.
Diagnosis is based mainly on the microscopic observation of parasites in blood smears (Figure \(5\)). Serologic and antibody detection by IFA can also be performed and PCR-based tests are available. Many people do not require clinical intervention for Babesia infections, however, serious infections can be cleared with a combination of atovaquone and azithromycin or a combination of clindamycin and quinine.
Chagas Disease
Also called American trypanosomiasis, Chagas disease is a zoonosis classified as a neglected tropical disease(NTD). It is caused by the flagellated protozoan Trypanosoma cruzi and is most commonly transmitted to animals and people through the feces of triatomine bugs. The triatomine bug is nicknamed the kissing bug because it frequently bites humans on the face or around the eyes; the insect often defecates near the bite and the infected fecal matter may be rubbed into the bite wound by the bitten individual (Figure \(6\)). The bite itself is painless and, initially, many people show no signs of the disease. Alternative modes of transmission include contaminated blood transfusions, organ transplants from infected donors, and congenital transmission from mother to fetus.
Chagas disease is endemic throughout much of Mexico, Central America, and South America, where, according to WHO, an estimated 6 million to 7 million people are infected.7 Currently, Chagas disease is not endemic in the US, even though triatomine bugs are found in the southern half of the country.
Triatomine bugs typically are active at night, when they take blood meals by biting the faces and lips of people or animals as they sleep and often defecate near the site of the bite. Infection occurs when the host rubs the feces into their eyes, mouth, the bite wound, or another break in the skin. The protozoan then enters the blood and invades tissues of the heart and central nervous system, as well as macrophages and monocytes. Nonhuman reservoirs of T. cruzi parasites include wild animals and domesticated animals such as dogs and cats, which also act as reservoirs of the pathogen.8
There are three phases of Chagas disease: acute, intermediate, and chronic. These phases can be either asymptomatic or life-threatening depending on the immunocompetence status of the patient.
In acute phase disease, symptoms include fever, headache, myalgia, rash, vomiting, diarrhea, and enlarged spleen, liver, and lymph nodes. In addition, a localized nodule called a chagoma may form at the portal of entry, and swelling of the eyelids or the side of the face, called Romaña's sign, may occur near the bite wound. Symptoms of the acute phase may resolve spontaneously, but if untreated, the infection can persist in tissues, causing irreversible damage to the heart or brain. In rare cases, young children may die of myocarditis or meningoencephalitis during the acute phase of Chagas disease.
Following the acute phase is a prolonged intermediate phase during which few or no parasites are found in the blood and most people are asymptomatic. Many patients will remain asymptomatic for life; however, decades after exposure, an estimated 20%–30% of infected people will develop chronic disease that can be debilitating and sometimes life threatening. In the chronic phase, patients may develop painful swelling of the colon, leading to severe twisting, constipation, and bowel obstruction; painful swelling of the esophagus, leading to dysphagia and malnutrition; and flaccid cardiomegaly (enlargement of the heart), which can lead to heart failure and sudden death.
Diagnosis can be confirmed through several different tests, including direct microscopic observation of trypanosomes in the blood, IFA, EIAs, PCR, and culturing in artificial media. In endemic regions, xenodiagnoses may be used; this method involves allowing uninfected kissing bugs to feed on the patient and then examining their feces for the presence of T. cruzi.
The medications nifurtimox and benznidazole are effective treatments during the acute phase of Chagas disease. The efficacy of these drugs is much lower when the disease is in the chronic phase. Avoiding exposure to the pathogen through vector control is the most effective method of limiting this disease.
Exercise \(3\)
How do kissing bugs infect humans with Trypanosoma cruzi?
Leishmaniasis
Although it is classified as an NTD, leishmaniasis is relatively widespread in tropical and subtropical regions, affecting people in more than 90 countries. It is caused by approximately 20 different species of Leishmania, protozoan parasites that are transmitted by sand fly vectors such as Phlebotomus spp. and Lutzomyia spp. Dogs, cats, sheep, horses, cattle rodents, and humans can all serve as reservoirs.
The Leishmania protozoan is phagocytosed by macrophages but uses virulence factors to avoid destruction within the phagolysosome. The virulence factors inhibit the phagolysosome enzymes that would otherwise destroy the parasite. The parasite reproduces within the macrophage, lyses it, and the progeny infect new macrophages (see Micro Connections: When Phagocytosis Fails).
The three major clinical forms of leishmaniasis are cutaneous (oriental sore, Delhi boil, Aleppo boil), visceral (kala-azar, Dumdum fever), and mucosal (espundia). The most common form of disease is cutaneous leishmaniasis, which is characterized by the formation of sores at the site of the insect bite that may start out as papules or nodules before becoming large ulcers (Figure \(7\)).
It may take visceral leishmaniasis months and sometimes years to develop, leading to enlargement of the lymph nodes, liver, spleen, and bone marrow. The damage to these body sites triggers fever, weight loss, and swelling of the spleen and liver. It also causes a decrease in the number of red blood cells (anemia), white blood cells (leukopenia), and platelets (thrombocytopenia), causing the patient to become immunocompromised and more susceptible to fatal infections of the lungs and gastrointestinal tract.
The mucosal form of leishmaniasis is one of the less common forms of the disease. It causes a lesion similar to the cutaneous form but mucosal leishmaniasis is associated with mucous membranes of the mouth, nares, or pharynx, and can be destructive and disfiguring. Mucosal leishmaniasis occurs less frequently when the original cutaneous (skin) infection is promptly treated.
Definitive diagnosis of leishmaniasis is made by visualizing organisms in Giemsa-stained smears, by isolating Leishmania protozoans in cultures, or by PCR-based assays of aspirates from infected tissues. Specific DNA probes or analysis of cultured parasites can help to distinguish Leishmania species that are causing simple cutaneous leishmaniasis from those capable of causing mucosal leishmaniasis.
Cutaneous leishmaniasis is usually not treated. The lesions will resolve after weeks (or several months), but may result in scarring. Recurrence rates are low for this disease. More serious infections can be treated with stibogluconate(antimony gluconate), amphotericin B, and miltefosine.
Exercise \(4\)
Compare the mucosal and cutaneous forms of leishmaniasis.
Schistosomiasis
Schistosomiasis (bilharzia) is an NTD caused by blood flukes in the genus Schistosoma that are native to the Caribbean, South America, Middle East, Asia, and Africa. Most human schistosomiasis cases are caused by Schistosoma mansoni, S. haematobium, or S. japonicum. Schistosoma are the only trematodes that invade through the skin; all other trematodes infect by ingestion. WHO estimates that at least 258 million people required preventive treatment for schistosomiasis in 2014.9
Infected human hosts shed Schistosoma eggs in urine and feces, which can contaminate freshwater habitats of snails that serve as intermediate hosts. The eggs hatch in the water, releasing miracidia, an intermediate growth stage of the Schistosoma that infect the snails. The miracidia mature and multiply inside the snails, transforming into cercariae that leave the snail and enter the water, where they can penetrate the skin of swimmers and bathers. The cercariae migrate through human tissue and enter the bloodstream, where they mature into adult male and female worms that mate and release fertilized eggs. The eggs travel through the bloodstream and penetrate various body sites, including the bladder or intestine, from which they are excreted in urine or stool to start the life cycle over again (Figure 5.2.4).
A few days after infection, patients may develop a rash or itchy skin associated with the site of cercariae penetration. Within 1–2 months of infection, symptoms may develop, including fever, chills, cough, and myalgia, as eggs that are not excreted circulate through the body. After years of infection, the eggs become lodged in tissues and trigger inflammation and scarring that can damage the liver, central nervous system, intestine, spleen, lungs, and bladder. This may cause abdominal pain, enlargement of the liver, blood in the urine or stool, and problems passing urine. Increased risk for bladder cancer is also associated with chronic Schistosoma infection. In addition, children who are repeatedly infected can develop malnutrition, anemia, and learning difficulties.
Diagnosis of schistosomiasis is made by the microscopic observation of eggs in feces or urine, intestine or bladder tissue specimens, or serologic tests. The drug praziquantel is effective for the treatment of all schistosome infections. Improving wastewater management and educating at-risk populations to limit exposure to contaminated water can help control the spread of the disease.
Cercarial Dermatitis
The cercaria of some species of Schistosoma can only transform into adult worms and complete their life cycle in animal hosts such as migratory birds and mammals. The cercaria of these worms are still capable of penetrating human skin, but they are unable to establish a productive infection in human tissue. Still, the presence of the cercaria in small blood vessels triggers an immune response, resulting in itchy raised bumps called cercarial dermatitis (also known as swimmer’s itch or clam digger's itch). Although it is uncomfortable, cercarial dermatitis is typically self-limiting and rarely serious. Antihistamines and antipruritics can be used to limit inflammation and itching, respectively.
Exercise \(5\)
How do schistosome infections in humans occur?
Common Eukaryotic Pathogens of the Human Circulatory System
Protozoan and helminthic infections are prevalent in the developing world. A few of the more important parasitic infections are summarized in Figure \(8\).
Clinical Focus: Resolution
Despite continued antibiotic treatment and the removal of the venous catheter, Barbara’s condition further declined. She began to show signs of shock and her blood pressure dropped to 77/50 mmHg. Anti-inflammatory drugs and drotrecogin-α were administered to combat sepsis. However, by the seventh day of hospitalization, Barbara experienced hepatic and renal failure and died.
Staphylococcus aureus most likely formed a biofilm on the surface of Barbara’s catheter. From there, the bacteria were chronically shed into her circulation and produced the initial clinical symptoms. The chemotherapeutic therapies failed in large part because of the drug-resistant MRSA isolate. Virulence factors like leukocidin and hemolysins also interfered with her immune response. Barbara’s ultimate decline may have been a consequence of the production of enterotoxins and toxic shock syndrome toxin (TSST), which can initiate toxic shock.
Venous catheters are common life-saving interventions for many patients requiring long-term administration of medication or fluids. However, they are also common sites of bloodstream infections. The World Health Organization estimates that there are up to 80,000 catheter-related bloodstream infections each year in the US, resulting in about 20,000 deaths.10
Key Concepts and Summary
• Malaria is a protozoan parasite that remains an important cause of death primarily in the tropics. Several species in the genus Plasmodium are responsible for malaria and all are transmitted by Anopheles mosquitoes. Plasmodium infects and destroys human red blood cells, leading to organ damage, anemia, blood vessel necrosis, and death. Malaria can be treated with various antimalarial drugs and prevented through vector control.
• Toxoplasmosis is a widespread protozoal infection that can cause serious infections in the immunocompromised and in developing fetuses. Domestic cats are the definitive host.
• Babesiosis is a generally asymptomatic infection of red blood cells that can causes malaria-like symptoms in elderly, immunocompromised, or asplenic patients.
• Chagas disease is a tropical disease transmitted by triatomine bugs. The trypanosome infects heart, neural tissues, monocytes, and phagocytes, often remaining latent for many years before causing serious and sometimes fatal damage to the digestive system and heart.
• Leishmaniasis is caused by the protozoan Leishmania and is transmitted by sand flies. Symptoms are generally mild, but serious cases may cause organ damage, anemia, and loss of immune competence.
• Schistosomiasis is caused by a fluke transmitted by snails. The fluke moves throughout the body in the blood stream and chronically infects various tissues, leading to organ damage.
Footnotes
1. 1 World Health Organization. “World Malaria Report 2015: Summary.” 2015. http://www.who.int/malaria/publicati...015/report/en/. Accessed July 28, 2016.
2. 2 A.M. Tenter et al.. “Toxoplasma gondii: From Animals to Humans.” International Journal for Parasitology 30 no. 12-13 (2000):1217–1258.
3. 3 Centers for Disease Control and Prevention. “Parasites - Toxoplasmosis (Toxoplasma Infection). Epidemiology & Risk Factors.” 2015 http://www.cdc.gov/parasites/toxoplasmosis/epi.html. Accessed July 28, 2016.
4. 4 J. Flegr. “Effects of Toxoplasma on Human Behavior.” Schizophrenia Bulletin 33, no. 3 (2007):757–760.
5. 5 Ibid
6. 6 Centers for Disease Control and Prevention. “Parasites - Toxoplasmosis (Toxoplasma infection). Toxoplasmosis Frequently Asked Questions (FAQs).” 2013. http://www.cdc.gov/parasites/toxopla...info/faqs.html. Accessed July 28, 2016.
7. 7 World Health Organization. “Chagas disease (American trypanosomiasis). Fact Sheet.” 2016. http://www.who.int/mediacentre/factsheets/fs340/en/. Accessed July 29, 2016.
8. 8 C.E. Reisenman et al. “Infection of Kissing Bugs With Trypanosoma cruzi, Tucson, Arizona, USA.” Emerging Infectious Diseases 16 no. 3 (2010):400–405.
9. 9 World Health Organization. “Schistosomiasis. Fact Sheet.” 2016. http://www.who.int/mediacentre/factsheets/fs115/en/. Accessed July 29, 2016.
10. 10 World Health Organization. “Patient Safety, Preventing Bloodstream Infections From Central Line Venous Catheters.” 2016. www.who.int/patientsafety/imp...tation/bsi/en/. Accessed July 29, 2016. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/25%3A_Circulatory_and_Lymphatic_System_Infections/25.04%3A_Parasitic_Infections_of_the_Circulatory_and_Lymphatic_Systems.txt |
25.1: Anatomy of the Circulatory and Lymphatic Systems
The circulatory and lymphatic systems are networks of vessels and a pump that transport blood and lymph, respectively, throughout the body. When these systems are infected with a microorganism, the network of vessels can facilitate the rapid dissemination of the microorganism to other regions of the body, sometimes with serious results. In this section, we examine some of the key anatomical features of the circulatory and lymphatic systems, as well as general signs and symptoms of infection.
Multiple Choice
Which term refers to an inflammation of the blood vessels?
1. lymphangitis
2. endocarditis
3. pericarditis
4. vasculitis
Answer
D
Which of the following is located in the interstitial spaces within tissues and releases nutrients, immune factors, and oxygen to those tissues?
1. lymphatics
2. arterioles
3. capillaries
4. veins
Answer
C
Which of these conditions results in the formation of a bubo?
1. lymphangitis
2. lymphadenitis
3. ischemia
4. vasculitis
Answer
B
Which of the following is where most microbes are filtered out of the fluids that accumulate in the body tissues?
1. spleen
2. lymph nodes
3. pericardium
4. blood capillaries
Answer
B
Fill in the Blank
Vasculitis can cause blood to leak from damaged vessels, forming purple spots called ________.
Answer
petechiae
The lymph reenters the vascular circulation at ________.
Answer
the subclavian veins
Short Answer
How do lymph nodes help to maintain a microbial-free circulatory and lymphatic system?
Critical Thinking
What term refers to the red streaks seen on this patient’s skin? What is likely causing this condition?
(credit: modification of work by Centers for Disease Control and Prevention)
Why would septicemia be considered a more serious condition than bacteremia?
25.2: Bacterial Infections of the Circulatory and Lymphatic Systems
Bacterial infections of the circulatory system are almost universally serious. Left untreated, most have high mortality rates. Bacterial pathogens usually require a breach in the immune defenses to colonize the circulatory system. Most often, this involves a wound or the bite of an arthropod vector, but it can also occur in hospital settings and result in nosocomial infections.
Multiple Choice
Which of the following diseases is caused by a spirochete?
1. tularemia
2. relapsing fever
3. rheumatic fever
4. Rocky Mountain spotted fever
Answer
B
Which of the following diseases is transmitted by body lice?
1. tularemia
2. bubonic plague
3. murine typhus
4. epidemic typhus
Answer
D
What disease is most associated with Clostridium perfringens?
1. endocarditis
2. osteomyelitis
3. gas gangrene
4. rat bite fever
Answer
C
Which bacterial pathogen causes plague?
1. Yersinia pestis
2. Bacillus moniliformis
3. Bartonella quintana
4. Rickettsia rickettsii
Answer
A
Fill in the Blank
Lyme disease is characterized by a(n) ________ that forms at the site of infection.
Answer
bull’s eye-rash
________ refers to a loss of blood pressure resulting from a system-wide infection.
Answer
Septic shock
Short Answer
What are the three forms of plague and how are they contracted?
Compare epidemic and murine typhus.
Critical Thinking
Why are most vascular pathogens poorly communicable from person to person?
How have human behaviors contributed to the spread or control of arthropod-borne vascular diseases?
25.3: Viral Infections of the Circulatory and Lymphatic Systems
Viral pathogens of the circulatory system vary tremendously both in their virulence and distribution worldwide. Some of these pathogens are practically global in their distribution. Fortunately, the most ubiquitous viruses tend to produce the mildest forms of disease. In the majority of cases, those infected remain asymptomatic. On the other hand, other viruses are associated with life-threatening diseases that have impacted human history.
Multiple Choice
Which of the following viruses is most widespread in the human population?
1. human immunodeficiency virus
2. Ebola virus
3. Epstein-Barr virus
4. hantavirus
Answer
C
Which of these viruses is spread through mouse urine or feces?
1. Epstein-Barr
2. hantavirus
3. human immunodeficiency virus
4. cytomegalovirus
Answer
B
A patient at a clinic has tested positive for HIV. Her blood contained 700/µL CD4 T cells and she does not have any apparent illness. Her infection is in which stage?
1. 1
2. 2
3. 3
Answer
A
Fill in the Blank
________ is a cancer that forms in patients with HHV-4 and malaria coinfections.
Answer
Burkitt lymphoma
________ are transmitted by vectors such as ticks or mosquitoes.
Answer
Arboviruses
Infectious mononucleosis is caused by ________ infections.
Answer
Epstein-Barr virus
Short Answer
Describe the progression of an HIV infection over time with regard to the number of circulating viruses, host antibodies, and CD4 T cells.
Describe the general types of diagnostic tests used to diagnose patients infected with HIV.
Identify the general categories of drugs used in ART used to treat patients infected with HIV.
Critical Thinking
Which is a bigger threat to the US population, Ebola or yellow fever? Why?
25.4: Parasitic Infections of the Circulatory and Lymphatic Systems
Some protozoa and parasitic flukes are also capable of causing infections of the human circulatory system. Although these infections are rare in the US, they continue to cause widespread suffering in the developing world today. Malaria, toxoplasmosis, babesiosis, Chagas disease, leishmaniasis, and schistosomiasis are discussed in this section.
Multiple Choice
Which of the following diseases is caused by a helminth?
1. leishmaniasis
2. malaria
3. Chagas disease
4. schistosomiasis
Answer
D
Which of these is the most common form of leishmaniasis?
1. cutaneous
2. mucosal
3. visceral
4. intestinal
Answer
A
Which of the following is a causative agent of malaria?
1. Trypanosoma cruzi
2. Toxoplasma gondii
3. Plasmodium falciparum
4. Schistosoma mansoni
Answer
C
Which of the following diseases does not involve an arthropod vector?
1. schistosomiasis
2. malaria
3. Chagas disease
4. babesiosis
Answer
A
Fill in the Blank
The ________ mosquito is the biological vector for malaria.
Answer
Anopheles
The kissing bug is the biological vector for ________.
Answer
Chagas disease
Cercarial dermatitis is also known as ________.
Answer
swimmer’s itch
Short Answer
Describe main cause of Plasmodium falciparum infection symptoms.
Why should pregnant women avoid cleaning their cat’s litter box or do so with protective gloves?
Critical Thinking
What measures can be taken to reduce the likelihood of malaria reemerging in the US? | textbooks/bio/Microbiology/Microbiology_(OpenStax)/25%3A_Circulatory_and_Lymphatic_System_Infections/25.E%3A_Circulatory_and_Lymphatic_System_Infections_%28Exercises%29.txt |
Few diseases inspire the kind of fear that rabies does. The name is derived from the Latin word for “madness” or “fury,” most likely because animals infected with rabies may behave with uncharacteristic rage and aggression. And while the thought of being attacked by a rabid animal is terrifying enough, the disease itself is even more frightful. Once symptoms appear, the disease is almost always fatal, even when treated.
Rabies is an example of a neurological disease caused by an acellular pathogen. The rabies virus enters nervous tissue shortly after transmission and makes its way to the central nervous system, where its presence leads to changes in behavior and motor function. Well-known symptoms associated with rabid animals include foaming at the mouth, hydrophobia (fear of water), and unusually aggressive behavior. Rabies claims tens of thousands of human lives worldwide, mainly in Africa and Asia. Most human cases result from dog bites, although many mammal species can become infected and transmit the disease. Human infection rates are low in the United States and many other countries as a result of control measures in animal populations. However, rabies is not the only disease with serious or fatal neurological effects. In this chapter, we examine the important microbial diseases of the nervous system.
• 26.1: Anatomy of the Nervous System
The human nervous system can be divided into two interacting subsystems: the peripheral nervous system (PNS) and the central nervous system (CNS). The CNS consists of the brain and spinal cord. The peripheral nervous system is an extensive network of nerves connecting the CNS to the muscles and sensory structures.
• 26.2: Bacterial Diseases of the Nervous System
Bacterial infections that affect the nervous system are serious and can be life-threatening. Fortunately, there are only a few bacterial species commonly associated with neurological infections.
• 26.3: Acellular Pathogenic Diseases of the Nervous System
A number of different viruses and subviral particles can cause diseases that affect the nervous system. Viral diseases tend to be more common than bacterial infections of the nervous system today. Fortunately, viral infections are generally milder than their bacterial counterparts and often spontaneously resolve. Some of the more important acellular pathogens of the nervous system are described in this section.
• 26.4: Neuromycoses and Parasitic Diseases of the Nervous System
Fungal infections of the nervous system, called neuromycoses, are rare in healthy individuals. However, neuromycoses can be devastating in immunocompromised or elderly patients. Several eukaryotic parasites are also capable of infecting the nervous system of human hosts. Although relatively uncommon, these infections can also be life-threatening in immunocompromised individuals. In this section, we will first discuss neuromycoses, followed by parasitic infections of the nervous system.
• 26.E: Nervous System Infections (Exercises)
Thumbnail: Sir Charles Bell’s portrait of a soldier dying of tetanus.
26: Nervous System Infections
Learning Objectives
• Describe the major anatomical features of the nervous system
• Explain why there is no normal microbiota of the nervous system
• Explain how microorganisms overcome defenses of the nervous system to cause infection
• Identify and describe general symptoms associated with various infections of the nervous system
Clinical Focus: Part 1
David is a 35-year-old carpenter from New Jersey. A year ago, he was diagnosed with Crohn’s disease, a chronic inflammatory bowel disease that has no known cause. He has been taking a prescription corticosteroid to manage the condition, and the drug has been highly effective in keeping his symptoms at bay. However, David recently fell ill and decided to visit his primary care physician. His symptoms included a fever, a persistent cough, and shortness of breath. His physician ordered a chest X-ray, which revealed consolidation of the right lung. The doctor prescribed a course of levofloxacin and told David to come back in a week if he did not feel better.
Exercise \(1\)
1. What type of drug is levofloxacin?
2. What type of microbes would this drug be effective against?
3. What type of infection is consistent with David’s symptoms?
The human nervous system can be divided into two interacting subsystems: the peripheral nervous system (PNS) and the central nervous system (CNS). The CNS consists of the brain and spinal cord. The peripheral nervous system is an extensive network of nerves connecting the CNS to the muscles and sensory structures. The relationship of these systems is illustrated in Figure \(1\).
The Central Nervous System
The brain is the most complex and sensitive organ in the body. It is responsible for all functions of the body, including serving as the coordinating center for all sensations, mobility, emotions, and intellect. Protection for the brain is provided by the bones of the skull, which in turn are covered by the scalp, as shown in Figure \(2\). The scalp is composed of an outer layer of skin, which is loosely attached to the aponeurosis, a flat, broad tendon layer that anchors the superficial layers of the skin. The periosteum, below the aponeurosis, firmly encases the bones of the skull and provides protection, nutrition to the bone, and the capacity for bone repair. Below the boney layer of the skull are three layers of membranes called meninges that surround the brain. The relative positions of these meninges are shown in Figure \(2\). The meningeal layer closest to the bones of the skull is called the dura mater (literally meaning tough mother). Below the dura mater lies the arachnoid mater (literally spider-like mother). The innermost meningeal layer is a delicate membrane called the pia mater (literally tender mother). Unlike the other meningeal layers, the pia mater firmly adheres to the convoluted surface of the brain. Between the arachnoid mater and pia mater is the subarachnoid space. The subarachnoid space within this region is filled with cerebrospinal fluid (CSF). This watery fluid is produced by cells of the choroid plexus—areas in each ventricle of the brain that consist of cuboidal epithelial cells surrounding dense capillary beds. The CSF serves to deliver nutrients and remove waste from neural tissues.
The Blood-Brain Barrier
The tissues of the CNS have extra protection in that they are not exposed to blood or the immune system in the same way as other tissues. The blood vessels that supply the brain with nutrients and other chemical substances lie on top of the pia mater. The capillaries associated with these blood vessels in the brain are less permeable than those in other locations in the body. The capillary endothelial cells form tight junctions that control the transfer of blood components to the brain. In addition, cranial capillaries have far fewer fenestra (pore-like structures that are sealed by a membrane) and pinocytotic vesicles than other capillaries. As a result, materials in the circulatory system have a very limited ability to interact with the CNS directly. This phenomenon is referred to as the blood-brain barrier.
The blood-brain barrier protects the cerebrospinal fluid from contamination, and can be quite effective at excluding potential microbial pathogens. As a consequence of these defenses, there is no normal microbiota in the cerebrospinal fluid. The blood-brain barrier also inhibits the movement of many drugs into the brain, particularly compounds that are not lipid soluble. This has profound ramifications for treatments involving infections of the CNS, because it is difficult for drugs to cross the blood-brain barrier to interact with pathogens that cause infections.
The spinal cord also has protective structures similar to those surrounding the brain. Within the bones of the vertebrae are meninges of dura mater (sometimes called the dural sheath), arachnoid mater, pia mater, and a blood-spinal cord barrier that controls the transfer of blood components from blood vessels associated with the spinal cord.
To cause an infection in the CNS, pathogens must successfully breach the blood-brain barrier or blood-spinal cord barrier. Various pathogens employ different virulence factors and mechanisms to achieve this, but they can generally be grouped into four categories: intercellular (also called paracellular), transcellular, leukocyte facilitated, and nonhematogenous. Intercellular entry involves the use of microbial virulence factors, toxins, or inflammation-mediated processes to pass between the cells of the blood-brain barrier. In transcellular entry, the pathogen passes through the cells of the blood-brain barrier using virulence factors that allow it to adhere to and trigger uptake by vacuole- or receptor-mediated mechanisms. Leukocyte-facilitated entry is a Trojan-horse mechanism that occurs when a pathogen infects peripheral blood leukocytes to directly enter the CNS. Nonhematogenous entry allows pathogens to enter the brain without encountering the blood-brain barrier; it occurs when pathogens travel along either the olfactory or trigeminal cranial nerves that lead directly into the CNS.
Link to Learning
View this video about the blood-brain barrier
Exercise \(2\)
What is the primary function of the blood-brain barrier?
The Peripheral Nervous System
The PNS is formed of the nerves that connect organs, limbs, and other anatomic structures of the body to the brain and spinal cord. Unlike the brain and spinal cord, the PNS is not protected by bone, meninges, or a blood barrier, and, as a consequence, the nerves of the PNS are much more susceptible to injury and infection. Microbial damage to peripheral nerves can lead to tingling or numbness known as neuropathy. These symptoms can also be produced by trauma and noninfectious causes such as drugs or chronic diseases like diabetes.
The Cells of the Nervous System
Tissues of the PNS and CNS are formed of cells called glial cells (neuroglial cells) and neurons (nerve cells). Glial cells assist in the organization of neurons, provide a scaffold for some aspects of neuronal function, and aid in recovery from neural injury.
Neurons are specialized cells found throughout the nervous system that transmit signals through the nervous system using electrochemical processes. The basic structure of a neuron is shown in Figure \(3\). The cell body (or soma) is the metabolic center of the neuron and contains the nucleus and most of the cell’s organelles. The many finely branched extensions from the soma are called dendrites. The soma also produces an elongated extension, called the axon, which is responsible for the transmission of electrochemical signals through elaborate ion transport processes. Axons of some types of neurons can extend up to one meter in length in the human body. To facilitate electrochemical signal transmission, some neurons have a myelin sheath surrounding the axon. Myelin, formed from the cell membranes of glial cells like the Schwann cells in the PNS and oligodendrocytes in the CNS, surrounds and insulates the axon, significantly increasing the speed of electrochemical signal transmission along the axon. The end of an axon forms numerous branches that end in bulbs called synaptic terminals. Neurons form junctions with other cells, such as another neuron, with which they exchange signals. The junctions, which are actually gaps between neurons, are referred to as synapses. At each synapse, there is a presynaptic neuron and a postsynaptic neuron (or other cell). The synaptic terminals of the axon of the presynaptic terminal form the synapse with the dendrites, soma, or sometimes the axon of the postsynaptic neuron, or a part of another type of cell such as a muscle cell. The synaptic terminals contain vesicles filled with chemicals called neurotransmitters. When the electrochemical signal moving down the axon reaches the synapse, the vesicles fuse with the membrane, and neurotransmitters are released, which diffuse across the synapse and bind to receptors on the membrane of the postsynaptic cell, potentially initiating a response in that cell. That response in the postsynaptic cell might include further propagation of an electrochemical signal to transmit information or contraction of a muscle fiber.
Exercise \(3\)
1. What cells are associated with neurons, and what is their function?
2. What is the structure and function of a synapse?
Meningitis and Encephalitis
Although the skull provides the brain with an excellent defense, it can also become problematic during infections. Any swelling of the brain or meninges that results from inflammation can cause intracranial pressure, leading to severe damage of the brain tissues, which have limited space to expand within the inflexible bones of the skull. The term meningitis is used to describe an inflammation of the meninges. Typical symptoms can include severe headache, fever, photophobia (increased sensitivity to light), stiff neck, convulsions, and confusion. An inflammation of brain tissue is called encephalitis, and patients exhibit signs and symptoms similar to those of meningitis in addition to lethargy, seizures, and personality changes. When inflammation affects both the meninges and the brain tissue, the condition is called meningoencephalitis. All three forms of inflammation are serious and can lead to blindness, deafness, coma, and death.
Meningitis and encephalitis can be caused by many different types of microbial pathogens. However, these conditions can also arise from noninfectious causes such as head trauma, some cancers, and certain drugs that trigger inflammation. To determine whether the inflammation is caused by a pathogen, a lumbar puncture is performed to obtain a sample of CSF. If the CSF contains increased levels of white blood cells and abnormal glucose and protein levels, this indicates that the inflammation is a response to an infectioninflinin.
Exercise \(4\)
1. What are the two types of inflammation that can impact the CNS?
2. Why do both forms of inflammation have such serious consequences?
Guillain-Barré Syndrome
Guillain-Barré syndrome (GBS) is a rare condition that can be preceded by a viral or bacterial infection that results in an autoimmune reaction against myelinated nerve cells. The destruction of the myelin sheath around these neurons results in a loss of sensation and function. The first symptoms of this condition are tingling and weakness in the affected tissues. The symptoms intensify over a period of several weeks and can culminate in complete paralysis. Severe cases can be life-threatening. Infections by several different microbial pathogens, including Campylobacter jejuni (the most common risk factor), cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, Mycoplasma pneumoniae,1 and Zika virus2 have been identified as triggers for GBS. Anti-myelin antibodies from patients with GBS have been demonstrated to also recognize C. jejuni. It is possible that cross-reactive antibodies, antibodies that react with similar antigenic sites on different proteins, might be formed during an infection and may lead to this autoimmune response.
GBS is solely identified by the appearance of clinical symptoms. There are no other diagnostic tests available. Fortunately, most cases spontaneously resolve within a few months with few permanent effects, as there is no available vaccine. GBS can be treated by plasmapheresis. In this procedure, the patient’s plasma is filtered from their blood, removing autoantibodies.
Key Concepts and Summary
• The nervous system consists of two subsystems: the central nervous system and peripheral nervous system.
• The skull and three meninges (the dura mater, arachnoid mater, and pia mater) protect the brain.
• Tissues of the PNS and CNS are formed of cells called glial cells and neurons.
• Since the blood-brain barrier excludes most microbes, there is no normal microbiota in the CNS.
• Some pathogens have specific virulence factors that allow them to breach the blood-brain barrier. Inflammation of the brain or meninges caused by infection is called encephalitis or meningitis, respectively. These conditions can lead to blindness, deafness, coma, and death.
Footnotes
1. Yuki, Nobuhiro and Hans-Peter Hartung, “Guillain–Barré Syndrome,” New England Journal of Medicine 366, no. 24 (2012): 2294-304.
2. Cao-Lormeau, Van-Mai, Alexandre Blake, Sandrine Mons, Stéphane Lastère, Claudine Roche, Jessica Vanhomwegen, Timothée Dub et al., “Guillain-Barré Syndrome Outbreak Associated with Zika Virus Infection in French Polynesia: A Case-Control Study,” The Lancet 387, no. 10027 (2016): 1531-9. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/26%3A_Nervous_System_Infections/26.01%3A_Anatomy_of_the_Nervous_System.txt |
Learning Objectives
• Identify the most common bacteria that can cause infections of the nervous system
• Compare the major characteristics of specific bacterial diseases affecting the nervous system
Bacterial infections that affect the nervous system are serious and can be life-threatening. Fortunately, there are only a few bacterial species commonly associated with neurological infections.
Bacterial Meningitis
Bacterial meningitis is one of the most serious forms of meningitis. Bacteria that cause meningitis often gain access to the CNS through the bloodstream after trauma or as a result of the action of bacterial toxins. Bacteria may also spread from structures in the upper respiratory tract, such as the oropharynx, nasopharynx, sinuses, and middle ear. Patients with head wounds or cochlear implants (an electronic device placed in the inner ear) are also at risk for developing meningitis.
Many of the bacteria that can cause meningitis are commonly found in healthy people. The most common causes of non-neonatal bacterial meningitis are Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. All three of these bacterial pathogens are spread from person to person by respiratory secretions. Each can colonize and cross through the mucous membranes of the oropharynx and nasopharynx, and enter the blood. Once in the blood, these pathogens can disseminate throughout the body and are capable of both establishing an infection and triggering inflammation in any body site, including the meninges (Figure \(1\)). Without appropriate systemic antibacterial therapy, the case-fatality rate can be as high as 70%, and 20% of those survivors may be left with irreversible nerve damage or tissue destruction, resulting in hearing loss, neurologic disability, or loss of a limb. Mortality rates are much lower (as low as 15%) in populations where appropriate therapeutic drugs and preventive vaccines are available.1
A variety of other bacteria, including Listeria monocytogenes and Escherichia coli, are also capable of causing meningitis. These bacteria cause infections of the arachnoid mater and CSF after spreading through the circulation in blood or by spreading from an infection of the sinuses or nasopharynx. Streptococcus agalactiae, commonly found in the microbiota of the vagina and gastrointestinal tract, can also cause bacterial meningitis in newborns after transmission from the mother either before or during birth.
The profound inflammation caused by these microbes can result in early symptoms that include severe headache, fever, confusion, nausea, vomiting, photophobia, and stiff neck. Systemic inflammatory responses associated with some types of bacterial meningitis can lead to hemorrhaging and purpuric lesions on skin, followed by even more severe conditions that include shock, convulsions, coma, and death—in some cases, in the span of just a few hours.
Diagnosis of bacterial meningitis is best confirmed by analysis of CSF obtained by a lumbar puncture. Abnormal levels of polymorphonuclear neutrophils (PMNs) (> 10 PMNs/mm3), glucose (< 45 mg/dL), and protein (> 45 mg/dL) in the CSF are suggestive of bacterial meningitis.2 Characteristics of specific forms of bacterial meningitis are detailed in the subsections that follow.
Meningococcal Meningitis
Meningococcal meningitis is a serious infection caused by the gram-negative coccus N. meningitidis. In some cases, death can occur within a few hours of the onset of symptoms. Nonfatal cases can result in irreversible nerve damage, resulting in hearing loss and brain damage, or amputation of extremities because of tissue necrosis.
Meningococcal meningitis can infect people of any age, but its prevalence is highest among infants, adolescents, and young adults.3 Meningococcal meningitis was once the most common cause of meningitis epidemics in human populations. This is still the case in a swath of sub-Saharan Africa known as the meningitis belt, but meningococcal meningitis epidemics have become rare in most other regions, thanks to meningococcal vaccines. However, outbreaks can still occur in communities, schools, colleges, prisons, and other populations where people are in close direct contact.
N. meningitidis has a high affinity for mucosal membranes in the oropharynx and nasopharynx. Contact with respiratory secretions containing N. meningitidis is an effective mode of transmission. The pathogenicity of N. meningitidis is enhanced by virulence factors that contribute to the rapid progression of the disease. These include lipooligosaccharide (LOS) endotoxin, type IV pili for attachment to host tissues, and polysaccharide capsules that help the cells avoid phagocytosis and complement-mediated killing. Additional virulence factors include IgA protease(which breaks down IgA antibodies), the invasion factors Opa, Opc, and porin (which facilitate transcellular entry through the blood-brain barrier), iron-uptake factors (which strip heme units from hemoglobin in host cells and use them for growth), and stress proteins that protect bacteria from reactive oxygen molecules.
A unique sign of meningococcal meningitis is the formation of a petechial rash on the skin or mucous membranes, characterized by tiny, red, flat, hemorrhagic lesions. This rash, which appears soon after disease onset, is a response to LOS endotoxin and adherence virulence factors that disrupt the endothelial cells of capillaries and small veins in the skin. The blood vessel disruption triggers the formation of tiny blood clots, causing blood to leak into the surrounding tissue. As the infection progresses, the levels of virulence factors increase, and the hemorrhagic lesions can increase in size as blood continues to leak into tissues. Lesions larger than 1.0 cm usually occur in patients developing shock, as virulence factors cause increased hemorrhage and clot formation. Sepsis, as a result of systemic damage from meningococcal virulence factors, can lead to rapid multiple organ failure, shock, disseminated intravascular coagulation, and death.
Because meningococcoal meningitis progresses so rapidly, a greater variety of clinical specimens are required for the timely detection of N. meningitidis. Required specimens can include blood, CSF, naso- and oropharyngeal swabs, urethral and endocervical swabs, petechial aspirates, and biopsies. Safety protocols for handling and transport of specimens suspected of containing N. meningitidis should always be followed, since cases of fatal meningococcal disease have occurred in healthcare workers exposed to droplets or aerosols from patient specimens. Prompt presumptive diagnosis of meningococcal meningitis can occur when CSF is directly evaluated by Gram stain, revealing extra- and intracellular gram-negative diplococci with a distinctive coffee-bean microscopic morphology associated with PMNs (Figure \(2\)). Identification can also be made directly from CSF using latex agglutination and immunochromatographic rapid diagnostic tests specific for N. meningitidis. Species identification can also be performed using DNA sequence-based typing schemes for hypervariable outer membrane proteins of N. meningitidis, which has replaced sero(sub)typing.
Meningococcal infections can be treated with antibiotic therapy, and third-generation cephalosporins are most often employed. However, because outcomes can be negative even with treatment, preventive vaccination is the best form of treatment. In 2010, countries in Africa’s meningitis belt began using a new serogroup A meningococcal conjugate vaccine. This program has dramatically reduced the number of cases of meningococcal meningitis by conferring individual and herd immunity.
Twelve different capsular serotypes of N. meningitidis are known to exist. Serotypes A, B, C, W, X, and Y are the most prevalent worldwide. The CDC recommends that children between 11–12 years of age be vaccinated with a single dose of a quadrivalent vaccine that protects against serotypes A, C, W, and Y, with a booster at age 16.4 An additional booster or injections of serogroup B meningococcal vaccine may be given to individuals in high-risk settings (such as epidemic outbreaks on college campuses).
Meningitis on Campus
College students living in dorms or communal housing are at increased risk for contracting epidemic meningitis. From 2011 to 2015, there have been at least nine meningococcal outbreaks on college campuses in the United States. These incidents involved a total of 43 students (of whom four died).5 In spite of rapid diagnosis and aggressive antimicrobial treatment, several of the survivors suffered from amputations or serious neurological problems.
Prophylactic vaccination of first-year college students living in dorms is recommended by the CDC, and insurance companies now cover meningococcal vaccination for students in college dorms. Some colleges have mandated vaccination with meningococcal conjugate vaccine for certain students entering college (Figure \(3\)).
Pneumococcal Meningitis
Pneumococcal meningitis is caused by the encapsulated gram-positive bacterium S. pneumoniae (pneumococcus, also called strep pneumo). This organism is commonly found in the microbiota of the pharynx of 30–70% of young children, depending on the sampling method, while S. pneumoniae can be found in fewer than 5% of healthy adults. Although it is often present without disease symptoms, this microbe can cross the blood-brain barrier in susceptible individuals. In some cases, it may also result in septicemia. Since the introduction of the Hib vaccine, S. pneumoniae has become the leading cause of meningitis in humans aged 2 months through adulthood.
S. pneumoniae can be identified in CSF samples using gram-stained specimens, latex agglutination, and immunochromatographic RDT specific for S. pneumoniae. In gram-stained samples, S. pneumoniae appears as gram-positive, lancet-shaped diplococci (Figure \(4\)). Identification of S. pneumoniae can also be achieved using cultures of CSF and blood, and at least 93 distinct serotypes can be identified based on the quellung reaction to unique capsular polysaccharides. PCR and RT-PCR assays are also available to confirm identification.
Major virulence factors produced by S. pneumoniae include PI-1 pilin for adherence to host cells (pneumococcal adherence) and virulence factor B (PavB) for attachment to cells of the respiratory tract; choline-binding proteins(cbpA) that bind to epithelial cells and interfere with immune factors IgA and C3; and the cytoplasmic bacterial toxin pneumolysin that triggers an inflammatory response.
With the emergence of drug-resistant strains of S. pneumoniae, pneumococcal meningitis is typically treated with broad-spectrum antibiotics, such as levofloxacin, cefotaxime, penicillin, or other β-lactam antibiotics. The two available pneumococcal vaccines are described in Bacterial Infections of the Respiratory Tract.
Haemophilus influenzae Type b
Meningitis due to H. influenzae serotype b (Hib), an encapsulated pleomorphic gram-negative coccobacilli, is now uncommon in most countries, because of the use of the effective Hib vaccine. Without the use of the Hib vaccine, H. influenzae can be the primary cause of meningitis in children 2 months thru 5 years of age. H. influenzae can be found in the throats of healthy individuals, including infants and young children. By five years of age, most children have developed immunity to this microbe. Infants older than 2 months of age, however, do not produce a sufficient protective antibody response and are susceptible to serious disease. The intracranial pressure caused by this infection leads to a 5% mortality rate and 20% incidence of deafness or brain damage in survivors.6
H. influenzae produces at least 16 different virulence factors, including LOS, which triggers inflammation, and Haemophilus adhesion and penetration factor (Hap), which aids in attachment and invasion into respiratory epithelial cells. The bacterium also has a polysaccharide capsule that helps it avoid phagocytosis, as well as factors such as IgA1 protease and P2 protein that allow it to evade antibodies secreted from mucous membranes. In addition, factors such as hemoglobin-binding protein (Hgp) and transferrin-binding protein (Tbp) acquire iron from hemoglobin and transferrin, respectively, for bacterial growth.
Preliminary diagnosis of H. influenzae infections can be made by direct PCR and a smear of CSF. Stained smears will reveal intracellular and extracellular PMNs with small, pleomorphic, gram-negative coccobacilli or filamentous forms that are characteristic of H. influenzae. Initial confirmation of this genus can be based on its fastidious growth on chocolate agar. Identification is confirmed with requirements for exogenous biochemical growth cofactors NAD and heme (by MALDI-TOF), latex agglutination, and RT-PCR.
Meningitis caused by H. influenzae is usually treated with doxycycline, fluoroquinolones, second- and third-generation cephalosporins, and carbapenems. The best means of preventing H. influenza infection is with the use of the Hib polysaccharide conjugate vaccine. It is recommended that all children receive this vaccine at 2, 4, and 6 months of age, with a final booster dose at 12 to 15 months of age.7
Neonatal Meningitis
S. agalactiae, Group B streptococcus (GBS), is an encapsulated gram-positive bacterium that is the most common cause of neonatal meningitis, a term that refers to meningitis occurring in babies up to 3 months of age.8 S. agalactiae can also cause meningitis in people of all ages and can be found in the urogenital and gastrointestinal microbiota of about 10–30% of humans.
Neonatal infection occurs as either early onset or late-onset disease. Early onset disease is defined as occurring in infants up to 7 days old. The infant initially becomes infected by S. agalactiae during childbirth, when the bacteria may be transferred from the mother’s vagina. Incidence of early onset neonatal meningitis can be greatly reduced by giving intravenous antibiotics to the mother during labor.
Late-onset neonatal meningitis occurs in infants between 1 week and 3 months of age. Infants born to mothers with S. agalactiae in the urogenital tract have a higher risk of late-onset menigitis, but late-onset infections can be transmitted from sources other than the mother; often, the source of infection is unknown. Infants who are born prematurely (before 37 weeks of pregnancy) or to mothers who develop a fever also have a greater risk of contracting late-onset neonatal meningitis.
Signs and symptoms of early onset disease include temperature instability, apnea (cessation of breathing), bradycardia(slow heart rate), hypotension, difficulty feeding, irritability, and limpness. When asleep, the baby may be difficult to wake up. Symptoms of late-onset disease are more likely to include seizures, bulging fontanel (soft spot), stiff neck, hemiparesis (weakness on one side of the body), and opisthotonos (rigid body with arched back and head thrown backward).
S. agalactiae produces at least 12 virulence factors that include FbsA that attaches to host cell surface proteins, PI-1 pilithat promotes the invasion of human endothelial cells, a polysaccharide capsule that prevents the activation of the alternative complement pathway and inhibits phagocytosis, and the toxin CAMP factor, which forms pores in host cell membranes and binds to IgG and IgM antibodies.
Diagnosis of neonatal meningitis is often, but not uniformly, confirmed by positive results from cultures of CSF or blood. Tests include routine culture, antigen detection by enzyme immunoassay, serotyping of different capsule types, PCR, and RT-PCR. It is typically treated with β-lactam antibiotics such as intravenous penicillin or ampicillin plus gentamicin. Even with treatment, roughly 10% mortality is seen in infected neonates.9
Exercise \(1\)
1. Which groups are most vulnerable to each of the bacterial meningitis diseases?
2. For which of the bacterial meningitis diseases are there vaccines presently available?
3. Which organism can cause epidemic meningitis?
Clostridium-Associated Diseases
Species in the genus Clostridium are gram-positive, endospore-forming rods that are obligate anaerobes. Endospores of Clostridium spp. are widespread in nature, commonly found in soil, water, feces, sewage, and marine sediments. Clostridium spp. produce more types of protein exotoxins than any other bacterial genus, including two exotoxins with protease activity that are the most potent known biological toxins: botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeNT). These two toxins have lethal doses of 0.2–10 ng per kg body weight.
BoNT can be produced by unique strains of C. butyricum, and C. baratii; however, it is primarily associated with C. botulinum and the condition of botulism. TeNT, which causes tetanus, is only produced by C. tetani. These powerful neural exotoxins are the primary virulence factors for these pathogens. The mode of action for these toxins was described in Virulence Factors of Bacterial and Viral Pathogens and illustrated in Figure 15.3.7.
Diagnosis of tetanus or botulism typically involves bioassays that detect the presence of BoNT and TeNT in fecal specimens, blood (serum), or suspect foods. In addition, both C. botulinum and C. tetani can be isolated and cultured using commercially available media for anaerobes. ELISA and RT-PCR tests are also available.
Tetanus
Tetanus is a noncommunicable disease characterized by uncontrollable muscle spasms (contractions) caused by the action of TeNT. It generally occurs when C. tetani infects a wound and produces TeNT, which rapidly binds to neural tissue, resulting in an intoxication (poisoning) of neurons. Depending on the site and extent of infection, cases of tetanus can be described as localized, cephalic, or generalized. Generalized tetanus that occurs in a newborn is called neonatal tetanus.
Localized tetanus occurs when TeNT only affects the muscle groups close to the injury site. There is no CNS involvement, and the symptoms are usually mild, with localized muscle spasms caused by a dysfunction in the surrounding neurons. Individuals with partial immunity—especially previously vaccinated individuals who neglect to get the recommended booster shots—are most likely to develop localized tetanus as a result of C. tetani infecting a puncture wound.
Cephalic tetanus is a rare, localized form of tetanus generally associated with wounds on the head or face. In rare cases, it has occurred in cases of otitis media (middle ear infection). Cephalic tetanus often results in patients seeing double images, because of the spasms affecting the muscles that control eye movement.
Both localized and cephalic tetanus may progress to generalized tetanus—a much more serious condition—if TeNT is able to spread further into body tissues. In generalized tetanus, TeNT enters neurons of the PNS. From there, TeNT travels from the site of the wound, usually on an extremity of the body, retrograde (back up) to inhibitory neurons in the CNS. There, it prevents the release of gamma aminobutyric acid (GABA), the neurotransmitter responsible for muscle relaxation. The resulting muscle spasms often first occur in the jaw muscles, leading to the characteristic symptom of lockjaw (inability to open the mouth). As the toxin progressively continues to block neurotransmitter release, other muscles become involved, resulting in uncontrollable, sudden muscle spasms that are powerful enough to cause tendons to rupture and bones to fracture. Spasms in the muscles in the neck, back, and legs may cause the body to form a rigid, stiff arch, a posture called opisthotonos (Figure \(5\)). Spasms in the larynx, diaphragm, and muscles of the chest restrict the patient’s ability to swallow and breathe, eventually leading to death by asphyxiation (insufficient supply of oxygen).
Neonatal tetanus typically occurs when the stump of the umbilical cord is contaminated with spores of C. tetani after delivery. Although this condition is rare in the United States, neonatal tetanus is a major cause of infant mortality in countries that lack maternal immunization for tetanus and where birth often occurs in unsanitary conditions. At the end of the first week of life, infected infants become irritable, feed poorly, and develop rigidity with spasms. Neonatal tetanus has a very poor prognosis with a mortality rate of 70%–100%.10
Treatment for patients with tetanus includes assisted breathing through the use of a ventilator, wound debridement, fluid balance, and antibiotic therapy with metronidazole or penicillin to halt the growth of C. tetani. In addition, patients are treated with TeNT antitoxin, preferably in the form of human immunoglobulin to neutralize nonfixed toxin and benzodiazepines to enhance the effect of GABA for muscle relaxation and anxiety.
A tetanus toxoid (TT) vaccine is available for protection and prevention of tetanus. It is the T component of vaccines such as DTaP, Tdap, and Td. The CDC recommends children receive doses of the DTaP vaccine at 2, 4, 6, and 15–18 months of age and another at 4–6 years of age. One dose of Td is recommended for adolescents and adults as a TT booster every 10 years.11
Botulism
Botulism is a rare but frequently fatal illness caused by intoxication by BoNT. It can occur either as the result of an infection by C. botulinum, in which case the bacteria produce BoNT in vivo, or as the result of a direct introduction of BoNT into tissues.
Infection and production of BoNT in vivo can result in wound botulism, infant botulism, and adult intestinal toxemia. Wound botulism typically occurs when C. botulinum is introduced directly into a wound after a traumatic injury, deep puncture wound, or injection site. Infant botulism, which occurs in infants younger than 1 year of age, and adult intestinal toxemia, which occurs in immunocompromised adults, results from ingesting C. botulinum endospores in food. The endospores germinate in the body, resulting in the production of BoNT in the intestinal tract.
Intoxications occur when BoNT is produced outside the body and then introduced directly into the body through food (foodborne botulism), air (inhalation botulism), or a clinical procedure (iatrogenic botulism). Foodborne botulism, the most common of these forms, occurs when BoNT is produced in contaminated food and then ingested along with the food (recall Case in Point: A Streak of Bad Potluck). Inhalation botulism is rare because BoNT is unstable as an aerosol and does not occur in nature; however, it can be produced in the laboratory and was used (unsuccessfully) as a bioweapon by terrorists in Japan in the 1990s. A few cases of accidental inhalation botulism have also occurred. Iatrogenic botulism is also rare; it is associated with injections of BoNT used for cosmetic purposes (see Micro Connections: Medicinal Uses of Botulinum Toxin).
When BoNT enters the bloodstream in the gastrointestinal tract, wound, or lungs, it is transferred to the neuromuscular junctions of motor neurons where it binds irreversibly to presynaptic membranes and prevents the release of acetylcholine from the presynaptic terminal of motor neurons into the neuromuscular junction. The consequence of preventing acetylcholine release is the loss of muscle activity, leading to muscle relaxation and eventually paralysis.
If BoNT is absorbed through the gastrointestinal tract, early symptoms of botulism include blurred vision, drooping eyelids, difficulty swallowing, abdominal cramps, nausea, vomiting, constipation, or possibly diarrhea. This is followed by progressive flaccid paralysis, a gradual weakening and loss of control over the muscles. A patient’s experience can be particularly terrifying, because hearing remains normal, consciousness is not lost, and he or she is fully aware of the progression of his or her condition. In infants, notable signs of botulism include weak cry, decreased ability to suckle, and hypotonia (limpness of head or body). Eventually, botulism ends in death from respiratory failure caused by the progressive paralysis of the muscles of the upper airway, diaphragm, and chest.
Botulism is treated with an antitoxin specific for BoNT. If administered in time, the antitoxin stops the progression of paralysis but does not reverse it. Once the antitoxin has been administered, the patient will slowly regain neurological function, but this may take several weeks or months, depending on the severity of the case. During recovery, patients generally must remain hospitalized and receive breathing assistance through a ventilator.
Exercise \(2\)
1. How frequently should the tetanus vaccination be updated in adults?
2. What are the most common causes of botulism?
3. Why is botulism not treated with an antibiotic?
Medicinal Uses of Botulinum Toxin
Although it is the most toxic biological material known to man, botulinum toxin is often intentionally injected into people to treat other conditions. Type A botulinum toxin is used cosmetically to reduce wrinkles. The injection of minute quantities of this toxin into the face causes the relaxation of facial muscles, thereby giving the skin a smoother appearance. Eyelid twitching and crossed eyes can also be treated with botulinum toxin injections. Other uses of this toxin include the treatment of hyperhidrosis (excessive sweating). In fact, botulinum toxin can be used to moderate the effects of several other apparently nonmicrobial diseases involving inappropriate nerve function. Such diseases include cerebral palsy, multiple sclerosis, and Parkinson’s disease. Each of these diseases is characterized by a loss of control over muscle contractions; treatment with botulinum toxin serves to relax contracted muscles.
Listeriosis
Listeria monocytogenes is a nonencapsulated, nonsporulating, gram-positive rod and a foodborne pathogen that causes listeriosis. At-risk groups include pregnant women, neonates, the elderly, and the immunocompromised (recall the Clinical Focus case studies in Microbial Growth and Microbial Mechanisms of Pathogenicity). Listeriosis leads to meningitis in about 20% of cases, particularly neonates and patients over the age of 60. The CDC identifies listeriosis as the third leading cause of death due to foodborne illness, with overall mortality rates reaching 16%.12 In pregnant women, listeriosis can cause also cause spontaneous abortion in pregnant women because of the pathogen’s unique ability to cross the placenta.
L. monocytogenes is generally introduced into food items by contamination with soil or animal manure used as fertilizer. Foods commonly associated with listeriosis include fresh fruits and vegetables, frozen vegetables, processed meats, soft cheeses, and raw milk.13 Unlike most other foodborne pathogens, Listeria is able to grow at temperatures between 0 °C and 50 °C, and can therefore continue to grow, even in refrigerated foods.
Ingestion of contaminated food leads initially to infection of the gastrointestinal tract. However, L. monocytogenes produces several unique virulence factors that allow it to cross the intestinal barrier and spread to other body systems. Surface proteins called internalins (InlA and InlB) help L. monocytogenes invade nonphagocytic cells and tissues, penetrating the intestinal wall and becoming disseminating through the circulatory and lymphatic systems. Internalins also enable L. monocytogenes to breach other important barriers, including the blood-brain barrier and the placenta. Within tissues, L. monocytogenes uses other proteins called listeriolysin O and ActA to facilitate intercellular movement, allowing the infection to spread from cell to cell (Figure \(6\)).
L. monocytogenes is usually identified by cultivation of samples from a normally sterile site (e.g., blood or CSF). Recovery of viable organisms can be enhanced using cold enrichment by incubating samples in a broth at 4 °C for a week or more. Distinguishing types and subtypes of L. monocytogenes—an important step for diagnosis and epidemiology—is typically done using pulsed-field gel electrophoresis. Identification can also be achieved using chemiluminescence DNA probe assays and MALDI-TOF.
Treatment for listeriosis involves antibiotic therapy, most commonly with ampicillin and gentamicin. There is no vaccine available.
Exercise \(3\)
How does Listeria enter the nervous system?
Hansen’s Disease (Leprosy)
Hansen’s disease (also known as leprosy) is caused by a long, thin, filamentous rod-shaped bacterium Mycobacterium leprae, an obligate intracellular pathogen. M. leprae is classified as gram-positive bacteria, but it is best visualized microscopically with an acid-fast stain and is generally referred to as an acid-fast bacterium. Hansen’s disease affects the PNS, leading to permanent damage and loss of appendages or other body parts.
Hansen’s disease is communicable but not highly contagious; approximately 95% of the human population cannot be easily infected because they have a natural immunity to M. leprae. Person-to-person transmission occurs by inhalation into nasal mucosa or prolonged and repeated contact with infected skin. Armadillos, one of only five mammals susceptible to Hansen’s disease, have also been implicated in transmission of some cases.14
In the human body, M. leprae grows best at the cooler temperatures found in peripheral tissues like the nose, toes, fingers, and ears. Some of the virulence factors that contribute to M. leprae’s pathogenicity are located on the capsule and cell wall of the bacterium. These virulence factors enable it to bind to and invade Schwann cells, resulting in progressive demyelination that gradually destroys neurons of the PNS. The loss of neuronal function leads to hypoesthesia (numbness) in infected lesions. M. leprae is readily phagocytized by macrophages but is able to survive within macrophages in part by neutralizing reactive oxygen species produced in the oxidative burst of the phagolysosome. Like L. monocytogenes, M. leprae also can move directly between macrophages to avoid clearance by immune factors.
The extent of the disease is related to the immune response of the patient. Initial symptoms may not appear for as long as 2 to 5 years after infection. These often begin with small, blanched, numb areas of the skin. In most individuals, these will resolve spontaneously, but some cases may progress to a more serious form of the disease. Tuberculoid (paucibacillary) Hansen’s disease is marked by the presence of relatively few (three or less) flat, blanched skin lesions with small nodules at the edges and few bacteria present in the lesion. Although these lesions can persist for years or decades, the bacteria are held in check by an effective immune response including cell-mediated cytotoxicity. Individuals who are unable to contain the infection may later develop lepromatous (multibacillary) Hansen’s disease. This is a progressive form of the disease characterized by nodules filled with acid-fast bacilli and macrophages. Impaired function of infected Schwann cells leads to peripheral nerve damage, resulting in sensory loss that leads to ulcers, deformities, and fractures. Damage to the ulnar nerve (in the wrist) by M. leprae is one of the most common causes of crippling of the hand. In some cases, chronic tissue damage can ultimately lead to loss of fingers or toes. When mucosal tissues are also involved, disfiguring lesions of the nose and face can also occur (Figure \(7\)).
Hansen’s disease is diagnosed on the basis of clinical signs and symptoms of the disease, and confirmed by the presence of acid-fast bacilli on skin smears or in skin biopsy specimens (Figure \(7\)). M. leprae does not grow in vitro on any known laboratory media, but it can be identified by culturing in vivo in the footpads of laboratory mice or armadillos. Where needed, PCR and genotyping of M. leprae DNA in infected human tissue may be performed for diagnosis and epidemiology.
Hansen’s disease responds well to treatment and, if diagnosed and treated early, does not cause disability. In the United States, most patients with Hansen’s disease are treated in ambulatory care clinics in major cities by the National Hansen’s Disease program, the only institution in the United States exclusively devoted to Hansen’s disease. Since 1995, WHO has made multidrug therapy for Hansen’s disease available free of charge to all patients worldwide. As a result, global prevalence of Hansen’s disease has declined from about 5.2 million cases in 1985 to roughly 176,000 in 2014.15 Multidrug therapy consists of dapsone and rifampicin for all patients and a third drug, clofazimin, for patients with multibacillary disease.
Currently, there is no universally accepted vaccine for Hansen’s disease. India and Brazil use a tuberculosis vaccineagainst Hansen’s disease because both diseases are caused by species of Mycobacterium. The effectiveness of this method is questionable, however, since it appears that the vaccine works in some populations but not in others.
Exercise \(4\)
1. What prevents the progression from tuberculoid to lepromatus leprosy?
2. Why does Hansen’s disease typically affect the nerves of the extremities?
Leper Colonies
Disfiguring, deadly diseases like leprosy have historically been stigmatized in many cultures. Before leprosy was understood, victims were often isolated in leper colonies, a practice mentioned frequently in ancient texts, including the Bible. But leper colonies are not just an artifact of the ancient world. In Hawaii, a leper colony established in the late nineteenth century persisted until the mid-twentieth century, its residents forced to live in deplorable conditions.16 Although leprosy is a communicable disease, it is not considered contagious (easily communicable), and it certainly does not pose enough of a threat to justify the permanent isolation of its victims. Today, we reserve the practices of isolation and quarantine to patients with more dangerous diseases, such as Ebola or multiple-drug-resistant bacteria like Mycobacterium tuberculosis and Staphylococcus aureus. The ethical argument for this practice is that isolating infected patients is necessary to prevent the transmission and spread of highly contagious diseases—even when it goes against the wishes of the patient.
Of course, it is much easier to justify the practice of temporary, clinical quarantining than permanent social segregation, as occurred in leper colonies. In the 1980s, there were calls by some groups to establish camps for people infected with AIDS. Although this idea was never actually implemented, it begs the question—where do we draw the line? Are permanent isolation camps or colonies ever medically or socially justifiable? Suppose there were an outbreak of a fatal, contagious disease for which there is no treatment. Would it be justifiable to impose social isolation on those afflicted with the disease? How would we balance the rights of the infected with the risk they pose to others? To what extent should society expect individuals to put their own health at risk for the sake of treating others humanely?
Bacterial Infections of the Nervous System
Despite the formidable defenses protecting the nervous system, a number of bacterial pathogens are known to cause serious infections of the CNS or PNS. Unfortunately, these infections are often serious and life threatening. Figure \(8\) summarizes some important infections of the nervous system.
Key Concepts and Summary
• Bacterial meningitis can be caused by several species of encapsulated bacteria, including Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, and Streptococcus agalactiae (group B streptococci). H. influenzae affects primarily young children and neonates, N. meningitidis is the only communicable pathogen and mostly affects children and young adults, S. pneumoniae affects mostly young children, and S. agalactiae affects newborns during or shortly after birth.
• Symptoms of bacterial meningitis include fever, neck stiffness, headache, confusion, convulsions, coma, and death.
• Diagnosis of bacterial meningitis is made through observations and culture of organisms in CSF. Bacterial meningitis is treated with antibiotics. H. influenzae and N. meningitidis have vaccines available.
• Clostridium species cause neurological diseases, including botulism and tetanus, by producing potent neurotoxins that interfere with neurotransmitter release. The PNS is typically affected. Treatment of Clostridium infection is effective only through early diagnosis with administration of antibiotics to control the infection and antitoxins to neutralize the endotoxin before they enter cells.
• Listeria monocytogenes is a foodborne pathogen that can infect the CNS, causing meningitis. The infection can be spread through the placenta to a fetus. Diagnosis is through culture of blood or CSF. Treatment is with antibiotics and there is no vaccine.
• Hansen’s disease (leprosy) is caused by the intracellular parasite Mycobacterium leprae. Infections cause demylenation of neurons, resulting in decreased sensation in peripheral appendages and body sites. Treatment is with multi-drug antibiotic therapy, and there is no universally recognized vaccine.
Footnotes
1. 1 Thigpen, Michael C., Cynthia G. Whitney, Nancy E. Messonnier, Elizabeth R. Zell, Ruth Lynfield, James L. Hadler, Lee H. Harrison et al., “Bacterial Meningitis in the United States, 1998–2007,” New England Journal of Medicine 364, no. 21 (2011): 2016-25.
2. 2 Popovic, T., et al. World Health Organization, “Laboratory Manual for the Diagnosis of Meningitis Caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenza,” 1999.
3. 3 US Centers for Disease Control and Prevention, “Meningococcal Disease,” August 5, 2015. Accessed June 28, 2015. www.cdc.gov/meningococcal/sur...nce/index.html.
4. 4 US Centers for Disease Control and Prevention, “Recommended Immunization Schedule for Persons Aged 0 Through 18 Years, United States, 2016,” February 1, 2016. Accessed on June 28, 2016. www.cdc.gov/vaccines/schedule...dolescent.html.
5. 5 National Meningitis Association, “Serogroup B Meningococcal Disease Outbreaks on U.S. College Campuses,” 2016. Accessed June 28, 2016. http://www.nmaus.org/disease-prevent...ase/outbreaks/.
6. 6 United States Department of Health and Human Services, “Hib (Haemophilus Influenzae Type B),” Accessed June 28, 2016. http://www.vaccines.gov/diseases/hib/#.
7. 7 US Centers for Disease Control and Prevention, “Meningococcal Disease, Disease Trends,” 2015. Accessed September 13, 2016. www.cdc.gov/meningococcal/sur...nce/index.html.
8. 8 Thigpen, Michael C., Cynthia G. Whitney, Nancy E. Messonnier, Elizabeth R. Zell, Ruth Lynfield, James L. Hadler, Lee H. Harrison et al., “Bacterial Meningitis in the United States, 1998–2007,” New England Journal of Medicine 364, no. 21 (2011): 2016-25.
9. 9 Thigpen, Michael C., Cynthia G. Whitney, Nancy E. Messonnier, Elizabeth R. Zell, Ruth Lynfield, James L. Hadler, Lee H. Harrison et al., “Bacterial Meningitis in the United States, 1998–2007,” New England Journal of Medicine 364, no. 21 (2011): 2016-25; Heath, Paul T., Gail Balfour, Abbie M. Weisner, Androulla Efstratiou, Theresa L. Lamagni, Helen Tighe, Liam AF O’Connell et al., “Group B Streptococcal Disease in UK and Irish Infants Younger than 90 Days,” The Lancet 363, no. 9405 (2004): 292-4.
10. 10 UNFPA, UNICEF WHO, “Maternal and Neonatal Tetanus Elimination by 2005,” 2000. www.unicef.org/immunization/f...tegy_paper.pdf.
11. 11 US Centers for Disease Control and Prevention, “Tetanus Vaccination,” 2013. Accessed June 29, 2016. http://www.cdc.gov/tetanus/vaccination.html.
12. 12 Scallan, Elaine, Robert M. Hoekstra, Frederick J. Angulo, Robert V. Tauxe, Marc-Alain Widdowson, Sharon L. Roy, Jeffery L. Jones, and Patricia M. Griffin, “Foodborne Illness Acquired in the United States—Major Pathogens,” Emerging Infectious Diseases 17, no. 1 (2011): 7-15.
13. 13 US Centers for Disease Control and Prevention, “Listeria Outbreaks,” 2016. Accessed June 29, 2016. https://www.cdc.gov/listeria/outbreaks/index.html.
14. 14 Sharma, Rahul, Pushpendra Singh, W. J. Loughry, J. Mitchell Lockhart, W. Barry Inman, Malcolm S. Duthie, Maria T. Pena et al., “Zoonotic Leprosy in the Southeastern United States,” Emerging Infectious Diseases 21, no. 12 (2015): 2127-34.
15. 15 World Health Organization, “Leprosy Fact Sheet,” 2016. Accessed September 13, 2016. http://www.who.int/mediacentre/factsheets/fs101/en/.
16. 16 National Park Service, “A Brief History of Kalaupapa,” Accessed February 2, 2016. www.nps.gov/kala/learn/histor...-kalaupapa.htm.
Contributor
• Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University) with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free at https://openstax.org/books/microbiology/pages/1-introduction) | textbooks/bio/Microbiology/Microbiology_(OpenStax)/26%3A_Nervous_System_Infections/26.02%3A_Bacterial_Diseases_of_the_Nervous_System.txt |
Learning Objectives
• Identify the most common acellular pathogens that can cause infections of the nervous system
• Compare the major characteristics of specific viral diseases affecting the nervous system
A number of different viruses and subviral particles can cause diseases that affect the nervous system. Viral diseases tend to be more common than bacterial infections of the nervous system today. Fortunately, viral infections are generally milder than their bacterial counterparts and often spontaneously resolve. Some of the more important acellular pathogens of the nervous system are described in this section.
Viral Meningitis
Although it is much more common than bacterial meningitis, viral meningitis is typically less severe. Many different viruses can lead to meningitis as a sequela of the primary infection, including those that cause herpes, influenza, measles, and mumps. Most cases of viral meningitis spontaneously resolve, but severe cases do occur.
Arboviral Encephalitis
Several types of insect-borne viruses can cause encephalitis. Collectively, these viruses are referred to as arboviruses (because they are arthropod-borne), and the diseases they cause are described as arboviral encephalitis. Most arboviruses are endemic to specific geographical regions. Arborviral encephalitis diseases found in the United States include eastern equine encephalitis (EEE), western equine encephalitis (WEE), St. Louis encephalitis, and West Nile encephalitis (WNE). Expansion of arboviruses beyond their endemic regions sometimes occurs, generally as a result of environmental changes that are favorable to the virus or its vector. Increased travel of infected humans, animals, or vectors has also allowed arboviruses to spread into new regions.
In most cases, arboviral infections are asymptomatic or lead to a mild disease. However, when symptoms do occur, they include high fever, chills, headaches, vomiting, diarrhea, and restlessness. In elderly patients, severe arboviral encephalitis can rapidly lead to convulsions, coma, and death.
Mosquitoes are the most common biological vectors for arboviruses, which tend to be enveloped ssRNA viruses. Thus, prevention of arboviral infections is best achieved by avoiding mosquitoes—using insect repellent, wearing long pants and sleeves, sleeping in well-screened rooms, using bed nets, etc.
Diagnosis of arboviral encephalitis is based on clinical symptoms and serologic testing of serum or CSF. There are no antiviral drugs to treat any of these arboviral diseases, so treatment consists of supportive care and management of symptoms.
Eastern equine encephalitis (EEE) is caused by eastern equine encephalitis virus (EEEV), which can cause severe disease in horses and humans. Birds are reservoirs for EEEV with accidental transmission to horses and humans by Aedes, Coquillettidia, and Culex species of mosquitoes. Neither horses nor humans serve as reservoirs. EEE is most common in US Gulf Coast and Atlantic states. EEE is one of the more severe mosquito-transmitted diseases in the United States, but fortunately, it is a very rare disease in the United States (Figure \(1\)).12
Western equine encephalitis (WEE) is caused by western equine encephalitis virus (WEEV). WEEV is usually transmitted to horses and humans by the Culex tarsalis mosquitoes and, in the past decade, has caused very few cases of encephalitis in humans in the United States. In humans, WEE symptoms are less severe than EEE and include fever, chills, and vomiting, with a mortality rate of 3–4%. Like EEEV, birds are the natural reservoir for WEEV. Periodically, for indeterminate reasons, epidemics in human cases have occurred in North America in the past. The largest on record was in 1941, with more than 3400 cases.3
St. Louis encephalitis (SLE), caused by St. Louis encephalitis virus (SLEV), is a rare form of encephalitis with symptoms occurring in fewer than 1% of infected patients. The natural reservoirs for SLEV are birds. SLEV is most often found in the Ohio-Mississippi River basin of the central United States and was named after a severe outbreak in Missouri in 1934. The worst outbreak of St. Louis encephalitis occurred in 1975, with over 2000 cases reported.4Humans become infected when bitten by C. tarsalis, C. quinquefasciatus, or C. pipiens mosquitoes carrying SLEV. Most patients are asymptomatic, but in a small number of individuals, symptoms range from mild flu-like syndromes to fatal encephalitis. The overall mortality rate for symptomatic patients is 5–15%.5
Japanese encephalitis, caused by Japanese encephalitis virus (JEV), is the leading cause of vaccine-preventable encephalitis in humans and is endemic to some of the most populous countries in the world, including China, India, Japan, and all of Southeast Asia. JEV is transmitted to humans by Culex mosquitoes, usually the species C. tritaeniorhynchus. The biological reservoirs for JEV include pigs and wading birds. Most patients with JEV infections are asymptomatic, with symptoms occurring in fewer than 1% of infected individuals. However, about 25% of those who do develop encephalitis die, and among those who recover, 30–50% have psychiatric, neurologic, or cognitive impairment.6 Fortunately, there is an effective vaccine that can prevent infection with JEV. The CDC recommends this vaccine for travelers who expect to spend more than one month in endemic areas.
As the name suggests, West Nile virus (WNV) and its associated disease, West Nile encephalitis (WNE), did not originate in North America. Until 1999, it was endemic in the Middle East, Africa, and Asia; however, the first US cases were identified in New York in 1999, and by 2004, the virus had spread across the entire continental United States. Over 35,000 cases, including 1400 deaths, were confirmed in the five-year period between 1999 and 2004. WNV infection remains reportable to the CDC.
WNV is transmitted to humans by Culex mosquitoes from its natural reservoir, infected birds, with 70–80% of infected patients experiencing no symptoms. Most symptomatic cases involve only mild, flu-like symptoms, but fewer than 1% of infected people develop severe and sometimes fatal encephalitis or meningitis. The mortality rate in WNV patients who develop neurological disease is about 10%. More information about West Nile virus can be found in Modes of Disease Transmission.
Link to Learning
This interactive map identifies cases of several arboviral diseases in humans and reservoir species by state and year for the United States.
Exercise \(1\)
1. Why is it unlikely that arboviral encephalitis viruses will be eradicated in the future?
2. Which is the most common form of viral encephalitis in the United States?
Clinical Focus: Part 2
Levofloxacin is a quinolone antibiotic that is often prescribed to treat bacterial infections of the respiratory tract, including pneumonia and bronchitis. But after taking the medication for a week, David returned to his physician sicker than before. He claimed that the antibiotic had no effect on his earlier symptoms. In addition, he now was experiencing headaches, a stiff neck, and difficulty focusing at work. He also showed the doctor a rash that had developed on his arms over the past week. His doctor, more concerned now, began to ask about David's activities over the past two weeks.
David explained that he had been recently working on a project to disassemble an old barn. His doctor collected sputum samples and scrapings from David’s rash for cultures. A spinal tap was also performed to examine David’s CSF. Microscopic examination of his CSF revealed encapsulated yeast cells. Based on this result, the doctor prescribed a new antimicrobial therapy using amphotericin B and flucytosine.
Exercise \(2\)
1. Why was the original treatment ineffective?
2. Why is the presence of a capsule clinically important?
Zika Virus Infection
Zika virus infection is an emerging arboviral disease associated with human illness in Africa, Southeast Asia, and South and Central America; however, its range is expanding as a result of the widespread range of its mosquito vector. The first cases originating in the United States were reported in 2016.The Zika virus was initially described in 1947 from monkeys in the Zika Forest of Uganda through a network that monitored yellow fever. It was not considered a serious human pathogen until the first large-scale outbreaks occurred in Micronesia in 2007;7 however, the virus has gained notoriety over the past decade, as it has emerged as a cause of symptoms similar to other arboviral infections that include fever, skin rashes, conjunctivitis, muscle and joint pain, malaise, and headache. Mosquitoes of the Aedes genus are the primary vectors, although the virus can also be transmitted sexually, from mother to baby during pregnancy, or through a blood transfusion.
Most Zika virus infections result in mild symptoms such as fever, a slight rash, or conjunctivitis. However, infections in pregnant women can adversely affect the developing fetus. Reports in 2015 indicate fetal infections can result in brain damage, including a serious birth defect called microcephaly, in which the infant is born with an abnormally small head (Figure \(2\)).8
Diagnosis of Zika is primarily based on clinical symptoms. However, the FDA recently authorized the use of a Zika virus RNA assay, Trioplex RT-PCR, and Zika MAC-ELISA to test patient blood and urine to confirm Zika virus disease. There are currently no antiviral treatments or vaccines for Zika virus, and treatment is limited to supportive care.
Exercise \(3\)
1. What are the signs and symptoms of Zika virus infection in adults?
2. Why is Zika virus infection considered a serious public health threat?
Rabies
Rabies is a deadly zoonotic disease that has been known since antiquity. The disease is caused by rabies virus (RV), a member of the family Rhabdoviridae, and is primarily transmitted through the bite of an infected mammal. Rhabdoviridae are enveloped RNA viruses that have a distinctive bullet shape (Figure \(3\)); they were first studied by Louis Pasteur, who obtained rabies virus from rabid dogs and cultivated the virus in rabbits. He successfully prepared a rabies vaccine using dried nerve tissues from infected animals. This vaccine was used to first treat an infected human in 1885.
The most common reservoirs in the United States are wild animals such as raccoons (30.2% of all animal cases during 2014), bats (29.1%), skunks (26.3%), and foxes (4.1%); collectively, these animals were responsible for a total of 92.6% of animal rabies cases in the United States in 2014. The remaining 7.4% of cases that year were in domesticated animals such as dogs, cats, horses, mules, sheep, goats, and llamas.9 While there are typically only one or two human cases per year in the United States, rabies still causes tens of thousands of human deaths per year worldwide, primarily in Asia and Africa.
The low incidence of rabies in the United States is primarily a result of the widespread vaccination of dogs and cats. An oral vaccine is also used to protect wild animals, such as raccoons and foxes, from infection. Oral vaccine programs tend to focus on geographic areas where rabies is endemic.10 The oral vaccine is usually delivered in a package of bait that is dropped by airplane, although baiting in urban areas is done by hand to maximize safety.11 Many countries require a quarantine or proof of rabies vaccination for domestic pets being brought into the country. These procedures are especially strict in island nations where rabies is not yet present, such as Australia.
The incubation period for rabies can be lengthy, ranging from several weeks or months to over a year. As the virus replicates, it moves from the site of the bite into motor and sensory axons of peripheral nerves and spreads from nerve to nerve using a process called retrograde transport, eventually making its way to the CNS through the spinal ganglia. Once rabies virus reaches the brain, the infection leads to encephalitis caused by the disruption of normal neurotransmitter function, resulting in the symptoms associated with rabies. The virions act in the synaptic spaces as competitors with a variety of neurotransmitters for acetylcholine, GABA, and glycine receptors. Thus, the action of rabies virus is neurotoxic rather than cytotoxic. After the rabies virus infects the brain, it can continue to spread through other neuronal pathways, traveling out of the CNS to tissues such as the salivary glands, where the virus can be released. As a result, as the disease progresses the virus can be found in many other tissues, including the salivary glands, taste buds, nasal cavity, and tears.
The early symptoms of rabies include discomfort at the site of the bite, fever, and headache. Once the virus reaches the brain and later symptoms appear, the disease is always fatal. Terminal rabies cases can end in one of two ways: either furious or paralytic rabies. Individuals with furious rabies become very agitated and hyperactive. Hydrophobia (a fear of water) is common in patients with furious rabies, which is caused by muscular spasms in the throat when swallowing or thinking about water. Excess salivation and a desire to bite can lead to foaming of the mouth. These behaviors serve to enhance the likelihood of viral transmission, although contact with infected secretions like saliva or tears alone is sufficient for infection. The disease culminates after just a few days with terror and confusion, followed by cardiovascular and respiratory arrest. In contrast, individuals with paralytic rabies generally follow a longer course of disease. The muscles at the site of infection become paralyzed. Over a period of time, the paralysis slowly spreads throughout the body. This paralytic form of disease culminates in coma and death.
Before present-day diagnostic methods were available, rabies diagnosis was made using a clinical case history and histopathological examination of biopsy or autopsy tissues, looking for the presence of Negri bodies. We now know these histologic changes cannot be used to confirm a rabies diagnosis. There are no tests that can detect rabies virus in humans at the time of the bite or shortly thereafter. Once the virus has begun to replicate (but before clinical symptoms occur), the virus can be detected using an immunofluorescence test on cutaneous nerves found at the base of hair follicles. Saliva can also be tested for viral genetic material by reverse transcription followed by polymerase chain reaction (RT-PCR). Even when these tests are performed, most suspected infections are treated as positive in the absence of contravening evidence. It is better that patients undergo unnecessary therapy because of a false-positiveresult, rather than die as the result of a false-negative result.
Human rabies infections are treated by immunization with multiple doses of an attenuated vaccine to develop active immunity in the patient (see the Clinical Focus feature in the chapter on Acellular Pathogens). Vaccination of an already-infected individual has the potential to work because of the slow progress of the disease, which allows time for the patient’s immune system to develop antibodies against the virus. Patients may also be treated with human rabies immune globulin (antibodies to the rabies virus) to encourage passive immunity. These antibodies will neutralize any free viral particles. Although the rabies infection progresses slowly in peripheral tissues, patients are not normally able to mount a protective immune response on their own.
Exercise \(4\)
1. How does the bite from an infected animal transmit rabies?
2. What is the goal of wildlife vaccination programs for rabies?
3. How is rabies treated in a human?
Poliomyelitis
Poliomyelitis (polio), caused by poliovirus, is a primarily intestinal disease that, in a small percentage of cases, proceeds to the nervous system, causing paralysis and, potentially, death. Poliovirus is highly contagious, with transmission occurring by the fecal-oral route or by aerosol or droplet transmission. Approximately 72% of all poliovirus infections are asymptomatic; another 25% result only in mild intestinal disease, producing nausea, fever, and headache.12 However, even in the absence of symptoms, patients infected with the virus can shed it in feces and oral secretions, potentially transmitting the virus to others. In about one case in every 200, the poliovirus affects cells in the CNS.13
After it enters through the mouth, initial replication of poliovirus occurs at the site of implantation in the pharynx and gastrointestinal tract. As the infection progresses, poliovirus is usually present in the throat and in the stool before the onset of symptoms. One week after the onset of symptoms, there is less poliovirus in the throat, but for several weeks, poliovirus continues to be excreted in the stool. Poliovirus invades local lymphoid tissue, enters the bloodstream, and then may infect cells of the CNS. Replication of poliovirus in motor neurons of the anterior horn cells in the spinal cord, brain stem, or motor cortex results in cell destruction and leads to flaccid paralysis. In severe cases, this can involve the respiratory system, leading to death. Patients with impaired respiratory function are treated using positive-pressure ventilation systems. In the past, patients were sometimes confined to Emerson respirators, also known as iron lungs (Figure \(4\)).
Direct detection of the poliovirus from the throat or feces can be achieved using reverse transcriptase PCR (RT-PCR) or genomic sequencing to identify the genotype of the poliovirus infecting the patient. Serological tests can be used to determine whether the patient has been previously vaccinated. There are no therapeutic measures for polio; treatment is limited to various supportive measures. These include pain relievers, rest, heat therapy to ease muscle spasms, physical therapy and corrective braces if necessary to help with walking, and mechanical ventilation to assist with breathing if necessary.
Two different vaccines were introduced in the 1950s that have led to the dramatic decrease in polio worldwide (Figure \(5\)). The Salk vaccine is an inactivated polio virus that was first introduced in 1955. This vaccine is delivered by intramuscular injection. The Sabin vaccine is an oral polio vaccine that contains an attenuated virus; it was licensed for use in 1962. There are three serotypes of poliovirus that cause disease in humans; both the Salk and the Sabin vaccines are effective against all three.
Attenuated viruses from the Sabin vaccine are shed in the feces of immunized individuals and thus have the potential to infect nonimmunized individuals. By the late 1990s, the few polio cases originating in the United States could be traced back to the Sabin vaccine. In these cases, mutations of the attenuated virus following vaccination likely allowed the microbe to revert to a virulent form. For this reason, the United States switched exclusively to the Salk vaccine in 2000. Because the Salk vaccine contains an inactivated virus, there is no risk of transmission to others (see Vaccines). Currently four doses of the vaccine are recommended for children: at 2, 4, and 6–18 months of age, and at 4–6 years of age.
In 1988, WHO launched the Global Polio Eradication Initiative with the goal of eradicating polio worldwide through immunization. That goal is now close to being realized. Polio is now endemic in only a few countries, including Afghanistan, Pakistan, and Nigeria, where vaccination efforts have been disrupted by military conflict or political instability.
The Terror of Polio
In the years after World War II, the United States and the Soviet Union entered a period known as the Cold War. Although there was no armed conflict, the two super powers were diplomatically and economically isolated from each other, as represented by the so-called Iron Curtain between the Soviet Union and the rest of the world. After 1950, migration or travel outside of the Soviet Union was exceedingly difficult, and it was equally difficult for foreigners to enter the Soviet Union. The United States also placed strict limits on Soviets entering the country. During the Eisenhower administration, only 20 graduate students from the Soviet Union were allowed to come to study in the United States per year.
Yet even the Iron Curtain was no match for polio. The Salk vaccine became widely available in the West in 1955, and by the time the Sabin vaccine was ready for clinical trials, most of the susceptible population in the United States and Canada had already been vaccinated against polio. Sabin needed to look elsewhere for study participants. At the height of the Cold War, Mikhail Chumakov was allowed to come to the United States to study Sabin’s work. Likewise, Sabin, an American microbiologist, was allowed to travel to the Soviet Union to begin clinical trials. Chumakov organized Soviet-based production and managed the experimental trials to test the new vaccine in the Soviet Union. By 1959, over ten million Soviet children had been safely treated with Sabin’s vaccine.
As a result of a global vaccination campaign with the Sabin vaccine, the overall incidence of polio has dropped dramatically. Today, polio has been nearly eliminated around the world and is only rarely seen in the United States. Perhaps one day soon, polio will become the third microbial disease to be eradicated from the general population [small pox and rinderpest (the cause of cattle plague) being the first two].
Exercise \(5\)
1. How is poliovirus transmitted?
2. Compare the pros and cons of each of the two polio vaccines.
Transmissible Spongiform Encephalopathies
Acellular infectious agents called prions are responsible for a group of related diseases known as transmissible spongiform encephalopathies (TSEs) that occurs in humans and other animals (see Viroids, Virusoids, and Prions). All TSEs are degenerative, fatal neurological diseases that occur when brain tissue becomes infected by prions. These diseases have a slow onset; symptoms may not become apparent until after an incubation period of years and perhaps decades, but death usually occurs within months to a few years after the first symptoms appear.
TSEs in animals include scrapie, a disease in sheep that has been known since the 1700s, and chronic wasting disease, a disease of deer and elk in the United States and Canada. Mad cow disease is seen in cattle and can be transmitted to humans through the consumption of infected nerve tissues. Human prion diseases include Creutzfeldt-Jakob disease and kuru, a rare disease endemic to Papua New Guinea.
Prions are infectious proteinaceous particles that are not viruses and do not contain nucleic acid. They are typically transmitted by exposure to and ingestion of infected nervous system tissues, tissue transplants, blood transfusions, or contaminated fomites. Prion proteins are normally found in a healthy brain tissue in a form called PrPC. However, if this protein is misfolded into a denatured form (PrPSc), it can cause disease. Although the exact function of PrPC is not currently understood, the protein folds into mostly alpha helices and binds copper. The rogue protein, on the other hand, folds predominantly into beta-pleated sheets and is resistant to proteolysis. In addition, PrPSc can induce PrPC to become misfolded and produce more rogue protein (Figure \(6\)).
As PrPSc accumulates, it aggregates and forms fibrils within nerve cells. These protein complexes ultimately cause the cells to die. As a consequence, brain tissues of infected individuals form masses of neurofibrillary tangles and amyloid plaques that give the brain a spongy appearance, which is why these diseases are called spongiform encephalopathy (Figure 6.4.3). Damage to brain tissue results in a variety of neurological symptoms. Most commonly, affected individuals suffer from memory loss, personality changes, blurred vision, uncoordinated movements, and insomnia. These symptoms gradually worsen over time and culminate in coma and death.
The gold standard for diagnosing TSE is the histological examination of brain biopsies for the presence of characteristic amyloid plaques, vacuoles, and prion proteins. Great care must be taken by clinicians when handling suspected prion-infected materials to avoid becoming infected themselves. Other tissue assays search for the presence of the 14-3-3 protein, a marker for prion diseases like Creutzfeldt-Jakob disease. New assays, like RT-QuIC (real-time quaking-induced conversion), offer new hope to effectively detect the abnormal prion proteins in tissues earlier in the course of infection. Prion diseases cannot be cured. However, some medications may help slow their progress. Medical support is focused on keeping patients as comfortable as possible despite progressive and debilitating symptoms.
Link to Learning
Because prion-contaminated materials are potential sources of infection for clinical scientists and physicians, both the World Health Organization and CDC provide information to inform, educate and minimize the risk of infections due to prions.
Exercise \(6\)
1. Do prions reproduce in the conventional sense?
2. What is the connection between prions and the removal of animal byproducts from the food of farm animals?
Acellular Infections of the Nervous System
Serious consequences are the common thread among these neurological diseases. Several cause debilitating paralysis, and some, such as Creutzfeldt-Jakob disease and rabies, are always or nearly always fatal. Since few drugs are available to combat these infections, vector control and vaccination are critical for prevention and containment. Figure \(7\) summarizes some important viral and prion infections of the nervous system.
Key Concepts and Summary
• Viral meningitis is more common and generally less severe than bacterial menigitis. It can result from secondary sequelae of many viruses or be caused by infections of arboviruses.
• Various types of arboviral encephalitis are concentrated in particular geographic locations throughout the world. These mosquito-borne viral infections of the nervous system are typically mild, but they can be life-threatening in some cases.
• Zika virus is an emerging arboviral infection with generally mild symptoms in most individuals, but infections of pregnant women can cause the birth defect microcephaly.
• Polio is typically a mild intestinal infection but can be damaging or fatal if it progresses to a neurological disease.
• Rabies is nearly always fatal when untreated and remains a significant problem worldwide.
• Transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease and kuru are caused by prions. These diseases are untreatable and ultimately fatal. Similar prion diseases are found in animals.
Footnotes
1. 1 US Centers for Disease Control and Prevention, “Eastern Equine Encephalitis Virus Disease Cases and Deaths Reported to CDC by Year and Clinical Presentation, 2004–2013,” 2014. www.cdc.gov/EasternEquineEnce..._2004-2013.pdf.
2. 2 US Centers for Disease Control and Prevention, “Eastern Equine Encephalitis, Symptoms & Treatment, 2016,” Accessed June 29, 2016. https://www.cdc.gov/easternequineenc.../symptoms.html.
3. 3 US Centers for Disease Control and Prevention, “Western Equine Encephalitis—United States and Canada, 1987,” Morbidity and Mortality Weekly Report 36, no. 39 (1987): 655.
4. 4 US Centers for Disease Control and Prevention, “Saint Louis encephalitis, Epidemiology & Geographic Distribution,” Accessed June 30, 2016. http://www.cdc.gov/sle/technical/epi.html.
5. 5 US Centers for Disease Control and Prevention, “Saint Louis encephalitis, Symptoms and Treatment,” Accessed June 30, 2016. http://www.cdc.gov/sle/technical/symptoms.html.
6. 6 US Centers for Disease Control and Prevention, “Japanese Encephalitis, Symptoms and Treatment,” Accessed June 30, 2016. www.cdc.gov/japaneseencephali...oms/index.html.
7. 7 Sikka, Veronica, Vijay Kumar Chattu, Raaj K. Popli, Sagar C. Galwankar, Dhanashree Kelkar, Stanley G. Sawicki, Stanislaw P. Stawicki, and Thomas J. Papadimos, “The Emergence of Zika Virus as a Global Health Security Threat: A Review and a Consensus Statement of the INDUSEM Joint Working Group (JWG),” Journal of Global Infectious Diseases 8, no. 1 (2016): 3.
8. 8 Mlakar, Jernej, Misa Korva, Nataša Tul, Mara Popović, Mateja Poljšak-Prijatelj, Jerica Mraz, Marko Kolenc et al., “Zika Virus Associated with Microcephaly,” New England Journal of Medicine 374, no. 10 (2016): 951-8.
9. 9 US Centers for Disease Control and Prevention, “Rabies, Wild Animals,” 2016. Accessed September 13, 2016. www.cdc.gov/rabies/location/u...d_animals.html.
10. 10 Slate, Dennis, Charles E. Rupprecht, Jane A. Rooney, Dennis Donovan, Donald H. Lein, and Richard B. Chipman, “Status of Oral Rabies Vaccination in Wild Carnivores in the United States,” Virus Research 111, no. 1 (2005): 68-76.
11. 11 Finnegan, Christopher J., Sharon M. Brookes, Nicholas Johnson, Jemma Smith, Karen L. Mansfield, Victoria L. Keene, Lorraine M. McElhinney, and Anthony R. Fooks, “Rabies in North America and Europe,” Journal of the Royal Society of Medicine 95, no. 1 (2002): 9-13. www.ncbi.nlm.nih.gov/pmc/articles/PMC1279140/.
12. 12 US Centers for Disease Control and Prevention, “Global Health – Polio,” 2014. Accessed June 30, 2016. http://www.cdc.gov/polio/about/index.htm.
13. 13 US Centers for Disease Control and Prevention, “Global Health – Polio,” 2014. Accessed June 30, 2016. http://www.cdc.gov/polio/about/index.htm. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/26%3A_Nervous_System_Infections/26.03%3A_Acellular_Pathogenic_Diseases_of_the_Nervous_System.txt |
Learning Objectives
• Identify the most common fungi that can cause infections of the nervous system
• Compare the major characteristics of specific fungal diseases affecting the nervous system
Fungal infections of the nervous system, called neuromycoses, are rare in healthy individuals. However, neuromycoses can be devastating in immunocompromised or elderly patients. Several eukaryotic parasites are also capable of infecting the nervous system of human hosts. Although relatively uncommon, these infections can also be life-threatening in immunocompromised individuals. In this section, we will first discuss neuromycoses, followed by parasitic infections of the nervous system.
Cryptococcocal Meningitis
Cryptococcus neoformans is a fungal pathogen that can cause meningitis. This yeast is commonly found in soils and is particularly associated with pigeon droppings. It has a thick capsule that serves as an important virulence factor, inhibiting clearance by phagocytosis. Most C. neoformans cases result in subclinical respiratory infections that, in healthy individuals, generally resolve spontaneously with no long-term consequences (see Respiratory Mycoses). In immunocompromised patients or those with other underlying illnesses, the infection can progress to cause meningitisand granuloma formation in brain tissues. Cryptococcus antigens can also serve to inhibit cell-mediated immunity and delayed-type hypersensitivity.
Cryptococcus can be easily cultured in the laboratory and identified based on its extensive capsule (Figure \(1\)). C. neoformans is frequently cultured from urine samples of patients with disseminated infections.
Prolonged treatment with antifungal drugs is required to treat cryptococcal infections. Combined therapy is required with amphotericin B plus flucytosine for at least 10 weeks. Many antifungal drugs have difficulty crossing the blood-brain barrier and have strong side effects that necessitate low doses; these factors contribute to the lengthy time of treatment. Patients with AIDS are particularly susceptible to Cryptococcus infections because of their compromised immune state. AIDS patients with cryptococcosis can also be treated with antifungal drugs, but they often have relapses; lifelong doses of fluconazole may be necessary to prevent reinfection.
Exercise \(1\)
1. Why are neuromycoses infections rare in the general population?
2. How is a cryptococcal infection acquired?
Neuromycoses
Neuromycoses typically occur only in immunocompromised individuals and usually only invade the nervous system after first infecting a different body system. As such, many diseases that sometimes affect the nervous system have already been discussed in previous chapters. Figure \(2\) presents some of the most common fungal infections associated with neurological disease. This table includes only the neurological aspects associated with these diseases; it does not include characteristics associated with other body systems.
Clinical Focus: Resolution
David’s new prescription for two antifungal drugs, amphotericin B and flucytosine, proved effective, and his condition began to improve. Culture results from David’s sputum, skin, and CSF samples confirmed a fungal infection. All were positive for C. neoformans. Serological tests of his tissues were also positive for the C. neoformans capsular polysaccharide antigen.
Since C. neoformans is known to occur in bird droppings, it is likely that David had been exposed to the fungus while working on the barn. Despite this exposure, David’s doctor explained to him that immunocompetent people rarely contract cryptococcal meningitis and that his immune system had likely been compromised by the anti-inflammatory medication he was taking to treat his Crohn’s disease. However, to rule out other possible causes of immunodeficiency, David’s doctor recommended that he be tested for HIV.
After David tested negative for HIV, his doctor took him off the corticosteroid he was using to manage his Crohn’s disease, replacing it with a different class of drug. After several weeks of antifungal treatments, David managed a full recovery.
Amoebic Meningitis
Primary amoebic meningoencephalitis (PAM) is caused by Naegleria fowleri. This amoeboflagellate is commonly found free-living in soils and water. It can exist in one of three forms—the infective amoebic trophozoite form, a motile flagellate form, and a resting cyst form. PAM is a rare disease that has been associated with young and otherwise healthy individuals. Individuals are typically infected by the amoeba while swimming in warm bodies of freshwater such as rivers, lakes, and hot springs. The pathogenic trophozoite infects the brain by initially entering through nasal passages to the sinuses; it then moves down olfactory nerve fibers to penetrate the submucosal nervous plexus, invades the cribriform plate, and reaches the subarachnoid space. The subarachnoid space is highly vascularized and is a route of dissemination of trophozoites to other areas of the CNS, including the brain (Figure \(3\)). Inflammation and destruction of gray matter leads to severe headaches and fever. Within days, confusion and convulsions occur and quickly progress to seizures, coma, and death. The progression can be very rapid, and the disease is often not diagnosed until autopsy.
N. fowleri infections can be confirmed by direct observation of CSF; the amoebae can often be seen moving while viewing a fresh CSF wet mount through a microscope. Flagellated forms can occasionally also be found in CSF. The amoebae can be stained with several stains for identification, including Giemsa-Wright or a modified trichrome stain. Detection of antigens with indirect immunofluorescence, or genetic analysis with PCR, can be used to confirm an initial diagnosis. N. fowleri infections are nearly always fatal; only 3 of 138 patients with PAM in the United States have survived.1 A new experimental drug called miltefosine shows some promise for treating these infections. This drug is a phosphotidylcholine derivative that is thought to inhibit membrane function in N. fowleri, triggering apoptosis and disturbance of lipid-dependent cell signaling pathways.2 When administered early in infection and coupled with therapeutic hypothermia (lowering the body’s core temperature to reduce the cerebral edema associated with infection), this drug has been successfully used to treat primary amoebic encephalitis.
Granulomatous Amoebic Encephalitis
Acanthamoeba and Balamuthia species are free-living amoebae found in many bodies of fresh water. Human infections by these amoebae are rare. However, they can cause amoebic keratitis in contact lens wearers (see Protozoan and Helminthic Infections of the Eyes), disseminated infections in immunocompromised patients, and granulomatous amoebic encephalitis (GAE) in severe cases. Compared to PAM, GAE tend to be subacute infections. The microbe is thought to enter through either the nasal sinuses or breaks in the skin. It is disseminated hematogenously and can invade the CNS. There, the infections lead to inflammation, formation of lesions, and development of typical neurological symptoms of encephalitis (Figure \(4\)). GAE is nearly always fatal.
GAE is often not diagnosed until late in the infection. Lesions caused by the infection can be detected using CT or MRI. The live amoebae can be directly detected in CSF or tissue biopsies. Serological tests are available but generally are not necessary to make a correct diagnosis, since the presence of the organism in CSF is definitive. Some antifungal drugs, like fluconazole, have been used to treat acanthamoebal infections. In addition, a combination of miltefosine and voriconazole (an inhibitor of ergosterol biosynthesis) has recently been used to successfully treat GAE. Even with treatment, however, the mortality rate for patients with these infections is high.
Exercise \(2\)
How is granulomatous amoebic encephalitis diagnosed?
Human African Trypanosomiasis
Human African trypanosomiasis (also known as African sleeping sickness) is a serious disease endemic to two distinct regions in sub-Saharan Africa. It is caused by the insect-borne hemoflagellate Trypanosoma brucei. The subspecies Trypanosoma brucei rhodesiense causes East African trypanosomiasis (EAT), and another subspecies, Trypanosoma brucei gambiense causes West African trypanosomiasis (WAT). A few hundred cases of EAT are currently reported each year.3 WAT is more commonly reported and tends to be a more chronic disease. Around 7000 to 10,000 new cases of WAT are identified each year.4
T. brucei is primarily transmitted to humans by the bite of the tsetse fly (Glossina spp.). Soon after the bite of a tsetse fly, a chancre forms at the site of infection. The flagellates then spread, moving into the circulatory system (Figure \(5\)). These systemic infections result in an undulating fever, during which symptoms persist for two or three days with remissions of about a week between bouts. As the disease enters its final phase, the pathogens move from the lymphatics into the CNS. Neurological symptoms include daytime sleepiness, insomnia, and mental deterioration. In EAT, the disease runs its course over a span of weeks to months. In contrast, WAT often occurs over a span of months to years.
Although a strong immune response is mounted against the trypanosome, it is not sufficient to eliminate the pathogen. Through antigenic variation, Trypanosoma can change their surface proteins into over 100 serological types. This variation leads to the undulating form of the initial disease. The initial septicemia caused by the infection leads to high fevers. As the immune system responds to the infection, the number of organisms decrease, and the clinical symptoms abate. However, a subpopulation of the pathogen then alters its surface coat antigens by antigenic variation and evades the immune response. These flagellates rapidly proliferate and cause another bout of disease. If untreated, these infections are usually fatal.
Clinical symptoms can be used to recognize the early signs of African trypanosomiasis. These include the formation of a chancre at the site of infection and Winterbottom’s sign. Winterbottom’s sign refers to the enlargement of lymph nodes on the back of the neck—often indicative of cerebral infections. Trypanosoma can be directly observed in stained samples including blood, lymph, CSF, and skin biopsies of chancres from patients. Antibodies against the parasite are found in most patients with acute or chronic disease. Serologic testing is generally not used for diagnosis, however, since the microscopic detection of the parasite is sufficient. Early diagnosis is important for treatment. Before the nervous system is involved, drugs like pentamidine (an inhibitor of nuclear metabolism) and suramin (mechanism unclear) can be used. These drugs have fewer side effects than the drugs needed to treat the second stage of the disease. Once the sleeping sickness phase has begun, harsher drugs including melarsoprol (an arsenic derivative) and eflornithine can be effective. Following successful treatment, patients still need to have follow-up examinations of their CSF for two years to detect possible relapses of the disease. The most effective means of preventing these diseases is to control the insect vector populations.
Exercise \(3\)
1. What is the symptom of a systemic Trypanosoma infection?
2. What are the symptoms of a neurological Trypanosoma infection?
3. Why are trypanosome infections so difficult to eradicate?
Neurotoxoplasmosis
Toxoplasma gondii is an ubiquitous intracellular parasite that can cause neonatal infections. Cats are the definitive host, and humans can become infected after eating infected meat or, more commonly, by ingesting oocysts shed in the feces of cats (see Parasitic Infections of the Circulatory and Lymphatic Systems). T. gondii enters the circulatory system by passing between the endothelial cells of blood vessels.5 Most cases of toxoplasmosis are asymptomatic. However, in immunocompromised patients, neurotoxoplasmosis caused by T. gondii infections are one of the most common causes of brain abscesses.6 The organism is able to cross the blood-brain barrier by infecting the endothelial cells of capillaries in the brain. The parasite reproduces within these cells, a step that appears to be necessary for entry to the brain, and then causes the endothelial cell to lyse, releasing the progeny into brain tissues. This mechanism is quite different than the method it uses to enter the bloodstream in the first place.7
The brain lesions associated with neurotoxoplasmosis can be detected radiographically using MRI or CAT scans (Figure \(6\)). Diagnosis can be confirmed by direct observation of the organism in CSF. RT-PCR assays can also be used to detect T. gondii through genetic markers.
Treatment of neurotoxoplasmosis caused by T. gondii infections requires six weeks of multi-drug therapy with pyrimethamine, sulfadiazine, and folinic acid. Long-term maintenance doses are often required to prevent recurrence.
Exercise \(4\)
1. Under what conditions is Toxoplasma infection serious?
2. How does Toxoplasma circumvent the blood-brain barrier?
Neurocysticercosis
Cysticercosis is a parasitic infection caused by the larval form of the pork tapeworm, Taenia solium. When the larvae invade the brain and spinal cord, the condition is referred to as neurocysticercosis. This condition affects millions of people worldwide and is the leading cause of adult onset epilepsy in the developing world.8
The life cycle of T. solium is discussed in Helminthic Infections of the Gastrointestinal Tract. Following ingestion, the eggs hatch in the intestine to form larvae called cysticerci. Adult tapeworms form in the small intestine and produce eggs that are shed in the feces. These eggs can infect other individuals through fecal contamination of food or other surfaces. Eggs can also hatch within the intestine of the original patient and lead to an ongoing autoinfection. The cystercerci, can migrate to the blood and invade many tissues in the body, including the CNS.
Neurocysticercosis is usually diagnosed through noninvasive techniques. Epidemiological information can be used as an initial screen; cysticercosis is endemic in Central and South America, Africa, and Asia. Radiological imaging (MRI and CT scans) is the primary method used to diagnose neurocysticercosis; imaging can be used to detect the one- to two-centimeter cysts that form around the parasites (Figure \(7\)). Elevated levels of eosinophils in the blood can also indicate a parasitic infection. EIA and ELISA are also used to detect antigens associated with the pathogen.
The treatment for neurocysticercosis depends on the location, number, size, and stage of cysticerci present. Antihelminthic chemotherapy includes albendazole and praziquantel. Because these drugs kill viable cysts, they may acutely increase symptoms by provoking an inflammatory response caused by the release of Taenia cysticerci antigens, as the cysts are destroyed by the drugs. To alleviate this response, corticosteroids that cross the blood-brain barrier(e.g., dexamethasone) can be used to mitigate these effects. Surgical intervention may be required to remove intraventricular cysts.
Parasitic Diseases of the Nervous System
Parasites that successfully invade the nervous system can cause a wide range of neurological signs and symptoms. Often, they inflict lesions that can be visualized through radiologic imaging. A number of these infections are fatal, but some can be treated (with varying levels of success) by antimicrobial drugs (Figure \(8\)).
Exercise \(5\)
1. What neurological condition is associated with neurocysticercosis?
2. How is neurocysticercosis diagnosed?
Key Concepts and Summary
• Neuromycoses are uncommon in immunocompetent people, but immunocompromised individuals with fungal infections have high mortality rates. Treatment of neuromycoses require prolonged therapy with antifungal drugs at low doses to avoid side effects and overcome the effect of the blood-brain barrier.
• Some protist infections of the nervous systems are fatal if not treated, including primary amoebic meningitis, granulomatous amoebic encephalitis, human African trypanosomiasis, and neurotoxoplasmosis.
• The various forms of ameobic encephalitis caused by the different amoebic infections are typically fatal even with treatment, but they are rare.
• African trypanosomiasis is a serious but treatable disease endemic to two distinct regions in sub-Saharan Africa caused by the insect-borne hemoflagellate Trypanosoma brucei.
• Neurocysticercosis is treated using antihelminthic drugs or surgery to remove the large cysts from the CNS.
Footnotes
1. 1 US Centers for Disease Control and Prevention, “Naegleria fowleri—Primary Amoebic Meningoencephalitis (PAM)—Amebic Encephalitis,” 2016. Accessed June 30, 2016. http://www.cdc.gov/parasites/naegleria/treatment.html.
2. 2 Dorlo, Thomas PC, Manica Balasegaram, Jos H. Beijnen, and Peter J. de Vries, “Miltefosine: A Review of Its Pharmacology and Therapeutic Efficacy in the Treatment of Leishmaniasis,” Journal of Antimicrobial Chemotherapy 67, no. 11 (2012): 2576-97.
3. 3 US Centers for Disease Control and Prevention, “Parasites – African Trypanosomiasis (also known as Sleeping Sickness), East African Trypanosomiasis FAQs,” 2012. Accessed June 30, 2016. www.cdc.gov/parasites/sleepin...faqs-east.html.
4. 4 US Centers for Disease Control and Prevention, “Parasites – African Trypanosomiasis (also known as Sleeping Sickness), Epidemiology & Risk Factors,” 2012. Accessed June 30, 2016. www.cdc.gov/parasites/sleepin...kness/epi.html.
5. 5 Carruthers, Vern B., and Yasuhiro Suzuki, “Effects of Toxoplasma gondii Infection on the Brain,” Schizophrenia Bulletin 33, no. 3 (2007): 745-51.
6. 6 Uppal, Gulshan, “CNS Toxoplasmosis in HIV,” 2015. Accessed June 30, 2016. emedicine.medscape.com/articl...98-overview#a3.
7. 7 Konradt, Christoph, Norikiyo Ueno, David A. Christian, Jonathan H. Delong, Gretchen Harms Pritchard, Jasmin Herz, David J. Bzik et al., “Endothelial Cells Are a Replicative Niche for Entry of Toxoplasma gondii to the Central Nervous System,” Nature Microbiology 1 (2016): 16001.
8. 8 DeGiorgio, Christopher M., Marco T. Medina, Reyna Durón, Chi Zee, and Susan Pietsch Escueta, “Neurocysticercosis,” Epilepsy Currents 4, no. 3 (2004): 107-11. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/26%3A_Nervous_System_Infections/26.04%3A_Neuromycoses_and_Parasitic_Diseases_of_the_Nervous_System.txt |
26.1: Anatomy of the Nervous System
The human nervous system can be divided into two interacting subsystems: the peripheral nervous system (PNS) and the central nervous system (CNS). The CNS consists of the brain and spinal cord. The peripheral nervous system is an extensive network of nerves connecting the CNS to the muscles and sensory structures.
Multiple Choice
What is the outermost membrane surrounding the brain called?
1. pia mater
2. arachnoid mater
3. dura mater
4. alma mater
Answer
C
What term refers to an inflammation of brain tissues?
1. encephalitis
2. meningitis
3. sinusitis
4. meningoencephalitis
Answer
A
Nerve cells form long projections called ________.
1. soma
2. axons
3. dendrites
4. synapses
Answer
B
Chemicals called ________ are stored in neurons and released when the cell is stimulated by a signal.
1. toxins
2. cytokines
3. chemokines
4. neurotransmitters
Answer
D
The central nervous system is made up of
1. sensory organs and muscles.
2. the brain and muscles.
3. the sensory organs and spinal cord.
4. the brain and spinal column.
Answer
D
Matching
Match each strategy for microbial invasion of the CNS with its description.
___intercellular entry A. pathogen gains entry by infecting peripheral white blood cells
___transcellular entry B. pathogen bypasses the blood-brain barrier by travel along the olfactory or trigeminal cranial nerves
___leukocyte-facilitated entry C. pathogen passes through the cells of the blood-brain barrier
___nonhematogenous entry D. pathogen passes between the cells of the blood-brain barrier
Answer
D, C, A, B
Fill in the Blank
The cell body of a neuron is called the ________.
Answer
soma
A signal is transmitted down the ________ of a nerve cell.
Answer
axon
The ________ is filled with cerebrospinal fluid.
Answer
subarachnoid space
The ________ ________ prevents access of microbes in the blood from gaining access to the central nervous system.
Answer
blood-brain barrier
The ________ are a set of membranes that cover and protect the brain.
Answer
meninges
Short Answer
Briefly describe the defenses of the brain against trauma and infection.
Describe how the blood-brain barrier is formed.
Identify the type of cell shown, as well as the following structures: axon, dendrite, myelin sheath, soma, and synapse.
Critical Thinking
What important function does the blood-brain barrier serve? How might this barrier be problematic at times?
26.2: Bacterial Diseases of the Nervous System
Bacterial infections that affect the nervous system are serious and can be life-threatening. Fortunately, there are only a few bacterial species commonly associated with neurological infections.
Multiple Choice
Which of the following organisms causes epidemic meningitis cases at college campuses?
1. Haemophilus influenzae type b
2. Neisseria meningitidis
3. Streptococcus pneumoniae
4. Listeria monocytogenes
Answer
B
Which of the following is the most common cause of neonatal meningitis?
1. Haemophilus influenzae b
2. Streptococcus agalactiae
3. Neisseria meningitidis
4. Streptococcus pneumoniae
Answer
B
What sign/symptom would NOT be associated with infant botulism?
1. difficulty suckling
2. limp body
3. stiff neck
4. weak cry
Answer
C
Which of the following can NOT be prevented with a vaccine?
1. tetanus
2. pneumococcal meningitis
3. meningococcal meningitis
4. listeriosis
Answer
D
How is leprosy primarily transmitted from person to person?
1. contaminated toilet seats
2. shaking hands
3. blowing nose
4. sexual intercourse
Answer
C
Fill in the Blank
The form of meningitis that can cause epidemics is caused by the pathogen ________.
Answer
Neisseria meningitidis
The symptoms of tetanus are caused by the neurotoxin ________.
Answer
tetanospasmin
________ is another name for leprosy.
Answer
Hansen’s disease
Botulism prevents the release of the neurotransmitter ________.
Answer
acetylcholine
________ is a neurological disease that can be prevented with the DTaP vaccine.
Answer
Tetanus
Tetanus patients exhibit ________ when muscle spasms causes them to arch their backs.
Answer
opisthotonos
Short Answer
A physician suspects the lesion and pustule pictured here are indicative of tuberculoid leprosy. If the diagnosis is correct, what microorganism would be found in a skin biopsy?
(credit: Centers for Disease Control and Prevention)
Critical Thinking
Explain how tetanospasmin functions to cause disease.
The most common causes of bacterial meningitis can be the result of infection by three very different bacteria. Which bacteria are they and how are these microbes similar to each other?
Explain how infant botulism is different than foodborne botulism.
26.3: Acellular Pathogenic Diseases of the Nervous System
A number of different viruses and subviral particles can cause diseases that affect the nervous system. Viral diseases tend to be more common than bacterial infections of the nervous system today. Fortunately, viral infections are generally milder than their bacterial counterparts and often spontaneously resolve. Some of the more important acellular pathogens of the nervous system are described in this section.
Multiple Choice
Which of these diseases can be prevented with a vaccine for humans?
1. eastern equine encephalitis
2. western equine encephalitis
3. West Nile encephalitis
4. Japanese encephalitis
Answer
D
Which of these diseases does NOT require the introduction of foreign nucleic acid?
1. kuru
2. polio
3. rabies
4. St. Louis encephalitis
Answer
A
Which of these is true of the Sabin but NOT the Salk polio vaccine?
1. requires four injections
2. currently administered in the United States
3. mimics the normal route of infection
4. is an inactivated vaccine
Answer
C
Which of the following animals is NOT a typical reservoir for the spread of rabies?
1. dog
2. bat
3. skunk
4. chicken
Answer
D
Fill in the Blank
The rogue form of the prion protein is called ________.
Answer
PrPSc
________ are the most common reservoir for the rabies virus worldwide.
Answer
Dogs
________ was the scientist who developed the inactivated polio vaccine.
Answer
Jonas Salk
________ is a prion disease of deer and elk.
Answer
Chronic wasting disease
The rogue form of prion protein exists primarily in the ________ conformation.
Answer
beta sheet
Short Answer
Explain how a person could contract variant Creutzfeldt-Jakob disease by consuming products from a cow with bovine spongiform encephalopathy (mad cow disease).
Critical Thinking
If the Sabin vaccine is being used to eliminate polio worldwide, explain why a country with a near zero infection rate would opt to use the Salk vaccine but not the Sabin vaccine?
26.4: Neuromycoses and Parasitic Diseases of the Nervous System
Fungal infections of the nervous system, called neuromycoses, are rare in healthy individuals. However, neuromycoses can be devastating in immunocompromised or elderly patients. Several eukaryotic parasites are also capable of infecting the nervous system of human hosts. Although relatively uncommon, these infections can also be life-threatening in immunocompromised individuals. In this section, we first discuss neuromycoses, followed by parasitic infections of the nervous system.
Multiple Choice
Which of these diseases results in meningitis caused by an encapsulated yeast?
1. cryptococcosis
2. histoplasmosis
3. candidiasis
4. coccidiomycosis
Answer
A
What kind of stain is most commonly used to visualize the capsule of cryptococcus?
1. Gram stain
2. simple stain
3. negative stain
4. fluorescent stain
Answer
C
Which of the following is the causative agent of East African trypanosomiasis?
1. Trypanosoma cruzi
2. Trypanosoma vivax
3. Trypanosoma brucei rhodanese
4. Trypanosoma brucei gambiense
Answer
C
Which of the following is the causative agent of primary amoebic meningoencephalitis?
1. Naegleria fowleri
2. Entameba histolyticum
3. Amoeba proteus
4. Acanthamoeba polyphaga
Answer
A
What is the biological vector for African sleeping sickness?
1. mosquito
2. tsetse fly
3. deer tick
4. sand fly
Answer
B
How do humans usually contract neurocysticercosis?
1. the bite of an infected arthropod
2. exposure to contaminated cat feces
3. swimming in contaminated water
4. ingestion of undercooked pork
Answer
D
Which of these is the most important cause of adult onset epilepsy?
1. neurocysticercosis
2. neurotoxoplasmosis
3. primary amoebic meningoencephalitis
4. African trypanosomiasis
Answer
A
Fill in the Blank
The ________ is the main virulence factor of Cryptococcus neoformans.
Answer
capsule
The drug of choice for fungal infections of the nervous system is ________.
Answer
Amphotericin B
The larval forms of a tapeworm are known as ________.
Answer
cysticerci
________ sign appears as swollen lymph nodes at the back of the neck in early African trypanosomiasis.
Answer
Winterbottom’s
________ African trypanosomiasis causes a chronic form of sleeping sickness.
Answer
West
The definitive host for Toxoplasma gondii is ________.
Answer
cats
Trypanosomes can evade the immune response through ________ variation.
Answer
antigenic
Short Answer
Why do nervous system infections by fungi require such long treatment times?
Briefly describe how humans are infected by Naegleria fowleri.
Briefly describe how humans can develop neurocysticercosis.
Critical Thinking
The graph shown tracks the body temperature of a patient infected with Trypanosoma brucei. How would you describe this pattern, and why does it occur?
(credit: modification of work by Wellcome Images)
Fungal meningoencephalitis is often the ultimate cause of death for AIDS patients. What factors make these infections more problematic than those of bacterial origin?
Compare East African trypanosomiasis with West African trypanosomiasis. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/26%3A_Nervous_System_Infections/26.E%3A_Nervous_System_Infections_%28Exercises%29.txt |
Learning Objectives
• Demonstrate awareness of the safety hazards present in the microbiology laboratory.
• Practice good laboratory safety.
Laboratory Safety Rules for Microbiology Lab Classes
There are many hazards associated with microbiology laboratories. Adherence to the following policies will help you, your classmates, and others who use the laboratory space to stay safe:
1. Do not eat, drink, store food, or smoke in the laboratory.
2. Decontaminate the workbenches at the beginning of laboratory.
3. Bring only necessary equipment to your work area.
4. Store personal belongings so no trip hazards are present in the laboratory.
5. Do not wear shirts with sleeves that hang down (fire hazard).
6. Tie or pull back long hair (fire hazard).
7. Do not carry flasks, test tubes, or bottles from their tops. Always carry from the base.
8. Never taste or smell anything in the laboratory unless your instructor has indicated that it is safe to do so.
9. Avoid touching your mouth and eyes during class unless you thoroughly wash your hands first.
10. Use hot gloves or appropriate glassware holders to handle hot glassware.
11. Report any spills or supply and equipment breakage to your instructor immediately.
12. Report any injuries to your instructor immediately.
13. Make sure all cuts are covered during laboratory.
14. Decontaminate the workbenches at the end of laboratory.
15. Thoroughly wash your hands at the end of class.
16. Follow all instructions for disposing of cultures and chemicals. Do not put any chemicals or cultures down the sink unless your instructor indicated it is safe to do so.
17. Cleanup all glassware, cultures, and supplies at the end of class. The classroom should look the same at the beginning and at the end of laboratory.
1.02: Media Preparation
Learning Objectives
• Define medium/media.
• Tell that microbes require nutrients to grow in the laboratory.
• Differentiate between broth, slant, deep, and petri plate.
• Calculate the amount of medium powder required to make a specific volume of medium.
• Successfully prepare microbiological media.
Microbiological Media
Just like all other living things, microbes require nutrients (food) to grow and live. Microbiological media (singular is "medium") is a mixture of water and nutrients necessary to grow microbes. Different types of media also can be used to provide information about the different characteristics microbes have.
Many types of microbiological media can be ordered in powder form from a science supplier. Instructions for preparing the medium are presented on the medium container. Typically, this involves dissolving a certain number of grams of the media powder in one liter of distilled or deionized water ("grams per liter" which is written as g/L). This does not mean that one must always make one liter of medium. The amount can be modified by calculating the amount of microbiological media powder required for the concentration (g/L) listed on the bottle)
Calculating Amount of Medium
Use the following formula to determine the amount of powdered medium to weigh and dissolve into DI or distilled water:
amount of solution you want to make (mL) x [concentration (g/1000 mL)] = amount of media powder to weigh out (g)
Because 1 L ("one liter") is the same thing as 1000 mL ("one thousand milliliters"), when the instructions on the media powder bottle tell to make a concentration in g/L ("grams per liter"), this is the same thing as g/1000 mL ("grams per thousand milliliters").
Calculation Example
Calculation
• In this example, you want to make 500 mL of medium. Instructions on the bottle say to make a concentration of 40 g/L.
• 40 g/L = 40 g/1000 mL
• Set up the formula:
• 500 mL x [40 g / 1000 mL]
• Calculate the amount to weigh out:
• Do the parentheses first: 500 mL x [40 g / 1000 mL]
• Multiply [note that mL units cancel out]: 500 mL x 0.04 g/mL
• Answer is the amount to weigh out in g ("grams"): 20 g
How to Make the Medium
1. Measure 500 mL of distilled or deionized water (not tap water) with a graduated cylinder.
2. Weigh 20 g of media powder using a balance.
3. Put a magnetic stir bar into a beaker, flask or bottle (must be larger capacity than 500 mL in for this example - 800 mL or 1000 mL) and add about half of the water.
4. Put the beaker, flask, or bottle onto a stir plate and turn it on so the magnetic stir bar is swirling the water enough to stir it well, but not too much that it is jumping and/or creating lots of bubbles in the water.
5. Gradually add the 20 g of media powder to the stirring water.
6. Add the reminder of the water. By adding the remaining water after the media powder, it will help dissolve any powder floating at the top of the water.
7. Allow to stir until the powder is completely dissolved.
8. If the medium is agar, the solution will need to be heated just to boiling (but not boiling over) starting after step 3. Keep stirring and heating until the solution is clear. Be careful not to over-heat since it is easy for the solution to boil over.
9. If the medium will be distributed into test tubes, measure amount into each test tube and add caps.
10. If the medium is for flasks or bottles, measure the amount into each flask or bottle and add caps or other covers (make sure screw-caps are not screwed on tightly before they are autoclaved).
11. If the medium is for petri plates, make sure the medium is in a container with a cover (make sure screw-caps are not screwed on tightly before they are autoclaved).
12. Autoclave.
13. If test tubes contain agar that will be for slants, prop the rack of test tubes so the agar slants as it cools.
14. If the medium is for petri plates, disinfect a workspace and pour agar into sterile petri plates and allow to cool completely.
Practice Calculations
Exercise 2.1
In order to make 400 mL of medium with a concentration of 15 g/L, how much medium powder would you weigh in grams?
Answer
Exercise 2.2
In order to make 650 mL of medium with a concentration of 20 g/L, how much medium powder would you weigh in grams?
Answer
Exercise 2.3
In order to make 200 mL of medium with a concentration of 30 g/L, how much medium powder would you weigh in grams?
Answer
Exercise 2.4
In order to make 300 mL of medium with a concentration of 45 g/L, how much medium powder would you weigh in grams?
Answer
Exercise 2.5
In order to make 150 mL of medium with a concentration of 35 g/L, how much medium powder would you weigh in grams?
Answer | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.01%3A_Laboratory_Safety.txt |
Learning Objectives
• Describe spontaneous generation theory and state that this is a theory that was once widely accepted and is an incorrect / false idea.
• Describe biogenesis theory and state that this theory is currently widely accepted.
• Apply the term "turbid" to microbiological cultures and the implications of turbidity.
• Describe Spallanzani's experiment that began to disprove spontaneous generation of microorganisms and why the scientific community did not reject spontaneous generation theory following his experiment.
• Interpret the meaning of Spallanzani's experimental results disproving spontaneous generation of microorganisms.
• Describe Pasteur's experiment that definitively disproved spontaneous generation of microorganisms and why the scientific community accepted the results of this experiment.
• Interpret the meaning of Pasteur's experimental results disproving spontaneous generation of microorganisms.
• Describe the achievements of Tyndall related to sterilization of media.
• Define endospore.
• Explain why endospores prevent sterilization of microbiological media by boiling only.
• Recreate and interpret Spallanzani's experiment disproving spontaneous generation of microorganisms.
• Recreate and interpret Pasteur's experiment disproving spontaneous generation of microorganisms.
The Myth of Spontaneous Generation
Spontaneous generation theory is a myth. It is a false idea that was once a widely accepted belief in the scientific community. Its ideas were so strong that it took multiple experiments by several different scientists to disprove the theory and start to shift the mindset about how living things arise.
Spontaneous generation theory says living things can arise or come from non-living things. In the microbial world, it was believed the microorganisms could be created from a fluid such as a beef broth. This is because a beef broth left out would inevitably begin growing microorganisms that scientists observed using their microscopes. These microbes did not come from the broth however as the scientists of the time believed. In reality, the microorganisms were either in the broth already (in small amounts) or landed in the broth from the air. Once even one cell was present in the beef broth, the broth could be used by the cells as a source of nutrients they used to be able to grow, divide, and produce a robust population of microorganisms that could then be seen with the microscope.
Instead of the theory of Spontaneous Generation, today the theory of biogenesis is currently accepted by the scientific community. This theory says that life comes from life. In other words, living things do not arise from non-living matter. A related theory that is also currently accepted is Cell Theory and it says that cells come from other cells (among other things).
Historical Experiments Disproving Spontaneous Generation of Microorganisms
Spallanzani's Experiment Disproving Spontaneous Generation Theory of Microorganisms
Lazzaro Spallanzani was an Italian priest who re-examined the spontaneous generation of microorganisms (e.g. bacteria) using a nutrient-rich broth such as a meat broth. He designed and conducted a famous experiment that began to question the validity of spontaneous generation theory.
A nutrient broth that is not sterile will eventually show microbial growth by becoming cloudy unless it is sterilized and microbes are prevented from entering the broth (usually by sealing the container). Cells present in the broth will use the broth as nutrients, divide, and produce a robust population of microorganisms. The broth can be examined using the microscope so that microorganisms growing there can be observed. A broth that begins clear that contains one or more cells (or is exposed to the environment where cells can enter the broth through the air), given time for microbial growth, will become turbid (cloudy or thick).
Spallanzani boiled nutrient broth to kill the microorganisms (sterilization). He compared covered and uncovered boiled nutrient broths to see if the broths would become turbid (cloudy), indicating microbial growth. What Spallanzani observed was the uncovered boiled broth became turbid over time since microorganisms were able to enter the broth from the air. The covered boiled broth however did not become turbid since microorganisms could not enter the sterile broth. This result indicated microorganisms were in the air and would contaminate a broth if exposed.
The scientific community, having believed that spontaneous generation was a fact of nature, still resisted the possibility that spontaneous generation is not possible for microorganisms. The argument was that the sealed flask in Spallanzani's experiment was closed off from an oxygen (O2). Perhaps microorganisms could not grow in the broth because there was not enough O2 present in the flask? As a result, spontaneous generation theory persisted.
Pasteur's Swan-Neck Experiment Disproving Spontaneous Generation Theory of Microorganisms
Louis Pasteur designed and conducted an experiment that provided strong evidence disproving spontaneous generation of microorganisms. Pasteur used a flask with a swan-neck that could prevent microbes, including those attached to dust particles in the air, from reaching the sterile nutrient broth while enabling O2 to pass into the flask. Since Spallanzani's experiment was scrutinized because O2 could not reach the nutrient broth. The fact that Pasteur developed a flask that allowed O2 to the broth and not microbes in the air created the conditions the scientific community needed to accept that spontaneous generation of microorganisms does not occur (microbes do not come from non-living matter).
In Pasteur's experiment, a nutrient broth is sterilized in a flask with a swan-neck tube attached. The swan-neck enabled O2 to reach the broth. The low dip in the swan neck traps dust and microbes and prevents them from reaching the broth. After sterilization, the flask with the swan-neck stayed sterile indefinitely, despite being open to the air. This design effectively trapped microbe-carrying dust particles that could contaminate a sterile growth medium (i.e. introduce microorganisms to the sterile growth medium). If the swan-neck tube was broken off, the result is a direct path for microbes in the air and attached to dust particles to enter the growth medium. Given time, a sterilized flask with the swan-neck broken off became turbid (cloudy), indicating microbial growth. These results indicated that microorganisms come from other microorganisms and that microorganisms do not come from non-living broth. This result conclusively settled the dispute about spontaneous generation: spontaneous generation theory was incorrect and biogenesis theory is correct.
Tyndall's Discovery and Endospores
Pasteur’s swan-necked experiment was then challenged when John Tyndall, an Irish scientist, repeated Pasteur’s experiment and found some boiled growth media remained sterile while others did not, despite very long boiling times. Through a series of experiments, Tyndall found that nutrient broth can contain heat-resistant microbes (now known as endospores – discovered by Ferdinand Cohn the same year as Tyndall’s work). Endospores can be produced from certain bacterial species (e.g. Bacillus sp. and Clostridium sp.) when growth/nutrient conditions are poor. Endospores are analagous to a survival bunker where bacterial cells transfer their DNA into a protective structure to survive the harsh conditions. Endospores can change back into normal bacterial cells (called vegetative cells [these are active bacterial cells whereas endospores are an inactive form]) if/when environmental conditions improve and are better for the microbes to grow.
Tyndall developed a process that he found completely sterilized growth media, including killing those containing heat-resistant microbes (endospores). This process is a lengthy series of steps involving repeatedly heating then resting the medium multiple times. This repeated heating and resting process insures sterilization of a medium.
More recently, it has been found endospores can also be killed when medium is heated under pressure. The typical method of sterilization used in laboratories today uses an autoclave, a device that places materials to be sterilized under pressure while heating. What used to take a few days to sterilize medium with Tyndall's process now can be sterilized in about an hour.
Re-creation of Lazzaro Spallanzani’s Spontaneous Generation Experiment
In this laboratory activity you will re-create Spallanzani's experiment. debunk the myth of spontaneous generation... again!
Laboratory Instructions
1. Write your group name, experiment name, and the date on two pieces of tape and place on the outside of two test tubes (put tape about half way down on the test tube so you can add a cap onto one of the test tubes).
2. Use a graduated cylinder to measure TSB.
3. Put 8 mL of TSB into each of the two test tubes.
4. Put a cap onto one of the test tubes and leave the other test tube uncapped.
5. Put test tubes in a central location indicated by your instructor. Your test tubes will be autoclaved by your instructor to sterilize them (kill all microbes) and then placed onto a bench.
6. Create a hypothesis with your group:
1. Do you expect that the covered, sterilized test tube will show microbial growth? Why or why not?
2. Do you expect that the uncovered, sterilized test tube will show microbial growth? Why or why not?
3. If you said that microbial growth will occur in one of the test tubes, where did the microbes come from?
7. You will examine your results next class. If the solution is turbid (foggy), that indicates microbial growth. If the solution is clear, that indicates no microbial growth.
8. Results:
1. Covered, sterilized tube:
2. Uncovered, sterilized tube:
9. Conclusions. What do the results you listed above mean about spontaneous generation? Consider your hypotheses above to help you answer.
Re-creation of Louis Pasteur’s Spontaneous Generation Experiment
In this laboratory activity you will re-create Pasteur's experiment. Debunk the myth of spontaneous generation... again!
Laboratory Instructions
1. Write your group name, experiment name, and the date on two pieces of tape and place on the outside of two 125 mL flasks.
2. Use a graduated cylinder to measure TSB.
3. Add 50 mL of TSB to each of the 125 mL flasks.
4. You may or may not be asked to bend glass tubing into a swan-neck shape using a Bunsen burner.
5. Put one swan-neck glass tube into one stopper and a straight glass tube into another stopper.
6. Place the two stoppers securely into the 125 mL flasks.
7. Put the flasks in a central location indicated by your instructor. Your flasks will be autoclaved by your instructor to sterilize them (kill all microbes) and then placed onto a bench.
8. Create a hypothesis with your group:
1. Do you expect that the sterilized flask with the swan-neck glass tube will show microbial growth? Why or why not?
2. Do you expect that the sterilized flask with the straight glass tube will show microbial growth? Why or why not?
3. If you said that microbial growth will occur in one of the test tubes, where did the microbes come from?
4. Why were scientists more convinced that spontaneous generation was incorrect by Pasteur's experiment and less convinced by Spallanzani's experiment?
9. You will examine your results next class. If the solution is turbid (foggy), that indicates microbial growth. If the solution is clear, that indicates no microbial growth.
10. Results:
1. Sterilized flask with swan-neck tube:
2. Sterilized flask with straight tube:
11. Conclusions. What do the results you listed above mean about spontaneous generation? Consider your hypotheses above to help you answer. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.03%3A_The_Myth_of_Spontaneous_Generation.txt |
Learning Objectives
• Tell the importance of using microscopes in microbiology.
• Successfully use the metric system for length including unit conversion calculations.
• Define resolution.
• Define magnification.
• Determine/calculate total magnification for each objective lens.
• Identify the structures of a light microscope.
• Identify the functions of the structures of a light microscope.
• Successfully use and care for a light microscope.
• Differentiate between light microscopy and electron microscopy.
• Differentiate between TEM and SEM and the appropriate uses for each.
Early Microscopy
The first microscope was developed in 1590 by Dutch lens grinders Hans and Zacharias Jansen. In 1667, Robert Hooke described the microscopic appearance of cork and used the term cell to describe the compartments he observed. Anton van Leeuwenhoek was the first person to observe living cells under the microscope in 1675—he described many types of cells, including bacteria. Since then more sophisticated and powerful scopes have been developed that allow for higher magnification and clearer images.
Microscopy is used by scientists and health care professionals for many purposes, including diagnosis of infectious diseases, identification of microorganisms (microscopic organisms) in environmental samples (including food and water), and determination of the effect of pathogenic (disease-causing) microbes on human cells. This exercise will familiarize you with the microscopes we will be using to look at various types of microorganisms throughout the semester.
Metric Units & Microscopy
Unlike typical length measurement units used in the United States (inches, feet, yards, miles), microscopy uses the metric system (nanometers, micrometers, millimeters). Scientific measurements use the metric system. The metric system is also used for measurement in every country in the world except in three countries: Liberia, Myanmar, and The United States. Although these measurements are likely new for you, they are very user-friendly since the metric system uses units that are all related to the meter (the base unit) and are different from each other by powers of 10.
Metric Units for Length
Since microscopes are concerned with the size (length) of structures and the magnification of these structures, we will consider the metric system as it relates to length. The base unit of length in the metric system is the meter (abbreviated as m). One meter is about 3.3 feet long. For units that are larger or smaller than meters, a prefix is added to the beginning of "meter" to indicate the magnitude of difference from the meter unit (base unit):
Unit of Length
Abbreviation
Size in relationship to a meter (meter is the base unit)
kilometer
km
1,000 or 103
hectometer
hm
100 or 102
decameter
dam
10 or 101
meter (base unit)
m
1 or 100
decimeter
dm
0.1 or 10-1
centimeter
cm
0.01 or 10-2
millimeter
mm
0.001 or 10-3
micrometer
µm
0.000001 or 10-6
nanometer
nm
0.000000001 or 10-9
picometer
pm
0.000000000001 or 10-12
Sizes of Cells & Microbes
Cells are typically measured using the micrometer (µm) unit, but some subcellular structures may be reported in nanometers (nm) measurements. To give you a sense of cell size, a typical human red blood cell is about eight millionths of a meter or eight micrometers (abbreviated as 8 μm) in diameter; the head of a pin of is about two thousandths of a meter (two mm) in diameter. That means about 250 red blood cells could fit on the head of a pin!
Use this interactive tool to get a sense of how big microbes are in comparison to some familiar objects.*
*microbes in this interactive tool include: amoeba proteus, paramecium, baker's yeast, E. coli bacterium, measles virus, hiv, phage, influenza virus, hepatitis virus, and rhinovirus
Converting Metric Length Units
Converting Units to Meters (the Base Unit)
Using the table above that distinguishes units by prefix, to convert unit measurements back to the base unit, simply multiply the number you are converting with the number in the "size relationship to a meter" column. For example:
Using your microscope you measure a cell as 20 µm ("twenty micrometers"). To convert 20 µm to meters, multiply 20 µm by 0.000001:
20 µm x 0.000001 = 0.00002 m
or
20 µm x 10-6 = 0.00002 m
You measure the distance it takes you to get home from school as 19 km ("nineteen kilometers"). To convert 19 km to meters, multiply 19 km by 1,000:
19 km x 1,000 = 19,000 m
or
19 km x 103 = 19,000 m
Exercise 4.1
Convert 2,500 µm to meters.
Answer
Exercise 4.2
Convert 12,000 nm to meters.
Answer
Exercise 4.3
Convert 35 mm to meters.
Answer
Converting Meters (the Base Unit) to Other Units
Using the table above that distinguishes units by prefix, to convert the base unit to another unit, simply divide the number you are converting with the number in the "size relationship to a meter" column. For example:
You are told that a certain very large cell is 0.0003 m long. To convert 0.0003 m to micrometers (µm), divide 0.0003 m by 0.000001:
0.0003 m ÷ 0.000001 = 300 µm
or
0.0003 m ÷ 10-6 = 300 µm
You read that running a marathon means running 42,000 m. To convert 42,000 m to kilometers (km), divide 42,000 m by 1,000:
42,000 m ÷ 1,000 = 42 km
or
42,000 m ÷ 103 = 42 km
Exercise 4.4
Convert 0.004 m to µm.
Answer
Exercise 4.5
Convert 0.3 m to mm.
Answer
Exercise 4.6
Convert 0.00000085 m to nm.
Answer
Light Microscopes vs. Electron Microscopes
Most microscopes used in college biology laboratories are classified as light microscopes (see the figure, part (a) below) and may also be called compound microscopes since they use two lenses whose magnifications compound (multiply). Visible light passes through the specimen and is bent through the lens system to enable the user to see the specimen. Light microscopes are advantageous since they are capable of viewing living organisms. However, since individual cells are generally transparent, their components are not distinguishable unless they are colored with special stains (colored chemicals that makes cells appear to have color such as pink, blue, or purple). Stained cells are dead cells, but staining is an important approach to better see cells and cell structures.
Due to the magnification limits of light microscopes, in order to gain a better understanding of cellular structure and function, scientists typically use electron microscopes that use electrons (subatomic particles) to carry the microscopic images instead of using light particles (photons). Electron microscopes also typically require staining the sample with heavy metals to be able to visualize the sample.
To see the difference in magnification and detail possible when comparing light microscopes and electron microscopes, compare the images of Salmonella (a type of bacteria) in the figure below.
The Light Microscope
What does it mean to be microscopic? Objects are said to be microscopic when they are too small to be seen with the unaided eye—they need to be magnified (enlarged) for the human eye to be able to see them. This includes human cells and many other types of cells that you will be studying in this class. The microscope you will be using uses visible light and two sets of lenses to produce a magnified image. The total magnification will depend on which objective lens you are using—the highest magnification possible on these microscopes is typically 1000X—meaning that objects appear 1000X larger than they actually are.
Resolution vs. Magnification
Magnification refers to the process of making an object appear larger than it is; whereas resolution is the ability to see objects clearly enough to tell two distinct objects apart. Although it is possible to magnify above 1000X, a higher magnification would result in a blurry image. (Think about magnifying a digital photograph beyond the point where you can see the image clearly). This is due to the limitations of visible light (details that are smaller than the wavelength of light used cannot be resolved).
The limit of resolution of the human eye is about 0.1 mm ("zero point one millimeters"), or 100 µm ("one-hundred micrometers"). Objects that are smaller than this cannot be seen clearly without magnification. Since most cells are much smaller than 100 µm, we need to use microscopes to see them.
The limit of resolution of a standard brightfield light microscope, also called the resolving power, is ~0.2 µm, or 200 nm. Biologists typically use microscopes to view all types of cells, including plant cells, animal cells, protozoa, algae, fungi, and bacteria. The nucleus and chloroplasts of eukaryotic cells can also be seen—however smaller organelles and viruses are beyond the limit of resolution of the light microscope.
Resolution is the ability of the lenses to distinguish between two adjacent objects as distinct and separate.
A compound light microscope has a maximum resolution of 0.2 µm, this means it can distinguish between two points ≥ 0.2 µm, any objects closer than 0.2um will be seen as 1 object. Shorter wavelengths of light provide greater resolution. This is why we often have a blue filter over our light source in the microscope, it helps to increase resolution since its wavelength is the shortest in the visible light spectrum. Without resolution, no matter how much the image is magnified, the amount of observable detail is fixed, and regardless of how much you increase the size of the image, no more detail can be seen. At this point, you will have reached the limit of resolution or the resolving power of the lens. This property of the lens is fixed by the design and construction of the lens. To change the resolution, a different lens is often the only answer.
Parts of a Light Microscope
The microscope is one of the microbiologist's most important tools. It allows for the visualization of small particles, including microbes, which are too small to be seen with the human eye. With the help of proper illumination, a microscope can magnify a specimen and optically resolve fine detail. This introduction to microscopy will include an explanation of features and adjustments of a compound brightfield light microscope, which magnifies images using a two lens system.
These microscopes combine an ocular lens (located in the eyepiece) and an objective lens (located above the stage and attached to a revolving nosepiece). Objective lenses can be selected based on the amount of magnification needed. Objective lenses are changed by turning the revolving nosepiece. The ocular lens remains constant and is not changed during normal microscope operation.
To view a sample placed on the microscope stage, the course focus knob and the fine focus knob are used to move the stage up and down. This will enable focusing on the sample.
Microscope Part
Description and Function of Microscope Part
eyepiece
location for looking into the microscope; holds ocular lenses (lenses magnify the image; typically have 10X magnification)
revolving nosepiece
contains the objective lenses; the nosepiece turns to change the objective lenses (changes the magnification)
objective lenses
mounted in the revolving nosepiece; typically microscopes have four objective lenses: 4X, 10X, 40X and 100X
stage clips
holds the microscope slide in place; located on the stage
stage
a platform located below the objectives where the slide is placed
stage controls
knobs that move the slide around the stage by moving the stage clips to position the specimen in the desired position
mechanical stage
the mechanism that moves the stage clips around the stage to move the slide; the stage control knobs control the mechanical stage
diaphragm
located beneath the condenser; regulates the amount of light by opening or closing its aperture
light source/ illuminator
a light source; usually a bulb built in the base of the microscope; there may be a dial to regulate the intensity of light depending on the model of microscope
condenser
a system that focuses the light coming up from the illuminator onto the specimen on the slide
arm
a structure of the microscope that connects the base to the head; it may be straight or curved depending on the model of microscope
base
the wide bottom of the microscope; supports the microscope on a bench or table
coarse focus
large knob located on both sides of the microscope below the stage; focuses the image by moving the stage rapidly; should only be used when using the 4X or 10X objective lenses; using the coarse focus with the 40X or 100X objective lenses could damage the lens or crack the slide
fine focus
small knob located on both sides of the microscope below the stage; focuses the image by moving the stage in small increments; used most with the 40X objective lens and 100X objective lens but can be used to fine-tune focus focus at lower objective lenses (4X and 10X)
Tips for Success Using a Light Microscope
1. Center the sample over the light shining up through the stage.
2. Begin with the lowest magnification objective lens in place (usually the 4X objective lens). Make sure it clicks into place over the sample and the light shining up through the stage.
3. Turn the course focus to bring the stage up as close as it can go to the objective lens. At this point, you should be close to being in focus.
4. Look into the eyepiece and slowly turn the course focus until you can see the sample. Many samples have been stained to have color (usually you can look for color - often pink, purple, or blue - you may be able to see the color on the slide with your naked eye).
5. Increase the objective lens magnification one step at a time. Focus the microscope each step (with course focus at 4X and 10X objective lenses and with fine focus at 40X and 100X objective lenses).
6. If you lose the sample, return back to the lowest magnification objective. This may seem like you are wasting time, but in reality it is the fastest way to find your sample again.
Detailed Instructions for Using a Light Microscope
Learning microscopy is very important in microbiology to examine the microscopic organisms that we are studying (bacteria, protozoa, fungi, etc.). Here is how we work with these microscopes:
Carrying a Compound Light Microscope
To take the microscope out of the cabinet or off of the cart using the following instructions:
1. Use your dominant hand to hold the arm of the microscope, and your non-dominant hand to hold the base of the microscope.
2. Carry the microscope upright. That way the ocular lenses located in the eyepiece do not fall out.
3. Make sure the electrical cord is wrapped or removed from the microscope, avoid tripping on the cord.
To return the microscope to the cabinet or to the cart use the following instructions:
1. Turn off the microscope.
2. Turn the revolving nosepiece to have lowest magnification objective, usually the 4X objective lens.
3. Disconnect from power source.
4. Wrap cord around the microscope.
5. If you used oil immersion, (the 100X objective), use lens paper only to remove any remnant of immersion oil on the 100X objective lens.
6. Place the microscope back in the cabinet or on the cart using the transport instructions above.
Focusing a Compound Light Microscope
1. Place the microscope on a table in front of you and plug it in such that the cord does not create a trip hazard for you or others in the laboratory.
2. Turn the revolving nosepiece to have lowest magnification objective in place, usually 4X. The objective should click when properly in place.
3. Place the slide on the stage of the microscope and properly position the stage clips such that the clips "hug" the slide on either side and are not on top of the slide since this could crack the slide (NOTE: there are some models of microscopes where stage clips do sit on top of the slide - ask your instructor about proper use of the stage clips for the model of microscope you are using).
4. Turn on the microscope and confirm that the light is on.
5. Move the stage control knobs to position the specimen on the slide directly over the path of the light.
6. With the 4X objective in place, turn the course focus knob to bring the stage as close to the objective lens as possible.
7. Look through the microscope and use the course focus knob to focus on the sample. Make sure the slide is in very sharp focus before moving on. Check with your instructor if you are uncertain.
8. To focus on the specimen using higher magnification than with the 4X objective, use the next steps:
9. With the specimen focused with the 4X objective, re-center the specimen using the stage adjustment knobs. DO NOT CHANGE THE FOCUS!
10. Change to the next highest objective lens without changing any other settings on the microscope.
11. Use ONLY the fine focus knob to modify the focus. The specimen should mostly be in focus and only minor adjustments should be necessary. Continue focusing until the image is sharp and clear.
12. Re-center the specimen using the stage adjustment knobs. DO NOT CHANGE THE FOCUS!
13. If moving to the next highest objective lens, change to the objective lens to the next highest without changing any other settings on the microscope.
14. Use ONLY the fine focus knob to adjust the focus. Continue focusing until the image is sharp and clear.
**If you get lost on your sample, go back to 40X magnification (objective says ‘4X’), re-focus, and follow the steps above again.**
Magnification
The optical system of a compound microscope consists of two lens systems: one found in the objective(s) lens(es) (Fig. 2, part 3); the other in the ocular (eyepiece) (Fig. 2 part 1). The objective lens system is found attached to a rotating nosepiece (Fig. 2, part 2). A microscope usually has three or four objectives that differ in their magnification and resolving power. Magnification is the apparent increase in size of an object. Resolving power is the term used to indicate the ability to distinguish two objects as separate. The most familiar example of resolving power is that of car headlights at night: at a long distance away, the headlights appear as one light; as the car approaches, the light becomes oblong, then barbell-shaped, and finally it becomes resolved into two separate lights. Both resolution and magnification are necessary in microscopy in order to give an apparently larger, finely detailed object to view.
Look at the engravings on the objective lenses and note both the magnification (for example: 10X, 40X, 100X) and the resolution given as N.A. = numerical aperture, from which the limit of resolution can be calculated:
microscope resolution limit = (wavelength)/(numerical aperture x 2)
At a wavelength of 550 nm (0.55 µm), the 100X objective lens with a N.A. of 1.25 has a resolving power of 0.22 µm. Visible light has of wavelength from about 400-750 nanometers (nm). Since the limit of resolution decreases at the shorter wavelengths, microscopes are usually fitted with a blue filter. The resolving power of the lens separates the details of the specimen, and the magnification increases the apparent size of these details so that they are visible to the human eye. Without both resolution and magnification, you would either see nothing (good resolution, no magnification) or a big blur (poor resolution, good magnification).
The objective lens system produces an image of the specimen, which is then further magnified by the ocular lens (eyepiece). The magnification of this lens is engraved on the ocular. The total magnification of the microscope is determined by the combination of the magnification of the objective lens and ocular lens that is in use, that is:
total magnification = (objective lens magnification) x (ocular lens/eyepiece magnification)
For example, with a 10X objective lens and a 10X ocular, the total magnification of the microscope is 100X. If the objective lens is changed to a 20X objective, then the total magnification is now 200X, whereas if a 10X objective is used with a 12.5X ocular lens, the total magnification is now 125X. When viewing a sample, magnifications are reported as the total magnification. Therefore, if making an illustration or taking a photograph of a magnified sample, report the magnified view as the total magnification (not as the magnification of the objective lens). Likewise, if you are examining a magnified image taken by another person, the magnification they report is the total magnification (not the objective lens). When reading laboratory instructions for viewing a sample with the microscope, total magnification will be given (not the objective lens).
The use of objective and ocular lenses with different magnifications allows greater flexibility when using the compound microscope. Due to the size of most bacteria (ranges widely from ~1 µm to over 100 µm), generally we require the use of the 100X oil immersion lens with a 10X ocular lense to view bacteria in a standard brightfield light microscope.
Sample Illumination
The objective lens and ocular lens systems can only perform well under optimal illumination conditions. To achieve these conditions, the light from the light source (bulb) must be centered on the specimen. The parallel light rays from the light source are focused on the specimen by the condenser lens system. The condenser can move up and down to affect this focus. Finally, the amount of light entering the condenser lens system is adjusted using the condenser diaphragm. It is critical that the amount of light be appropriate for the size of the objective lens receiving the light. This is important to give sufficient light, while minimizing glare from stray light, which could otherwise reduce image detail. The higher the magnification and resolving power of the lens, the more light is needed to view the specimen.
Oil Immersion
Objective lenses used for observing very small objects such as bacteria are almost always oil immersion lenses. With an oil immersion lens, a drop of oil is placed between the specimen and the objective lens so that the image light passes through the oil. Without the oil, light passing through the glass microscope slide and specimen would be refracted (bent) when it entered the air between the slide and the objective lens. This refracted light might still be able to contribute to the image of the specimen if the objective lens is large. However, at the higher magnification, the objective lens is small, so is unable to capture this light. The loss of this light leads to loss of image detail. Therefore, at higher magnifications, the area between the slide and the lens is modified to have the same (or nearly the same) refracting qualities (refractive index) as the glass and specimen by the addition of immersion oil. Watch the video below and read this article for more information about oil immersion.
To use an oil immersion lens, place a drop of oil on top of the dried specimen on the slide and carefully focus the microscope so that the objective lens is immersed in the oil. Any lens, which requires oil, is marked "oil" or "oil immersion." Conversely, any lens not marked "oil" should not be used with oil and is generally not sealed against oil seeping into and ruining the objective.
Electron Microscopes (EM)
In contrast to light microscopes, electron microscopes use a beam of electrons instead of a beam of light. Not only does this allow for higher magnification and, thus, more detail, it also provides higher resolving power. The method used to prepare the specimen for viewing with an electron microscope kills the specimen. Electrons have short wavelengths (shorter than photons of light) that move best in a vacuum, so living cells cannot be viewed with an electron microscope.
The best type of EM to examine a cell's internal structures such as vesicles, storage granules, ribosomes, mitochondria, and nuclei is transmission electron microscopy (TEM) since it is designed to allow electrons to pass through a thin sample. Samples must be prepared for TEM by embedding the sample, such as a cell, in a tiny plastic block, and using either a glass knife or a diamond knife to thin-section the sample using a microtome (laboratory equipment that makes thin sections). As a result, thin slices of cells can be observed with TEM, exposing intracellular details. An example of a thin-sectioned skin cell magnified by TEM is shown in the figure, part (a) below.
Another common type of EM is scanning electron microscopy (SEM). SEM works much differently than TEM since SEM radiates electrons onto a sample’s surface and these electrons are reflected back by the sample to elucidate the surface details of the sample. As a result, SEM is an excellent technique for examining the three-dimensional surface of an un-sectioned specimen and viewing only the external surfaces of cells and tissues. An example of an SEM image of skin cells with yeast infecting them is shown in the figure, part (b), below. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.04%3A_Microscopy.txt |
Learning Objectives
• Determine/calculate total magnification for each objective lens.
• Identify the structures of a light microscope.
• Identify the functions of the structures of a light microscope.
• Successfully use and care for a light microscope.
• Successfully examine different classifications of microbes using a light microscope.
• Illustrate different classifications of microbes.
• Compare the appearances of different classifications of microbes that were examined and illustrated using a light microscope.
Introduction
Learning to use a microscope is critical to your success in microbiology. This tool will provide the magnification and clarity necessary for you to observe and record quality images of microbes that cannot ordinarily be viewed with the naked eye.
Total Magnification
To better understand the compound light microscope (brightfield), it is important to have an understanding of magnification. Commonly, compound microscopes have 4 different objective lenses:
• 4X
• 10X
• 40X
• 100X
The object lens magnification is increased by the ocular lenses (located in the eyepieces), which has a 10X magnification. To calculate the total magnification at each objective, multiple the ocular lens magnification with the objective lens magnification.
Exercise 5.1
When the 4X objective lens is used, what is the total magnification of the light microscope?
Answer
Exercise 5.2
When the 10X objective lens is used, what is the total magnification of the light microscope?
Answer
Exercise 5.3
When the 40X objective lens is used, what is the total magnification of the light microscope?
Answer
Exercise 5.4
When the 100X objective lens is used, what is the total magnification of the light microscope?
Answer
Whenever you make an illustration or take a photo of a magnified specimen, the total magnification used should be given with the image. You can expect when you examine microscopic images that the magnification of the image is given as the total magnification (not the objective lens magnification).
Identify Parts of the Light Microscope
Exercise 5.5
Name the components of a compound microscope labeled A-L.
Answers:
A
B
C
D
E
F
G
H
I
J
K
L
Match the Functions of the Parts of the Light Microscope
Exercise 5.6
Match the parts of the light microscope with their functions.
ocular lens A. Adjusts the position of a slide on the stage.
revolving nosepiece B. Adjusts the focus of the microscope by moving the stage up and down in large increments.
arm C. A surface where a slide is positioned under the objective lens and over the light source.
stage control D. A structure capable of rotating to change the objective lens being used to magnify the sample.
base E. A magnifying lens located inside the microscope part where a person looks into the microscope.
coarse focus F. Provides light that shines on the sample and carries the image of the specimen through the magnifying lenses.
fine focus G. A structure capable of changing the amount of light passing through the specimen.
light source / illuminator H. A structural component that serves to support the weight of the microscope from underneath.
objective lens I. Secures a slide in place on the microscope.
stage J. Where the microscope user looks into the microscope to view a magnified image of the specimen.
stage clip K. Adjusts the focus of the microscope by moving the stage up and down in smaller increments.
diaphragm L. A structural component that serves to support the eyepiece, revolving nosepiece, and stage.
eyepiece M. A magnifying lens which increases magnification of the specimen that can easily be changed by rotating between lenses of different magnifications.
Answers:
ocular lens
revolving nosepiece
arm
stage control
base
coarse focus
fine focus
light source / illuminator
objective lens
stage
stage clip
diaphragm
eyepiece
Examine Specimen with Low Magnification
Place The letter ‘e’ slide on the microscope stage and focus the slide using the 4x objective.
1. Draw the ‘e’ as you see it on the slide with the naked eye (not looking through the microscope).
2. Look through the microscope and now draw the ‘e’ as you see.
3. In addition to being able to see the 'e' larger and with more detail, what else about the image of the 'e' has changed when comparing your illustration from 1. and from 2.?
Examine Microbes at Higher Magnifications
"Microbes" include viruses, bacteria, archaea, fungi, protozoa, and helminths. Viruses are too small to see with the light microscope and must be imaged with an electron microscope (not used in class). Bacteria and archaea appear as tiny specks at the highest magnifications with a light microscope, protozoa and fungi and their details can be clearly imaged with a light microscope, and helminths (worms) can, depending on the species, be too long to see the entire worm at once with the light microscope.
Today, you will examine an example of a helminth, a fungus, protozoa, and bacteria. Choose the magnification the enables you to view the microbe well and clearly. Remember the relative sizes of these microbes. Smaller microbes will require higher magnifications.
Helminth
Examine an example of a species of helminth. Illustrate the magnified sample in detail, write down the name of the specimen, and indicate the total magnification you made the illustration at. The goal is to distinguish the different categories of microbes based on your illustrations.
Fungus
Examine an example of a species of fungus. Illustrate the magnified sample in detail, write down the name of the specimen, and indicate the total magnification you made the illustration at. The goal is to distinguish the different categories of microbes based on your illustrations.
Protozoa
Examine an example of a species of protozoa. Illustrate the magnified sample in detail, write down the name of the specimen, and indicate the total magnification you made the illustration at. The goal is to distinguish the different categories of microbes based on your illustrations.
Bacteria
Examine an example of a species of bacteria. Illustrate the magnified sample in detail, write down the name of the specimen, and indicate the total magnification you made the illustration at. The goal is to distinguish the different categories of microbes based on your illustrations.
Compare and Contrast Different Types of Microbes
1. What were some notable similarities among the different microbe classifications examined with the light microscope?
2. What were some notable differences among the different microbe classifications examined with the light microscope?
3. Do all microbes have the same structure? Explain your answer. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.05%3A_Get_to_Know_the_Microscope_and_Microbes.txt |
Learning Objectives
• Identify that all matter is composed of atoms and that atoms build molecules.
• Define organic molecules and inorganic molecules and be able to identify whether a molecule is organic or inorganic based on its chemical structure.
• List the four types of biological molecules, their monomers, and their functions.
• Define dehydration synthesis and hydrolysis and describe the importance of these reactions for biological molecules.
• Describe a burn test and interpret results of a burn test.
• Successfully build molecular molecules biological molecule monomers and conduct dehydration synthesis and hydrolysis reactions with these models.
• Test food items for a significant presence of proteins, carbohydrates, and lipids.
Atoms & Molecules
All matter (anything with physical substance) is made atoms; this includes all living things from microorganisms to humans to blue whales. Atoms, composed of protons, neutrons, and electrons, are the smallest structures that makes up elements of the periodic table. Therefore, the periodic table lists different types of atoms (also known as elements). Each type of element is different because the number of protons it has (the number of protons is the same as the atomic number).
Use this interactive tool to get a sense of how big microbes are in comparison to some familiar objects.*
*microbes in this interactive tool include: amoeba proteus, paramecium, baker's yeast, E. coli bacterium, measles virus, hiv, phage, influenza virus, hepatitis virus, and rhinovirus
Organic vs. Inorganic Molecules
All molecules can be classified as either organic or inorganic. Organic molecules are types of molecules typically found in living things and molecules that came from living things. In contrast, inorganic molecules are considered to be molecules not produced by living things. While organic molecules are considered the molecules of life/biological molecules, inorganic molecules can still be necessary for life. For example, water (H2O) is a molecule that is required by all living things, yet it is considered inorganic. A straight-forward way to tell determine if a molecule is organic or inorganic is based on the types of elements it has:
• organic molecules always contain carbon atoms (C) and hydrogen atoms (H).
• inorganic molecules can contain carbon atoms (C) and can contain hydrogen atoms (H), but inorganic molecules will not contain both carbon atoms (C) and hydrogen atoms (H).
Note
Inorganic molecules usually do not contain carbon atoms (C). Also, organic molecules are formed only by covalent bonds, whereas inorganic molecules can be formed by covalent bonds or ionic bonds.
Exercise 6.1
Below are empirical formulas for a variety of different molecules. Based on the elements found in each molecule, identify whether each is an organic molecule or an inorganic molecule:
Click each above for the answer.
What if we have a sample of a substance (and perhaps do not know the elements it contains) and would like to determine if the substance is organic or inorganic?
A simple laboratory test that can determine if a sample is organic or inorganic is called the burn test. This test is very simple: place the substance over heat - if it burns/turns black, it is likely organic. If it does not burn/does not turn black, it is likely inorganic.
Molecules that Make up Living Things
Biological molecules are classified into four types:
• proteins
• carbohydrates
• lipids
• nucleic acids
Each category of biological molecule has distinct features, including their building blocks and the types of functions they can serve. Biological molecules tend to be very large and are built of repeating units of smaller molecules. These small subunits that make up larger biological molecules are called monomers. The monomers of proteins are called amino acids, the monomers of carbohydrates are called monosaccharides, the monomers of lipids are called fatty acids, and the monomers of nucleic acids are called nucleotides.
Monomers are joined together by covalent bonds through dehydration synthesis reactions to form larger biological molecules made of multiple monomers. The number of monomers bonded together is apparent in the generalized naming scheme for biological molecules:
number of monomers bonded together commonly used prefix protein name carbohydrate name lipid name nucleic acid name
1
(no bonds between monomers)
mono- amino acid monosaccharide fatty acid nucleotide
2 di- dipeptide disaccharide diglyceride dinucleotide
3 tri- tripeptide trisaccharide triglyceride trinucleotide
"a few" (2, 3, or 4) oligo- oligopeptide oligosaccharide n/a oligonucleotide
many
(could be hundreds or more)
poly- polypeptide polysaccharide n/a polynucleotide
Note
Lipids are limited to triglycerides and cannot get bigger. This is due to the glycerol structure fatty acids bond with that can only attach to three fatty acids. There are other types of lipids that do not fit this model, such as steroid molecules like cholesterol.
Polymers and other monomers bonded together can also be broken down into smaller pieces by breaking covalent bonds through hydrolysis reactions.
Proteins
Proteins are one of the most abundant organic molecules in living systems and have the most diverse range of functions of all macromolecules. Proteins may be structural, regulatory, contractile, or protective; they may serve in transport, storage, or membranes; or they may be toxins or enzymes. Each cell in a living system may contain thousands of proteins, each with a unique function. Their structures, like their functions, vary greatly. They are all, however, polymers of amino acids, arranged in a linear sequence.
Type Examples Functions
digestive enzymes amylase, lipase, pepsin, trypsin Help in digestion of food by catabolizing nutrients into monomeric units
transport hemoglobin, albumin Carry substances in the blood or lymph throughout the body
structural actin, tubulin, keratin Construct different structures, like the cytoskeleton
hormones insulin, thyroxine Coordinate the activity of different body systems
defense immunoglobulins Protect the body from foreign pathogens
contractile actin, myosin Effect muscle contraction
storage legume storage proteins, egg white (albumin) Provide nourishment in early development of the embryo and the seedling
Proteins have different shapes and molecular weights; some proteins are globular in shape whereas others are fibrous in nature. For example, hemoglobin is a globular protein, but collagen, found in our skin, is a fibrous protein. Protein shape is critical to its function, and this shape is maintained by many different types of chemical bonds. Changes in temperature, pH, and exposure to chemicals may lead to permanent changes in the shape of the protein, leading to loss of function, known as denaturation. All proteins are made up of different arrangements of the same 20 types of amino acids.
Amino acids are the monomers that make up proteins. Each amino acid has the same fundamental structure, which consists of a central carbon atom, also known as the alpha (α) carbon, bonded to an amino group (NH2), a carboxyl group (COOH), and to a hydrogen atom. Every amino acid also has another atom or group of atoms bonded to the central atom known as the R group.
The name "amino acid" is derived from the fact that they contain both amino group (NH2) and carboxylic acid group (COOH) in their basic structure. As mentioned, there are 20 amino acids present in proteins. Ten of these are considered essential amino acids in humans because the human body cannot produce them and they are obtained from the diet. For each amino acid, the R group (or side chain) is different.
The chemical nature of the side chain determines the nature of the amino acid (that is, whether it is acidic, basic, polar, or nonpolar). For example, the amino acid glycine has a hydrogen atom as the R group. Amino acids such as valine, methionine, and alanine are nonpolar or hydrophobic in nature, while amino acids such as serine, threonine, and cysteine are polar and have hydrophilic side chains. The side chains of lysine and arginine are positively charged, and therefore these amino acids are also known as basic amino acids. Proline has an R group that is linked to the amino group, forming a ring-like structure. Proline is an exception to the standard structure of an animo acid since its amino group is not separate from the side chain.
The sequence and the number of amino acids ultimately determine the protein's shape, size, and function. Each amino acid is attached to another amino acid by a covalent bond, known as a peptide bond, which is formed by a dehydration reaction. The carboxyl group of one amino acid and the amino group of the incoming amino acid combine, releasing a molecule of water. The resulting bond is the peptide bond.
The products formed by such linkages are called peptides. As more amino acids join to this growing chain, the resulting chain is known as a polypeptide. Each polypeptide has a free amino group at one end. This end is called the N terminal, or the amino terminal, and the other end has a free carboxyl group, also known as the C or carboxyl terminal. While the terms polypeptide and protein are sometimes used interchangeably, a polypeptide is technically a polymer of amino acids, whereas the term protein is used for a polypeptide or polypeptides that have combined together, often have bound non-peptide prosthetic groups, have a distinct shape, and have a unique function. After protein synthesis (translation), most proteins are modified. These are known as post-translational modifications. They may undergo cleavage, phosphorylation, or may require the addition of other chemical groups. Only after these modifications is the protein completely functional.
Carbohydrates
Carbohydrates are a group of macromolecules that are a vital energy source for the cell and provide structural support
Most people are familiar with carbohydrates, one type of macromolecule, especially when it comes to what we eat. To lose weight, some individuals adhere to “low-carb” diets. Athletes, in contrast, often “carb-load” before important competitions to ensure that they have enough energy to compete at a high level. Carbohydrates are, in fact, an essential part of our diet; grains, fruits, and vegetables are all natural sources of carbohydrates. Carbohydrates provide energy to the body, particularly through glucose, a simple sugar that is a component of starch and an ingredient in many staple foods. Carbohydrates also have other important functions in humans, animals, and plants.
Carbohydrates can be represented by the stoichiometric formula (CH2O)n, where n is the number of carbons in the molecule. In other words, the ratio of carbon to hydrogen to oxygen is 1:2:1 in carbohydrate molecules. This formula also explains the origin of the term “carbohydrate”: the components are carbon (“carbo”) and the components of water (hence, “hydrate”). Carbohydrates are classified into three subtypes: monosaccharides, disaccharides, and polysaccharides.
Monosaccharides (mono- = “one”; sacchar- = “sweet”) are simple sugars, the most common of which is glucose. In monosaccharides, the number of carbons usually ranges from three to seven. Most monosaccharide names end with the suffix -ose. If the sugar has an aldehyde group (the functional group with the structure R-CHO), it is known as an aldose, and if it has a ketone group (the functional group with the structure RC(=O)R'), it is known as a ketose. Depending on the number of carbons in the sugar, they also may be known as trioses (three carbons), pentoses (five carbons), and or hexoses (six carbons).
Disaccharides (di- = “two”) form when two monosaccharides undergo a dehydration reaction (also known as a condensation reaction or dehydration synthesis). During this process, the hydroxyl group of one monosaccharide combines with the hydrogen of another monosaccharide, releasing a molecule of water and forming a covalent bond. A covalent bond formed between a carbohydrate molecule and another molecule (in this case, between two monosaccharides) is known as a glycosidic bond. Glycosidic bonds (also called glycosidic linkages) can be of the alpha or the beta type.
Common disaccharides include lactose, maltose, and sucrose. Lactose is a disaccharide consisting of the monomers glucose and galactose. It is found naturally in milk. Maltose, or malt sugar, is a disaccharide formed by a dehydration reaction between two glucose molecules. The most common disaccharide is sucrose, or table sugar, which is composed of the monomers glucose and fructose.
A long chain of monosaccharides linked by glycosidic bonds is known as a polysaccharide (poly- = “many”). The chain may be branched or unbranched, and it may contain different types of monosaccharides. The molecular weight may be 100,000 daltons or more depending on the number of monomers joined. Starch, glycogen, cellulose, and chitin are primary examples of polysaccharides.
Lipids
Lipids include a diverse group of compounds that are largely nonpolar in nature. This is because they are hydrocarbons that include mostly nonpolar carbon–carbon or carbon–hydrogen bonds. Non-polar molecules are hydrophobic (“water fearing”), or insoluble in water. Lipids perform many different functions in a cell. Cells store energy for long-term use in the form of fats. Lipids are also the building blocks of many hormones and are an important constituent of all cellular membranes. Lipids include fats, oils, waxes, phospholipids, and steroids.
A fat molecule consists of two main components—glycerol and fatty acids. Glycerol is an organic compound (alcohol) with three carbons, five hydrogens, and three hydroxyl (OH) groups. Fatty acids have a long chain of hydrocarbons to which a carboxyl group is attached, hence the name “fatty acid.” The number of carbons in the fatty acid may range from 4 to 36; most common are those containing 12–18 carbons. In a fat molecule, the fatty acids are attached to each of the three carbons of the glycerol molecule with an ester bond through an oxygen atom.
Phospholipids are major constituents of the plasma membrane, the outermost layer of animal cells. Like fats, they are composed of fatty acid chains attached to a glycerol or sphingosine backbone. Instead of three fatty acids attached as in triglycerides, however, there are two fatty acids forming diacylglycerol, and the third carbon of the glycerol backbone is occupied by a modified phosphate group. A phosphate group alone attached to a diaglycerol does not qualify as a phospholipid; it is phosphatidate (diacylglycerol 3-phosphate), the precursor of phospholipids. The phosphate group is modified by an alcohol. Phosphatidylcholine and phosphatidylserine are two important phospholipids that are found in plasma membranes.
Unlike the phospholipids and fats discussed earlier, steroids have a fused ring structure. Although they do not resemble the other lipids, they are grouped with them because they are also hydrophobic and insoluble in water. All steroids have four linked carbon rings and several of them, like cholesterol, have a short tail.
Nucleic Acids
Nucleic acids are the most important macromolecules for the continuity of life. They carry the genetic blueprint of a cell and carry instructions for the functioning of the cell.
The two main types of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is the genetic material found in all living organisms, ranging from single-celled bacteria to multicellular mammals. It is found in the nucleus of eukaryotes and in the organelles, chloroplasts, and mitochondria. In prokaryotes, the DNA is not enclosed in a membranous envelope.
DNA and RNA are made up of monomers known as nucleotides. The nucleotides combine with each other to form a polynucleotide, DNA or RNA. Each nucleotide is made up of three components: a nitrogenous base, a pentose (five-carbon) sugar, and a phosphate group. Each nitrogenous base in a nucleotide is attached to a sugar molecule, which is attached to one or more phosphate groups.
Laboratory Instructions
Burn Test
Conduct the Burn Test
1. Put safety goggles on.
2. Put a small amount of baking soda into a test tube.
3. Hold the test tube with a test tube holder.
4. Ignite a Bunsen burner.
5. Angle the test tube so the open end is not facing yourself or any other person. Place the bottom of the test tube into the flame to heat the baking soda.
6. Continue burning until the baking soda is clearly very hot and smoking.
7. Observe the baking soda color.
8. Write results in the table below.
9. Repeat steps 2-8 for each of the following substances (with new test tubes each time): hair, salt, starch, sugar, vegetable oil, water.
Alternatively, your instructor may have completed these burn tests for you. Observe the results and record the results in the table below.
Burn Test Results & Questions
Substance Tested in the Burn Test Appearance of Substance after Burn Test Is the Substance Organic or Inorganic? If Substance is Organic, Give the Type of Biological Molecule (protein, carbohydrate, lipid, or nucleic acid)
baking soda
hair
salt
starch
sugar
vegetable oil
water
1. Complete the table above based on results of the burn test.
2. What type(s) of biological molecules were not tested using the burn test? Give examples of these types of biological molecules.
3. True or False: Every type of molecule found within a living thing is organic. Explain your answer.
4. True or False: It is possible for inorganic molecules to be essential for life.
5. What is the burn test and what can it show us?
Building Monomers of Biological Molecules
To become familiar with the molecular modeling kits, examine the colored balls that represent different elements common in biological molecules. Each colored ball has one or more holes. These holes represent the ability of these elements to form bonds:
element element abbreviation molecular model ball color bonding capacity
carbon C black
oxygen O red
hydrogen H white
nitrogen N blue
phosphorus P orange
Based on the holes in each of the model element balls, fill in the column that indicates the bonding capacity of the element. Bonding capacity is based on the number of protons the element has and the number of valence electrons the element has.
Protein Monomers: Amino Acids
1. Your instructor will assign you to build one of the two amino acids shown above.
2. Use the diagram above and the table indicating the molecular model element ball colors to build the amino acid you are assigned.
3. With your partner, conduct a dehydration synthesis reaction as directed by your instructor to form a dipeptide from your amino acids. This will form a molecule of water.
4. Conduct a hydrolysis reaction by breaking the water molecule to break down the dipeptide into the two amino acids.
Carbohydrate Monomers: Monosaccharides
1. Your instructor will assign you to build one of the two monosaccharides shown above.
2. Use the diagram above and the table indicating the molecular model element ball colors to build the monosaccharide you are assigned.
3. With your partner, conduct a dehydration synthesis reaction as directed by your instructor to form a disaccharide from your monosaccharides. This will form a molecule of water.
4. Conduct a hydrolysis reaction by breaking the water molecule to break down the disaccharide into the two monosaccharides.
Hint
Start this structure by building the circle of carbons (they have red numbers 1-5 in glucose and 2-5 in fructose) with the one oxygen that helps make up the ring. Once you have the circle, then build off of that circle.
Lipids: Fatty Acids
1. Build the fatty acid shown above.
2. Work with your partner to build one molecule of glycerol.
3. With your partner, conduct two dehydration synthesis reactions as directed by your instructor to form a diglyceride from your two fatty acids and the glycerol. This will form two molecules of water.
4. Conduct a hydrolysis reaction by breaking the water molecules to break down the diglycerides into the two fatty acids and one glycerol.
Nucleic Acids: Nucleotides
1. Work together with your partner to build the nucleotide above.
• You can each build one of the pieces of the nucleotide: phosphate group, sugar, or base.
• When the three parts are completed, form bonds between the parts as shown in the diagram above.
Building Monomers of Biological Molecules Questions
classification of biological molecule monomer of this classification of biological molecule elements found in this type of biological molecule function(s) of this type of biological molecule name a specific example of a molecule in this category of biological molecules
1. Fill out the table above to summarize information about the four categories of biological molecules.
2. Define the following:
• atom:
• element:
• organic molecule:
• inorganic molecule:
• monomer:
• polymer:
• dehydration synthesis:
• hydrolysis:
3. Catabolic reactions break down larger biological molecules into smaller biological molecules. Anabolic reactions form bonds to join together smaller biological molecules into larger biological molecules.
• Fill in the blanks with the bold words above: Dehydration synthesis is a type of reaction that must be a(n) _________________________ and hydrolysis is a type of reaction that must be a(n) ____________________________.
Foods and their Biological Molecules
Foods are Made of Biological Molecules
1. Discuss with your group the types of foods you are familiar with that fall into the categories of biological molecules listed above. Fill in the table.
proteins
(list protein-rich food below)
carbohydrates
(list carbohydrate-rich foods below)
lipids (fats)
(list lipid-rich foods/cooking ingredients below)
Note
Just like humans need food, so do microorganisms. Microorganisms also "eat." They, like us humans, require nutrients to survive.
Note
Pathogenic (disease-causing) microorganisms use their host (e.g. humans) as a source of nutrients.
Test Food Samples for Proteins
1. Put on laboratory safety goggles/glasses.
2. Label five test tubes with the samples to be tested: green banana, black banana, muscle, egg, DI or distilled water.
3. To each test tube, add 5 drops of the samples to be tested as labeled on the test tubes.
4. Add 3 drops of biuret reagent and mix well by swirling.
5. Wait 2 minutes.
6. Blue color is negative for significant protein content; purple/violet is positive for significant protein content
7. Record results in the table below.
Test Food Samples for Sugars (Small Carbohydrates)
1. Empty and thoroughly rinse the test tubes after the protein test.
2. To each test tube, add 5 drops of the samples to be tested as labeled on the test tubes.
3. Add 3 drops of Benedict's solution and mix well by swirling.
4. Gently place test tubes in a beaker of boiling or nearly boiling water.
5. Wait 2 minutes.
6. Use a test tube holder to remove the test tubes from the hot water and allow to cool.
7. Examine the liquid in the test tubes and interpret the results:
• green to yellow - very low to low levels (positive) of monosaccharides and disaccharides
• yellow-orange to orange - moderate to high levels (positive) of monosaccharides and disaccharides
• orange-red - very high levels (positive) of monosaccharides and disaccharides
• blue, grey, or any other color - negative for detectable monosaccharides and disaccharides
8. Record results in the table below.
Test Food Samples for Starch (A Large Carbohydrate)
1. Empty and thoroughly rinse the test tubes after the sugars test.
2. To each test tube, add 5 drops of the samples to be tested as labeled on the test tubes.
3. Add 3 drops of iodine and mix well by swirling.
4. Examine the liquid in the test tubes and interpret the results:
• black liquid or amber liquid with black chunks - positive for starch
• amber liquid (no black color) - negative for starch
5. Record the color of the test result in the table below.
Test Food Samples for Lipids (Fats)
1. On a piece of brown paper, draw five circles and label with with the samples to be tested: green banana, black banana, muscle, egg, DI or distilled water.
2. Drop one drop of each substance to be tested into the appropriate circle and allow to dry for at least 15 minutes.
3. Examine the dried spots. An oily spot (similar to the oil you would see on a brown napkin after french fries or potato chips) on the paper indicates a positive result for lipids.
4. Record your results in the table below.
Foods & their Biological Molecules Results & Questions
Type of Cells Blended with DI or distilled Water protein (+/-) sugar (small carbohydrates) (+/-) starch (large carbohydrates) (+/-) lipid (fats) (+/-)
green (under-ripe) banana cells
black (over-ripe) banana cells
muscle cells (chicken breast)
egg (chicken egg)
DI or distilled water (the control)
1. Complete the table above to summarize results from testing food items for biological molecules.
2. Of the four categories of biological molecules, which ones did we test for in these experiments?
3. Which category of biological molecule was not tested in these experiments?
4. Nucleic acids include DNA molecules. All cells have DNA (with only very few exceptions). If we tested the following, should the be positive or negative for nucleic acids? Briefly explain your answer for each.
• banana cells:
• muscle cells:
• egg cells:
• DI water or distilled water:
5. Each of the samples tested were prepared by mixing the food item with DI water or distilled water and mixing it in a blender. Think about the integrity of the experiment. Why did we need to also separately test the DI water or distilled water? | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.06%3A_Molecules_of_Life.txt |
Learning Objectives
• Differentiate between the types of microbiological media.
• Define aseptic, aseptic technique, pure culture, contamination, sterilization, autoclave, disinfectant, and antiseptic.
• Successfully use aseptic technique in microbiology transfers.
• Describe good aseptic technique in microbiology transfers.
• Recognize examples of good and bad aseptic technique and possible sources of contamination.
Microbiological Media
To study bacteria and other microorganisms, it is necessary to grow them in controlled conditions. Microbes are grown in substances that provide the nutrients necessary to sustain their metabolic activities and reproduction called "growth media" or simply "media" (singular is "medium"). Growth media can be either liquid or solid.
A liquid medium is called a broth. Broths can be used to determine growth patterns in a liquid medium, and for certain types of inoculations and metabolic tests. They are also the method of choice for growing large quantities of bacteria.
Solid growth media usually contains agar, which is a mixture of polysaccharides derived from red algae. It is used as a solidification agent because it (1) is not broken down by bacteria, (2) contains no nutrients that can be used by bacteria and (3) melts at high temperatures, and yet is solid at temperatures used for most bacterial growth. Solid growth media is used in the following forms: agar plates, agar slants and agar deeps. Making solid media is similar to making Jell-O, where a powder is mixed into water and heated to fully dissolve the powder. When the solution cools it solidifies. Melted agar is poured into a test tube and then allowed to solidify vertically for an agar deep, or at an angle for an agar slant. Agar plates are made by pouring melted agar into a petri dish. (Petersen, 2016)
Because of the relatively small tube opening (less opportunity to dry out or become contaminated) and the surface area available for growth, agar slants are commonly used to culture and store bacteria for intermediate periods of time (weeks). These types of cultures are called stocks. Deeps are often used to for certain differential metabolic tests.
In contrast to deeps and slants, agar plates have a large surface area for growth. Bacterial cells can be spread out over the surface so that they form discrete colonies which can be characterized. In a few weeks, you will be using a series of plate cultures to separate two different microbes from a mixture. In addition, specialized media in plate form is used for certain biochemical tests. (Petersen, 2016)
Media Contamination
Microbiologists generally study the organisms in pure culture, a culture that contains a single microbial species. If an unintended microorganism is introduced into a pure culture, the culture becomes contaminated. Aseptic technique is the collection of procedures and techniques designed to prevent the introduction of unwanted organisms into a pure culture or into the laboratory environment.
The term “aseptic” literally means “without contamination.” These procedures are as important for the experimenter’s safety as they are for maintaining culture purity.
Sterilization is the complete removal all vegetative cells, endospores, and viruses from an item (OpenStax CNX, 2018). Sterilization is all or none; something is either sterile or it is not sterile. In this course, all media, the substance in which the cells are grown, is sterilized by autoclave.
An autoclave uses moist heat (steam) under pressure to destroy all life forms. Whereas most vegetative cells can be killed at temperatures between 60 and 80oC, bacterial spores require temperatures above boiling (>100oC) for destruction. With a pressure of 15-20 lbs./in2, the autoclave can achieve a temperature of 121-132oC. Media under these conditions for at least 20 minutes will kill all spores as well as vegetative cells. Larger volumes require longer exposure times to ensure sufficient heat transfer to the materials being sterilized. The steam must directly contact the liquids or dry materials being sterilized, so containers are left loosely closed and instruments are loosely wrapped in paper or foil. The key to autoclaving is achieving a temperature high enough to kill spores for complete sterilization (OpenStax CNX, 2018).
Disinfection is the killing or growth inhibition of vegetative microbes. Generally, spores and some hearty cells will survive disinfection. Chemical disinfectants, such as chlorine bleach or products containing chlorine, are used to clean nonliving surfaces such as laboratory benches, clinical surfaces, and bathroom sinks (OpenStax CNX, 2018). We will use a chorine-based disinfectant to clean our work surfaces and to clean up any culture spills. Note that sterilization and disinfection are not interchangeable! (Why?) Spraying your bench top with disinfectant does not make it sterile.
Antiseptics are antimicrobial chemicals safe for use on living skin or tissues. Examples include hydrogen peroxide and isopropyl alcohol (OpenStax CNX, 2018).
When working in a microbiology laboratory, you must always remember that bacteria are present on all surfaces in the lab, as well as on your own hands and clothing. Aseptic techniques are designed to prevent the transfer of bacteria from the surrounding environment into a culture medium and from a culture to the environment. These techniques require care, concentration and practice. (Petersen, 2016)
Transfer Procedures
Because these procedures are completely new to most students, I strongly recommend that you watch the video at least twice. Keep in mind the following principles. (Some of these have been covered in the Laboratory Safety Exercise. They bear repeating because they are very important to keep you safe.)
Always begin by preparing your work area and making the necessary labels. Make sure you are clear about what transfers need to be made. The incinerator should be turned on HI for at least 20 minutes prior to using.
A transfer can be thought of in two parts, obtaining the cells (inoculum) from the source/parent culture and inoculating the new sterile tube or plate. Transfers, with very few exceptions, are performed by a single individual. You should not be holding the tube while your partner inoculates it.
Before you Start
1. Culture media must initially be sterile. Inspect your media before you start. If a culture medium appears cloudy or you observe unwanted growth, consult with your TA or instructor to be sure it is not contaminated before using it.
2. Label your tubes white label tape. Masking tape is provided for other uses.
3. Label plates on the bottom.
4. Inspect the parent cultures. If the cells have fallen to the bottom, be sure to re-suspend them by flicking the tube gently to mix. Never shake a tube.
5. Disinfect the lab bench.
6. Light the Bunsen burner. The flame show be blue and a moderate height (not to tall, not too short).
Sterilizing the Inoculating Loop or Needle
1. Hold the inoculating loop in your dominant hand like a pencil. To sterilize, place it in the Bunsen burner for at least 10 seconds. The entire wire must be heated red hot. Use the center blue region of the flame (not the top or bottom of the flame). Watch the clock for the time. Students tend to count too fast.
2. Do not let the loop sit in the incinerator more than 15 seconds.
3. Hold the instrument in the air allowing the wire to cool for about 15 seconds before making any transfers. Please do not wave it around to cool it.
4. The wire is now sterile. If at this time, you set it down on the bench top, which is not sterile, it must be incinerated again before going into any culture. If a sterile instrument is touched to anything not sterile including your hand, sleeve, the outside of a tube or plate, a slide or the bench top, it becomes contaminated and cannot be used in an aseptic transfer.
Obtaining the Inoculum from a Tube Culture
1. With your non-dominate hand, pick up the parent tube by grasping the tube just below the cap and lifting it out of the rack.
2. Grasp the cap with the pinky and ring finger of your dominate hand and gently twist the tube out of the cap keeping your dominate hand still. See Figures 3. The cap is kept in your hand and never placed on the bench top.
3. Heat the mouth of the open tube by passing it through the flame of the Bunsen burner. Heating creates convection currents, which carry airborne particles away from the mouth of the tube, preventing contamination of the culture or medium within.
4. For a broth parent culture: Place the cooled loop into the broth and remove making sure that you have a thin film of liquid filling the loop. Jiggling the loop in the broth is not needed and can result in the formation of tiny aerosol droplets. Please do not jiggle the wire.
5. For a slant parent culture: Touch the cooled loop to the growth. Do not break the agar surface. Refrain from “swiping” a large mass of cells. You do not need to see cells on the loop to have millions!
6. Again, heat the mouth of the tube after withdrawing the transfer instrument. This step incinerates any microbes that may have been deposited on the lip of the tube during the transfer.
7. Replace the cap and set the parent tube back in the test tube rack.
8. Keep the inoculating instrument in your hand.
To obtain the inoculum from a plate culture
1. Turn the culture plate with bacteria growing on it right side up.
2. Lift the lid a short distance, with your non-dominate hand, so that the lid acts at a shield protecting the agar surface from falling microbes in the air. See Figure 4.
3. Touch the cooled loop to the growth. Do not breath the agar surface. Refrain from “swiping” a large mass of cells. You do not need to see cells on the loop to have millions!
4. Replace the lid immediately after withdrawing the transfer instrument and turn the plate upside-down again.
5. Keep the inoculating instrument in your hand.
Inoculating a slant
1. With your non-dominate hand, pick up the parent tube by grasping the tube just below the cap and lifting it out of the rack.
2. Grasp the cap with the pinky and ring finger of your dominate hand and gently twist the tube out of the cap. Keeping your dominate hand still is especially important because there are cells on the loop at this point. Keep the cap in your hand.
3. Pass the mouth of the tube through the flame.
4. Insert the loop all the way to the bottom of the slant surface.
5. Drag the loop on the agar “snaking” your way up the slant creating a “fishtail pattern.” This is called a fishtail inoculation. See Figure 5.
6. Again, heat the mouth of the tube after withdrawing the transfer instrument. Replace the cap and set the parent tube back in the test tube rack.
7. Immediately flame the inoculating loop and wire for a full 10 seconds before setting it down.
Inoculating a broth
1. With your non-dominate hand, pick up the parent tube by grasping the tube just below the cap and lifting it out of the rack.
2. Grasp the cap with the pinky and ring finger of your dominate hand and gently twist the tube out of the cap. Keeping your dominate hand still is especially important because there are cells on the loop at this point.
3. Pass the mouth of the tube through the flame.
4. Insert the loop to the bottom of the broth liquid and then remove the loop. Jiggling is not necessary to dislodge cells.
5. Again, heat the mouth of the tube after withdrawing the transfer instrument. Replace the cap and set the parent tube back in the test tube rack.
6. Immediately flame the inoculating loop and wire for a full 10 seconds before setting it down.
After all inoculations
1. Cultures to be incubated should be placed in the designated area for culture incubation. Otherwise, a student’s culture may be disposed of accidentally.
2. Be sure to turn it off the Bunsen burner when you are finished with it.
Because there is so much to remember, the first time you make transfers many of the above actions are repeated in context. After a few weeks practice, the repetition will no longer be necessary and it will be assumed that you will adhere to the procedures above without reminder.
Practice Aseptic Technique
1. Aseptically transfer a loop of sterile TSB to another test tube of sterile TSB:
1. Flame loop.
2. Remove cap from one test tube of sterile TSB and hold it in your hand (don't put it down and don't touch the open end).
3. Pass the mouth of the test tube through the flame.
4. Insert the flamed loop into the sterile TSB.
5. There should be a film of liquid across the loop (similar to how a bubble wand will have a film across it).
6. Pull the loop out of the test tube.
7. Pass the mouth of the test tube through the flame.
8. Put the cap back onto the test tube.
9. Remove cap from the other test tube of sterile TSB and hold it in your hand (don't put it down and don't touch the open end).
10. Pass the mouth of the test tube through the flame.
11. Insert the loop with the film of sterile TSB.
12. Pass the mouth of the test tube through the flame.
13. Put the cap back onto the test tube.
14. Flame the loop.
15. Let each person in your group repeat with the same two test tubes.
16. If every member of the group successfully transferred the TSB aseptically, there will be no growth (no turbidity) next class.
2. Put labels on both culture tubes with your name and “aseptic.”
3. Check the culture tubes next class for turbidity to determine whether or not your aseptic transfer was successful. A successful transfer would result in both tubes being clear (no growth).
Aseptic Technique Results
1. Record your results in the table below.
Growth (+) or No Growth (-)
TSB culture tube 1
TSB culture tube 2
1. If you observed growth in the TSB culture tubes, what might have gone wrong? If you were successful in keeping both sterile, what are some possible sources of error that could cause contamination?
2. Explain the importance of aseptic technique in microbiology.
3. What is the purpose of flaming in aseptic technique?
4. What are some signs of growth in a liquid medium?
Works Cited
• Petersen, J. a. (2016). Laboratory Excercises in Microbiology: Discovering the Unseen World Through Hands-On Investigation. CUNY Academic Works. Retrieved from http://academicworks.cuny.edu/qb_oers/16 | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.07%3A_Aseptic_Technique.txt |
Learning Objectives
• Define "colony" and describe how colonies arise.
• Successfully identify isolated colonies.
• Characterize colony appearances and distinguish different microbial species based on colony appearance.
• Successfully execute a zig-zag streak plate and describe how and when this approach is used.
• Successfully execute a quadrant streak plate and describe how and when this approach is used.
• Describe and interpret how streak plate approaches result in the petri plate growth patterns.
Bacterial Colonies
If a single bacterial cell is placed on the surface of a TSA agar plate and allowed to multiply for 24 to 48 hours, it would grow into a mass of cells visible to the human eye called a colony. Colonies formed by the same microbial species growing on the same medium will all look alike. This is because the cell shape, pigmentation, division plane, rate of cell division and other characteristics of the organism result in the progeny cells stacking on one another in a pattern resulting in a characteristic colony form.
When identifying unknown microorganisms, to separate a mixture of microbial species, an isolated colony must be obtained. Since a single colony began with the growth of a single cell, all of the cells in that colony should be of a single species. Therefore, an isolated colony can be transferred to a sterile medium to isolate the species at the beginning of the identification process. Isolated colonies are found on their own away from other growth. A colony that is touching other colonies is not considered isolated (see Figure 1).
Characteristics of Colonies on a Petri Plates
Even on general purpose growth media, bacteria can exhibit characteristic patterns of growth. Some examples are shown below. While these growth patterns are an important piece of information when identifying a bacterial species, they are not sufficient for a positive identification. Staining procedures and metabolic tests must be used for a definitive identification.
Zig Zag Streak Plate
If you swab a door handle, where bacteria are likely to be present, and then pass the swab across the surface of a TSA plate, cells would be deposited onto the surface from the swab. Initially, the swab may have a fairly high concentration of cells and the area touched by it will have lots of different cell types placed close together. After these grow up, the cells’ progeny will crowd together and overlap with other cells’ progeny forming areas called confluent growth.
As the swab moves across the agar leaving cells on the agar in a zig zag pattern as shown in Figure 4, regions touched later in the process will have fewer and fewer cells. Individual cells are far enough apart that each one would grow into a discrete isolated colony. The result may look something like Figure 3. Because the door handle likely has a variety of microbes on it, there are numerous colony forms. This technique is called a zig zag streak plate and is one type of isolation streak method.
The steps of the zig zag streak plate are:
1. Aseptically obtain a sample using an inoculating loop or sterile cotton swab.
2. Turn the agar plate right-side-up.
3. Hold the plate lid so that it acts as a shield protecting the agar surface from microbes falling from the air.
4. Starting the streak on the side of the plate farthest from your dominant hand, pass the loop on the surface of the agar in a zig zag pattern filling the surface of the plate. See Figure 4.
5. Replace the lid, and immediately incinerate the loop or dispose of the cotton swab.
6. Place the plate upside-down for incubation.
Some tips for a good zigzag streak: Use as much of the agar surface as you can. Make broad strokes that span the width of the plate.
Quadrant Streak Plate
Now consider streaking a sample from a pure TSA broth culture prepared for you. If there is visible turbidity, there will be a high density of cells. If you used the same zig zag streaking pattern, the cells would never be reduced in concentration such that you could get isolated colonies. In order to reduce the cell density on the surface of the plate, we can use a quadrant streak plate.
The quadrant streak plate reduces or dilutes the number of cells using successive streaking zones on the petri plate and flaming the inoculation loop between zones. The petri plate is divided into four sections or zones. Bacteria are deposited in the first section at full strength from the source. Then the inoculating loop is sterilized. From this point on, no additional cells are added to the agar surface. The sterile loop is used to spread out cells that have already been placed on the plate. After spreading cells from the first area to the second, the loop is sterilized again. This eliminates extra cells from the loop. The sterile loop is used to spread some cells from the second area into the third area diluting them further. Cells are spread into a fourth area from the third area after the loop is sterilized. After all the regions have been inoculated, the hope is that in the last section cells are far enough apart so that they grow up into isolated colonies.
This quadrant streak plate technique allows one to observe isolated colonies and characterize them and determine if your observations are consistent with our expectations for the organism you are working with. If you are working with a pure culture, you would expect that all the colonies would look the same, similar size, color, shape etc. One or more different looking colonies indicates your culture was contaminated or you created contamination by poor aseptic technique.
The steps of the quadrant streak plate technique are as follows:
1. Aseptically obtain a loopful of the culture and set the tube back in a rack.
2. Turn the plate right-side up and place it on a piece of white scratch paper so that the lines can be seen.
3. Rotate the plate so that area/zone 1 is farthest from your dominate hand.
4. Holding the plate lid so that it acts as a shield protecting the agar surface from microbes falling from the air, pass the loop on the surface of the agar in area 1 in the pattern shown.
5. Replace the plate lid on the petri plate.
6. Flame the loop and allow it to cool.
7. Rotate the petri plate.
8. Spread the cells already on the petri plate agar surface in area/zone 1 with the sterile loop by streaking in the pattern shown. This step pulls highly concentrated cells from area/zone 1 into area/zone 2 spreading them out.
9. Replace the plate lid on the petri plate.
10. Flame the loop and allow it to cool.
11. Rotate the petri plate.
12. Again, spread some of the cells from area/zone 2 into area/zone 3 by streaking in the pattern shown.
13. Replace the plate lid on the petri plate.
14. Flame the loop and allow it to cool.
15. Rotate the petri plate.
16. Again, spread some of the cells from area/zone 3 into area/zone 4 by streaking in the pattern shown.
17. Replace the plate lid and flame the loop again before setting it down.
18. Turn the plate upside-down for incubation and storage.
In this technique it is essential that you flame the loop after each area is inoculated. This is what reduces the cell density because you are spreading cells already on the plate. You are not adding additional cells by using a loop with cells on it. You eliminate additional cells by flaming. After incubation, the goal is at least 3 well isolated colonies.
Keep in mind that you want to use as much of the agar surface as possible. Your streaks should span the width of the plate. If you keep touching the previous high density streak, you will pull too many cells into the next area and will not reduce the number enough to get isolated colonies. If you do not cross over the previous area enough, you will not have enough cells in the next one.
If you choose to cool your loop in the agar, always use a spot close to the edge and away from any previous streak. The resulting growth pattern should be dense growth in area/zone 1, more diffuse in area/zone 2 and less growth in area/zone 3, and the least growth in area/zone 4.
Lab Instructions
1. Each person will label 1 TSA plate with their name, “quadrant streak plate,” and with the bacterial sample they will be streaking (one of the following - divide the following three cultures so that each person in your group does a streak plate of a different culture):
1. Escherichia coli
2. Serratia marcescens
3. Escherichia coli & Serratia marcescens (mixture)
2. Follow the instructions above for the quadrant streak plate technique.
3. Invert the Petri plate and incubate at 30 °C.
4. Next class, examine the colonies and characterize the colony form, elevation, margin, diameter, and color.
5. Either make a drawing of the plates of your group or take photos of the plates.
Streak Plate Results
1. Fill out the table below to describe the types of colonies observed:
Escherichia coli & Serratia marcescens
Escherichia coli
Serratia marcescens
Colony type 1
Colony type 2
Colony form
Colony elevation
Colony margin
Colony color
Colony diameter (mm)
2. Make detailed drawings of your plates (using color). Alternatively, you can take images of the plates and attach them below.
1. Did you obtain isolated colonies on each of the plates?
Escherichia coli:
Serratia marcescens:
Escherichia coli & Serratia marcescens (mixture):
1. Explain how the streak-plate technique dilutes and spreads out microorganisms to form individual colonies.
2. Considering how you streaked the plates, which area of the streak plate will always contain:
the greatest amount of growth:
the least amount of growth:
1. Does each individual colony represent the growth of one cell? Explain your answer.
2. Why can a single colony on a plate be used to start a pure culture?
Works Cited
Franklund, C. (n.d.). Microbiology Laboratory Manual, Observing and Recording the Microbial World. Farris State University. Retrieved 2018, from https://github.com/WeeBeasties/microbiology- laboratory-manual | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.08%3A_Plating_on_Petri_Plates_for_Isolation.txt |
Learning Objectives
• Define simple stain.
• Tell the purpose of simple staining and what bacterial feature(s) can and cannot be ascertained when using a simple stain.
• Prepare a bacterial smear and properly heat fix the smear.
• Tell the importance of fixation in preparing a bacterial sample for staining.
• Describe how bacterial smears are prepared differently when using a liquid culture versus using a solid culture.
• Prepare a simple stain.
• Examine the bacterial smear with a microscope and make an accurate illustration of the bacterial cells.
• Use the microscope to identify bacterial features (cell shape and cell arrangements).
• Identify bacterial cell shapes and cell arrangements given examples.
• Identify flaws in a simple stain and ascertain ways to improve future stains.
Simple Staining
Most types of cells do not have much natural pigment and are therefore difficult to see under the light microscope unless they are stained. Several types of stains can be used to make bacterial cells more visible. Some staining techniques can be used to determine the cells’ biochemical or structural properties, such as cell wall type and presence or absence of endospores. This type of information can help scientists identify and classify microorganisms, and can be used by health care providers to diagnose the cause of a bacterial infection.
One type of staining procedure that can be used is the simple stain, in which only one stain is used, and all types of bacteria appear as the color of that stain when viewed under the microscope. Some stains commonly used for simple staining include crystal violet, safranin, and methylene blue. Simple stains can be used to determine a bacterial species’ morphology (cell shape) and arrangement (single, chains, clusters, etc.), but they do not give any additional information. Other staining approaches utilize more than one stain (differential stains) and can be used to determine other differences of between different bacterial species (e.g. cell wall structures, presence or absence of endospores, presence or absence of capsules, presence or absence and arrangement of flagella, etc.). These features cannot be distingushed using a simple stain.
Table 1: Common simple stain types include basic stains (high pH stains), acidic stains (low pH stains), and negative stains (stains the surroundings of cells instead of the cells themselves). Basic stains include methylene blue, crystal violet, malachite green, basic fuchsin, carbolfuchsin, and safranin. Acidic stains include eosin, acid fuchsin, rose bengal, and congo red. Basic stains are attracted to negatively charged molecules in the cell including nucleic acids (DNA and RNA) and some proteins. Acidic stains can be positive stains (stain the cells) or they can be negative stains (stain the background and not the cells). Acidic stains can stain positively charged molecules in cells including some proteins. Negative stains include India ink and nigrosin. Negative stains produce a darker background and cells appear light and unstained. When a simple stain is done, only one stain is applied to the cells to better see the cells using the microscope.
A good stained smear should be somewhat difficult to see with the naked eye on the surface of a microscope slide. If there is a dark spot of color visible, it means that the bacteria are stacked too densely for adequate evaluation. This is a common mistake made by students learning to make bacterial smears.
Bacterial Cell Morphology & Arrangement
Two characteristics that can be used to distinguish different types of bacteria is their microscopic shapes and their microscopic arrangements. Identification of these features can lead to a diagnosis and aid in treatment of a patient. The cell shape describes the overall shape of a single bacterial cell. Arrangements describe how bacterial cells are grouped (or not grouped) with each other. Both of these characteristics must be determined using a microscope and typically occurs after staining the cells.
Table 2: Summary of the most common bacterial cell shapes and bacterial cell arrangements with cartoon-like illustrations of each, descriptions, and microscopic images.
Cell Shape
(singular / plural)
Cell Arrangement
(singular / plural)
Illustration
Description
Image
(color of cells is based on the type of stain used)
coccus / cocci single or solitary Cells are alone and round or spherical.
coccus / cocci diplococcus / diplococci Cells are in pairs and round or spherical.
coccus / cocci tetrad Cells are grouped in fours and are round or spherical.
coccus / cocci sarcina Cells are group in eights as two groups of four on top of each other. Cells are round.
coccus / cocci streptococcus / streptococci Cells are in a chain and are round or spherical.
coccus / cocci staphylococcus / staphylococci Cells are in bunches or clusters and are round or spherical.
spirochete / spirochetes single or solitary Cells are alone and long and thin with a three-dimensional corkscrew forming a helical shape.
spirillum / spirilla single or solitary Cells are alone and long and thin with a wavy appearance but are not helical in shape.
vibrio / vibrios single or solitary Cells are alone and have a rod shape with an arch or curve.
bacillus / bacilli single or solitary Cells are alone and rod shaped.
bacillus / bacilli diplobacillus / diplobacilli Cells are in pairs and are rod shaped.
bacillus / bacilli streptobacillus / streptobacilli Cells are in chains and are rod shaped.
bacillus / bacilli palisades Cells are rod shaped and lined up with each other long-ways resembling the pickets in a picket fence.
coccobacillus / coccobacilli single or solitary Cells are alone and are short rods.
Note
There are other less common bacterial morphologies (e.g., filamentous, squares, etc.) that are not shown here.
Note
It may be difficult to tell the difference between spirochetes and spirilla using a common light microscope. The difference is more obvious with an electron microscope (see images below) or using other types of light microscopes. Please note that spirilla typically have one flagellum at each end or pole of the cell. The spirilla image does not show flagella.
To learn more about comparing spirilla and spirochetes, see this article Difference Between Spirilla and Spirochetes.
Preparing Bacteria for Staining (Bacterial Smear and Fixation)
Bacterial Smear
Prior to staining bacterial cells they first must be applied to the microscope slide in what is called a bacterial smear. If bacteria are transferred from a liquid culture, a loopful of culture can be aseptically removed from the culture and then spread on the surface of the microscope slide. If bacteria are transferred from a solid culture such as a slant or a petri plate, a single drop of DI or distilled water is added to a microscope slide and then a small amount of growth is obtained aseptically with a loop. The loop is then used to spread the water drop around the slide so it becomes a thin layer of water containing bacteria.
Note
Chunks of bacteria on the microscope slide surface means that too much bacterial growth was transferred to the slide and it will be difficult to distinguish individual cells when viewing the sample with a microscope.
After bacteria have been applied to a microscope slide, the smear is dried thoroughly before fixation. The smear can be allowed to air dry or can be put on a slide warmer to speed up the drying process.
Warning
After preparation of the smear you must fix the sample (see the Fixation section below). The bacteria will wash off the slide during staining if you do not fix them to the slide.
Fixation
Before cells are stained with a dye, they must be fixed to the slide so that they do not wash away with the excess stain. The “fixing” of a sample refers to the process of attaching cells to a slide. In addition to adhering the specimen to the slide, fixation also kills microorganisms in the specimen, stopping their movement and metabolism while preserving the integrity of their cellular components for observation.
Fixation is often achieved either by heating (heat fixing) or chemically treating the specimen. To heat-fix a sample, after a thin layer of the specimen is spread on the slide (called a smear or emulsion), and the slide is then briefly heated over a heat source. Done correctly, this will attach cells to the microscope slide and kill the cells.
Note
A heat-fixed slide can be stained immediately or kept for months. If you wish to keep heat-fixed, unstained slides for further work, you can wrap the slides in a paper towel or put them in a slide box and store them in your lab drawer.
Laboratory Instructions
Prepare a Bacterial Smear
Note
You may find it helpful to draw a circle (wax pencil is best) on the opposite side of the slide where you will spread your smear. This will help you later in locating the smear. The wax pencil is better than a marker because it will not wash off easily from the glass.
Note
If you are using a liquid culture, gently mix the culture until you get an even, cloudy mixture (it should look somewhat like skim milk). If you mix too aggressively, you will lose the bacterial morphology.
1. Prepare a bacterial smear on the slide:
• If you are taking bacteria from a solid culture (slant or petri plate), place a small drop (only 1 drop) of saline, deionized (DI) water, or distilled water onto a microscope slide and use a loop to aseptically add bacteria to the water (avoid taking a large chunk of bacteria since the cells will be too dense to view individual cells). Use the loop to spread the bacteria in the water drop and to spread the water drop out to make it thinner (it will dry faster).
• If you are taking a bacteria from a a liquid culture (broth), place 1 or 2 loopfuls of bacteria directly onto a microscope slide (no saline or water added).
2. Allow the slide(s) to air dry on the slide warmer (or air dry if a slide warmer is not available).
Heat Fix the Bacterial Smear
Once the liquid has completely evaporated on the surface of the slide, heat fix by passing the slide:
1. Attach a wooden clip to the microscope slide to hold it.
2. Pass the underside of the microscope slide through a flame three times.
3. Allow the slide to cool and then continue with your staining protocol.
Note
If you heat fix too little, the bacteria will wash off the slide. If you heat fix too much, you will cook the bacteria and denature them.
Simple Stain
1. Use the slide(s) that you already prepared when creating bacterial smears and heat fixing (see above).
2. Grip the microscope slide in wood clip over a waste container bucket.
3. Add methylene blue stain to the heat-fixed smear. There is no reason to cover the entire slide with stain. Just make sure to cover the smear with stain.
4. Set a timer for 1.5 or 2 minutes. The stain will remain on the smear during this time.
5. Angle the slide and gently rinse the slide with DI or distilled water using a squirt bottle. Water is not applied directly to the smear. Instead, apply the water to the area of the slide just above the smear so that water will rinse across the smear and into the waste bucket. Continue rinsing until water running off the slide appears clear.
6. Blot gently with bibulous paper. Do not wipe. Just gently blot with bibulous paper to get rid of excess water.
7. Place the stained slide on a microscope and examine. Be sure to begin at the lowest power (look for the blue color of the methylene blue dye). Focus at lowest power and then increase one objective magnification at a time.
Results & Questions
1. Draw the cells you observe after the simple stain in the space above.
2. Critique your cell drawings:
• Does the drawing accurately show how the cells appear when you look into the microscope (including cell shape and arrangement)?
• Does the drawing accurately show the cell size you see in the microscope?
• Does the drawing accurately show the color(s) you see in the microscope?
3. What microbial characteristics can one ascertain from a simple stain?
4. What were the shapes of the cells you observed? Use microbiological terminology.
5. What was the arrangement of the cells you observed? Use microbiological terminology.
6. A successful simple stain would enable you to observe individual cells (cells not on top of each other) and a sample where you can clearly see the cells, their undistorted shapes, and their undistorted arrangements. Reflect on your results. In what way(s) could your simple stain be improved?
7. What might you do to improve the results of the simple stain as mentioned in 6.
8. Fixing a bacterial smear is an essential step in preparing for staining. Why? What might happen if cells are not fixed to the microscope slide?
9. Fixing also has another effect on the cells other than your answer to 8. What is it?
10. Define simple stain.
11. How is the procedure different when taking cells from a solid medium compared to taking cells from a liquid medium? Why is it important that your smear be thin? | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.09%3A_Simple_Stain.txt |
Learning Objectives
• Explain the importance of Gram stains in health care and microbiology.
• Define "differential stain" and contrast with "simple stain."
• Identify the Gram stain as a type of differential stain
• Tell what the Gram stain tells us about different species of bacteria.
• Examine Gram-stained cells and interpret whether the cells are Gram-positive or Gram-negative.
• Identify cell morphology of bacteria.
• Describe the structure of the cell walls of Gram-positive cells.
• Describe the structure of the cell walls of Gram-negative cells.
• Identify structures of Gram-positive cell walls and Gram-negative cell walls in diagrams models of cell walls.
• Successfully conduct a Gram stain and differentiate cells as Gram-positive and Gram-negative.
• Name each stain/reagent of the Gram stain and explain its function and how it will interacts with Gram-positive and Gram-negative cell walls during the Gram stain procedure.
• Troubleshoot unsuccessful Gram stains and explain how errors might be fixed.
Differential Stains
Microbiologists commonly perform differential stains, as this allows them to gather additional information about the bacteria they are working with. Differential stains use more than one stain, and cells of different bacterial species can have different appearances based on their chemical or structural properties. Some examples of differential stains are the Gram stain, acid-fast stain, and endospore stain.
Table 1: Summary of some common differential stains used in microbiology. The Gram stain uses the following dyes/reagents: crystal violet, Gram's iodine, ethanol, and safranin. The Gram stain distinguishes cells by cell wall type (Gram-positive or Gram negative). Gram-positive cells stain purple/violet. Gram-negative cells stain pink. The acid fast stain uses the following dyes: basic fuchsin and methylene blue. The acid fast stain is used to distinguish acid fast bacteria (reveals the cell wall structure of certain types of bacteria beyond Gram-positive and Gram-negative). Acid fast bacteria appear read and non-acid fast bacteria appear blue. The endospore stain uses the following: heat, malachite green, and safranin. The endospore stain is used to distinguish between bacterial species that do and do not form endospores. Endospores (if present) appear bluish-green and all other cells/cell structures appear pink. The flagella stain uses the following dyes/reagents: tannic acid or potassium alum mordant, pararosaline or basic fuchsin. The flagella stain is used to visualize flagella (if present). The capsule stain uses the following dyes: India ink or nigrosin (stains the background, not cells) and a counterstain. The result shows the background as dark, the cells the color of the counterstain, and the capsule (if present) appearing clear. The capsule stain determines if a bacterial species has capsules or not. Capsules appear as transparent halos around dyed cells.
The Gram Stain Shows Differences in Cell Wall Structures
The Gram stain, developed by Christian Gram in 1884, is the most widely used differential stain in bacteriology. Most bacteria are divided into two major groups- Gram-positive bacteria and Gram-negative bacteria based on the cell wall composition. Knowing the Gram reaction of a clinical isolate (isolated bacterial species from a patient) can help the health care professional make a diagnosis and choose the appropriate antibiotic for treatment.
Figure 1: (Top) Microscopic images of bacteria stained with the Gram stain. (Top right) Gram-positive bacterial species stain purple with the Gram stain. (Top left) Gram-negative bacterial species stain pink with the Gram stain. (Bottom) Diagrams illustrating major structural components of Gram-positive bacterial cell walls and Gram-negative bacterial cell walls. (Bottom right) Gram-positive bacterial species have a thick layer of peptidoglycan outside of the plasma membrane. (Bottom left) Gram-negative bacterial species have a thin layer of peptidoglycan outside of the plasma membrane with an outer membrane outside of the peptidoglycan layer. Embedded in the outer membrane of Gram-negative cell walls are lipopolysaccharides (LPS) that are not shown in this diagram.
The results of the Gram stain reflect differences in cell wall composition. Gram-positive cells have thick layers of peptidoglycan (a substance composed of carbohydrates and protein subunits) in their cell walls. Gram-negative bacteria have very little peptidoglycan. Gram-positive bacteria also have teichoic acids, whereas Gram-negative bacteria do not. Gram-negative cells have an outer membrane that resembles the phospholipid bilayer of the plasma membrane. The outer membrane contains lipopolysaccharides (LPS), which are released as endotoxins when Gram-negative cells die. This can be of concern to a person with an infection caused by a Gram-negative organism.
Figure 2: Detailed view of the structure of bacterial cell walls for Gram-positive species and for Gram-negative species. (Left) Gram-positive cell walls consist of a thick layer of peptidoglycan (PG) outside of the cell's plasma membrane (PM) with teichoic acid (TA) and lipoteichoic acid embedded across the peptidoglycan layer. (Right) Gram-negative cell walls consist of a thin layer of peptidoglycan (PG) outside of the cell's plasma membrane (PM). Gram-negative cell walls have an outer membrane (OM) beyond the peptidoglycan layer containing lipopolysaccharide (LPS) as well as porin channels that enable some materials to pass across the outer membrane. In Gram-positive and Gram-negative cell wall types lipoprotein (LP) is also found.
Gram stains are best performed on fresh culture. Older cells may have damaged cell walls and not yield the correct Gram reaction. Also, some species are known as Gram-variable, and so both Gram-positive and Gram-negative reactions may be visible on your slide.
Although the vast majority of bacteria are either Gram-positive or Gram-negative, it is important to remember that not all bacteria can be stained with this procedure (for example, Mycoplasma sp., which have no cell wall, stain poorly with the Gram stain).
How the Gram Stain Works
The Gram stain uses four stains/reagents: crystal violet, Gram's iodine, ethanol, and safranin. Crystal violet (the primary stain), enters the peptidoglycan of all bacteria giving them a purple color. The next stain is Gram’s iodine, the mordant, which combines with the crystal violet to make a bigger complex in the peptidoglycan wall. The next step is the most critical. Ethanol is used as a decolorizer will remove the crystal violet/iodine complexes from the thin peptidoglycan layers of Gram-negative cell walls, but not the thick peptidoglycan layers of Gram-positive cell walls. The length of time given for the decolorization step with ethanol will be important in obtaining the best results from the Gram stain. After the decolorization step, Gram-positive cells will still hold onto the purple crystal violet color, but Gram-negative cells will lose the purple color and be transparent. We still want to see the Gram-negative cells and to be able to distinguish them from the Gram-positive cells. Safranin is called a counterstain since it is used to stain the now colorless Gram-negative cells a pink color.
Figure 3: Overview of the Gram stain procedure and how each step changes cell color. (1) Applying crystal violet turns all cells purple. (2) Applying Gram's iodine does not change cell colors (all cells stay purple), but it will cause the crystal violet to firmly adhere to Gram-positive cell walls, but not Gram-negative cell walls. (3) The alcohol (ethanol) step is a decolorization step. Ethanol will remove crystal violet from Gram-negative cells, but not Gram-positive cells. After this step, Gram-positive cells will remain purple and Gram-negative cells will be transparent. (4) Safranin is a reddish-pink stain that will stain transparent cells (Gram-negative cells) pink. After this step, Gram-positive cells will still be purple and Gram-negative cells will be pink.
Exercise 10.1
You conduct a Gram stain and when you observe the bacterial cells with the microscope, you observe pink colored cells. What does this tell you?
Answer
Exercise 10.2
You conduct a Gram stain and when you observe the bacterial cells with the microscope, you observe purple colored cells. What does this tell you?
Answer
Exercise 10.3
You conduct a Gram stain and when you observe the bacterial cells with the microscope, you observe purple colored cells and pink colored cells mixed together. What does this tell you?
Answer
The Gram stain has proven to be very useful in the identification of bacteria and in predicting which antibiotics are most likely to be effective to kill bacteria. There are some problems with the technique, however. If the procedure is not performed properly, the results may be erroneous. Thus, you will need to practice the technique until your results are satisfactory. There are some bacterial species, such as Mycobacterium tuberculosis or Legionella pneumophila that usually do not stain with the Gram stain at all. Nevertheless, this technique has become one that microbiologists rely heavily upon.
Table 2: Stains and reagents used during the Gram stain.
Stain/Reagent use in the Gram stain Color of the Stain/Reagent Role of the Stain/Reagent What the Stain/Reagent Does
crystal violet purple primary stain stains all cells purple
Gram's iodine amber mordant attaches to the crystal violet to form larger complexes that prevent crystal violet from leaving thick peptidoglycan layers in Gram-positive cell walls
ethanol clear decolorizer removes crystal violet from thin peptidoglycan layers in Gram-negative cell walls leaving Gram-negative cells appearing transparent
safranin red/pink counterstain colors transparent Gram-negative cells pink
Figure 4: Gram-positive cell walls have a single membrane (the plasma membrane) enclosed by a thick, cross-linked peptidoglycan layer. Gram-negative cell walls have a thin layer of peptidoglycan between the plasma membrane and an outer membrane. When the Gram-positive cells and Gram-negative cells are saturated with crystal violet (first step of the Gram stain), they become purple. When Gram's iodine is applied to the cells, it causes the crystal violet to adhere firmly in the thick peptidoglycan layer of Gram-positive cell walls. In Gram-negative cell walls, the mordant does not firmly adhere the crystal violet within the thin peptidoglycan layer (even though iodine and crystal violet still react and form large complexes). In the third step of the Gram stain, the decolorizer, ethanol, is added to the cells. The ethanol removes the crystal violet from Gram-negative cells since crystal violet did not adhere strongly to the thin peptidoglycan layer and the cells become transparent. The crystal violet is not removed from the Gram-positive cells since the iodine-crystal violet complexes adhere to the thick peptidoglycan layer. Gram-positive cells remain purple. In the last step of the Gram stain, safranin is used as a counterstain to stain transparent cells (Gram-negative cells) pink. The end result is Gram-positive cells that are purple and Gram-negative cells that are pink.
Exercise 10.4
1. How many species can you see in this Gram stain?
2. What is the Gram (positive or negative) of the cells?
3. What is the cell shape of the cells (see Bacterial Cell Morphology & Arrangement for assistance)?
Answer
Exercise 10.5
1. How many species can you see in this Gram stain?
2. What is the Gram (positive or negative) of the cells?
3. What is the cell shape of the cells (see Bacterial Cell Morphology & Arrangement for assistance)?
Answer
Exercise 10.6
1. How many species can you see in this Gram stain?
2. What is the Gram (positive or negative) of the cells?
3. What is the cell shape of the cells (see Bacterial Cell Morphology & Arrangement for assistance)?
Answer
Improving and Troubleshooting your Gram Stain Approach
Gram staining requires practice. It is an art as much as a science. If your results do not come out as they should, adjust your procedure to correct the problem for future stains.
Figure 5: If you are staining a mixture of Gram-negative and Gram-positive bacteria and you do not see cells, see all cells purple, or see all cells pink, it is likely due to one of these common errors. (1) If you do not see anything (no cells are visible), it is likely because the smear was not properly heat-fixed and the bacteria washed off the slide during staining. (2) If you see all cells appear purple, it indicates that the decolorization step was too short. The next time you do a Gram stain, increase the time for decolorization. (3) If you see all cells appear pink, it indicates that the decolorization step was too long. The next time you do a Gram stain, decrease the time for decolorization.
Factors to control to obtain a proper Gram stain result:
1. Smear preparation. Beginning students tend to make the smears too thick by putting too much inoculum on the slide. This will make it harder to properly decolorize the bacteria. Always put saline or water on the slide before or after adding bacteria when using a culture from solid medium (slant or plate). The amount of inoculum from a solid media should be a “pinpoint” amount. You should just barely touch the organisms you want to stain with your loop. NOTE: You do not need to put saline or water on the slide when making smears from a liquid media.
2. Allow the smear to air dry. This means the slide must sit until it has no longer appears moist. Alternatively, you may use a slide warmer to dry your smear. The slide warmer will dry the slide faster. When using the slide warmer, do not take your eyes off the smear! As soon as the slide is dry, remove it from the slide warmer. If too much heat is used, the cell wall of the organisms may be distorted or rupture and release the crystal violet dye when it should retain it. Extreme overheating may cause the entire cell to lyse, and you will see nothing under the microscope.
3. Heat fixing will adhere the bacteria to the slide and kill the bacteria. If you do not have any cells on the slide, likely the heat fixing step was skipped or not enough to fix the cells to the slide.
4. Freshness and concentration of reagents. Reagents must be fresh, and concentrations must be accurate.
5. Failure to drain slides before adding next reagent. After you wash a slide with water and before you add the next reagent, you must be sure to tilt the slide to allow excess water to drain off it. If you leave a bubble of water over the bacteria, the reagent you add may never reach the bacteria or it will be greatly diluted by the water present on the slide.
6. Age of the bacterial culture. Older Gram-positive organisms lose their ability to retain the violet dye as they move out of the exponential growth phase.
7. Timing of the decolorizing step. This is the most critical stage of the procedure. If the decolorizer is left on too long, Gram-positive organisms will appear Gram-negative. If the decolorizer is not left on long enough, Gram-negative organisms will appear Gram-positive. The thickness of the smear will dictate how long you will need to decolorize. It is impossible to give an exact time. You must decolorize one smear at a time and watch it closely. When the color begins to lift off the smear, you should wash off the decolorizer.
8.
Note
If you mix too aggressively, you will lose the bacterial morphology.
Note
If you heat fix too little, the bacteria will wash off the slide. If you heat fix too much, you will cook the bacteria and denature them
Laboratory Instructions
Prepare a Bacterial Smear
Note
You may find it helpful to draw a circle (wax pencil is best) on the opposite side of the slide where you will spread your smear. This will help you later in locating the smear. The wax pencil is better than a marker because it will not wash off easily from the glass.
Note
If you are using a liquid culture, gently mix the culture until you get an even, cloudy mixture (it should look somewhat like skim milk). If you mix too aggressively, you will lose the bacterial morphology.
1. Prepare a bacterial smear on the slide:
• If you are taking bacteria from a solid culture (slant or petri plate), place a small drop (only 1 drop) of saline, deionized (DI) water, or distilled water onto a microscope slide and use a loop to aseptically add bacteria to the water (avoid taking a large chunk of bacteria since the cells will be too dense to view individual cells). Use the loop to spread the bacteria in the water drop and to spread the water drop out to make it thinner (it will dry faster).
• If you are taking a bacteria from a a liquid culture (broth), place 1 or 2 loopfuls of bacteria directly onto a microscope slide (no saline or water added).
2. Allow the slide(s) to air dry on the slide warmer (or air dry if a slide warmer is not available).
Heat Fix the Bacterial Smear
Once the liquid has completely evaporated on the surface of the slide, heat fix by passing the slide:
1. Attach a wooden clip to the microscope slide to hold it.
2. Pass the underside of the microscope slide through a flame three times.
3. Allow the slide to cool and then continue with your staining protocol.
Note
If you heat fix too little, the bacteria will wash off the slide. If you heat fix too much, you will cook the bacteria and denature them.
Gram Stain
1. Use the slide(s) that you already prepared when creating bacterial smears and heat fixing (see above).
2. Grip the microscope slide in wood clip over a waste container bucket.
3. Primary stain: Add crystal violet (primary stain) onto the smear (there is no reason to cover the entire slide with stain). Allow stain to remain on the smear for 1 minute.
4. Rinse the smear with deionized (DI) water or distilled water.
5. Shake excess water off the smear.
6. Mordant: Add Gram’s iodine onto the smear (there is no reason to cover the entire slide with iodine). Leave for 1 minute.
7. Rinse with deionized (DI) water or distilled water.
8. Shake excess water off the smear.
9. Decolorizer: Tilt the slide at a 45° angle and let the ethanol run over surface of slide until no more crystal violet color comes out of the smear (time varies — no more than 5-15 seconds).
10. Tilt the slide at a 45° angle and rinse the smear with deionized (DI) water or distilled water.
11. Shake excess water off the smear.
12. Counterstain: Add safranin onto the smear (there is no reason to cover the entire slide with stain) for 1 minute.
13. Rinse with deionized water.
14. Shake excess water off the smear.
15. Gently blot (do not wipe) the excess water from the slide using bibulous paper.
16. Place the stained slide on a microscope and examine. Be sure to begin at the lowest power (look for the purple and pink colors of the stains). Focus at lowest power and then increase one objective magnification at a time.
Results & Questions
1. Draw the cells you observe after the Gram stain in the space above.
2. Critique your cell drawings:
• Does the drawing accurately show how the cells appear when you look into the microscope (including cell shape and arrangement)?
• Does the drawing accurately show the cell size you see in the microscope?
• Does the drawing accurately show the color(s) you see in the microscope?
3. After you examined your slide, did it show the Gram stain was successful? If you saw both Gram-positive and Gram-negative cells in the mixture of bacteria then your Gram stain was successful. If not, what did you see (or not see)?
4. If your answer to question 3. indicated that your Gram stain was not successful, what might have occurred? See the section Improving and Troubleshooting your Gram Stain Approach for assistance.
5. What was the cell shape of the Gram-positive cells you observed (see Bacterial Cell Morphology & Arrangement for assistance)?
6. What was the cell shape of the Gram-negative cells you observed (see Bacterial Cell Morphology & Arrangement for assistance)?
7. Staphylococcus aureus is the Gram-positive species you observed today. What does being "Gram-positive" mean about the cell wall structure of S. aureus?
8. Escherichia coli is the Gram-negative species you observed today. What does being "Gram-negative" mean about the cell wall structure of E. coli?
9. Some species of bacteria are Gram-variable. What does this mean?
10. What might occur if the heat fixation step is skipped?
11. What might occur if decolorization is too long?
12. What might occur if decolorization is too short?
13. What is the difference between a simple stain and a differential stain?
14. Is the Gram stain a simple stain or a differential stain? | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.10%3A_Gram_Stain.txt |
Learning Objectives
• Distinguish between prokaryotic cells and eukaryotic cells in terms of structure, size, and the types of organisms that have these cell types.
• Identify structures of bacterial cells in models and diagrams, including details of Gram-positive and Gram-negative cell walls and flagella.
• Describe the functions of the structures found in prokaryotic cells.
• Define endospore.
• Identify the structures and explain the functions of endospores.
• Identify and name bacterial cell shapes and arrangements using correct terminologies.
• Name the correct terms of flagellar arrangements from descriptions, illustrations, and images.
Bacteria are Prokaryotic Cells
Cells fall into one of two broad categories: prokaryotic and eukaryotic. Prokaryotic cells do not have a nucleus (membrane-bound structure that surrounds the cell's DNA). Only eukaryotic cells have a nucleus. Bacteria and archaea are the forms of life that are composed of prokaryotic cells, whereas plants, animals, fungi, and protists (including protozoa) are all composed of eukaryotic cells. In addition to prokaryotic and eukaryotic cells differing from each other based on absence or presence of a nucleus, prokaryotic cells are typically much smaller than eukaryotic cells and also have fewer organelle structures inside of their cells.
All cells (both prokaryotic and eukaryotic) share four common components:
1. a plasma membrane, an outer covering that separates the cell’s interior from its surrounding environment
2. cytoplasm, consisting of a jelly-like cytosol within the cell in which other cellular components are found
3. DNA, the genetic material of the cell
4. ribosomes, which synthesize proteins (prokaryotic ribosomes differ from eukaryotic ribosomes in several ways)
A prokaryote is a simple, mostly single-celled (unicellular) organism that lacks a nucleus, or any other membrane-bound organelle. Prokaryotic DNA is found in a central part of the cell: the nucleoid
prokaryotic cell structure function of this cell structure
capsule enables the cell to attach to surfaces in its environment (including attachment to a host in pathogenic [disease-causing] species)
cell wall acts as an extra layer of protection, helps the cell maintain its shape, and prevents dehydration
cytoplasm semifluid inside of the cell that holds cell components and is the site of the cell's metabolism (its chemical reactions that keep it alive)
flagellum (singular) / flagella (plural) a whip-like tail that rotates to move a cell; prokaryotic cells can have no flagella, one flagellum, or multiple flagella depending on the species
nucleoid a central region within a prokaryotic cell composed of the cell's chromosome (its DNA); the chromosome is a single DNA molecule that is in a circular and comprises the genome of the cell; this chromosome wraps and twist onto itself to form the nucleoid
plasma membrane a fluid membrane that separates the inside of the cell from the outside of the cell; this structure is semipermeable (some materials can pass through the membrane and others cannot)
plasmid (not shown in the figure) a small DNA molecule (often circular) found in the cytoplasm; plasmids are much smaller than the DNA chromosome in the nucleoid; plasmids can be exchanged by cells or picked up by cells to acquire new traits; plasmids can carry antibiotic resistance genes and therefore create challenges for treating some infections when they are shared between cells; prokaryotic cells can survive without plasmids and do not always contain a plasmid, while some may contain multiple plasmids
pilus (singular) / pili (plural) important for attachment to surfaces or other cells, including host cells for pathogenic (disease-causing) cells; some pili, called "sex pili," can be used to exchange genetic material (DNA) between cells during a process called conjugation
ribosome (singular) / ribosomes (plural) small structures found in the cytoplasm that build proteins; proteins are needed to conduct a cell's metabolism, form structures, and do so many other things within a cell; instructions for building different types of proteins come through chemical messages originating in the DNA
Bacterial Cell Shapes & Arrangements
Bacterial cells can have a variety of cell shapes that differ for different species. Each cell shape has a specific terminology used to describe it:
• cocci - round
• bacilli - rods
• vibrios - curved rods
• spirochetes - long, thin, wavy, and helical/corkscrew
• spirilla - long, thin wavy, and not helical/corkscrew
Bacteria that are cocci can be found arranged as:
• solitary - single cells unattached to other cells (may still be seen attached to another cell during division process)
• diplococci - cells attached as pairs
• tetrads - cells are grouped as four attached cells
• sarcina - cells are grouped as eight attached cells
• streptococci - cells are arranged in long chains resembling a string of pearls
• staphylococci - cells are arranged in large bunches
Bacteria that are bacilli can be found arranged as:
• solitary - single cells unattached to other cells (may still be seen attached to another cell during division process)
• diplobacilli - cells attached as pairs
• streptobacilli - cells are arranged in long chains end-to-end
• palisades - cells arranged lined up next to each other long-ways and resembles pickets in a picket fence
To see illustrations and images of cell shapes and cell arrangements as well as more details see Bacterial Cell Morphology & Arrangement in the Simple Stain chapter.
Bacterial Cell Wall Structures
Most bacteria are divided into two major groups: Gram-positive bacteria and Gram-negative bacteria based on the cell wall composition (can be differentiated by using the Gram stain procedure on the bacteria). Knowing the Gram reaction of a clinical isolate (isolated bacterial species from a patient) can help the health care professional make a diagnosis and choose the appropriate antibiotic for treatment.
The results of the Gram stain reflect differences in cell wall composition. Gram-positive cells have thick layers of peptidoglycan (a substance composed of carbohydrates and protein subunits) in their cell walls. Gram-negative bacteria have very little peptidoglycan. Gram-positive bacteria also have teichoic acids, whereas Gram-negative bacteria do not. Gram-negative cells have an outer membrane that resembles the phospholipid bilayer of the plasma membrane. The outer membrane contains lipopolysaccharides (LPS), which are released as endotoxins when Gram-negative cells die. This can be of concern to a person with an infection caused by a Gram-negative organism.
Some species are known as Gram-variable, and so both Gram-positive and Gram-negative reactions may occur when a Gram-variable species is stained using the Gram stain. The vast majority of bacteria are either Gram-positive or Gram-negative. However, not all bacteria can be stained with the Gram stain (for example, Mycoplasma sp., which have no cell wall, stains poorly with the Gram stain).
Bacterial Endospores
An endospore is a form of a bacterial cell. Only some species of bacteria can produce endospores. Endospores are made so the cell can survive poor growth or poor survival conditions. An endospore is a mainly inactive version of a cell that is analagous to a survival bunker. When environmental conditions are poor, or even deadly, bacteria that are able to will produce endospores to survive. When environmental conditions improve, endospores can produce the active cells again (active cells are called vegetative cells.) Bacterial endospores are the most resistant structures of all living organisms, and they can live in this dormant dehydrated state for hundreds of years (even some documented at thousands of years). The stimulus that triggers sporulation (formatio of spores) can vary and may include nutrient depletion, desiccation, chemicals, heat, etc.
Note
Endospores are not for reproduction. One spore forms inside of one vegetative cell (vegetative = metabolically active cell). When environmental conditions improve, one spore germinates to produce one vegetative cell.
Endospore production is a very important characteristic of some bacteria, allowing them to resist adverse environmental conditions such as desiccation, chemical exposure, extreme heat, etc. Endospores were first identified in the 1800s (John Tyndall developed a process for destroying them with an intermittent heating procedure), although the stain procedures to identify them did not develop until the early twentieth century.
The identification of endospores is very important for the clinical microbiologist who is analyzing a patient's body fluid or tissue since there are not that many spore-forming genera. Knowing if the species of bacteria causing an infection forms spores or not helps to narrow down the possible bacterial species causing the infection. In fact, there are two major pathogenic (disease-causing) spore-forming genera: Bacillus and Clostridium. Bacillus and Clostridium species cause a number of dangerous and lethal diseases such as botulism, gangrene, tetanus, c-diff, and anthrax, to name a few.
Some bacteria have to be put into unfavorable situations (high cell density and starvation are two key triggers) to go into sporulation. Other species will make spores easily without much provocation (e.g. Bacillus subtilis). Vegetative cells that have not yet made spores may be in the process of making the spore or will not make them at all. The vegetative cell is metabolically active, whereas the spore is not. The location where an endospore is within a vegetative cell is also useful for distinguishing bacterial species. Endospores may be located in a terminal (end of the cell), subterminal (near the end of the cell), or central (middle of the cell) position. A particular species of the genus will form spores in a specific area, producing another useful taxonomic identification tool and therefore useful in identifying the species of bacteria.
Bacterial Flagella
Flagella are structures used by cells to move in aqueous environments. Bacterial flagella act like propellers. They are stiff spiral filaments composed of flagellin protein subunits that extend outward from the cell and spin in solution. The basal body is the motor for the flagellum and is embedded in the plasma membrane. A hook region connects the basal body to the filament. Gram-positive and gram-negative bacteria have different basal body configurations due to differences in cell wall structure.
Different types of motile bacteria exhibit different arrangements of flagella. A bacterium with a singular flagellum, typically located at one end of the cell (polar), is said to have a monotrichous flagellum. An example of a monotrichously flagellated bacterial pathogen is Vibrio cholerae, the gram-negative bacterium that causes cholera. Cells with amphitrichous flagella have a flagellum or tufts of flagella at each end. An example is Spirillum minor, the cause of spirillary (Asian) rat-bite fever or sodoku. Cells with lophotrichous flagella have a tuft at one end of the cell or both ends of the cell. The gram-negative bacillus Pseudomonas aeruginosa, an opportunistic pathogen known for causing many infections, including “swimmer’s ear” and burn wound infections, has lophotrichous flagella. Flagella that cover the entire surface of a bacterial cell are called peritrichous flagella. The gram-negative bacterium E. coli shows a peritrichous arrangement of flagella.
Laboratory Instructions
Identify Prokaryotic Cell Model Structures
1. Carefully examine cell models of bacterial cells.
2. Identify the following structures on the cell models:
• Prokaryotic structure
Diagram letter
basal body (of flagellum)
capsule
cell wall
cytoplasm
filament (of flagellum)
flagellum
Gram-negative cell wall
Gram-positive cell wall
hook (of flagellum)
lipopolysaccharide
nucleoid
outer membrane
peptidoglycan (thick layer)
peptidoglycan (thin layer)
periplasm (2 places)
pilus
plasma membrane (3 places)
plasmid
ribosome
teichoic acid
Collaboratively Study Prokaryotic Cell Structures
1. Work with your group. You will point to each structure on the prokaryotic cell model to ask each member of your group its name. Wait for the person who you are asking to come up with the correct name of the structure on the model.
2. Your group members will conduct the same process in step 1 so you and the rest of the group get practice recalling the prokaryotic cell structures on the model.
3. Continue this practice until everyone in the group can make the cell structures without looking at the handout (i.e. from memory).
4. You will be quizzed on the prokaryotic cell model by your instructor. They will initial the checkpoint below when you and your group successfully identify structures on the prokaryotic cell model from memory.
Checkpoint: __________
If you are waiting for assistance from your instructor, move on to the next section(s) until they are available.
Prokaryotic Cell Shapes
1. Use a microscope to examine the three different bacterial types on the Bacteria: Three Types slide (there are three different sections to the slide – move the slide around to see all three).
2. Make detailed and clear illustrations of all three types of bacteria in the spaces provided below clearly showing the cell shapes/arrangements.
3. In the spaces provided, indicate the appropriate term or terms that describe the cell shapes (and arrangements if appropriate).
Bacterial Type #1
Bacterial shape/arrangement term(s) appropriate for this bacterial species: ______________________
Bacterial Type #2
Bacterial shape/arrangement term(s) appropriate for this bacterial species: ______________________
Bacterial Type #3
Bacterial shape/arrangement term(s) appropriate for this bacterial species: ______________________
Endospores
1. Examine a slide that shows bacterial endospores.
2. Make a clear illustration of the sample.
3. In the illustration, label an endospore that is forming inside of a vegetative cell as “endospore forming inside vegetative cell”
4. In the illustration, label a free spore as “endospore.”
Flagellar Arrangements
Some bacterial species do not have any flagella. Those bacterial species that do have flagella exhibit flagella in different arrangements. Make illustrations in each box below to show what the terms describing flagellar arrangements look like. If you are uncertain, use the lab manual for help.
monotrichous
amphitrichous
lophotrichous
(draw two different types of arrangements)
peritrichous | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.11%3A_Prokaryotic_Cells.txt |
Learning Objectives
• Describe what an endospore/spore is and why they are important for the bacterial species that form them.
• Identify/give examples of environmental conditions that can stimulate spore formation.
• Tell that Bacillus species and Clostridium species can be clinically important endospore-forming species.
• Compare and contrast "vegetative cell" and "spore."
• Successfully conduct an endospore stain.
• Interpret results of an endospore stain.
• Identify when endospores are terminal, subterminal, and central in microscopic images, diagrams, and descriptions.
• Tell how the endospore stain works including the stains involved and how the stains penetrate cells and do or do not wash out of cells.
• Apply the concept of bacterial endospores to healthcare settings and how spores can make treatment of infections by spore-forming species challenging.
• Tell that endospores are only produced by only some bacterial species, and therefore their presence/absence can be useful in identifying bacterial species.
Endospores
An endospore is a form of a bacterial cell. Only some species of bacteria can produce endospores. Endospores are made so the cell can survive poor growth or poor survival conditions. An endospore is a mainly inactive version of a cell that is analagous to a survival bunker. When environmental conditions are poor, or even deadly, bacteria that are able to will produce endospores to survive. When environmental conditions improve, endospores can produce the active cells again (active cells are called vegetative cells.) Bacterial endospores are the most resistant structures of all living organisms, and they can live in this dormant dehydrated state for hundreds of years (even some documented at thousands of years). The stimulus that triggers sporulation (formatio of spores) can vary and may include nutrient depletion, desiccation, chemicals, heat, etc.
Note
Endospores are not for reproduction. One spore forms inside of one vegetative cell (vegetative = metabolically active cell). When environmental conditions improve, one spore germinates to produce one vegetative cell.
Endospore production is a very important characteristic of some bacteria, allowing them to resist adverse environmental conditions such as desiccation, chemical exposure, extreme heat, etc. Endospores were first identified in the 1800s (John Tyndall developed a process for destroying them with an intermittent heating procedure), although the stain procedures to identify them did not develop until the early twentieth century.
The identification of endospores is very important for the clinical microbiologist who is analyzing a patient's body fluid or tissue since there are not that many spore-forming genera. Knowing if the species of bacteria causing an infection forms spores or not helps to narrow down the possible bacterial species causing the infection. In fact, there are two major pathogenic (disease-causing) spore-forming genera: Bacillus and Clostridium. Bacillus and Clostridium species cause a number of dangerous and lethal diseases such as botulism, gangrene, tetanus, c-diff, and anthrax, to name a few.
Some bacteria have to be put into unfavorable situations (high cell density and starvation are two key triggers) to go into sporulation. Other species will make spores easily without much provocation (e.g. Bacillus subtilis). Vegetative cells that have not yet made spores may be in the process of making the spore or will not make them at all. The vegetative cell is metabolically active, whereas the spore is not. The location where an endospore is within a vegetative cell is also useful for distinguishing bacterial species. Endospores may be located in a terminal (end of the cell), subterminal (near the end of the cell), or central (middle of the cell) position. A particular species of the genus will form spores in a specific area, producing another useful taxonomic identification tool and therefore useful in identifying the species of bacteria.
Endospore Stain
The endospore stain is a differential stain that enables visualization of endospores and differentiation of spores from vegetative cells. The primary stain, malachite green, is a relatively weakly binding stain that attaches to the cell wall of vegetative cells and the spore wall of endospores and mature spores. In fact, if washed well with water, the stain comes right out of the cell walls of vegetative cells. In contrast, malachite green will not wash from the spore wall. Once the stain is in the spore wall, it is locked in and will not wash out. This is why there does not need to be a decolorizer in this stain procedure.
It is difficult to stain the tough spores. Heat is used to enable the malachite green to penetrate the low-permeability spore walls. A variety of chemicals make up the spore wall (keratin protein, calcium), but deeper in the wall is peptidoglycan. The keratin forming the outer portion of the endospore wall resists stain. Heating the cells will make the spore wall more permeable to the malachite green so it can then attach to the peptidoglycan. Once in, the malachite green will not come out because the overlying spore wall becomes less permeable when the smear cools.
When the malachite green is washed out of the vegetative cells they become transparent. A counterstain, safranin, is used to stain the vegetative cells pink. The result is endospores that appear green and vegetative cells that appear pink.
Laboratory Instructions
Prepare a Bacterial Smear
Note
You may find it helpful to draw a circle (wax pencil is best) on the opposite side of the slide where you will spread your smear. This will help you later in locating the smear. The wax pencil is better than a marker because it will not wash off easily from the glass.
Note
If you are using a liquid culture, gently mix the culture until you get an even, cloudy mixture (it should look somewhat like skim milk). If you mix too aggressively, you will lose the bacterial morphology.
1. Prepare a bacterial smear on the slide:
• If you are taking bacteria from a solid culture (slant or petri plate), place a small drop (only 1 drop) of saline, deionized (DI) water, or distilled water onto a microscope slide and use a loop to aseptically add bacteria to the water (avoid taking a large chunk of bacteria since the cells will be too dense to view individual cells). Use the loop to spread the bacteria in the water drop and to spread the water drop out to make it thinner (it will dry faster).
• If you are taking a bacteria from a a liquid culture (broth), place 1 or 2 loopfuls of bacteria directly onto a microscope slide (no saline or water added).
2. Allow the slide(s) to air dry on the slide warmer (or air dry if a slide warmer is not available).
Heat Fix the Bacterial Smear
Once the liquid has completely evaporated on the surface of the slide, heat fix by passing the slide:
Attach a wooden clip to the microscope slide to hold it.
1. Pass the underside of the microscope slide through a flame three times.
2. Allow the slide to cool and then continue with your staining protocol.
Note
If you heat fix too little, the bacteria will wash off the slide. If you heat fix too much, you will cook the bacteria and denature them.
Endospore Stain
1. Put a beaker of water on the hot plate and boil until steam is coming up from the water.
2. Turn the hot plate heat down so that the water is barely boiling.
3. Place a wire stain rack over the beaker. Steam should be coming up through the wire rack.
4. Cut a small piece of paper towel and place it on top of the smear on the slide. The towel will keep the dye from evaporating too quickly, thereby giving more contact time between the dye and the bacterial walls.
5. Flood the smear with the primary dye, malachite green, and leave for 5 minutes. Keep the paper towel moist with the malachite green. DO NOT let the dye dry on the towel.
6. Remove and discard the small paper towel piece.
7. Wash the slide really well with water.
8. Move the slide off the wire rack. The next steps are done without the steam of the hot water.
9. Use a wooden clip and attach it to the slide and put the slide over a small chemical waste bucket.
10. Apply safranin to the smear and leave for 1 minute.
11. Wash the slide well with water allowing the water to run off the slide into the bucket.
12. Blot the dry (do not wipe) with bibulous paper.
13. Place the stained slide on a microscope and examine. Be sure to begin at the lowest power (look for the pink and green colors of the stains). Focus at lowest power and then increase one objective magnification at a time.
Results & Questions
1. Draw the cells you observe after the endospore in the space above.
2. Critique your cell drawings:
• Does the drawing accurately show how the cells appear when you look into the microscope (including cell shape and arrangement)?
• Does the drawing accurately show the cell size you see in the microscope?
• Does the drawing accurately show the color(s) you see in the microscope?
3. The species of bacteria you stained is Bacillus subtilis. Based on the results of your endospore stain, does Bacillus subtilis form endospores? How can you tell?
4. If you observed endospores in your stain, were these endospores terminal, subterminal, or central in the vegetative cells? Explain your answer.
5. What color are the endospores/spores in the endospore stain?
6. What color are the vegetative cells in the endospore stain?
7. Fill in the blanks: The ___________________ are the metabolically active cells that stain ___________________ colored in the endospore stain. The ___________________ are mainly metabolically inactive cells that stain ___________________ colored in the endospore stain.
8. Why might a species of bacteria form endospores? What is the advantage of forming spores for bacteria?
9. What genera of bacteria are known to have pathogenic (disease-causing) species and are endospore-forming genera?
10. Why might infections with species of bacteria that produce spores be more difficult to treat in a healthcare setting than species of bacteria that do not produce spores?
11. What is the purpose of the steam in the endospore stain?
12. Why does there not have to be a decolorizer in this stain? | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.12%3A_Endospore_Stain.txt |
Learning Objectives
• Describe what bacterial capsules are and where they are found in bacterial cells.
• Tell how the capsule stain works.
• Give at least three ways bacterial capsules benefit bacterial cells.
• Successfully conduct a capsule stain.
• Examine, illustrate, label, and interpret results of a capsule stain.
Bacterial Capsules
Bacterial capsules are typically composed of a polysaccharide layer which is thick, detectable, and discrete layer outside the cell wall. Capsules do not stain well for microscopic examination. Because of this, to visualize bacterial capsules, stains/reagents are used in a way that will stain the environment surrounding the bacterial cells and stains the bacterial cell itself, but not the capsule. This creates a non-stained area that is the capsule. Capsules appear as uncolored halos surrounding the bacterial cell.
The ability to produce a capsule is coded in the DNA of the bacteria and can therefore be species-dependent or even strain-dependent. The capsule is not an absolutely essential cellular component for bacteria and some species and strains do not form capsules. Capsules are often produced only under specific growth conditions. The thickness of the capsule can vary depending on the bacterial species, its age, and the medium in which the bacterium is growing. Even though not essential for life, capsules can help bacteria to survive.
Capsules protect pathogenic bacteria from the phagocytic action of immune cells and allow pathogens to invade the body by enabling them to avoid being engulfed by white blood cells. If a pathogenic bacterium loses its ability to form capsules, it often ceases to be pathogenic. Some bacterial species of the microbiome also have capsules to protect them from phagocytosis. Environmental bacteria living in soil and water are protected by their capsules from being engulfed by free-living protozoa.
In addition to protection from phagocytosis, the capsule protects cells against desiccation (drying out). Capsules also enhances the ability of cells to attach. For example, pathogenic bacterial cells with capsules aid in attachment to host cells to establish their growth/infection. Further, capsules enable bacterial cells can attach to other bacteria in biofilms. Bacteria that establish the first layer of a biofilm may utilize their capsules to adhere to a surface and enable attachment of other bacterial cells for biofilm development.
Laboratory Instructions
Capsule Stain (Anthony's Capsule Stain)
1. Gently stir the skim milk broth culture with your loop and place 2-3 loops of Enterobacter aerogenes or Serratia marcescens (whichever is available) on a microscope slide.
2. Using your inoculating loop, spread the sample out to cover about one inch of the slide.
3. Let it completely air dry. Do not heat fix or use the slide warmer. Capsules stick well to glass, and heat may destroy the capsule.
4. Stain with 1% crystal violet for two minutes (do not use the same crystal violet solution as the Gram stain).
5. Gently wash off the excess crystal violet stain with 20% copper sulfate solution.
6. Gently shake off the excess copper sulfate solution and let air dry (do not blot or use slide warmer).
7. Examine the slide with the microscope. The bacterial cell and the milk dried on the slide will pick up the purple dye while the capsule will remain colorless.
Results & Questions
1. Illustrate the bacterial cells viewed in the microscope. Label a bacterial cell and a capsule in your illustration.
2. Where are bacterial capsules located in a bacterial cell?
3. What type of molecule are bacterial capsules typically composed of?
4. Why do bacterial capsules appear white/clear after staining?
5. Give three different ways bacterial capsules give species/strains that have them an advantage.
6. Do all bacteria have/need a capsule? Explain your answer. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.13%3A_Capsule_Stain.txt |
Learning Objectives
• Describe the difference between acid-fast bacteria and non-acid-fast bacteria.
• Explain how the acid-fast stain works comparing acid-fast and non-acid fast bacteria.
• Identify the genera of bacteria that are acid-fast and two examples of diseases caused by these species.
• Differentiate the cell wall structures of acid-fast and non-acid fast bacteria.
• Successfully execute an acid-fast stain and interpret the results.
Acid Fast Stain
Acid fast stain is a differential stain used to identify acid-fast organisms such as members of the genus Mycobacterium. Acid-fast microorganisms are characterized by wax-like, nearly impermeable cell walls; they contain mycolic acid and large amounts of fatty acids, waxes, and complex lipids. This type of cell wall is resistant to most compounds, therefore acid-fast microorganisms require a special staining technique.
The ability of the bacteria to resist decolorization with acid-alcohol confers acid-fastness to the bacterium. Acid-fast bacteria, of which there are very few---the major genus Mycobacterium, have a high concentration of mycolic acid, a lipid, in their cell walls. Although difficult to stain, once the stain goes into the cell wall, the cell will not de-stain or decolorize easily. The ability of the bacteria to resist decolorization with acid-alcohol confers acid-fastness to the bacterium. The phenol in the carbol fuchsin facilitates the dye going into the waxy wall of the bacterium. Acid-fast bacteria stain poorly with the Gram stain procedure, appearing weakly Gram-positive or Gram-variable. They are usually characterized using the acid-fast staining procedure.
Steam is used to get the carbol fuchsin primary dye to go into the cell wall. Once in, it will not come out: But the acid-alcohol decolorizer will take it out of the non-acid fast cell walls since the primary dye does not bind strongly to the cell wall. Non-acid fast bacteria will also take up the carbol fuchsin, but the acid alcohol decolorizer will remove it from wall since the primary dye does not bind strongly to the cell wall.
The primary stain used in acid fast staining, carbol fuchsin, is lipid-soluble and contains phenol, which helps the stain penetrate the cell wall. This is further assisted by the addition of heat in the form of heat (steam). Steam helps to loosen up the waxy layer and promotes entry of the primary stain inside the cell. The smear is then rinsed with a very strong decolorizer (acid-alcohol), which strips the stain from all non-acid-fast cells but does not permeate the cell wall of acid-fast organisms. The decolorized non-acid-fast cells then take up the counterstain, which in our case is methylene blue.
Acid-fastness is an uncommon characteristic shared by the genera Mycobacterium and Nocardia (weakly acid-fast). Because of this feature, this stain is extremely helpful in identification in diseases caused by acid-fast bacteria, particularly tuberculosis and leprosy. In addition, the stain is used to determine the presence of acid-fast bacteria from lung tissue in patients undergoing antibiotic therapy.
Structure and Composition of the Acid-Fast Cell Wall
The mycobacterial cell wall resembles both the Gram-positive and Gram-negative cell walls by having a peptidoglycan layer nearly as thick as Gram-positive cell walls and an outer, waxy layer mimicking the outer membrane of Gram negative cell walls. The cell wall of mycobacteria plays a key role in intrinsic antibiotic resistance and virulence (Forrellad et al. 2013; Becker and Sander 2016).
Acid-fast bacteria are Gram-positive, but in addition to peptidoglycan, the outer membrane or envelope of the acid-fast cell wall of contains large amounts of glycolipids, especially mycolic acids that make up approximately 60% of the acid fast cell wall in the genus Mycobacterium.
The following is a list of the layers of the acid-fast cell wall beginning with the innermost layer (nearest to the plasma membrane):
• Layer 1: Layer of peptidoglycan
• Layer 2: Layer of arabinogalactan (a long carbohydrate composed of the sugars arabinose and galactose)
• Layer 3: Outer membrane containing mycolic acids
• Layer 4: Outer surface is studded with surface proteins that differ with the strain and species of the bacterium
Like the outer membrane of the Gram negative cell wall, porins are required to transport small hydrophilic molecules through the outer membrane of the acid-fast cell wall.
Lab Instructions
1. Label a slide and draw a circle on the center of the slide with a wax pencil.
2. Prepare an emulsion on the slide with 4 loopfuls of Staphylococcus epidermidis from a broth culture onto the slide (these will be your non-acid-fast bacteria).
3. Then, add one loopful of Mycobacterium chelonae (these are your acid-fast bacteria) and mix the two bacteria together.
4. Allow the slide(s) to air dry on the slide warmers (while these slides are drying).
5. Once the liquid has completely evaporated, heat fix the bacteria by passing it through your flame three times.
6. Make sure the slide rack on top of your beaker is completely level. Then, bring your water to boil while the slides are drying. You only need about 200 milliliters of water. If you add more, you will be waiting all lab period for your water to boil.
7. Once the water is boiling, place your slide on the slide rack above the boiling water.
8. Cover the area of your smear on the slide with a square piece of paper towel. Cut the paper towel to make sure none of the paper is hanging off the slide.
9. Carefully apply the carbol fuchsin stain to the paper towel. If a stain appears in the water you are boiling, please stop and discard the stained water in the liquid waste disposal. The fumes from carbol fuchsin can be toxic.
10. Steam with the stain on the slide for 7 minutes while continuously applying more stain so the paper square never dries out.
11. Gently remove the paper with forceps and discard it in the small waste paper cup that will be provided on your bench. Then, rinse the slide with water.
12. Put the slide over the staining basin and gently rinse with water.
13. Decolorize with 6 drops of acid-alcohol (not ethanol from the Gram stain kit), then rinse with water.
14. Counterstain with methylene blue for 2 minutes.
15. Rinse with water and blot dry with bibulous paper (do not use the slide warmer).
16. Examine under the 100x objective lens with oil immersion and record your results.
Results & Analysis
1. Take a photo of the cells or make an illustration of them.
2. Was your acid-fast stain successful? How do you know?
3. What color are the acid-fast bacteria?
4. What color are non-acid-fast negative bacteria?
5. Describe the structure of the cell walls of bacteria that appear fuchsia with the acid-fast stain.
6. Describe what you know about the structure of the cell walls of bacteria that appear blue/purple with the acid-fast stain.
7. What is the medical importance of using the acid-fast stain (what diseases/infections can it help to identify and how might it relate to virulence and antibiotic resistance)?
Works Cited
• Becker K, Sander P. Mycobacterium tuberculosis lipoproteins in virulence and immunity–fighting with a double‐edged sword. FEBS Lett. 2016;590:3800–19.
• Forrellad MA, Klepp LI, Gioffré A et al. . Virulence factors of the Mycobacterium tuberculosis complex. Virulence. 2013;4:3–66. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.14%3A_Acid-Fast_Stain.txt |
Learning Objectives
• Give at least three real-world examples why determining bacterial numbers is an important technique.
• Explain how the standard plate count approach works.
• Calculate CFU of an original sample.
• Explain how absorbance can be used as is a measure of sample turbidity and cell numbers.
• Tell what a spectrophotometer is and how it works.
• Compare and contrast the standard plate count approach and absorbance measurements as it relates to determining cell density in a sample and the type of information these techniques provide about the cells.
• Describe why a standard line correlating CFU and absorbance is important and how it can be used.
• Use a standard line as well as a standard line equation to determine CFU of a sample from absorbance readings.
• Successful conduct plate counts and absorbance readings.
• Graph CFU vs. absorbance and create a standard line.
• Calculate CFU for samples and compare with safety standards to make conclusions about the safety of the samples.
What is the Determination of Bacterial Numbers Approach and What is it Used for?
Many microbiological studies require determining the concentration of viable bacterial cells within a sample. In addition to microbiological research, the concentration of viable bacterial cells is also important to examine to:
• make sure water is safe to drink
• make sure treated municipal wastewater is safe to release into the environment
• make sure water is safe to swim in (ocean, lakes, ponds, pools etc.)
• make sure food products are safe
Professionals in government and industry frequently test the safety of water and foods using the same type of approach described below to ascertain bacterial concentrations.
How does the Determination of Bacterial Numbers Approach Work?
The determination of bacterial numbers approach is used to answer one main question: what is the concentration of viable bacterial cells in this sample? This approach utilizes petri plates to conduct a standard plate count. The plate count will make it possible to determine cell concentrations, but it will take about 24 hours before we have those results.
Pairing the standard plate count technique with a faster method for estimating cell concentrations, absorbance measurements, can provide accurate cell concentrations within a couple minutes (rather than waiting 24 hours for cultures to grow). This involves creating a standard curve using plate count results and absorbance readings. Once a standard curve is produced, the number of bacteria in a sample can be determined using only absorbance readings and the standard curve (without the need to grow petri plate cultures for 24 hours).
Plate Count
Microbiologists use a technique called the standard plate count to estimate the population density of bacteria in a sample by plating a small and dilute portion of the sample and counting the number of bacteria colonies.
Note
A single bacterial colony originates from a single bacterial cell. When a single cell is deposited on a petri plate, it divides to form a colony that is visible with the naked eye. Therefore, when we count then number of isolated bacterial colonies on a petri plate, we are counting the number of cells present in that sample.
We use serial dilutions to create decreasing concentrations of the original sample that are then plated so that a plate will be created with a low enough number of bacteria that we can count individual colonies. From that number, we can calculate the original cell density in the broth. A turbid broth culture of bacteria can have millions or billions of bacterial cells in a single milliliter! If we transferred bacteria directly from that tube to a petri plate, it would be impossible to count colonies since there would be an overwhelming amount of growth on the petri plate (often called a lawn of bacteria).
Since we do not know how many bacteria are in a given sample or culture, we do not know how much we need to dilute the culture in order to grow a petri plate where the number of bacterial colonies is "countable" (we count a plate that has between 25-250 bacterial colonies). Therefore, we do a dilution series to create a number of plates, with a range of dilutions, in the hopes we will produce a countable plate. The number of colonies we count on a petri plate enables us to calculate the CFU or colony forming units. CFU is a measure of the concentration of the live, viable bacterial cells capable of reproducing when grown on a petri plate in cells per milliliter (cells/mL).
To Calculate CFU (cells/mL)
Step 1: Determine the concentration of cells in the diluted sample:
(# of colonies counted on the petri plate) ÷ (amount of diluted sample added to the petri plate in mL) = CFU in diluted sample (cells/mL)
Note: 100 μL = 0.1 mL; 200 μL = 0.2 mL
Step 2: Determine the concentration of cells in the original sample:
(CFU in diluted sample) ÷ (dilution of the petri plate counted) = CFU in original sample (cells/mL)
Example of CFU calculation
You conduct a standard plate count where 200 μL of each dilution is added to each petri plate. The petri plates diluted to 10-2 and 10-4 are lawns and cannot be counted. The 10-6 diluted petri plate results in 129 colonies. The 10-8 diluted petri plate has 12 colonies on it (so it is not used for the CFU calculation since it is not within 25-250 colonies). Calculate the CFU in cells/mL of the original sample based on these results.
Solution
• # of colonies counted = 129
• amount of diluted sample added to the petri plate in mL = 200 μL = 0.2 mL
• dilution of the petri plate counted = 10-6
Step 1: Determine the concentration of cells in the diluted sample:
(# of colonies counted on the petri plate) ÷ (amount of diluted sample added to the petri plate in mL) = CFU in diluted sample (cells/mL)
(129 colonies) ÷ (0.2 mL diluted sample added to petri plate) = 645 cells/mL in the diluted sample
Step 2: Determine the concentration of cells in the original sample:
(CFU in diluted sample) ÷ (dilution of the petri plate counted) = CFU in original sample (cells/mL)
(645 cells/mL in diluted sample) ÷ (10-6 dilution of the petri plate counted) = 645,000,000 cells/mL in original sample
CFU = 645,000,000 cells/mL in original sample
Exercise 15.1
You conduct a standard plate count where 100 μL of each dilution is added to each petri plate. The petri plates diluted to 10-2 and 10-4 and 10-6 are lawns and cannot be counted. The 10-8 diluted petri plate results in 211 colonies. Calculate the CFU in cells/mL of the original sample based on these results.
Answer
Exercise 15.2
You conduct a standard plate count where 50 μL of each dilution is added to each petri plate. The petri plates diluted to 10-2 is a lawn and cannot be counted. The 10-4 diluted petri plate results in 162 colonies. The 10-6 and 10-8 diluted petri plates both had less than 25 colonies on it (so it is not used for the CFU calculation since it is not within 25-250 colonies). Calculate the CFU in cells/mL of the original sample based on these results.
Answer
Exercise 15.3
You conduct a standard plate count where 250 μL of each dilution is added to each petri plate. The petri plates diluted to 10-2 and 10-4 are lawns and cannot be counted. The 10-6 diluted petri plate results in 89 colonies. The 10-8 diluted petri plate has 3 colonies on it (so it is not used for the CFU calculation since it is not within 25-250 colonies). Calculate the CFU in cells/mL of the original sample based on these results.
Answer
Absorbance Readings
Cells can absorb, transmit, and/or reflect light. Spectrophotometry is a technique where the amount of light a substance absorbs, or the amount of light that transmits through the sample, is measured.
A spectrophotometer is an instrument that measures the amount of photons (the intensity of light) absorbed after it passes through sample solution.
Spectrophotometric (turbidimetric) analysis can be used to determine the quantity of cells in a sample based on the amount of light that is absorbed by the sample. It measures how turbid (cloudy) the solution is. When photons of light hit a cell, the light is absorbed, reflected, or diverted in a different direction. Therefore, the amount of light passing through a sample can be used to measure cell density in a sample.
Although using spectrophotometry to measure cell density in a sample can be quite effective, it will yield results that can be different from plate counts. For example, the standard plate count method is an indirect measurement of cell density and reveals information related only to live bacteria. The spectrophotometric analysis is based on turbidity and indirectly measures all bacteria (cell biomass), dead and alive.
Increased turbidity in a culture is another index of bacterial growth and cell numbers (biomass). By using a spectrophotometer, the amount of transmitted light decreases as the cell population increases. The transmitted light is converted to electrical energy, and this is indicated on a galvanometer. The reading, called absorbance or optical density, indirectly reflects the number of bacteria. This method is faster than the standard plate count but is limited because sensitivity is restricted to bacterial suspensions of 107 cells/mL or greater. Samples can be diluted to obtain accurate results when samples contain more than 107 cells/mL or more.
Putting it Together: Using Absorbance as a Quick Way to Determine Cell Concentrations
The CFU concentration determined in the standard plate count can be correlated with absorbance readings. This is done by setting up a graph with absorbance readings of different dilutions on one axis and cell concentrations of that culture at those dilutions on the other axis. Then the CFU is calculated different dilutions and correlated with the corresponding absorbance readings at those same dilutions. These data points are plotted on a graph and a line is drawn the best fits these data. This line, called a trend line or standard line, can be used to determine the cell concentrations of samples with unknown cell concentrations using absorbance only. This enables a microbiologist to quickly determine cell concentrations within a couple minutes rather than taking the time and materials to conduct plate counts for every sample.
Example of Using Position of Standard Line to Determine CFU of a Sample
You have a liquid sample containing the same species of bacteria used to make the standard line in the graph above. You want to find out the cell concentration of the sample in CFU without conducting a standard plate count because you need an answer right away (and not in 24-48 hours). You read the absorbance of the sample in the spectrophotometer. The absorbance reading is 0.1. Use the standard line above to determine the CFU.
Answer
The CFU at 0.1 absorbance is approximately 22.5 x 106 cells/mL or 2.3 x 107 cells/mL
Solution
1. Find the number corresponding to the absorbance on the graph's axis (in this case 0.1 on the x-axis).
2. On the graph, draw a straight line along the absorbance reading on the axis until it reaches the standard line. In this case, draw straight up from 0.1 until it hits the dotted standard line.
3. On the graph, draw a straight line from the point identified in step 2. to the other axis (in this case away toward the y-axis).
4. Where you intersect the other axis, determine the value of this point on the graph. This will be the approximate CFU at this absorbance. At 0.1 absorbance, the standard line is about half way between 25 and 20 (22.5 is halfway between 25 and 20), but the unit given on the graph indicates these numbers should be multiplied by 106. This is how 22.5 x 106 cells/mL was determined as the CFU at this absorbance. We may adjust how we present the number to 2.3 x 107 cells/mL.
Exercise 15.4
You have a liquid sample containing the same species of bacteria used to make the standard line in Figure 6. The absorbance reading of the sample is 0.04. Determine the cell concentration of the sample in CFU without conducting a standard plate count.
Answer
An alternate way, and more accurate way, to determine the CFU from a standard line is to use the equation for the standard line. The equation for the standard line on the graph above is y = 226.21x. The way this graph is set up is with CFU on the y-axis, so the y in this equation is equal to CFU. The way this graph is set up is with absorbance on the x-acis, so the x in this equation is equal to absorbance. If we have an absorbance value, we just plug that number into the location where the x is and calculate what y is. y will be the CFU (but in this case because of the label on the y-axis, the answer will be x 106)
Example of Using Standard Line Equation to Determine CFU of a Sample
The absorbance of a sample is measured at 0.2. Use the equation from the standard line created from data in Figure 6.
Solution
• Equation for standard line: y = 226.21x
• The absorbance (which is x): 0.2
• Set up the equation: y = (226.21) x (0.2)
• Calculate y: 45.242
• Recall that the graph axis indicates y is x 106, so the final answer is: 45.242 x 106 cells/mL or 4.5 x 107 cells/mL
Exercise 15.5
The absorbance of a sample is measured at 0.31. Use the equation from the standard line created from data in Figure 6.
Answer
Exercise 15.6
The absorbance of a sample is measured at 0.18. Use the equation from the standard line created from data in Figure 6.
Answer
Laboratory Instructions
Plate Counts of a Bacterial Culture for Determination of Bacterial Numbers
Dilution Series
1. Obtain 4 microcentrifuge tubes and label them #1-4.
2. Aseptically add 990 μL of sterile water to the tubes labeled #1-4.
3. Mix the broth culture, then aseptically add 10 μL of the culture to tube #1 and vortex.
4. Change the pipette tip. Aseptically transfer 10 μL from tube #1 to tube #2, close the lid and vortex.
5. Change the pipette tip. Aseptically transfer 10 μL from tube #2 to tube #3, close the lid and vortex.
6. Change the pipette tip. Aseptically transfer 10 μL from tube #3 to tube #4, close the lid and vortex.
Plate Culture Dilutions
1. Obtain 4 TSA plates and label them with the dilution of the sample you will add to the plate (10-2, 10-4, 10-6, and 10-8) as well as your group name/number.
2. Add 100 μL from tube #1 onto the center of the petri plate labeled 10-2.
3. Change the pipette tip. Add 100 μL from tube #2 onto onto the center of the petri plate labeled 10-4.
4. Change the pipette tip. Add 100 μL from tube #3 onto the center of the petri plate labeled 10-6.
5. Change the pipette tip. Add 100 μL from tube #4 onto the center of the petri plate labeled 10-8.
6. Put a small amount of ethanol in one half of an empty petri plate (not one of the ones you labeled).
7. Dip the spreader tool in the ethanol.
8. Wave the spreader through the flame of a Bunsen burner. You should see the ethanol burning off the spreader.
9. Pause a moment to allow the spreader tool to cool down.
10. Partially open the petri plate labeled 10-2 and touch the spreader to the agar in a location away from the 100 μL you added to the plate. This will help to cool the spreader and avoid frying the bacteria with the hot spreader.
11. Once cooled, use the spreader to evenly spread the 100 μL you added to the plate all over the surface of the plate. Allow the spreader to gently skid along the surface of the medium. Rotate the petri plate to make it easier to spread the liquid evenly over the surface.
12. Close the petri plate and set aside to dry.
13. Repeat steps 7-12 for each of the petri plates.
14. Put the spreader back in the ethanol to kill any bacteria on it.
15. Allow the petri plates to dry for 5-10 minutes.
16. Invert the petri plates and incubate at 37°C for 24 hours.
17. Count the colonies where appropriate (only between 25 and 250) and record results in the table below.
18. Calculate CFU for the original culture using the colony count that falls between 25 and 250.
Absorbance of a Bacterial Culture for Determination of Bacterial Numbers
Dilution Series
1. Place the original (non-diluted) culture in a test tube rack with four test tubes containing 5 mL of sterile TSB. Label the four test tubes of sterile media as "1/2," "1/4," "1/8," and "1/16."
2. Transfer 5 mL of the original (non-diluted culture), to the tube labeled 1/2. Thoroughly mix.
3. Transfer 5 mL from the 1/2 tube to the tube labeled 1/4. Thoroughly mix.
4. Transfer 5 mL from the 1/4 tube to the tube labeled 1/8. Thoroughly mix.
5. Transfer 5 mL from the 1/8 tube to the tube labeled 1/16. Thoroughly mix.
6. Record your values, along with the dilutions that they came from. Using the plate count data, calculate the colony-forming units per milliliter for each dilution.
Measure Absorbance of Diluted Samples
1. Set the spectrophotometer wavelength at 600 nm.
2. Transfer sterile TSB to a cuvette and place in the spectrophotometer and close the lid.
3. Zero the spectrophotometer.
4. Add 2 mL of the original (non-diluted) culture in a cuvette. Record the absorbance.
5. Add 2 mL of the 1/2 dilution to a cuvette. Record the absorbance.
6. Add 2 mL of the 1/4 dilution to a cuvette. Record the absorbance.
7. Add 2 mL of the 1/8 dilution to a cuvette. Record the absorbance.
8. Add 2 mL of the 1/16 dilution to a cuvette. Record the absorbance.
Results & Questions
Plate Counts of a Bacterial Culture for Determination of Bacterial Numbers
Dilution Plated Amount of the Dilution Plated Number of Colonies
10-2 100 μL
10-4 100 μL
10-6 100 μL
10-8 100 μL
1. Examine the petri plates. Count colonies where you can. If there are more than 250 colonies then simply write "250+." If you cannot distinguish individual colonies write "lawn." If there are less than 25 colonies then simply write "-25." Only plates with 25-250 colonies are used for determining bacterial numbers.
2. Calculate the original CFU in cells/mL in the original culture (before dilution).
3. A plate has 72 colonies with a total dilution factor of 10-7. 100 μL was pipetted onto the plate. What was the CFU concentration the original sample?
Absorbance of a Bacterial Culture for Determination of Bacterial Numbers
Culture Dilution Absorbance (600 nm)
1 (undiluted)
1/2
1/4
1/8
1/16
1. Record the absorbance readings in the table above.
2. Examine any trends in the table able. Finish this sentence: As the culture becomes more dilute, the absorbance _____________.
3. What is an absorbance reading? What does it measure?
4. Briefly explain what a spectrophotometer is and what it does.
5. Describe how an absorbance can indicate the density of cells within a culture.
6. How is the information about the cells in a culture indicated by an absorbance reading different from the information about the cells in a culture indicated by a plate count?
Creating a Standard Line between Plate Counts and Absorbance
Culture Dilution Absorbance (600 nm) CFU (cells/mL)
1 (no dilution)
1/2
1/4
1/8
1/16
1. Complete the table above:
1. Transfer data from absorbance readings in the previous section to the "Absorbance (600 nm)" column in the table above.
2. Transfer the CFU of the original culture calculated in the plate count section to the "CFU (cells/mL)" column in the dilution row that says "1 (no dilution)."
3. Using the original culture CFU, calculate the CFU at a 1/2 dilution and put this value in the "CFU (cells/mL)" column in the dilution row that says 1/2: (CFU) x (1/2)
4. Using the original culture CFU, calculate the CFU at a 1/4 dilution and put this value in the "CFU (cells/mL)" column in the dilution row that says 1/4: (CFU) x (1/4)
5. Using the original culture CFU, calculate the CFU at a 1/8 dilution and put this value in the "CFU (cells/mL)" column in the dilution row that says 1/8: (CFU) x (1/8)
6. Using the original culture CFU, calculate the CFU at a 1/16 dilution and put this value in the "CFU (cells/mL)" column in the dilution row that says 1/4: (CFU) x (1/16)
2. Create a graph with absorbance on the x-axis and CFU (cells/mL) on the y-axis. You can number the y-axis indicating that the number on the axis is x 106, x 107, or x 108 as appropriate for your data. You may graph data using Excel or other graphing software or using graph paper. Follow the instruction of your instructor. Plot each data point as a dot on the graph.
1. Important!
If you are graphing using graph paper, make sure that when you label the axes that each line or box on the graph has a defined value and that every box on that axis counts as that same value. There examples given below showing how to number axes evenly so that each box has a certain unit and remains consistent for that entire side of the graph. In example 1, the y-axis has each box worth 1 and the x-axis has each box worth 0.02 (and only every five boxes are labeled). In example 2, the y-axis has each box worth 0.4 (and only every five boxes are labeled) and the x-axis has each box worth 0.01.
In the "Do not Graph This Way!" column below, an example of what not to do is shown. Notice that the boxes on the axes have been labeled with numbers, but each box does not have a defined unit and the numbering is not consistent. Do not do this.
Graph This Way (Every Box on the Axis has a Defined Value that is Consistent on that Axis) Do not Graph This Way! (Boxes on the Axis Do Not have a Defined Value)
Example 1:
Example 2:
Example of what NOT to do:
3. If you used graphing software add a trend line to your data and set the y-intercept at zero. Also make sure to have the software place the equation for the line on the graph. If you are using graph paper, use a ruler and it place along the data points making sure the ruler passes through the bottom left corner of the graph corresponding with zero on the x-axis and the y-axis. Draw a straight line using the ruler that best matches the data points. Only draw one straight line. This line does not have to touch every data point (and it probably will not). Do not connect each data point with the ruler with multiple lines. There should only be one straight line on the graph (see Figure 6 for an example).
4. After creating the standard curve above you continue your research with this same bacterial species, but now instead of doing the plate counts (which took a lot of time and materials) you simply use a spectrophotometer to measure absorbance to determine CFU (cells/mL) using the standard curve. As you continue your research, you measure the bacterial samples using the spectrophotometer. Use the standard curve you created to determine the amount of CFUs in each sample in the table below based on the absorbance. If you used graphing software, determine CFUs using the trend line equation (see example of using trend line equation to determine CFU from absorbance). If you used graph paper, use the position of the trend line as it relates to the absorbance reading to determine CFUs (see example of using position of the trend line to determine CFU from absorbance).
1. Absorbance (600 nm) CFU (cells/mL)
0.15
0.02
0.3
0.25
5. Explain the purpose of constructing the calibration curve.
6. You count colonies on a plate where 100 μL was added of a 10-3 dilution. The colony count is 53. How many cells per milliliter are in your original sample?
7. You count colonies on plate where 100 μL was added of a 10-5 final dilution. Your count is 129. How many cells per milliliter are in your original sample?
8. You are testing a sample of water from a local swimming pool. An undiluted sample (100) where 100 μL was added to a petri plate produced 38 colonies. The safety standards for swimming pools state that the water cannot contain more than 200 CFU per milliliter. Is the pool you are testing safe to swim in? Why or why not?
9. As part of an ongoing recreational waters safety monitoring program, you are testing a sample of water from swimming beaches in Morro Bay, CA. An undiluted sample (100) where 100 μL was added to a petri plate produced 1 Escherichia coli colony. The EPA safety standards for recreational water cannot contain more than 235 E. coli per 100 mL. Is the beach safe to swim in? Why or why not?
10. You are testing a sample of milk from a local dairy that has been cited for some recent safety violations. A 10-1 dilution where 100 μL was added to a petri plate produced 201 colonies on it. The FDA safety standard for milk of the type you sampled is 3 x 104 per milliliter (above this threshold and the milk is unsafe). Is this milk sample safe to drink? Why or why not? | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.15%3A_Determination_of_Bacterial_Numbers.txt |
Learning Objectives
• Differentiate between eukaryotic cell and prokaryotic cell structures.
• Identify the structures and functions of components of eukaryotic cells.
• Name the categories of microorganisms that are eukaryotic.
• Provide a description that differentiates each type of eukaryotic microorganism from the other types of eukaryotic microorganisms.
• Examine specimens of different types of eukaryotic microorganisms and identify structures of these microorganisms, especially the nucleus.
Eukaryotic Cells
The cells of eukaryotic organisms have several distinguishing characteristics. Above all, eukaryotic cells are defined by the presence of a nucleus surrounded by a complex nuclear membrane. Also, eukaryotic cells are characterized by the presence of membrane-bound organelles in the cytoplasm. Organelles such as mitochondria, the endoplasmic reticulum (ER), Golgi apparatus, lysosomes, and peroxisomes are held in place by the cytoskeleton, an internal network that supports transport of intracellular components and helps maintain cell shape. The genome of eukaryotic cells is packaged in multiple, rod-shaped chromosomes as opposed to the single, circular-shaped chromosome that characterizes most prokaryotic cells.
Table 1: A comparison of prokaryotic cells and eukaryotic cells.
Summary of Cell Structures
Prokaryotes Eukaryotes
Bacteria Archaea
size ~0.5–1 μm ~0.5–1 μm ~5–20 μm
surface area-to-volume ratio High High Low
nucleus No No Yes
genome characteristics
• Single chromosome
• Circular
• Haploid
• Lacks histones
• Single chromosome
• Circular
• Haploid
• Contains histones
• Multiple chromosomes
• Linear
• Haploid or diploid
• Contains histones
cell division Binary fission Binary fission Mitosis, meiosis
membrane lipid composition
• Ester-linked
• Straight-chain fatty acids
• Bilayer
• Ether-linked
• Branched isoprenoids
• Bilayer or monolayer
• Ester-linked
• Straight-chain fatty acids
• Sterols
• Bilayer
cell wall composition
• Peptidoglycan, or
• None
• Pseudopeptidoglycan, or
• Glycopeptide, or
• Polysaccharide, or
• Protein (S-layer), or
• None
• Cellulose (plants, some algae)
• Chitin (fungi)
• Silica (some algae)
• Most others lack cell walls
motility structures Rigid spiral flagella composed of flagellin Rigid spiral flagella composed of archaeal flagellins Flexible flagella and cilia composed of microtubules
membrane-bound organelles No No Yes
endomembrane system No No Yes (ER, Golgi, lysosomes)
ribosomes 70S 70S
• 80S in cytoplasm and rough ER
• 70S in mitochondria, chloroplasts
Table 2: Functions of Eukaryotic Cell Structures.
Cell Structure Function
cell wall structure outside of the plasma membrane in some types of species; maintains cell shape
centrioles / centrosome organizes microtubules (particularly important during cell division to move the chromosomes and separate them correctly into the two daughter cells)
chloroplast conducts photosynthesis
chromatin DNA with its associated proteins
cilia external-facing protein fibers that wave to move a cell
cytoplasm semi-fluid surrounding cellular structures with dissolved molecules; location of many cellular metabolic reactions
cytoskeleton microtubules, intermediate filaments, and microfilaments (provides a cell shape, structure, can be used for cell movement or moving materials around the cell)
flagellum / flagella a whip-like tail that moves a cell
Golgi complex / apparatus / body modifies proteins and packages them into vesicles for transport to their destinations
lysosome digests food and waste materials
mitochondria / mitochondrion makes ATP (an energy-rich molecule) using nutrients
nuclear membrane / nuclear envelope membrane enclosing the nucleus; protein-lined pores allow materials to move in and out of the nucleus
nuclear pore protein-lined pores allow materials to move in and out of the nucleus
nucleolus a condensed region within the cell nucleus where ribosomes are formed
nucleus contains chromatin, a nuclear envelope, and a nucleolus
peroxisomes metabolizes oxygen-containing waste
ribosomes makes proteins
rough endoplasmic reticulum membranes associated with ribosomes where the ribosomes make membrane proteins and proteins for secretion out of the cell
smooth endoplasmic reticulum membranes without ribosomes; make lipids; detoxification
Microbes with Eukaryotic Cells
Eukaryotic organisms include protozoans, algae, fungi, plants, and animals. Some eukaryotic cells are independent, single-celled microorganisms, whereas others are part of multicellular organisms.
Eukaryotic microbes are an extraordinarily diverse group, including species with a wide range of life cycles, morphological specializations, and nutritional needs. Although more diseases are caused by viruses and bacteria than by microscopic eukaryotes, these eukaryotes are responsible for some diseases of great public health importance. For example, the protozoal disease malaria was responsible for 584,000 deaths worldwide (primarily children in Africa) in 2013, according to the World Health Organization (WHO). The protozoan parasite Giardia causes a diarrheal illness (giardiasis) that is easily transmitted through contaminated water supplies. In the United States, Giardia is the most common human intestinal parasite. Although it may seem surprising, parasitic worms are included within the study of microbiology because identification depends on observation of microscopic adult worms or eggs. Even in developed countries, these worms are important parasites of humans and of domestic animals. There are fewer fungal pathogens, but these are important causes of illness, as well. On the other hand, fungi have been important in producing antimicrobial substances such as penicillin.
Protozoa
Protozoa are nonphotosynthetic, motile eukaryotic organisms that are always unicellular. Historically, these microorganisms have been classified as protozoa because they were "animal like" unicellular eukaryotic organisms that did not fit with other eukaryotic taxonomic groupings (plants, fungi, or animals).
Protozoans inhabit a wide variety of habitats, both aquatic and terrestrial. Many are free-living, while others are parasitic, carrying out a life cycle within a host or hosts and potentially causing illness. There are also beneficial symbionts that provide metabolic services to their hosts. During the feeding and growth part of their life cycle, they are called trophozoites; these feed on small particulate food sources such as bacteria. While some types of protozoa exist exclusively in the trophozoite form, others can develop from trophozoite to an encapsulated cyst stage when environmental conditions are too harsh for the trophozoite. A cyst is a cell with a protective wall, and the process by which a trophozoite becomes a cyst is called encystment. When conditions become more favorable, these cysts are triggered by environmental cues to become active again through excystment.
Protozoans have a variety of reproductive mechanisms. Some protozoans reproduce asexually and others reproduce sexually; still others are capable of both sexual and asexual reproduction. In protozoans, asexual reproduction occurs by binary fission, budding, or schizogony. In schizogony, the nucleus of a cell divides multiple times before the cell divides into many smaller cells. The products of schizogony are called merozoites and they are stored in structures known as schizonts. Protozoans may also reproduce sexually, which increases genetic diversity and can lead to complex life cycles. Protozoans can produce haploid gametes that fuse through syngamy. However, they can also exchange genetic material by joining to exchange DNA in a process called conjugation. This is a different process than the conjugation that occurs in bacteria. The term protist conjugation refers to a true form of eukaryotic sexual reproduction between two cells of different mating types. It is found in ciliates, a group of protozoans, and is described later in this subsection.
All protozoans have a plasma membrane, or plasmalemma, and some have bands of protein just inside the membrane that add rigidity, forming a structure called the pellicle. Some protists, including protozoans, have distinct layers of cytoplasm under the membrane. In these protists, the outer gel layer (with microfilaments of actin) is called the ectoplasm. Inside this layer is a sol (fluid) region of cytoplasm called the endoplasm. These structures contribute to complex cell shapes in some protozoans, whereas others (such as amoebas) have more flexible shapes.
Figure 4: Amoeba motion involves formation of pseudopodia ("false feet"). These extensions enable the cell to creep along in a liquid environmental and also means that the shape of the cell is constantly changing.
Different groups of protozoans have specialized feeding structures. They may have a specialized structure for taking in food through phagocytosis, called a cytostome, and a specialized structure for the exocytosis of wastes called a cytoproct. Oral grooves leading to cytostomes are lined with hair-like cilia to sweep in food particles. Protozoans are heterotrophic. Protozoans that are holozoic ingest whole food particles through phagocytosis. Forms that are saprozoic ingest small, soluble food molecules.
Many protists have whip-like flagella or hair-like cilia made of microtubules that can be used for locomotion. Other protists use cytoplasmic extensions known as pseudopodia (“false feet”) to attach the cell to a surface; they then allow cytoplasm to flow into the extension, thus moving themselves forward.
Protozoans have a variety of unique organelles and sometimes lack organelles found in other cells. Some have contractile vacuoles, organelles that can be used to move water out of the cell for osmotic regulation (salt and water balance). Mitochondria may be absent in parasites or altered to kinetoplastids (modified mitochondria) or hydrogenosomes.
Figure 6: A free-living ciliated cell with contractile vacuole pulsating to regulate water and salt balance in the cell.
Algae
The algae are autotrophic eukaryotes that can be unicellular or multicellular and are distinct from other eukaryotic groupings (plants, fungi, and animals). These organisms are found in the supergroups Chromalveolata (dinoflagellates, diatoms, golden algae, and brown algae) and Archaeplastida (red algae and green algae). They are important ecologically and environmentally because they are responsible for the production of approximately 70% of the oxygen and organic matter in aquatic environments. Some types of algae, even those that are microscopic, are regularly eaten by humans and other animals. Additionally, algae are the source for agar, agarose, and carrageenan, solidifying agents used in laboratories and in food production. Although algae are typically not pathogenic, some produce toxins. Harmful algal blooms, which occur when algae grow quickly and produce dense populations, can produce high concentrations of toxins that impair liver and nervous-system function in aquatic animals and humans.
Like protozoans, algae often have complex cell structures. For instance, algal cells can have one or more chloroplasts that contain structures called pyrenoids to synthesize and store starch. The chloroplasts themselves differ in their number of membranes, indicative of secondary or rare tertiary endosymbiotic events. Primary chloroplasts have two membranes—one from the original cyanobacteria that the ancestral eukaryotic cell engulfed, and one from the plasma membrane of the engulfing cell. Chloroplasts in some lineages appear to have resulted from secondary endosymbiosis, in which another cell engulfed a green or red algal cell that already had a primary chloroplast within it. The engulfing cell destroyed everything except the chloroplast and possibly the cell membrane of its original cell, leaving three or four membranes around the chloroplast. Different algal groups have different pigments, which are reflected in common names such as red algae, brown algae, and green algae.
Some algae, the seaweeds, are macroscopic and may be confused with plants. Seaweeds can be red, brown, or green, depending on their photosynthetic pigments. Green algae, in particular, share some important similarities with land plants; however, there are also important distinctions. For example, seaweeds do not have true tissues or organs like plants do. Additionally, seaweeds do not have a waxy cuticle to prevent desiccation. Algae can also be confused with cyanobacteria, photosynthetic bacteria that bear a resemblance to algae; however, cyanobacteria are prokaryotes.
Algae have a variety of life cycles. Reproduction may be asexual by mitosis or sexual using gametes.
Fungi
The fungi comprise a diverse group of organisms that are heterotrophic and typically saprozoic (consume dead organic matter). In addition to the well-known macroscopic fungi (such as mushrooms and molds), many unicellular yeasts and spores of macroscopic fungi are microscopic. For this reason, fungi are included within the field of microbiology.
Fungi are important to humans in a variety of ways. Both microscopic and macroscopic fungi have medical relevance, with some pathogenic species that can cause mycoses (illnesses caused by fungi). Some pathogenic fungi are opportunistic, meaning that they mainly cause infections when the host’s immune defenses are compromised and do not normally cause illness in healthy individuals. Fungi are important in other ways. They act as decomposers in the environment, and they are critical for the production of certain foods such as cheeses. Fungi are also major sources of antibiotics, such as penicillin from the fungus Penicillium.
Fungi have well-defined characteristics that set them apart from other organisms. Most multicellular fungal bodies, commonly called molds, are made up of filaments called hyphae. Hyphae can form a tangled network called a mycelium and form the thallus (body) of fleshy fungi. Hyphae that have walls between the cells are called septate hyphae; hyphae that lack walls and cell membranes between the cells are called nonseptate or coenocytic hyphae).
In contrast to molds, yeasts are unicellular fungi. The budding yeasts reproduce asexually by budding off a smaller daughter cell; the resulting cells may sometimes stick together as a short chain or pseudohypha.
Some fungi are dimorphic, having more than one appearance during their life cycle. These dimorphic fungi may be able to appear as yeasts or molds, which can be important for infectivity. They are capable of changing their appearance in response to environmental changes such as nutrient availability or fluctuations in temperature, growing as a mold, for example, at 25 °C (77 °F), and as yeast cells at 37 °C (98.6 °F). This ability helps dimorphic fungi to survive in diverse environments. Two examples of dimorphic yeasts are the human pathogens Histoplasma capsulatum and Candida albicans. H. capsulatum causes the lung disease histoplasmosis, and C. albicans is associated with vaginal yeast infections, oral thrush, and candidiasis of the skin.
There are notable unique features in fungal cell walls and membranes. Fungal cell walls contain chitin, as opposed to the cellulose found in the cell walls of plants and many protists. Additionally, whereas animals have cholesterol in their cell membranes, fungal cell membranes have different sterols called ergosterols. Ergosterols are often exploited as targets for antifungal drugs.
Fungal life cycles are unique and complex. Fungi reproduce sexually either through cross- or self-fertilization. Haploid fungi form hyphae that have gametes at the tips. Two different mating types (represented as “+ type” and “– type”) are involved. The cytoplasms of the + and – type gametes fuse (in an event called plasmogamy), producing a cell with two distinct nuclei (a dikaryotic cell). Later, the nuclei fuse (in an event called karyogamy) to create a diploid zygote. The zygote undergoes meiosis to form spores that germinate to start the haploid stage, which eventually creates more haploid mycelia. Depending on the taxonomic group, these sexually produced spores are known as zygospores (in Zygomycota), ascospores (in Ascomycota), or basidiospores (in Basidiomycota).
Fungi may also exhibit asexual reproduction by mitosis, mitosis with budding, fragmentation of hyphae, and formation of asexual spores by mitosis. These spores are specialized cells that, depending on the organism, may have unique characteristics for survival, reproduction, and dispersal. Fungi exhibit several types of asexual spores and these can be important in classification.
Helminths
Parasitic helminths are animals that are often included within the study of microbiology because many species of these worms are identified by their microscopic eggs and larvae. There are two major groups of parasitic helminths: the roundworms (Nematoda) and flatworms (Platyhelminthes). Of the many species that exist in these groups, about half are parasitic and some are important human pathogens. As animals, they are multicellular and have organ systems. However, the parasitic species often have limited digestive tracts, nervous systems, and locomotor abilities. Parasitic forms may have complex reproductive cycles with several different life stages and more than one type of host. Some are monoecious, having both male and female reproductive organs in a single individual, while others are dioecious, each having either male or female reproductive organs.
Nematoda (Roundworms)
Phylum Nematoda (the roundworms) is a diverse group containing more than 15,000 species, of which several are important human parasites. These unsegmented worms have a full digestive system even when parasitic. Some are common intestinal parasites, and their eggs can sometimes be identified in feces or around the anus of infected individuals. Ascaris lumbricoides is the largest nematode intestinal parasite found in humans; females may reach lengths greater than 1 meter. A. lumbricoides is also very widespread, even in developed nations, although it is now a relatively uncommon problem in the United States. It may cause symptoms ranging from relatively mild (such as a cough and mild abdominal pain) to severe (such as intestinal blockage and impaired growth).
Figure 10: Enterobius vermicularis is a type of parasitic roundworm.
Of all nematode infections in the United States, pinworm (caused by Enterobius vermicularis) is the most common. Pinworm causes sleeplessness and itching around the anus, where the female worms lay their eggs during the night. Toxocara canis and T. cati are nematodes found in dogs and cats, respectively, that can be transmitted to humans, causing toxocariasis. Antibodies to these parasites have been found in approximately 13.9% of the U.S. population, suggesting that exposure is common.7 Infection can cause larval migrans, which can result in vision loss and eye inflammation, or fever, fatigue, coughing, and abdominal pain, depending on whether the organism infects the eye or the viscera. Another common nematode infection is hookworm, which is caused by Necator americanus (the New World or North American hookworm) and Ancylostoma duodenale (the Old World hookworm). Symptoms of hookworm infection can include abdominal pain, diarrhea, loss of appetite, weight loss, fatigue, and anemia.
Trichinellosis, also called trichinosis, caused by Trichinella spiralis, is contracted by consuming undercooked meat, which releases the larvae and allows them to encyst in muscles. Infection can cause fever, muscle pains, and digestive system problems; severe infections can lead to lack of coordination, breathing and heart problems, and even death. Finally, heartworm in dogs and other animals is caused by the nematode Dirofilaria immitis, which is transmitted by mosquitoes. Symptoms include fatigue and cough; when left untreated, death may result.
Platyhelminths (Flatworms)
Phylum Platyhelminthes (the platyhelminths) are flatworms. This group includes the flukes, tapeworms, and the turbellarians, which include planarians. The flukes and tapeworms are medically important parasites.
The flukes (trematodes) are nonsegmented flatworms that have an oral sucker (and sometimes a second ventral sucker) and attach to the inner walls of intestines, lungs, large blood vessels, or the liver. Trematodes have complex life cycles, often with multiple hosts. Several important examples are the liver flukes (Clonorchis and Opisthorchis), the intestinal fluke (Fasciolopsis buski), and the oriental lung fluke (Paragonimus westermani). Schistosomiasis is a serious parasitic disease, considered second in the scale of its impact on human populations only to malaria. The parasites Schistosoma mansoni, S. haematobium, and S. japonicum, which are found in freshwater snails, are responsible for schistosomiasis. Immature forms burrow through the skin into the blood. They migrate to the lungs, then to the liver and, later, other organs. Symptoms include anemia, malnutrition, fever, abdominal pain, fluid buildup, and sometimes death.
The other medically important group of platyhelminths are commonly known as tapeworms (cestodes) and are segmented flatworms that may have suckers or hooks at the scolex (head region). Tapeworms use these suckers or hooks to attach to the wall of the small intestine. The body of the worm is made up of segments called proglottids that contain reproductive structures; these detach when the gametes are fertilized, releasing gravid proglottids with eggs. Tapeworms often have an intermediate host that consumes the eggs, which then hatch into a larval form called an oncosphere. The oncosphere migrates to a particular tissue or organ in the intermediate host, where it forms cysticerci. After being eaten by the definitive host, the cysticerci develop into adult tapeworms in the host's digestive system. Taenia saginata (the beef tapeworm) and T. solium (the pork tapeworm) enter humans through ingestion of undercooked, contaminated meat. The adult worms develop and reside in the intestine, but the larval stage may migrate and be found in other body locations such as skeletal and smooth muscle. The beef tapeworm is relatively benign, although it can cause digestive problems and, occasionally, allergic reactions. The pork tapeworm can cause more serious problems when the larvae leave the intestine and colonize other tissues, including those of the central nervous system. Diphylobothrium latum is the largest human tapeworm and can be ingested in undercooked fish. It can grow to a length of 15 meters. Echinococcus granulosus, the dog tapeworm, can parasitize humans and uses dogs as an important host.
Laboratory Activities
Eukaryotic Cell Structures
A: ______________________________
B: ______________________________
C: ______________________________
D: ______________________________
E: ______________________________
F: ______________________________
G: ______________________________
H: ______________________________
I: ______________________________
J: ______________________________
K: ______________________________
L: ______________________________
M: ______________________________
N: ______________________________
O: ______________________________
1. Examine the eukaryotic cell model and name the cell structures labeled with letters A-O.
2. Match the descriptions with the corresponding eukaryotic cell structures.
1. _____ rigid structure outside of the plasma membrane that creates cell structure, support and shape
2. _____ short protein fibers on the outside of the cell that wave to move the cell
3. _____ dense region inside of the nucleus where ribosomes are put together
4. _____ membrane sphere where oxygen-containing toxins are destroyed
5. _____ complex of DNA and protein found in the nucleus
6. _____ a cell structure where photosynthesis occurs
7. _____ a network of different types of protein strands that support the cell structure, organize the cell, can enable cell movement, and help transport materials around the cell
8. _____ tiny structures that make proteins
9. _____ small passageways in the nuclear membrane where materials can pass in and out of the nucleus
10. _____ a membrane composed of phospholipids and other structures that separates the inside and outside of the cell and controls what materials move in and out of the cell
11. _____ a series of membranes where lipids are made and some detoxification activities take place
12. _____ location where many cellular molecules are dissolved in a semi-fluid and cellular reactions take place
13. _____ membrane sphere where food and waste materials are broken down
14. _____ long and slender external protein filament that move in order to move a cell around its environment
15. _____ a membrane that separates the nucleus from the rest of the cell
16. _____ a large cell structure separated by a membrane where DNA is stored and protected
17. _____ a series of membranes with ribosomes attached that produce membrane proteins and external proteins
18. _____ a cell structure where ATP (an energy-rich molecule) is produced using nutrients
19. _____ a series of membranes where proteins are modified, packaged, and transported to their destinations
A. nucleolus
B. cytoplasm
C. ribosomes
D. nuclear membrane
E. rough endoplasmic reticulum
F. plasma membrane
G. cytoskeleton
H. lysosome
I. peroxisome
J. nucleus
K. nuclear pore
L. Golgi complex
M. chloroplast
N. mitochondria
O. cilia
P. flagella
Q. cell wall
R. smooth endoplasmic reticulum
S. chromatin
Examine Microbes that have Eukaryotic Cells
Protozoa
Your instructor will provide you with a slide of a species of protozoan. Examine the protozoan with the microscope, make an illustration in the location below, label the nucleus of the protozoan cell, and label any additional cell structures you can identify.
Algae
Your instructor will provide you with a slide of a species of algae. Examine the algae with the microscope, make an illustration in the location below, label the nucleus of the algal cell, and label any additional cell structures you can identify.
Fungi
Your instructor will provide you with a slide of a species of fungus. Examine the fungus with the microscope, make an illustration in the location below, label the nucleus of the fungal cell, and label any additional cell structures you can identify.
Helminth
Your instructor will provide you with a slide of a species of helminth. Examine the helminth with the microscope, make an illustration in the location below and label any structures you can identify. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.16%3A_Eukaryotic_Cells.txt |
Learning Objectives
• Explain what the starch hydrolysis test is and how it works.
• Explain how starch hydrolysis relates to the amylase gene and the enzyme amylase.
• State the chemical reaction that occurs when bacteria hydrolyze starch.
• Successfully conduct the starch hydrolysis test.
• Interpret the results of the starch hydrolysis test.
• State why starch hydrolysis is a beneficial characteristic for bacteria.
• Tell that starch hydrolysis is a characteristic that only some bacterial species have and that is therefore useful for species identification and characterization.
Starch Hydrolysis Test
Starch is a long carbohydrate molecule made of hundreds of glucose molecules bonded together into a very long chain. There is a lot of energy in starch! If bacteria are able to break down starch, this is an advantage because that means they can access the sugars in starch and the energy in those sugars (as well as the carbon).
Some species of bacteria can break down starch and some species of bacteria cannot break down starch. Whether a bacterial species can break down starch or not is based on whether they have a gene (a segment of DNA) that codes for the enzyme amylase. Amylase is an enzyme that breaks down starch into smaller sugars.
Starch is too large to pass through the plasma membrane of a cell and must be split into individual glucose molecules. Bacteria that can produce the exoenzyme amylase are able to hydrolyze starch by secreting these enzymes into the environment around them. The enzyme amylase is secreted out of the cells (amylase is considered an exoenzyme, meaning it is an enzyme secreted outside of the cells ["exo-" means "outside"]) into the surrounding media, catalyzing the breakdown of starch into smaller sugars. These smaller sugars can then be absorbed by the cells and the cells can use the sugars as a source of energy and carbon.
Since only some species of bacteria are capable of breaking down starch (aka starch hydrolysis) testing if bacteria can break down starch or not is a way we can differentiate one bacterial species from another. Therefore, examining starch hydrolysis of a bacterial species is useful for identifying and characterizing bacterial species. Starch can be added to petri plate media in order to determine if bacteria are capable of breaking down the starch.
Starch inside of a petri plate is clear and cannot be seen without using iodine. Iodine reacts with starch, producing a black or bluish-black color. As starch is hydrolyzed by bacterial amylase and is converted to sugars, there will be less and less starch to react with the iodine. Strong amylase producers may convert all of the starch in the agar to sugars, while weak amylase producers may convert the starch surrounding the growth areas only.
After bacteria are allowed to grow, iodine is added to the petri plate to detect the presence and absence of starch.
• Where starch is on the petri plate will appear blue-black. Iodine reacts with starch to produce blue-black coloration when starch is present.
• Where starch isn't on the petri plate will appear clear or yellowish. Iodine will not produce a blue-black color where starch is absent. These regions therefore appear a clear or yellowish color on the petri plate when starch is not present.
Since the entire petri plate contained starch at the start of the test, clear halos surrounding bacterial growth indicates that the bacterial species was able to hydrolyze the starch resulting in the clear zone without starch (we would say that this bacterial species is starch hydrolysis positive). If the growth does not exhibit clear zones surrounding it, the starch is intact throughout the plate (no starch hydrolysis or we say that the bacterial species is starch hydrolysis negative).
Laboratory Instructions
Inoculating Starch Plates
1. Obtain a starch agar petri plate.
2. Write on the bottom of the starch agar petri plate to separate the plate into four sections. Label as shown above.
3. Aseptically make a single line streak of the corresponding bacterial species in each of the sections on the petri plate. The "control" region will remain empty (no bacterial streak).
4. Invert the petri plate and incubate at either 25º C or 37º C for 24-28 hours.
Adding Iodine to Visualize Location of Starch
1. After the starch plate has incubated and bacteria is growing on the surface, placing the agar plate on a white piece of paper or white background since this will really help you to distinguish whether or not clear zones occurred.
2. Cover the agar and growth with iodine.
3. Examine the petri plate. Species that hydrolyze starch will have a region around the bacterial growth that is yellowish or clear around the growth (the growth may appear light in color - look for a clear zone surrounding the growth [the growth is raised on the plate]). When starch is not hydrolyzed, the growth may appear lighter in color, but there is no clear zone surrounding the growth (unless the clear zone is extending from a bacterial species that does hydrolyze starch.
Results & Questions
Bacterial Species Result for Starch Hydrolysis (+ or -) Does this species produce amylase? Does this species have the amylase gene?
Escherichia coli
Bacillus subtilis
Proteus vulgaris
1. Complete the table above to indicate the results of this experiment.
2. What does it mean, in the starch hydrolysis test, when there is a clear zone surrounding bacterial growth?
3. What does it mean, in the starch hydrolysis test, when there is no clear zone surrounding bacterial growth?
4. What is hydrolysis?
5. What is starch hydrolysis?
6. What is amylase?
7. Give the chemical reaction catalyzed by amylase.
8. Why is it an advantage for a bacterial species to be able to hydrolyze starch?
9. Do all bacteria hydrolyze starch? Explain your answer.
10. Fill in the blank. Whether a species of bacteria is capable of producing amylase is dependent on _________.
11. The starch hydrolysis test is useful for identifying and characterizing bacteria. Why? | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.17%3A_Starch_Hydrolysis.txt |
Learning Objectives
• Describe what catalase is and why it is important for bacterial survival.
• Give the chemical reaction catalyzed by catalase.
• Explain how testing for catalase is useful for characterizing and identifying bacterial species.
• Tell the types of bacterial species that typically produce catalase and those that typically do not produce catalase.
• Successfully conduct a catalase test.
• Interpret results for a catalase test.
Catalase
Catalase is an enzyme produced by some species of bacteria. This enzyme protects bacteria from hydrogen peroxide (H2O2) that can damage and kill them. Catalase will convert hydrogen peroxide into liquid water (H2O) and oxygen gas (O2). As a result, if catalase is very active due to an abundance of hydrogen peroxide, the rapid production of oxygen gas (O2) will produce bubbles.
Bacteria that conduct aerobic metabolism (biological reactions that require O2) produce hydrogen peroxide (H2O2) as a toxic byproduct of their metabolism. Toxic hydrogen peroxide can cause intracellular damage, such as damage to DNA, lipids, and proteins. To remove H2O2 and other similar compounds, cells produce enzymes to break them down, such as catalase.
Bacteria can only make catalase if they have the gene for catalase in their DNA. When the catalase gene is expressed by the cell (DNA-->RNA-->protein), the bacteria produce catalase. Only species that have the catalase gene will make catalase and will test catalase positive. Bacterial species that do not have the catalase gene cannot make catalase and will test catalase negative. Therefore, testing for the presence or absence of catalase is a way bacterial species can be characterized and another tool for classifying and identifying bacterial species.
A simple test to determine if bacteria produce catalase is to add hydrogen peroxide to bacteria. This may be done by adding the hydrogen peroxide to bacteria placed on a slide or adding it to bacteria growing on an agar slant. If catalase is present, the hydrogen peroxide will be broken down into water and oxygen gas, resulting in the production of bubbles (catalase positive). This test does not require any special type of medium, however it should never be performed on organisms that have been grown on blood agar (a medium that contains blood). This is because there is a catalase activity in blood that would produce a false positive result.
Most aerobic bacteria (bacteria that require O2) and facultatively anaerobic bacteria (bacteria that live and metabolize O2 or in an environment without O2) produce catalase. Obligate anaerobe bacterial species (bacteria that must live in an environment without O2 to survive) lack catalase, which is why they cannot survive in an atmosphere containing oxygen. However, some of them have modified versions of catalase to deal with any possible exposure to oxygen.
Laboratory Instructions
Catalase Test
1. Obtain a glass slide and a bottle of hydrogen peroxide.
2. Using a sterilized inoculating loop, smear a small amount of bacteria onto the dry slide.
3. Place a drop of hydrogen peroxide on top of the bacteria.
4. Look for bubbles immediately:
• bubbles = catalase positive
• no bubbles = catalase negative
Results & Questions
Catalase (+/-)
Streptococcus pyogenes
Staphylococcus aureus
1. Complete the table above with results from the catalase test.
2. What is catalase?
3. Why is catalase production an advantage for bacteria to survive?
4. What types of bacteria typically produce catalase?
5. What types of bacteria typically do not produce catalase?
6. Give the chemical reaction catalyzed by the catalase enzyme.
7. When a bacterial species produces the catalase enzyme, what does it tell us about this species genes?
8. When a bacterial species does not produce the catalase enzyme, what does this tell us about this species genes?
9. Why can the catalase test be used to help with bacterial identification? | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.18%3A_Catalase_Test.txt |
Learning Objectives
• Describe the role of cytochrome c oxidase in bacterial cells.
• Tell that cytochrome c oxidase is found in only certain species of bacteria and is therefore useful for bacterial species identification and characterization.
• Explain what being "oxidase positive" means about a bacterial species metabolism, electron transport chain, and genetics.
• Explain that being "oxidase negative" does not mean the species is anaerobic, but just that it does not contain the cytochrome c oxidase enzyme and gene.
• Successfully conduct and interpret an oxidase test.
Cytochrome c Oxidase
Aerobic respiration is an O2-requiring process that uses energy from nutrient molecules to produce ATP molecules to provide for the cell's energy needs. During aerobic respiration, the electron transport chain transfers high-energy electrons from protein to protein and uses that energy to build up a H+ gradient that is utilized by ATP synthase to make ATP. At the end of the aerobic electron transport chain, an enzyme transfers the electron from the electron transport chain to O2 (the electrons at the end of the chain are low energy). In some bacteria capable of aerobic respiration, that enzyme that transfers electrons to O2, the final electron acceptor for aerobic respiration, is called cytochrome c oxidase.
A test called the oxidase test can be used in the laboratory to determine if a bacterial species has cytochrome c oxidase. Those species that have cytochrome c oxidase are called oxidase positive and those species that do not have cytochrome c oxidase are called oxidase negative. The ability of a bacterial species to produce cytochrome c oxidase is coded in its DNA. If the cytochrome c oxidase gene is present in the bacterial species, it will produce this enzyme. If the gene is absent, that bacterial species cannot produce cytochrome c oxidase. The oxidase test is therefore useful for characterizing bacterial species and differentiating bacterial species from each other for identification purposes.
Note
Bacterial species that contain cytochrome c oxidase are capable of aerobic respiration. Oxidase positive bacterial species are all aerobic.
However, oxidase negative species are not necessarily anaerobic. Oxidase negative species may still be aerobic species or facultative anaerobes, but they produce and use a different enzyme, other than cytochrome c oxidase, to transfer electrons from the electron transport chain to O2.
The Oxidase Test
The oxidase test is a key test to differentiate between the bacterial families Pseudomonadaceae (oxidase positive species) and Enterobacteriaceae (oxidase negative species), and is useful for speciation and identification of many other bacteria.
The oxidase test utilizes a special reagent called oxidase reagent. This reagent is colorless. However, in the presence of cytochrome c oxidase, the oxidase reagent will transfer its electrons to cytochrome c oxidase and exhibit a bluish or purplish color in 30 seconds or less.
• oxidase positive bacteria contain cytochrome c oxidase and produce a change in color of the reagent from colorless to bluish or purplish in less than 30 seconds.
• oxidase negative bacteria do not contain cytochrome c oxidase and do not change the color of oxidase reagent in less than 30 seconds.
Laboratory Instructions
Oxidase Test
1. Obtain an oxidase test strip and half of an empty petri plate.
2. Place the oxidase test strips face up on the half empty petri plate.
3. Lightly moisten the oxidase test strip with a small drop of water. Do not over-moisten the strip!
4. Using a sterile swab or sterile loop, obtain a large amount of bacteria from a petri plate.
5. Rub the bacteria on the swab or sterile loop onto the moistened region of the oxidase test strip.
6. Start a timer for 30 seconds.
7. Watch for purple color to develop on the oxidase test strip within 30 seconds.
• purple or bluish color change in less than 30 seconds is oxidase positive
• no color change within 30 seconds is oxidase negative (color change after 30 seconds is considered oxidase negative)
8. Repeat for each bacterial species you are testing.
Results & Questions
Oxidase (+/-)
Species produces cytochrome c oxidase (+/-)
Escherichia coli
Pseudomonas aeruginosa
1. Complete the table above with results of the oxidase test.
2. What metabolic pathway is cytochrome c oxidase important in?
3. What would happen to a bacterial cell that lost its ability to produce cytochrome c oxidase?
4. Is a bacterial species that produces cytochrome c oxidase aerobic? Explain your answer.
5. Is a bacterial species that does not produce cytochrome c oxidase anaerobic? Explain your answer.
6. Is the oxidase test useful for bacterial species identification and characterization? Explain your answer.
1.20: Citrate Test
Learning Objectives
• Explain what the citrate test indicates about a bacteria species' genes, enzymes, and metabolism.
• Tell what citrate permease is and what it does for bacterial species that have it.
• Tell that the citrate test is useful for bacterial identification and characterization.
• Successfully conduct a citrate test and interpret the results.
Citrate Test
All living things need carbon to survive. The carbon-containing molecules that bacteria can utilize as a carbon source differs based on the bacterial species and is dependent on their genes. Their genes dictate what enzymes the bacterial species can produce. Since enzymes are necessary for a cell's metabolic reactions, the genes of a bacterial species dictate their abilities to use different carbon sources.
Some bacterial species, but not all bacterial species, can utilize citrate as a source of carbon. Organisms that can survive using citrate as the sole source of carbon have a citrate permease enzyme that can transport citrate molecules into the cell. The citrate is then made into pyruvate, which can be converted into a variety of different products in the cell.
Simmons' citrate is a chemically defined microbiological medium that contains sodium citrate as the sole carbon source. A pH indicator, bromothymol blue, is also included in Simmons' citrate medium. Bacteria that can grow on this medium (i.e., that can survive on citrate as the sole source of carbon) produce alkaline byproducts that will change the medium color from green (neutral pH) to blue (alkaline pH).
Bacterial species that are capable of metabolizing citrate are considered citrate positive and will cause Simmons' citrate agar to change from green to blue. Bacterial species that are incapable of metabolizing citrate are considered citrate negative and will result in Simmons' citrate agar remaining green (no color change). Since only some bacterial species can metabolize citrate, the citrate test is useful for bacterial identification and characterization.
Laboratory Instructions
Citrate Test
1. Obtain two test tubes with Simmons' citrate agar slants.
2. Use tape to label the test tubes with your group name, bacteria name, and the type of test medium.
3. Using a sterile inoculating loop, aseptically obtain a small amount of bacteria and streak the entire slant of the citrate agar. Repeat for both species.
4. Incubate the inoculated tube at 37 °C until next lab session.
5. Observe the color of the Simmons’s citrate slant.
• bright blue is citrate positive
• no color change (agar is still green is citrate negative
Results & Questions
citrate (+/-)
bacterial species has citrate permease (+/-) bacterial species has citrate permease gene (+/-)
Escherichia coli
Serratia marcescens
1. Complete the table above with the results observed.
2. How does the citrate test relate to understanding the metabolism of a bacterial species?
3. What does the color change from green to blue mean about the pH of the culture?
4. What does the color change from green to blue mean about the bacterial species in the culture?
5. Can the citrate test be used for bacterial identification and characterization? Explain your answer. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.19%3A_Cytochrome_c_Oxidase.txt |
Learning Objectives
• Explain how aerobic and anaerobic respiration differ.
• Define and recognize descriptions of obligate aerobes, obligate anaerobes, facultative anaerobes, microaerophiles, and aerotolerant anaerobes.
• Tell that bacterial species' oxygen requirements are useful for species identification and characterization.
• Describe how thioglycollate agar tubes work and how they can be used to determine bacterial species' oxygen requirements.
• Tell how anaerobic chambers and GasPak systems work to culture obligate anaerobe species.
• Successfully conduct and interpret thyioglycollate cultures to determine oxygen requirements of bacteria.
• Successfully grow bacteria in aerobic and anaerobic conditions and interpet the results.
O2 and Bacterial Metabolism & Growth
Bacteria can differ dramatically in their ability to utilize oxygen (O2). Under aerobic conditions, if the bacterial species can conduct aerobic respiration, oxygen acts as the final electron acceptor for the electron transport chain located in the plasma membrane of prokaryotes. Bacteria use the electron transport chain to generate a H+ gradient that is used by ATP synthase to make ATP. ATP is the energy source for most cellular processes and therefore essentially for keeping cells alive. In the absence of oxygen (O2), some bacteria can use alternative metabolic pathways including anaerobic respiration and/or fermentation. During anaerobic respiration, other alternative molecules are used as the final electron acceptor for the electron transport chain such as nitrate (NO3), sulfate (SO4), and carbonate (CO3).
The presence or absence of molecular oxygen can be a critical factor in the ability of bacteria to grow in each environment. When bacteria use oxygen in cellular respiration and other chemical reactions, toxic superoxide and peroxides are produced. These highly reactive byproducts damage the cell unless they are quickly neutralized. Aerobic bacteria (grow in O2 environments) produce enzymes such as catalase, peroxidase and/or superoxide dismutase that break down toxic forms of oxygen and their intermediate byproducts. Bacteria called anaerobes produce ATP via anaerobic means (anaerobic respiration and/or fermentation). Anaerobes have no tolerance for oxygen since they cannot produce catalase, peroxidase and/or superoxide dismutase to remove toxic byproducts of O2.
Bacterial species are classified by their oxygen requirements as follows:
• obligate aerobes: Produce ATP via aerobic respiration. Require around 20% atmospheric oxygen.
• microaerophiles: Produce ATP via aerobic respiration or fermentation. Require between 5-15% atmospheric oxygen for growth.
• aerotolerant anaerobes: Produce ATP via anaerobic respiration and can conduct fermentation. Oxygen can be present, but they do not utilize it for ATP production or fermentation.
• facultative anaerobes: Produce ATP via aerobic respiration, anaerobic respiration, and/or fermentation. These organisms grow equally well in aerobic or anaerobic environments.
• obligate anaerobes: Produce ATP via anaerobic respiration or fermentation. These bacteria die in the presence of O2 because they lack the enzymes needed to break down toxic forms of oxygen and their intermediate byproducts.
Since bacterial species differ in their oxygen requirements, testing this feature is useful for identifying and characterizing bacterial species.
Determining Bacterial Oxygen Requirements with Thioglycollate Medium
Thioglycollate is a medium designed to test the aerotolerance (tolerance to O2) of bacteria. Along with nutrients, it contains a reducing agent, sodium thioglycollate, which combines with oxygen to produce water. Thioglycollate also contains a small amount of agar which helps reduce oxygen diffusion and helps maintain the stratification of organisms growing in different layers of the broth. Because the thioglycollate can eliminate the oxygen in the bottom of the tube, but not at the surface, varying concentrations of oxygen are found within the tube. On occasion, an indicator is added to the media to indicate the presence or absence of oxygen and shows where the aerobic and anaerobic zones separate. For example, resazurin is pink in the presence of oxygen and colorless when reduced.
One can determine a bacterium's oxygen requirements by cultivating them in a special medium called thioglycollate agar tubes. Based on the location and distribution of the bacteria in these tubes, a species can be classified as obligate aerobe, microaerophile, facultative anaerobe, aerotolerant anaerobe, or obligate anaerobe.
Growing Bacteria in Strict Anaerobic Conditions
Anaerobic Chambers
The cultivation of anaerobic bacterial species requires an anaerobic chamber. This is a special chamber is a closed environment without O2 where the microbiologist can work with and cultivate obligate anaerobes without exposing them to oxygen. Anaerobic chambers contain a hydrogen (H2) gas mixture that is circulated through a heated palladium catalyst to remove oxygen (O2) by forming water (H2O). Anaerobic chambers use a gas mixture of H2 and nitrogen gas (N2) (5/95%) or N2/carbon dioxide (CO2)/H2 (85/10/5 %) to remove oxygen. An airlock is used to reduce O2 levels prior to the transfer of samples in and out of the chamber.
GasPak Anaerobic Systems
A Gas Pak jar or bag is an alternative way to grow strict anaerobes that must be grown in an atmosphere without oxygen. Plates or tubes are placed in a sealed jar or bag along with a GasPak envelopes that functions as a hydrogen and carbon dioxide generator. The hydrogen combines with the oxygen in the jar to produce water. A palladium catalyst in the chamber or bag catalyzes the formation of water from hydrogen and oxygen, thereby removing the O2. To insure anaerobic conditions are effectively produced in the GasPak jar or bag, a strip of paper soaked in methylene blue dye is included in the jar or bag. Methylene blue is colorless in an anaerobic environment and blue in an aerobic environment. These systems are compact, easy to use, and less expensive than an anaerobic chamber.
Laboratory Instructions
Determining Bacterial Oxygen Requirements with Thioglycollate Medium
1. The thioglycollate broth should be either boiled first before inoculation OR recently made so that the oxygen content is very low. (Your instructor will tell you if it needs to be boiled).
2. Use tape to label the test tube with your group name, the bacterial species, and the type of medium.
3. Inoculate a tube of thioglycollate broth with the assigned bacterial species. Make sure that the loop or needle goes down to the very bottom of the broth, but do not get the metal holder region of the loop in the sterile broth since it will contaminate it.
4. Incubate at 25 °C or 37 °C.
Growing Bacteria With & Without O2
1. On the underside of two TSA Petri plates, use a marker to create 3 sections on the Petri plate (see image above) with the sections labeled as Escherichia coli, Pseudomonas aeruginosa, and Control. Depending on the circumstances, your instructor may also choose to include another section with a Clostridium species as well. If that is the case, create an additional section and label it with the Clostridium species name. Label one plate as "aerobic" and the other plate as "anaerobic."
2. Aseptically inoculate each section of the petri plates with the corresponding bacterial species. You may use a streak in a straight line or a zig-zag pattern within the labeled section for each. Leave the Control section without any added bacteria.
3. Incubate the aerobic plate as usual at 37 °C. The anaerobic plate will be placed in an anaerobic jar or anaerobic bag and a GasPak pouch. THe GasPak will be activated by your instructor to generate an anaerobic environment and then incubated at 37 °C.
Results & Discussion
Determining Bacterial Oxygen Requirements with Thioglycollate Medium
growth in the aerobic region of the thioglycollate medium
(+/-)
growth in the microaerobic region of the thioglycollate medium
(+/-)
growth in the anaerobic region of the thioglycollate medium
(+/-)
distribution of growth in the medium (even, uneven); if uneven, where was most of the growth? classify this species by its oxygen requirement (e.g. obligate aerobe, facultative anaerobe, etc.)
Escherichia coli
Pseudomonas aeruginosa
Clostridium sp. (if used)
1. Examine the thioglycollate medium. Do not shake or stir. Complete the table above to summarize results and determine the oxygen requirement for each species.
2. Explain how these results relate to aerobic respiration and anaerobic respiration?
3. How does the thioglycollate test tube create aerobic, microaerobic, and anaerobic conditions to test oxygen tolerances of bacterial species?
4. True or False. All bacterial species can grow in the presence of oxygen. Explain your answer.
5. True or False. All bacterial species can grow in the absence of oxygen. Explain your answer.
6. Can testing the oxygen tolerances of bacterial species be useful for species identification and characterization? Explain your answer.
Growing Bacteria With & Without O2
aerobic growth
(+/-)
anaerobic growth
(+/-)
classify this species by its oxygen requirement (e.g. obligate aerobe, facultative anaerobe, etc.)
Escherichia coli
Pseudomonas aeruginosa
Clostridium sp. (if used)
1. Examine the aerobic and anaerobic petri plates and fill out the table above with the results.
2. Explain how these results relate to aerobic respiration and anaerobic respiration?
3. True or False. All bacterial species can grow in the presence of oxygen. Explain your answer.
4. True or False. All bacterial species can grow in the absence of oxygen. Explain your answer.
5. How does the GasPak system produce an anaerobic environment?
6. Can testing the oxygen tolerances of bacterial species be useful for species identification and characterization? Explain your answer. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.21%3A_Bacterial_Oxygen_Requirements.txt |
Learning Objectives
• Explain what fermentation is and why it is important for microorganisms.
• Give examples of types of fermentation products, including fermentation products used by humans.
• Tell how fermentation tests can be useful in identification and characterization of bacterial species.
• Describe how the fermentation test works including the functions of phenol red and Durham tubes.
• Tell that fermentation can utilize different carbohydrates resulting in different fermentation reactions.
• Successfully conduct and interpret fermentation tests.
Fermentation is a Metabolic Process
Fermentation is a metabolic process that some microorganisms use to break down glucose and other sugars when O2 is not available or could not be used by the microorganism. Fermentation is a way bacteria can produce ATP to meet their energy needs (although fermentation produces significantly less ATP than aerobic respiration or anaerobic respiration). Fermentation includes the metabolic pathway glycolysis (where a single molecule of glucose is broken down into 2 molecules of pyruvate), as well as additional fermentation reactions that produce a variety of end products (acids, alcohols, gases). The end products are characteristic of individual bacterial species. Fermentation is also possible from non-sugar molecules. Even unusual compounds like aromatics (benzoate), glycerol (sugar-alcohol), and acetylene (hydrocarbons) may be fermented by some bacterial species!
Note that fermentation is mainly a mechanism used by cells to regenerate NAD+ so that NAD+ is available for glycolysis to continue when cellular respiration is not occurring. Fermentation also tends to produce waste products that can accumulate in the extracellular environment. By contrast, the waste left over after ATP production by aerobic respiration are limited to CO2 and H2O. There can be numerous end products from fermentation, many of which is useful for us humans, but not necessarily the microbes. We use many fermentation products--as diverse as antibiotics, alcohols, and a variety of foods. Microbes such as yeast (e.g. Saccharomyces cerevisiae) and bacteria are genetically engineered to produce valuable fermentation products.
Much of the original energy in the substrate remains within the chemical bonds of organic end products such as lactic acid or ethanol. For example, ethanol has so much stored energy it can be used in gasoline solutions to be combusted/burned to release that energy stored in its chemical bonds!
Fermentation of a Variety of Carbohydrates
Bacteria, depending on the species, can ferment different carbohydrates. Although glycolysis (the pathway leading to fermentation) begins with glucose, some bacteria have the enzymes needed for additional chemical reactions to convert other monosaccharides (e.g. fructose and mannose) as well as disaccharides (e.g. lactose and sucrose) so they can enter the glycolysis pathway. These bacteria therefore require the genes in their DNA that code for enzymes capable of converting these sugars into molecules that can enter glycolysis. For example, the enzyme beta-galactosidase is necessary to break down lactose. This enzyme is coded in the DNA of microbes that can metabolize lactose so they can produce this enzyme.
Therefore bacteria can be differentiated both based on their ability to ferment various carbohydrates, as well as the end products that result from the fermentation process. As a result, examining the ability of bacterial species to ferment a variety of carbohydrates is an approach used to characterize bacterial species and is useful for species identification.
Fermentation Test
Some bacteria will produce gases when fermenting a carbohydrate. To detect these gases, a Durham tube is used. This is a small inverted glass tube that is placed within the larger glass tube containing the fermentation medium. If gases (typically CO2) are produced during the fermentation process, a bubble will form at the top of the Durham tube. If you see a bubble in the Durham tube, this means fermentation occurred and gas was produced during fermentation.
The medium used to test carbohydrate fermentation is a nutrient broth that contains a fermentable carbohydrate (usually a monosaccharide or a disaccharide), peptone (amino acids) as well as a pH indicator. The pH of the medium is adjusted to approximately 7.5, so it appears orange/red with a phenol red pH indicator. These types of carbohydrate fermentation tubes are therefore called phenol red (sugar) broths. For example, if the fermentation test is being done to test fermentation of glucose, glucose is added to phenol red medium and the medium is called phenol red glucose. If the fermentation test is being done to test fermentation of lactose, lactose is added to phenol red medium and the medium is called phenol red lactose.
If the carbohydrate in the medium is fermented and acidic end products are formed, the color of the medium changes from red-orange to yellow. Occasionally, bacteria will not ferment the carbohydrate, but instead will break down proteins producing ammonia (NH3) in the growth medium. In this case, the medium will become more alkaline and appear red.
Results of a fermentation can be interpreted as follows:
• red-orange color indicates no acid was produced
• yellow color indicates acid was produced during fermentation
• a gas bubble trapped in the Durham tube indicates gas was produced during fermentation
• no gas bubble trapped in the Durham tube indicates no gas was produced
• if medium is red-orange and no gas bubble is trapped in the Durham tube, no fermentation occurred
Exercise 22.1
Interpret the result of this fermentation test:
• Did this bacterial species produce gas?
• Did this bacterial species produce acid?
• Did fermentation occur?
Answer
Exercise 22.2
Interpret the result of this fermentation test:
• Did this bacterial species produce gas?
• Did this bacterial species produce acid?
• Did fermentation occur?
Answer
Laboratory Instructions
Fermentation Test
In this experiment, fermentation of two different carbohydrates will be tested: glucose and lactose.
1. Label three phenol red glucose tubes each with a species names to be tested (Escherichia coli, Bacillus subtilis, and Proteus vulgaris), group name, and medium name. Repeat for three phenol red lactose tubes.
2. Use aseptic technique to transfer a loop of the appropriate cultures to each of the culture broths.
3. Incubate cultures at 37 °C for 48 hours.
Results & Questions
medium color with glucose
(red or yellow)
bubble in Durham tube with glucose
(+/-)
acid production with glucose
(+/-)
gas production with glucose
(+/-)
fermentation of glucose
(+/-)
Escherichia coli
Bacillus subtilis
Proteus vulgaris
medium color with lactose
(red or yellow)
bubble in Durham tube with lactose
(+/-)
acid production with lactose
(+/-)
gas production with lactose
(+/-)
fermentation of lactose
(+/-)
Escherichia coli
Bacillus subtilis
Proteus vulgaris
1. Complete the tables above based on the results observed from the fermentation tests.
2. Was there a difference in fermentation/fermentation products produced by E. coli with glucose versus lactose? Explain your answer.
3. Was there a difference in fermentation/fermentation products produced by B. subtilis with glucose versus lactose? Explain your answer.
4. Was there a difference in fermentation/fermentation products produced by P. vulgaris with glucose versus lactose? Explain your answer.
5. What is fermentation?
6. Do all bacterial species ferment in the same way and produce the same end products? Explain your answer.
7. Why is fermentation an important process in some bacterial species?
8. Are fermentation tests useful for bacterial species identification and characterization? Explain your answer.
9. What is the purpose of phenol red in the fermentation medium?
10. What is the purpose of the Durham tube in the fermentation tube? | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.22%3A_Fermentation.txt |
Learning Objectives
• Explain what a SIM deep is and tell the tests that can be conducted in a SIM deep.
• List possible metabolic reasons why a species of bacteria could be producing hydrogen sulfide.
• Tell what the indole test examines and what enzyme it tests for.
• Explain what the motility is and that is indicates whether or not bacterial species produce flagella for motility.
• Successfully conduct SIM tests and Interpore results of these tests.
SIM Medium
SIM (sulfur reduction, indole, motility) medium is an example combination medium, meaning that one can determine several bacterial activities/characteristics through the use of one medium. SIM medium tests for sulfur reduction, indole production and motility. SIM is an example of a The form of medium used for this test is an agar deep. SIM Medium contains the following: pancreatic digest of casein, peptic digest of animal tissue, ferrous ammonium sulfate Fe(NH4)2(SO4), sodium thiosulfate Na2S2O3, agar (3.5 g/L) and distilled or deionized water.
SIM - Sulfur Reduction
Sulfur can be reduced producing hydrogen sulfide (H2S) by bacteria in two unrelated ways:
1. One process occurs during putrefaction. When proteins putrefy, the resulting foul “rotten egg” smell is due to the production of hydrogen sulfide gas (H2S). Hydrogen sulfide is a byproduct of the conversion of the amino acid cysteine to pyruvate by the enzyme cysteine desulfurase.
2. The second mode of H2S generation involves anaerobic respiration. In some prokaryotes, thiosulfate (S2O32-) is the terminal electron acceptor in an anaerobic respiration. When thiosulfate is reduced (picks up electrons) the result is H2S gas. In either case, invisible H2S gas is produced.
Because hydrogen sulfide gas is colorless (though not odorless!), SIM medium uses an indicator reaction. Iron (in the form of ferrous ammonium sulfate) in the medium combines with H2S gas to form iron sulfide, FeS, a black precipitate. Any black color in the medium indicates the bacterial species is positive for sulfur reduction. If there is no black color in the medium, the bacterial species is negative for sulfur reduction.
Unfortunately, this test does not distinguish between the hydrogen sulfide produced as a result of putrefaction and hydrogen sulfide produced at the end of an anaerobic respiration.
SIM - Indole
Tryptophan is an amino acid found in most proteins. Some bacteria produce tryptophanase, an enzyme that breaks tryptophan down into indole, ammonia and pyruvate (see below). Not all bacterial species produce tryptophanase. Whether a bacterial species produces tryptophanase is dependent on its genes. Testing for the activity of tryptophanase using the indole test is an effective way to differentiate one bacterial species from another and to characterize bacteria.
The pyruvate and ammonia (NH3) are converted into other molecules, but the indole accumulates, and thus can be detected in the media.
The presence of indole therefore indicates that an organism produces the enzyme tryptophanase. Indole can be detected using a chemical known as Kovac’s reagent. Indole forms a red ring with the addition of Kovac’s reagent indicating the bacterial species is indole positive and that it produces tryptophanase. When a bacterial species is indole negative (indicating no tryptophanase activity), the Kovac's reagent will produce a dark yellow ring.
SIM - Motility
Motility is the ability of a microbe to “swim” using flagella. The reduced agar content of this medium, 3.5 g/L compared to 12-15 g/L in most solid media, creates a semi liquid environment allowing motile cells to spread from their original placement. The stab technique deposits cells in a straight line down the center of the deep using an inoculation needle rather than an inoculation loop. If growth is observed beyond the stab line into the periphery of the tube, the test is positive for motility. Avoid confusing growth produced by the lateral movement of the needle during an imperfect stab inoculation with actual motility. Rotating the tube for a side view will help you determine if growth is confined to the original inoculation line or has truly spread into the periphery of the tube.
Motility is indicated by the ability of the organism to ‘fan’ away from the streak. Or, the entire tube may appear cloudy when compared to an un-inoculated control. If the organism is non-motile, the growth will only appear along the stab line.
Laboratory Instructions
1. Obtain a deep of SIM medium.
2. Label the test tube with your group name, the medium name, and the bacterial species name.
3. Using an inoculating needle, stab the medium about 2/3 of the way down and out the same pathway as quickly as possible with the bacterial species provided by your instructor.
4. Repeat steps 1-3 if testing multiple bacterial species.
5. Incubate the tube for at least 48 hours.
6. After the incubation period, examine your tube.
Indole Test
1. After the SIM deep has incubated for at least 48 hours, examine results for motility and hydrogen sulfide production and record results.
2. Add 10 drops of Kovac’s reagent to the top of the SIM meidum tube.
• if Kovac's reagent produces a dark yellow ring indicates the species is indole negative
• if Kovac's reagent produces a red ring indicates the species is indole positive
Results & Questions
bacterial species medium color hydrogen sulfide production (+/-) Kovac's reagent color indole (+/-) location of growth (along stab / fanned out) motility (+/-)
Escherichia coli
Proteus vulgaris
Staphylococcus aureus
1. Complete the table above with results from the SIM deep tests.
2. A bacterial species that is positive for hydrogen sulfide production could be producing H2S in one of two ways. What are these?
3. Is hydrogen sulfide black? Explain your answer.
4. A bacterial species that is positive for indole produces what enzyme? What does this enzyme do?
5. What is the role of Kovac's reagent in the indole test?
6. A bacterial species that is positive motility has what type of bacterial cell structure found only in some cells?
7. Is using a SIM deep useful for bacterial species identification and characterization? Explain your answer. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.23%3A_SIM_Deep_Tests.txt |
Learning Objectives
• Describe what coagulase is and what this enzyme does.
• Tell that bacterial species that produce coagulase are pathogenic.
• Identify that the coagulase test is useful for differentiating between Staphylococcus sp.
• Tell how the two types of coagulase tests work and which one is more definitive.
• Successfully conduct and interpret the coagulase test.
Coagulase
The enzyme coagulase, produced by a few of the Staphylococcus species (but not all of them), is a key feature of pathogenic Staphylococcus species. The coagulase enzyme produces coagulation of blood, allowing the microorganism to "wall" its infection off from the host's protective mechanisms rather effectively. As with other enzymes, whether or not a species can produce an enzyme is dependent upon whether that species has the gene for the enzyme. Therefore, testing for coagulase activity tells us about the genes and DNA of a bacterial species.
This test determines the ability of the bacteria to produce bound coagulase. This exoenzyme will cause the conversion of fibrinogen in blood plasma to fibrin and form a clot. The fibrin covers the surface of the bacteria, making it resistant to phagocytosis.
There are 2 methods to test for coagulase: (1) slide agglutination test and (2) tube agglutination test. The tube agglutination test is more accurate, but the slide agglutination test is faster. If you have an unknown Staphylococcus species, you might consider running the slide test, and, if negative, running the tube test. The tube method is the definitive test.
Pathogenic Staphylococcus species (e.g. Staphylococcus aureus) can be confirmed using the coagulase test. Since there are 2 kinds of coagulase enzymes—bound and free---there are 2 different tests that can be used to identify these enzymes. Both of the enzymes activate fibrinogen in plasma, in different ways. Your instructor will direct you as to which procedure to use, but the tube test is the definitive test: if you get a negative test result for the slide test, run the tube test.
Laboratory Instructions
Slide Agglutination Test
1. Make a 1 inch diameter circle on a clean glass slide using a wax pencil.
2. Place two drops of thawed rabbit plasma into the circle, using a wooden pick or a clean loop.
3. Add a HEAVY inoculum and emulsify it in the plasma (should be milky-looking).
4. Fibrin threads form between the cells, causing them to agglutinate, or clump.
5. There will a visible clumping of cells within 10-15 seconds.
6. This test is for the bound coagulase enzyme.
Tube Agglutination Test
If the slide coagulase test reaction is negative, inoculate a tube of rabbit plasma overnight and check for a clot in the tube. This is considered a positive reaction for free coagulase.
1. Inoculate a tube containing ½ ml of rabbit plasma with the bacterial inoculum.
2. Place at 37º C and check at ½ hour or at next lab period (some strains will give a + reaction in a few hours, other strains take longer) by tipping the slide at an angle.
3. Any degree of coagulation is considered a positive test for the free coagulase enzyme.
Results & Questions
coagulase (+/-) species produces a coagulase enzyme (+/-) species contains a coagulase gene (+/-)
Staphylococcus aureus
Staphylococcus epidermidis
1. Complete the table above to indicate results of the coagulase test.
2. A coagulase positive result indicates what about the bacterial species' pathogenicity?
3. When a bacterial species living inside of a host (such as a human) produces coagulase, what does coagulase do? Be specific.
4. Can testing for coagulase activity be useful for identifying and characterizing bacterial species? Explain your answer.
5. The coagulase test is useful for differentiation bacterial species with the genus ____________________.
1.25: Gelatin Hydrolysis
Learning Objectives
• Explain what gelatin is and what gelatin hydrolysis is.
• Tell that species that can hydrolyze gelatin have a gelatinase gene and produce a gelatinase enzyme.
• Explain how the gelatin hydrolysis test works.
• Successfully conduct and interpret the gelatin hydrolysis test.
• Tell that gelatin hydrolysis is useful for characterizing and identifying bacterial species.
Gelatin & Gelatin Hydrolysis
Gelatin is a protein derived from collagen, a connective tissue of animals. When chilled on ice, gelatin forms cross-links to itself to create a semi-solid state (gelatin makes Jello! have its unique form and texture). Gelatin provides a rich source of amino acids and peptides for bacteria, but its structure is too large to be transported inside the cell directly. Therefore, it must first be broken down (gelatin hydrolysis) by exoenzyme proteins called gelatinases. Not all species of bacteria have a gene for a gelatinase enzyme. Only those species of bacteria that do have a gelatinase gene are capable of producing a gelatinase and are capable of breaking down gelatin and using it as a nutrient source. As such, the gelatin hydrolysis test is used to differentiate between bacteria that do and do not hydrolyze gelatin, and therefore is useful for characterizing and identifying bacterial species.
To determine if a bacterial species hydrolyzes gelatin (and therefore has the a gelatinase gene and produces a gelatinase enzyme), the gelatin hydrolysis test is used. When bacteria that have this enzyme are inoculated into a nutrient medium containing gelatin, they will produce a gelatinase enzyme and break down the gelatin. After incubation of the bacteria in the medium that hydrolyze gelatin, the medium will no longer form a semi-solid state, even after chilling. Those species that do not hydrolyze gelatin will not have an effect on the gelatin and it will maintain its semi-solid state after chilling.
Laboratory Instructions
1. For each bacterial species being tested, use aseptic technique to collect the bacteria using an inoculation needle and stab the gelatin deep with a needle all the way to the bottom (being careful NOT to get the metal holder into the agar).
2. Incubate at 25º C for a couple days, up to a week. Keep the medium for a week if negative.
3. Examine results by placing the test tubes on ice for 15 minutes or in the fridge for 30 minutes.
Results & Questions
Bacterial Species Gelatin medium liquid after incubation and chilling (+/-)
Species hydrolyzes gelatin (+/-)
Species has a gelatinase enzyme (+/-) Species has a gelatinase gene (+/-)
1. Complete the table above with experimental results and interpretations.
2. Why is it that when gelatin is broken down the gelatin medium no longer forms a semi-solid even after chilling?
3. What is gelatin?
4. Why might the ability to hydrolyze gelatin be an advantage for some species of bacteria?
5. Why might gelatin hydrolysis be associated with pathogenic species and not non-pathogenic species? | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.24%3A_Coagulase_Test.txt |
Learning Objectives
• Explain how different bacterial species can conduct nitrate reduction including the possible reactions, possible products, and the name of the enzyme that reduces nitrate.
• Tell the role of nitrate in anaerobic respiration of the species that reduce nitrate.
• Successfully conduct and interpret results of the nitrate reduction test.
• Explain why the nitrate reduction test is useful for characterizing and differentiating species of bacteria.
Nitrate Reduction
Nitrate (NO3) is a nitrogen-containing molecule that can be reduced by some bacterial species, but not others. Therefore, examining the ability of bacterial species to conduct nitrogen reduction is useful for characterizing and identifying bacterial species.
Reduction of nitrate generally occurs during anaerobic respiration in which an organism derives its oxygen from nitrate to serve as the final electron acceptor to remove electrons from the electron transport chain. In some species, nitrate is reduced to nitrite leaving nitrite as evidence of the process:
NO3 (nitrate) → NO2 (nitrite)
In other species, nitrate is first reduced to nitrite and then to ammonia:
NO3 (nitrate) → NO2 (nitrite) → NH3 (ammonia)
In still other bacterial species, denitrification occurs where nitrate is reduced completely to N2 gas and becomes largely unavailable to most living things as a nitrogen source:
NO3 (nitrate) → NO2 (nitrite) → N2 (nitrogen gas)
Nitrate broth is used to determine if an organism can reduce nitrate. Some bacteria can reduce nitrate (NO3) to nitrite (NO2) by producing the enzyme nitrate reductase. Other bacteria can reduce nitrate to nitrogen gas by also producing the enzyme nitrite reductase which reduces nitrite to nitrogen gas. Other organisms do not have the ability to reduce nitrate at all.
Nitrate Reduction Test
The nitrate reduction test determines if bacteria conduct nitrate reduction, and therefore if they have the gene for the nitrate reductase enzyme, resulting in the reduction of nitrate (NO3). To determine if nitrite is produce, nitrite (NO2) in the medium will form nitrous acid that reacts with the reagent sulfanilic acid (added to the medium), and that reacts with the another reagent naphthylamine (also added to the medium) to form a red color. To determine if denitrification occurs (N2 is produced), a Durham tube is used to capture the gas. If N2 or NO2 are not detected, rather than testing for ammonia (NH3) directly, zinc is used to determine if there is nitrate remaining in the medium. If there is nitrate remaining in the medium, this indicates that no nitrate reduction occurred and will eliminate the possibility that nitrate was reduced to ammonia. However, if there is no nitrate remaining and N2 or NO2 was not detected, this indicates that nitrate reduction occurred and ammonia was produced.
Interpretation of the Nitrate Reduction Test
Step 1: Examine Cultures for N2 Production
There are various ways that a bacterium can utilize nitrate as the final electron acceptor in anaerobic respiration. Some species will completely reduce nitrate to N2 gas, a process called denitrification. If denitrification occurred and therefore N2 was produced, there will be a pocket of gas (this is N2 gas) in the top of the Durham tube. The test tube is examined for the presence or absence of N2 in the Durham tube before any reagents are added.
Step 2: Examine Cultures for NO2 Production
If there is no nitrogen gas (N2), it is still possible that nitrate (NO3) was reduced to nitrate (NO2). There are still a couple of possible outcomes that need examining to determine whether or not nitrate (NO3) was reduced:
• possibility 1: nitrate (NO3) reduction to nitrite (NO2)
• possibility 2: nitrate (NO3) reduction to ammonia (NH3)
• possibility 3: no reduction of nitrate (NO3)
To determine if nitrate was reduced to nitrate (possibility 1), reagents are added to the culture medium. A red color will be produced in the medium only when nitrite (NO2) is present in the medium, indicating nitrate reduction to nitrite.
Step 3: Examine Cultures for NH3 Production
Even if no N2 or NO2 were produced in the culture, it is still possible that nitrate was reduced in the culture. The following two possibilities still exist:
1. possibility 2: nitrate (NO3) reduction to ammonia (NH3)
2. possibility 3: no reduction of nitrate (NO3)
To differentiate between the above two possibilities, powdered zinc is added. The zinc will not show ammonia production. Instead, the zinc will show if the nitrate is still present in the medium. If the nitrate is still present in the medium, then the nitrate was not reduced and was therefore not reduced to ammonia (nitrate reduction negative).
After zinc is added to the medium, there are two possible outcomes:
• no pink color forms: this means that possibility 2 occurred and nitrate was reduced to ammonia.
• pink color forms: this means that possibility 3 occurred and no nitrate was reduced in the culture. If nitrate (NO3), is still be present in the tube, then zinc reduces the nitrate to nitrite, which then reacts with the 2 reagents already added to the tube producing a pink color.
REACTION N2 gas Color after adding reagents Color after adding zinc
NO3 to NO2 none red (Zn not added)
NO3 to N2 yes no color (Zn not added)
NO3 to NH3 none no color no color
no NO3 reduction none no color pink-red
Laboratory Instructions
1. Inoculate the nitrate broths with the assigned bacterial species.
2. Incubate at the optimal temperature, 30º C or 37º C, for these bacterial species.
3. After Incubation: Look for N2 gas first before adding reagents and record results for whether or not N2 gas was produced.
4. Add 6-8 drops of nitrite reagent A.
5. Add the same number of drops of nitrite reagent B.
6. You should see a reaction within a minute or less. Record results for whether or not nitrite was produced.
7. If you have not seen evidence of either nitrite (NO2) or N2 gas, add a bit of powdered zinc to the culture medium. A bit of zinc is about the amount that sticks to the end of a wood stick.
8. The reduction of unused nitrate (NO3) by zinc takes a couple of minutes. Record results for whether or not ammonia was produced.
Results & Questions
NO3 present/absent (+/-) NO2 present/absent (+/-) N2 present/absent (+/-) nitrate reduction (+/-)
Alcaligenes faecalis
Escherichia coli
Pseudomonas aeruginosa
1. Complete the table above with experimental results from the nitrate reduction cultures.
2. Name the 2 major end products of nitrate reduction.
3. What does the gas pocket in the Durham tube indicate about nitrate reduction by that bacterial species? Be specific.
4. What does the red color after adding reagents to the medium mean about nitrate reduction by that bacterial species? Be specific.
5. What does a lack of pink color (no pink) after adding zinc mean about nitrate reduction by that bacterial species? Be specific.
6. What does a pink color after adding zinc mean about nitrate reduction by that bacterial species? Be specific.
7. Species that CAN conduct nitrate reduction may conduct the process differently. Explain this statement.
8. Define denitrification.
9. Is nitrate reduction an aerobic pathway or an anaerobic pathway? | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.26%3A_Nitrate_Reduction.txt |
Learning Objectives
• Describe the purpose and usefulness of the MR-VP test.
• Compare and contrast the MR test and the VP test.
• Tell how the MR test and VP test are conducted and what the results mean.
• Define fermentation and describe its importance.
• Name the metabolic pathway that occurs in MR positive bacterial species.
• Name the metabolic pathway that occurs in VP positive bacterial species.
• Successfully conduct and interpret the MR-VP test.
Methyl Red–Voges Proskauer (MR-VP) Test
The MR-VP test is actually a set of two separate tests that uses one broth medium containing glucose to determine the possible types of glucose fermentation being conducted by a bacterial species. Some bacterial species may ferment glucose a using mixed acid fermentation pathway (detected in the MR test or methyl red test) while some other bacterial species may ferment glucose via the butanediol fermentation pathway (detected in the VP test or Voges-Proskauer test).
Since the type of fermentation conducted differs by bacterial species (or strain), testing the type of fermentation pathway is useful for characterizing bacteria and identifying them. Metabolic reactions are dependent on the enzymes that species (or strain) has, which is dependent on the genes that species (or strain) carries in its DNA. Therefore, testing the types of metabolisms species conduct provides insights into differences in their genetics (their DNA).
Fermentation
Fermentation is a type of metabolic pathway some bacterial species conduct to produce ATP (ATP is used as an energy source to keep the cell alive). Fermentation is an anaerobic process (no O2 is used) and species that conduct fermentation will produce a small amount of ATP that has been derived from the energy in a glucose molecule.
Fermentation begins with glycolysis, the metabolic pathway that breaks down glucose into two pyruvate (or pyruvic acid) molecules and will produce a net gain (overall gain) of 2 ATP. Then, in a fermentation pathway (the pathways differ depending on the species), pyruvate is converted into other molecules to regenerate NAD+ so the glycolysis pathway can continue (NAD+ is a cofactor in a glycolysis reaction that must be available for glycolysis to occur). Additionally, fermentation pathways utilize organic molecules, derived from pyruvate, as final electron acceptors.
Methyl Red (MR) Test Detects Mixed Acid Fermentation
Mixed-acid fermentation pathways are fermentation pathways that some bacterial species conduct to produce a mixture of fermentation products. There is not just one way bacteria conduct mixed acid fermentation, but a number of them. The end-products produced through mixed-acid fermentation is species-dependent and sometimes strain-dependent (depends on the genes that the species has; genes dictate the enzymes produced and therefore the chemical reactions occurring in the species' or strains' metabolism). Below shows one example of glycolysis with a mixed acid fermentation pathway that produces acetoin, formate, lactate, succinate, acetate, and ethanol as byproducts of the process.
The methyl red test (MR) utilizes a liquid medium containing glucose. After this medium is inoculated and incubated to enable bacteria to grow and conduct fermentation, the reagent methyl red is added to the medium. Methyl red is a pH indicator that will detect mixed acid production by changing the medium to a red color (methyl red positive). In the absence of mixed acid fermentation, methyl red will not produce a red color (methyl red negative).
MR-VP Broth (glucose) –> pyruvate –> mixed acid fermentation pathway (red color with methyl red)
Voges-Proskauer (VP) Test Detects Butanediol Fermentation
The Voges-Proskauer test determines if 2,3-butanediol is a product of glucose fermentation by a bacterial species. This fermentation byproduct typically is produced by species of Klebsiella and Enterobacter.
The Voges-Proskauer test removes some of the MR-VP medium into a separate test tube prior to conducting the methyl red test and adds Barritt’s A reagent followed by Barritt's B reagent to the medium. After mixing and a rest period, a liquid layer forms at the top of the test tube indicating the test results. If the top liquid layer is red or red-brown, the bacteria is Voges-Proskauer positive indicating it produces 2,3-butanediol as a byproduct of fermentation. If the top liquid layer is yellow, the bacteria is Voges-Proskauer negative indicating it does not produce 2,3-butanediol as a byproduct of fermentation.
MR-VP Broth (glucose) –> pyruvate –>butanediol fermentation pathway (red color in top layers of medium after adding reagents, mixing, and resting)
Laboratory Instructions
1. Obtain a MR-VP broth tube. Use tape to label the tube with your name, your assigned organism(s) and the name of the test media.
2. Aseptically transfer bacteria with a loop to inoculate the MR-VP broth.
3. Incubate the inoculated tube in the class test tube rack until next lab session.
4. After incubation, using a transfer pipette, transfer half of the inoculated MR-VP broth to a clean test tube. Label this tube “VP.” Leave the other half of the inoculated broth in the original MR-VP broth test tube. Label this tube “MR.”
5. Methyl red test:
1. Add 10 drops of methyl red reagent to “MR” tube.
2. Examine the color of the medium.
3. Record the results.
6. Voges-Proskauer test:
1. First add 15 drops of Barritt’s A reagent (alpha-napththol) to the "VP" tube.
2. Add 5 drops of Barritt’s B reagent (40% KOH) to the “VP” tube. NOTE: A reversal in the order of the reagents may result in a weak-positive or false-negative reaction.
3. Hold the test tube from the glass and mix the tube well by flicking it with your fingers.
4. Let the test tube sit undisturbed in a test tube rack for 20 minutes.
5. Examine the color of the top layer of liquid and record the results.
Results & Questions
bacterial species tested color of medium for MR test results from methyl red test (+/-) species conducts mixed-acid fermentation (+/-) color of top liquid layer for VP test results from Voges-Proskauer test (+/-) species conducts butanediol fermentation (+/-)
Escherichia coli
Enterobacter aerogenes
1. Complete the table above to summarize results and interpretations of those results.
2. What is fermentation and why is it important for bacterial species that conduct fermentation?
3. What are the interrelationships between glycolysis and fermentation (there are two main ones)?
4. What is mixed-acid fermentation?
5. What is butanediol fermentation?
6. Explain why the MR-VP test is useful for characterizing and identifying bacterial species. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.27%3A_MR-VP_Tests.txt |
Learning Objectives
• Identify that EMB medium is both selective and differential.
• Explain how EMB is a selective medium and how this works.
• Explain how EMB is differential and how this works.
• Define coliform.
• Successfully utilize EMB medium and interpret its results.
Eosin-Methylene Blue (EMB) Medium
Eosin-methylene blue (EMB) medium is a selective medium and a differential medium used for isolation of Gram-negative rods in a variety of specimen types, including being used frequently in clinical laboratories. EMB is a selective medium because it inhibits Gram-positive bacterial growth. The two dyes in this medium, eosin and methylene blue, act as selective agents to inhibit Gram-positive bacteria, but allow for the growth of Gram-negative bacteria.
EMB enables differentiation of bacterial species that are coliforms due to its dyes and its lactose content. EMB contains two types of sugars: lactose and sucrose. It is the lactose that is the key to this medium's ability to differentiate coliform bacteria. Lactose-fermenting bacteria (Escherichia coli and other coliforms) produce acid using lactose. When acids are produced by bacteria, the combination of the dyes (which serve as pH indicators in this medium) produces color variations in the colonies because of the acidity. Strong acidity produces a deep purple colony with a green metallic sheen, whereas less acidity may produce a brown-pink coloration of colony. Nonlactose fermenters appear as translucent or pink.
Definition
coliform: A classification of bacterial species that is typically associated with animal digestive tracts and fecal contamination in the environment. These bacterial species are Gram-negative bacilli that do not produce endospores and have the ß-galactosidase gene, and can therefore break down lactose. Coliform bacteria break down lactose to produce acid and gas.
Laboratory Instructions
1. To observe the effects of EMB medium as selective and differential, aseptically conduct quadrant streak plates on three different EMB plates with:
• Escherichia coli - a Gram-negative coliform species
• Pseudomonas aeruginosa - a Gram-negative non-coliform species
• Staphylococcus aureus - a Gram-positive species
2. Invert and incubate the plates for 24-48 hours.
3. Observe, record, and interpret the results.
Results & Questions
bacterial species growth (+/-) species is likely Gram (+/-) colony color(s) lactose utilization produces acid (+/-) species is a coliform (+/-)
Escherichia coli
Pseudomonas aeruginosa
Staphylococcus aureus
1. Record the results and interpretations from the EMB plates in the table above.
2. What types of bacterial species is EMB used to identify?
3. How is EMB a selective medium?
4. How is EMB a differential medium?
5. Define coliform.
6. Explain the roles of the dyes in EMB (there are two).
7. Explain the role of lactose in EMB.
1.29: Mannitol Salt Agar
Learning Objectives
• Define selective medium and differential medium.
• Explain how mannitol salts agar is selective.
• Explain how mannitol salts agar is differential.
• Successfully utilize mannitol salts agar to characterize/identify bacterial species and interpret results.
Mannitol Salts Agar
Mannitol salts agar is a microbiological medium that is both selective and differential. Selective means the medium will only allow certain microorganisms to grow. Differential means the medium will show some characteristic of the microorganisms growing on it and can be used to differentiate between different species.
Mannitol salts agar is selective since it has a high salt concentration and will only allow halophilic (salt-loving species) or halotolerant (salt-tolerant) species to grow on it. This medium contains 7.5% NaCl (salt), whereas typical media contains about 0.5% NaCl.
• species that grow on mannitol salts agar: mannitol utilization and salt resistance positive
• species that do not grow on mannitol salts agar: mannitol utilization/salt resistance negative
Mannitol salts agar is differential since it will detect acid production as a result of fermentation of mannitol (a sugar in the medium) in species that can ferment mannitol to produce acid. The medium contains a pH indicator, phenol red, that is red-orange when the pH is neutral (around pH 7). If a species ferments mannitol and produces acid, the pH of the medium decreases and phenol red will become yellow.
• medium remains red-orange after growth and growth observed: mannitol fermentation negative
• medium is yellow after growth: mannitol fermentation positive
Use of mannitol salts agar is useful for differentiation of species of Staphylococcus and Micrococcus. Mannitol fermentation by pathogenic staphylococci, such as Staphylococcus aureus, is indicated by the media changing to yellow, whereas Staphylococcus epidermidis, a non-pathogenic species of staphylococci, does not produce a yellow color.
Laboratory Instructions
1. Obtain a mannitol salts agar petri plate and label with your name/group name or number and "mannitol salts agar."
2. Draw a straight line on the underside of the petri plate to separate the plate into two halves. Label one side Staphylococcus aureus and the other side Staphylococcus epidermidis.
3. Aseptically inoculate each half of the plate with the corresponding bacterial species with a streak that is a straight line in the center of each half of the petri plate.
4. Invert the petri plate and incubate for 24 hours.
5. Observe, record, and interpret results.
Results & Questions
bacterial species growth (+/-) salt resistance and mannitol utilization (+/-) medium color surrounding growth mannitol fermentation (+/-)
Staphylococcus aureus
Staphylococcus epidermidis
1. Fill in the table above with results and interpretations.
2. Define selective medium.
3. Explain how mannitol salts agar is a selective medium.
4. Define differential medium.
5. Explain how mannitol salts agar is a differential medium.
6. Explain how phenol red, a component of mannitol salts agar medium, detects if a species conducts mannitol fermentation. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.28%3A_EMB_Agar.txt |
Learning Objectives
• Describe in detail / identify the structures of DNA from nucleotide components to double helix.
• Describe in detail / identify the structures of RNA from nucleotide components to single strand.
• Compare the structures of DNA and RNA.
• Utilize and identify 5' and 3' directionality of DNA and RNA nucleotides and molecules.
• Explain the process of DNA replication in detail and identify components of the process in diagrams and figures.
• Build DNA and RNA nucleotides using puzzle pieces and compare their structures.
• Build DNA and RNA nucleic acid sequences using puzzle pieces and identify and compare structures of these molecules.
• Replicate the process of DNA replication using a DNA puzzle model.
• Compare the puzzle re-creation of DNA replication with events that occur in the actual process of DNA replication.
Importance of Understanding DNA Structure
Understanding the structure of DNA is fundamental to a better understanding of biology overall, but particularly microbiology, disease, and modern medical approaches. Here are a few ways that having a thorough understanding of DNA structure will impact your ability to understand additional microbiological topics:
• the DNA of a microbe dictates if that microbe is pathogenic or not and the degree of its pathogenicity
• the DNA of a microbe is the basic blueprint for that microbe's metabolism, characteristics, abilities, structure, and survival approaches
• DNA information is used during the process of gene expression
• understanding DNA structure is essential to better understand gene expression
• understanding how gene expression works enables us to understand the link between DNA and a microbe's characteristics
• how pathogens (disease-causing microbes) evolve/change over time (think of the seasonal flu strains or new variants of the virus that causes COVID-19) is related to its DNA (or the closely related molecule RNA in the case of the virus that causes COVID-19) and how that DNA changes over time
• DNA is used to identify microbes (e.g. what microbe is causing an infection) in diagnostic approaches such as PCR testing
• understanding how PCR works for diagnostic techniques requires a firm understanding of DNA structure and DNA replication
• understanding modern biological science is impossible without a firm understanding of DNA structure and DNA replication (and PCR too)
Note
PCR has a huge number of applications beyond medical diagnostics!
DNA Structure
Nucleotides are the Monomers of DNA
The building blocks of DNA are nucleotides. The important components of the nucleotide are a nitrogenous (nitrogen-bearing) base, a 5-carbon sugar (pentose), and a phosphate group. The nucleotide is named depending on the nitrogenous base. The nitrogenous base can be a purine such as adenine (A) and guanine (G), or a pyrimidine such as cytosine (C) and thymine (T).
The purines have a double ring structure with a six-membered ring fused to a five-membered ring. Pyrimidines are smaller in size; they have a single six-membered ring structure.
Important
The carbon atoms of the pentose sugar are numbered 1', 2', 3', 4', and 5' (1' is read as “one prime,” 2' is read "two prime," etc.). These prime numbers are used to describe the direction of DNA strands using 5' to designate one side of the DNA molecule and using 3' to designate the other side of the DNA molecule.
When nucleotides are built in processes such as DNA replication and gene expression, these directions are incredibly important since nucleic acids can only form new bonds on the 3' end. As a result, it is often stated that nucleic acids (DNA and RNA) are built from 5' to 3'.
The sugar is deoxyribose in DNA and ribose in RNA. The phosphate, which makes DNA and RNA acidic, is connected to the 5' carbon of the sugar by the formation of an ester linkage between phosphoric acid and the 5'-OH group (an ester is an acid + an alcohol). In DNA nucleotides, the 3' carbon of the sugar deoxyribose is attached to a hydroxyl (OH) group. In RNA nucleotides, the 2' carbon of the sugar ribose also contains a hydroxyl group. The base is attached to the 1' carbon of the sugar.
Overall DNA Structure
The nucleotides form covalent bonds with each other to produce phosphodiester bonds (a fancy science name for the covalent bonds joining nucleotides together). The phosphate group forms a covalent bond with the hydroxyl group of the 3' carbon of the sugar of the next nucleotide, thereby forming a 5'-3' phosphodiester bond. In a polynucleotide, one end of the chain has a free 5' phosphate, and the other end has a free 3'-OH. These are called the 5' and 3' ends of the chain.
The result of nucleotides bonding together is a sugar-phosphate backbone with the nitrogenous bases hanging off of the side. This structure resembles one half of a ladder. The side support rails of the ladder is analagous to the sugar-phosphate backbone, and the half-rung in the middle of the ladder is analagous to the bases hanging off the side. In order for DNA to be a complete double-stranded molecule, the single DNA strand pairs with another DNA strand. The result is a complete ladder with the side-supports rails being sugar-phosphate backbones and the rungs in the middle are the bases of the two DNA strands interacting with each other in the middle.
The two DNA strands can only pair together when the two strands are antiparallel, that is, the strands are parallel, but in opposite directions. One DNA strand will be in the 5' to 3' direction, and it will pair with a strand upside-down to it in the 3' to 5' position. This arrangement enables complementary base pairs to hydrogen bond with each other. The DNA bases that meet in the middle of the double-stranded DNA molecule pair such that adenine (A) always pairs with thymine (T) and guanine (G) always pairs with cytosine (C). The A-T pair is held together by two hydrogen bonds and the G-C pair is held together by three hydrogen bonds.
The entire DNA ladder twists in three-dimensions to produce a helical formation to the molecule.
RNA Structure
DNA structure is very similar to DNA structure. RNA is composed of nucleotides bonded together with phosphodiester bonds forming a sugar-phosphate backbone with bases hanging off of the side. There are three main differences between DNA and RNA:
1. RNA nucleotides contain ribose as the pentose sugar instead of deoxyribose (ribose has an -OH on the 2' carbon whereas deoxyribose has an -H on the 2' carbon)
2. RNA nucleotides will not contain the base thymine, but will instead contain uracil; uracil base-pairs with adenine in the same way thymine does
3. RNA is a single-stranded molecule
DNA Replication
DNA replication has been well studied in prokaryotes primarily because of the small size of the genome and because of the large variety of mutants that are available. E. coli has 4.6 million base pairs in a single circular chromosome and all of it gets replicated in approximately 42 minutes, starting from a single site along the chromosome and proceeding around the circle in both directions. This means that approximately 1000 nucleotides are added per second. Thus, the process is quite rapid and occurs without many mistakes.
DNA replication employs a large number of structural proteins and enzymes, each of which plays a critical role during the process. One of the key players is the enzyme DNA polymerase, also known as DNA pol, which adds nucleotides one-by-one to the growing DNA chain that is complementary to the template strand. The addition of nucleotides requires energy; this energy is obtained from the nucleoside triphosphates ATP, GTP, TTP and CTP. Like ATP, the other NTPs (nucleoside triphosphates) are high-energy molecules that can serve both as the source of DNA nucleotides and the source of energy to drive the polymerization. When the bond between the phosphates is “broken,” the energy released is used to form the phosphodiester bond between the incoming nucleotide and the growing chain. In prokaryotes, three main types of polymerases are known: DNA pol I, DNA pol II, and DNA pol III. It is now known that DNA pol III is the enzyme required for DNA synthesis; DNA pol I is an important accessory enzyme in DNA replication, and along with DNA pol II, is primarily required for repair.
How does the replication machinery know where to begin? It turns out that there are specific nucleotide sequences called origins of replication where replication begins. In E. coli, which has a single origin of replication on its one chromosome (as do most prokaryotes), this origin of replication is approximately 245 base pairs long and is rich in AT sequences. The origin of replication is recognized by certain proteins that bind to this site. An enzyme called helicase unwinds the DNA by breaking the hydrogen bonds between the nitrogenous base pairs. ATP hydrolysis is required for this process. As the DNA opens up, Y-shaped structures called replication forks are formed. Two replication forks are formed at the origin of replication and these get extended bi-directionally as replication proceeds. Single-strand binding proteins coat the single strands of DNA near the replication fork to prevent the single-stranded DNA from winding back into a double helix.
DNA polymerase has two important restrictions: it is able to add nucleotides only in the 5' to 3' direction (a new DNA strand can be only extended in this direction). It also requires a free 3'-OH group to which it can add nucleotides by forming a phosphodiester bond between the 3'-OH end and the 5' phosphate of the next nucleotide. This essentially means that it cannot add nucleotides if a free 3'-OH group is not available. Then how does it add the first nucleotide? The problem is solved with the help of a primer that provides the free 3'-OH end. Another enzyme, RNA primase, synthesizes an RNA segment that is about five to ten nucleotides long and complementary to the template DNA. Because this sequence primes the DNA synthesis, it is appropriately called the primer. DNA polymerase can now extend this RNA primer, adding nucleotides one-by-one that are complementary to the template strand.
The replication fork moves at the rate of 1000 nucleotides per second. Topoisomerase prevents the over-winding of the DNA double helix ahead of the replication fork as the DNA is opening up; it does so by causing temporary nicks in the DNA helix and then resealing it. Because DNA polymerase can only extend in the 5' to 3' direction, and because the DNA double helix is antiparallel, there is a slight problem at the replication fork. The two template DNA strands have opposing orientations: one strand is in the 5' to 3' direction and the other is oriented in the 3' to 5' direction. Only one new DNA strand, the one that is complementary to the 3' to 5' parental DNA strand, can be synthesized continuously towards the replication fork. This continuously synthesized strand is known as the leading strand. The other strand, complementary to the 5' to 3' parental DNA, is extended away from the replication fork, in small fragments known as Okazaki fragments, each requiring a primer to start the synthesis. New primer segments are laid down in the direction of the replication fork, but each pointing away from it. (Okazaki fragments are named after the Japanese scientist who first discovered them. The strand with the Okazaki fragments is known as the lagging strand.)
The leading strand can be extended from a single primer, whereas the lagging strand needs a new primer for each of the short Okazaki fragments. The overall direction of the lagging strand will be 3' to 5', and that of the leading strand 5' to 3'. A protein called the sliding clamp holds the DNA polymerase in place as it continues to add nucleotides. The sliding clamp is a ring-shaped protein that binds to the DNA and holds the polymerase in place. As synthesis proceeds, the RNA primers are replaced by DNA. The primers are removed by the exonuclease activity of DNA pol I, which uses DNA behind the RNA as its own primer and fills in the gaps left by removal of the RNA nucleotides by the addition of DNA nucleotides. The nicks that remain between the newly synthesized DNA (that replaced the RNA primer) and the previously synthesized DNA are sealed by the enzyme DNA ligase, which catalyzes the formation of phosphodiester linkages between the 3'-OH end of one nucleotide and the 5' phosphate end of the other fragment.
Once the chromosome has been completely replicated, the two DNA copies move into two different cells during cell division.
The process of DNA replication can be summarized as follows:
1. DNA unwinds at the origin of replication.
2. Helicase opens up the DNA-forming replication forks; these are extended bidirectionally.
3. Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA.
4. Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling.
5. Primase synthesizes RNA primers complementary to the DNA strand.
6. DNA polymerase III starts adding nucleotides to the 3'-OH end of the primer.
7. Elongation of both the lagging and the leading strand continues.
8. RNA primers are removed by exonuclease activity.
9. Gaps are filled by DNA pol I by adding dNTPs.
10. The gap between the two DNA fragments is sealed by DNA ligase, which helps in the formation of phosphodiester bonds.
Enzyme/protein Specific Function
DNA pol I Removes RNA primer and replaces it with newly synthesized DNA
DNA pol III Main enzyme that adds nucleotides in the 5'-3' direction
Helicase Opens the DNA helix by breaking hydrogen bonds between the nitrogenous bases
Ligase Seals the gaps between the Okazaki fragments to create one continuous DNA strand
Primase Synthesizes RNA primers needed to start replication
Sliding Clamp Helps to hold the DNA polymerase in place when nucleotides are being added
Topoisomerase Helps relieve the strain on DNA when unwinding by causing breaks, and then resealing the DNA
Single-strand binding proteins (SSB) Binds to single-stranded DNA to prevent DNA from rewinding back.
Laboratory Instructions
DNA Structure & RNA Structure
1. Organize the puzzle pieces so that all of the puzzle pieces and in piles by type (e.g. one pile for phosphate groups, one pile for deoxyribose, one pile for guanine, etc.).
2. Answer questions 1-5 in the "Results & Questions" section under "DNA Structure."
3. Build eight DNA bases: one with adenine, one with thymine, one with guanine, and one with cytosine.
4. Build four RNA bases: one with adenine, one with uracil, one with guanine, and one with cytosine.
5. Answer questions 6-8 in the "Results & Questions" section under "DNA Structure."
6. Connect DNA nucleotides to each other to form the sequence: 5'-GTAC-3'
7. Connect RNA nucleotides to each other to form the sequence: 5'-GUAC-3'
8. Answer questions 9-11 in the "Results & Questions" section under "DNA Structure."
9. Build a complementary strand of DNA to the DNA sequence already built and pair them together.
10. Answer questions 12-17 in the "Results & Questions" section under "DNA Structure."
DNA Replication
1. Build the following DNA nucleotides:
• 4 adenine DNA nucleotides
• 4 thymine DNA nucleotides
• 8 guanine DNA nucleotides
• 8 cytosine DNA nucleotides
2. Build a single strand of DNA with the following structure: 5'-AGCCTG-3'
3. Build the complementary DNA strand to the sequence above and pair it with the DNA molecule you made in step 2.
4. Answer questions 1-6 in the "Results & Questions" section under "DNA Replication."
5. The segment of DNA you built is the origin of replication. Presto-change-o! You are now the enzyme helicase! Do what helicase would do.
6. Answer questions 7-9 in the "Results & Questions" section under "DNA Replication."
7. Presto-change-o! You are now the enzyme DNA pol III (a DNA polymerase the elongates growing DNA strands during DNA replication). Build new DNA molecules to complement the template DNA strands starting at the 5' end and ending at the 3' end of the new strands (just like DNA pol III would do).
8. Answer questions 10-13 in the "Results & Questions" section under "DNA Replication."
9. Disassemble all of the puzzle pieces and return to the box/bag where they came from.
Results & Questions
DNA Structure & RNA Structure
1. DNA nucleotides contain deoxyribose as the pentose (5-carbon) sugar and RNA nucleotides contain ribose as the pentose sugar. Compare the chemical structures written on the deoxyribose and ribose puzzle pieces. What is the difference between ribose and deoxyribose?
2. Consider your answer to question 1. What do you suppose "deoxy-" is referring to in the name "deoxyribose?"
3. Identify the different base puzzle pieces (there are 5 types). Examine the chemical structures of the bases written on these puzzle pieces. Why do you suppose that DNA and RNA bases are often referred to as "nitrogenous bases?"
4. Adenine and guanine are classified as "purines" and thymine, cytosine, and uracil are classified "pyrimidines." Looking at the chemical structures written on these puzzle pieces, what pattern do you notice that distinguishes purines from pyrimidines?
5. Adenine pairs with thymine in DNA and with uracil in RNA. Guanine pairs with cytosine in both DNA and RNA. What generalization can you make about base-pairing and whether bases are purines or pyrimidines (e.g. do purines pair with purines, to pyrimidines pair with pyrimidines, or do purines pair with pyrimidines)?
6. Compare the the DNA nucleotides with the RNA nucleotides. What similarities are there?
7. Compare the the DNA nucleotides with the RNA nucleotides. What differences are there?
8. On the image above showing a DNA nucleotide, write in 5' to show the 5' side of the nucleotide and write in 3' to show the 3' side of the nucleotide.
9. Covalent bonds were formed between the nucleotides you joined together. These covalent bonds have a special name. What are these bonds called?
10. Identify the 5' end and the 3' end of the DNA strand. Identify the 5' end of the RNA strand and the 3' end of the DNA strand. What component of the nucleotides do you find at the 5' end of these strands?
11. What is missing from the DNA puzzle?
12. Compare the DNA and RNA structures. What new difference is apparent in their structures?
13. Examine the two DNA strands paired together. Notice that one DNA strand faces 5' to 3' and the other DNA strand is in the reverse direction. What is this arrangement called?
14. The two DNA strands can easily be pulled apart from each other at the bases (unlike other locations in the puzzle, such as between the phosphates and the bases). Why is this? What types of interactions hold the bases of two DNA strands together?
15. What are hydrogen bonds?
16. How many hydrogen bonds are formed in the A-T pair?
17. How many hydrogen bonds are formed in the G-C pair?
DNA Replication
1. Fill in the diagram above with the following:
• phos. = phosphate group
• deoxy. = deoxyribose
• A = adenine
• T = thymine
• G = guanine
• C = cytoskne
• 5' - show the 5' end on both DNA strands
• 3' - show the 3' end on both DNA strands
2. The two DNA strands interact at the bases with hydrogen bonds. Are hydrogen bonds weak or strong attractions?
3. How might it be possible for the two DNA strands to separate at the bases?
4. What is the purpose of DNA replication?
5. Where on a DNA molecule does DNA replication begin?
6. What DNA replication enzyme breaks the hydrogen bonds between the nitrogenous bases to create single-stranded sections of DNA?
7. What did helicase do to the double-stranded DNA puzzle?
8. What enzyme elongates a growing DNA strand during DNA replication?
9. What end of a growing DNA molecule can the enzyme named in question 8 add new nucleotides to (5' or 3')?
10. What features of the actual process of DNA replication are missing from this re-creation of DNA replication? There are at least three.
11. Would DNA pol III be able to create a new DNA molecule such as we did in this puzzle re-creation of DNA replication? Explain your answer.
12. How is DNA replication a semiconservative process and how did we show this in the puzzle re-creation of DNA replication?
13. Compare the newly replicated DNA double-strands in your puzzle pieces. Closely examine the sequences of the DNA and the directions of the strands. Are the two double strands identical? Give the DNA sequences with 5' and 3' directionality to explain. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.30%3A_DNA_RNA_and_DNA_Replication.txt |
Learning Objectives
• Define the following: PCR, amplify, Taq polymerase, primer, thermal cycler, denaturation, annealing, extension.
• Give at least three applications of PCR in microbiology.
• Tell what PCR does.
• Explain how PCR works including the three steps of PCR and what happens in each of those steps.
• Successfully conduct PCR in the laboratory.
• Successfully interpret results from a PCR experiment.
Introduction to PCR
Polymerase chain reaction (PCR) is a technique that scientists use to amplify (make many copies of) specific DNA regions for further analysis. PCR has a huge variety of applications including:
• diagnosis of a microbial infection
• studying disease-causing organisms
• determining the presence of difficult to culture, or unculturable, microorganisms in humans or environmental samples
• amplifying a target region of DNA for cloning into a plasmid vector
• detecting genetic diseases
• cloning gene fragments to analyze genetic diseases
• identifying contaminant foreign DNA in a sample
• preparing DNA for sequencing
• determining paternity
• identifying the source of a DNA sample left at a crime scene
• comparing samples of ancient DNA with modern organisms
Most methods of DNA analysis, such as restriction enzyme digestion and agarose gel electrophoresis, or DNA sequencing require large amounts of a specific DNA fragment. In the past, large amounts of DNA were produced by growing the host cells of a genomic library. However, libraries take time and effort to prepare and DNA samples of interest often come in minute quantities. The polymerase chain reaction (PCR) permits rapid amplification in the number of copies of specific DNA sequences for further analysis. One of the most powerful techniques in molecular biology, PCR was developed in 1983 by Kary Mullis while at Cetus Corporation.
Taq Polymerase: An Enzyme Making PCR Possible
PCR is an in vitro laboratory technique (done outside of cells - in this case in a small centrifuge tube) that takes advantage of the natural process of DNA replication. Recall that DNA replication requires a DNA polymerase enzyme to build a DNA molecule complementary to a template DNA molecule. In PCR, A heat-stable DNA polymerase enzyme is used since PCR requires high temperatures to denature DNA (separate double-stranded DNA to make the DNA single-stranded). The heat-stable DNA polymerase does not denature in these conditions since it is derived from a hyperthermophilic bacterial species ("loves" hot temperatures) called Thermus aquaticus. Taq polymerase is a DNA polymerase from T. aquaticus (taking "T" from the genus and "aq" from the first two letters of the specific epithet). T. aquaticus was first isolated from a hot spring in Yellowstone National Park and thrives in very hot temperatures.
DNA Replication (and PCR) Require Primers
DNA replication requires the use of primers for the initiation of replication. Recall that DNA polymerases can only elongate a DNA molecule and cannot build a new strand from the start. In living cells, DNA replication uses the enzyme primase to build primers composed of RNA that DNA polymerase can elongate to build new DNA strands. In the laboratory setting, RNA is not very stable stable, and therefore, DNA primers are used for PCR. Primers not only are necessary for a DNA polymerase to elongate and produce DNA copies, primers are used in PCR to target a specific DNA sequence that we want to copy. Primer sequences are specifically designed and engineered with a specific sequence in order to target a specific DNA region. This insures that only the DNA sequence we want to copy gets copied and not other places in the DNA template molecule(s).
For example, if PCR is used for diagnosis of a microbial infection in a human, primers would be designed with sequences that match a specific region of DNA in that microbe, and does not match DNA in other microbes or in humans (human DNA will be mixed with any samples taken from a human). If the DNA is successfully copied (amplified), and matches positive controls (samples that contain the microbe the test is looking for), that microbe is causing infection in that human.
PCR Uses Temperature Cycles to Amplify (Copy) DNA
PCR occurs over multiple cycles. Each cycle containing three steps: denaturation, annealing, and extension. Machines called thermal cyclers are used for PCR; these machines can be programmed to automatically cycle through the temperatures required at each of the denaturation, annealing, and extension steps.
1. denaturation: First, double-stranded template DNA containing the target sequence is denatured at approximately 95 °C. The high temperature required to physically (rather than enzymatically) separate the DNA strands is the reason the heat-stable DNA polymerase is required.
2. annealing: Next, the temperature is lowered to approximately 50 °C (although this can vary based on the PCR protocol and primers). This allows the DNA primers complementary to the ends of the target sequence to anneal (stick) to the template strands, with one primer annealing to each strand.
3. extension: Finally, the temperature is raised to 72 °C, the optimal temperature for the activity of Taq polymerase, allowing for the addition of nucleotides to the primer using the single-stranded target as a template.
Each cycle (denaturation, annealing, and extension) doubles the number of double-stranded target DNA copies. Typically, PCR protocols include 25–40 cycles, allowing for the amplification of a single target sequence by tens of millions to over a trillion.
Video 1: Animation showing what occurs inside of a PCR tube during the PCR reaction. Pay careful attention to the thermometer in the top left corner since it is showing how the temperature changes created by the thermal cycler stimulates each of the PCR steps to occur.
Analysis of PCR Products
In order to analyze the PCR products after the DNA region of interest has been amplified, it is common for gel electrophoresis to be used to separate DNA based on size. The PCR products can then be compared with controls and other samples to make conclusions the sample. See the chapter on DNA Fingerprinting to see how gel electrophoresis works.
Laboratory Instructions
Laboratory instructions will vary based on the protocol your instructor is following. Your instructor will provide specific instructions for this laboratory.
Questions
1. What does PCR stand for?
2. Give three examples of applications of PCR.
3. What does PCR do?
4. What cellular process does PCR mimic inside of a laboratory tube?
5. What is Taq polymerase and where does it come from?
6. How is Taq polymerase different from other DNA polymerases?
7. Why does PCR require a thermostable DNA polymerase?
8. Give two reasons why primers are used in PCR.
9. Explain how primers target a specific DNA sequence.
10. Why are DNA primers used in PCR when RNA primers are used inside cells?
11. What instrument is necessary for PCR and what does it do?
12. List the three steps of PCR and what happens in each step.
13. The three steps you listed in the previous question, how many times are they repeated during the PCR process?
14. How many DNA copies are produced of the primer-targeted DNA during PCR? | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.31%3A_PCR.txt |
Learning Objectives
• Give at least three applications for DNA fingerprinting.
• Explain/apply how restriction enzymes work, including be able to identify recognition sites/sequences and predict DNA fragment sizes from examples.
• Define and use the following terms: restriction enzyme, recognition site/sequence, sticky ends, blunt ends, restriction fragment length polymorphism (RFLP), gel electrophoresis.
• Explain/apply how gel electrophoresis works.
• Successfully load and run an electrophoresis gel.
• Analyze and interpret results from an electrophoresis gel.
• Analyze a viral outbreak scenario and determine the best course of action.
Applications of DNA Fingerprinting
DNA fingerprinting is a way to identify using DNA. Some applications of DNA fingerprinting include:
• identifying a microbe causing an infection (diagnostic test)
• identifying microbes for scientific research
• paternity testing
• forensic DNA analysis to match DNA to criminal suspects
• a wide variety of genetic research
DNA fingerprinting involves multiple biotechnologies, including PCR (see the chapter on PCR), but here this laboratory focuses on creating DNA fingerprints using restriction enzymes and visualizing the DNA fingerprints using gel electrophoresis.
Creating a DNA Fingerprint
Restriction Enzymes
DNA fingerprints are created by first isolating DNA from an unknown sample to be identified and compared with known samples. If the samples match, it enables identification. The isolated DNA (i.e. DNA that has been removed from cells and other cell components) is mixed with a restriction enzyme to create a fingerprint. The restriction enzyme will cut the DNA in a pattern that will differ from DNA from other sources, unless the identify of the DNA is the same (matching known and unknown samples enables identification).
The DNA fragments produced by the restriction enzyme are separated by size using an approach called gel electrophoresis (see the Gel Electrophoresis section below). The result is a pattern of bands that can be compared with other patterns from known samples. If fingerprints match, it likely means that the DNA originated from the same organism. For paternity testing, half of the fingerprint will originate from the biological mother and half of the fingerprint will originate from the biological father.
Restriction enzymes are found in some bacteria and have been isolated to use for a variety of biotechnologies such as DNA fingerprinting. These enzymes cut DNA at a characteristic recognition site. Recognition sites are different for each restriction enzyme. Typically, recognition sites are palindromic, that is they read the same backwards and forwards. Ordinary words that are palindromic include "mom," "dad," "wow," and "racecar." With DNA, a palindrome is based on reading one DNA strand 5' to 3' and comparing it with its complement DNA strand as read 5' to 3'. For example:
5'-GAATTC-3'
3'-CTTAAG-5'
Notice that the complementary DNA strands above, if reading from the 5' end, have the same sequence: 5'-GAATTC-3'. This example is a recognition sequence from the restriction enzyme known as EcoRI. The "Eco" part of the enzyme name comes from the fact that this enzyme originates from Escherichia coli (E. coli). The rest of the restriction enzyme name comes from the strain of the organism (in this case the R strain) and the order in which the enzyme was discovered (in this case it is the first restriction enzyme discovered from the R strain of E. coli - I is the Roman numeral for 1).
In the example of EcoRI, this enzyme cuts the DNA between the "G" and the "A" on the 5' side. As a result, both the top and bottom DNA strands are cut and only held together by the few hydrogen bonds between the 5'-AATT-3' on both strands. Because of this (and that hydrogen bonds are rather weak attractions), the two DNA strands fully separate leaving the 5'-AATT-3' overhangs on both broken strands. These overhangs are called sticky ends.
Restriction enzymes will identify every location on a DNA molecule with the recognition sequence and cut the DNA there. This means the a restriction enzyme will likely make multiple cuts in the DNA. This will produce DNA fragments of different numbers of fragments with different sizes based on the base sequence of the DNA. The fragment sizes and number of fragments produces the DNA fingerprint used for identification purposes in DNA fingerprinting.
DNA Fingerprinting is also called Restriction Fragment Length Polymorphisms (RFLPs) Analysis
Restriction enzyme recognition sites are short (only a few nucleotides long), sequence-specific palindromes, and may be found throughout the genome. Thus, differences in DNA sequences in the genomes of individuals will lead to differences in distribution of restriction enzyme recognition sites that can be visualized as distinct fingerprints using a technique called gel electrophoresis (see the seciton on Gel Electrophoresis below). Restriction fragment length polymorphism (RFLP) analysis compares DNA banding patterns of different DNA samples after restriction digestion.
RFLP analysis has many practical applications in both medicine and forensic science. For example, epidemiologists use RFLP analysis to track and identify the source of specific microorganisms implicated in outbreaks of food poisoning or certain infectious diseases. RFLP analysis can also be used on human DNA to determine inheritance patterns of chromosomes with variant genes, including those associated with heritable diseases or to establish paternity.
Forensic scientists use RFLP analysis as a form of DNA fingerprinting, which is useful for analyzing DNA obtained from crime scenes, suspects, and victims. DNA samples are collected, the numbers of copies of the sample DNA molecules are increased using PCR, and then subjected to restriction enzyme digestion and agarose gel electrophoresis to generate specific banding patterns. By comparing the banding patterns of samples collected from the crime scene against those collected from suspects or victims, investigators can definitively determine whether DNA evidence collected at the scene was left behind by suspects or victims.
Gel Electrophoresis
Gel electrophoresis is a technique commonly used to separate biological molecules based on size and biochemical characteristics, such as charge and polarity. Agarose gel electrophoresis is widely used to separate DNA (or RNA) of varying sizes that may be generated by restriction enzyme digestion (such as DNA fingerprinting / RFLP analysis) or by other means, such as the PCR.
Important
DNA molecules have an overall negative charge. This is due to negative charges on the phosphate groups of its nucleotides. As a result, DNA can be pulled toward a positive charge. This is how gel electrophoresis pulls DNA through an agarose gel.
Due to its negatively charged backbone, DNA is strongly attracted to a positive electrode. In agarose gel electrophoresis, the gel is oriented horizontally in a buffer solution. Samples are loaded into sample wells on the side of the gel closest to the negative electrode, then drawn through the molecular sieve of the agarose matrix toward the positive electrode. The agarose matrix impedes the movement of larger molecules through the gel, whereas smaller molecules pass through more readily. Thus, the distance of migration is inversely correlated to the size of the DNA fragment, with smaller fragments traveling a longer distance through the gel. Sizes of DNA fragments within a sample can be estimated by comparison to fragments of known size in a DNA ladder also run on the same gel.
Important
Small DNA fragments travel farther through the electrophoresis gel than larger DNA fragments.
You can think about this as being analagous to rocks in a river. Large boulders do not move very far, even if the current is swift, but small pebbles are capable of moving great distances in river's current. Similarly, large DNA cannot move well through the gel and will remain closer to the wells and smaller DNA can move easily through the gel and will move farther away from the wells.
Laboratory Instructions
DNA Fingerprinting Scenario
You are an epidemiologist responsible for tracking outbreaks of potentially deadly disease and coordinating responses to help contain the spread of these diseases. It has been reported that a new viral infection is spreading in the Midwestern United States. This infection has symptoms very similar to two pervious viral outbreaks:
1. the East Coast viral outbreak produced disease the progressed to multiple organ failure and death
2. the West Coast viral outbreak produced disease with manageable symptoms and did not progress to organ failure or death
With this new outbreak in the Midwest, you believe that the virus causing the infections is either the a virus similar to the East Coast outbreak or the West Coast outbreak. How you organize the response to this outbreak is going to be dependent on whether the virus in the Midwest is the more severe strain or the less severe strain.
To determine if the virus is the East Coast virus or the West Coast virus, you conduct RFLP analysis (aka DNA fingerprinting) by extracting the viral DNA and digesting it with a restriction enzyme. To visualize the DNA fingerprint and compare it with the DNA fingerprint of the East Coast and West Coast viruses, you must use gel electrophoresis and then analyze the results.
Load and Run an Electrophoresis Gel
1. If the agarose gel has been prepared for you, place it into an electrophoresis chamber, making sure that the wells are positioned on the negative side (black-colored electrode).
2. Cover the gel completely with running buffer.
3. Use a micropipette and sterile tip to carefully load 20 μl of DNA with loading dye into the wells of the gel as instructed by your instructor. Here are some important things to consider:
• Make sure the micropipette tip is inside the well before pushing down the plunger to put the DNA in the well.
• Careful that the micropipette tip does not go too far into the well since it can break the bottom of the well open and release your DNA into the buffer rather than into the well.
• When you are ready, push down the plunger slowly so the DNA does not get pushed out of the well from the force of it being expelled out of the micropipette.
• Careful not to release the plunger while still inside of the well since this will suck the DNA back up into the micropipette tip.
4. When the gel is loaded, close the electrophoresis chamber and plug it into a power source, making sure that black is plugged into black and red is plugged into red.
5. Set the voltage of the power source as instructed by your instructor and turn the power on.
6. Watch as bubbles are generated in the buffer as the electrical current passes through the buffer.
7. Check back in 10-15 minutes to see how the loading dye has moved across the gel.
8. At the time instructed by your instructor, turn off the power supply.
9. Unplug the leads from the power supply.
10. Remove the top of the electrophoresis chamber.
11. Carefully remove the gel in its plastic holder and follow your instructor's instructions for staining (if there is time / if you are proceeding to staining in class).
Results & Questions
Table 1: Results from gel electrophoresis of DNA fingerprints of East coast virus, West coast virus, and Midwest virus. Some of the columns and rows may not be used in the table below, depending on how your gel was set up and the number of bands in each lane.
well 1 well 2 well 3 well 4 well 5 well 6
sample in this well:
band 1 migration (mm)
band 2 migration (mm)
band 3 migration (mm)
band 4 migration (mm)
band 5 migration (mm)
band 6 migration (mm)
band 7 migration (mm)
band 8 migration (mm)
band 9 migration (mm)
band 10 migration (mm)
1. Analyze the banding pattern on the gel by measuring the distance each band traveled from the well in millimeters (mm). See the diagram above to assist with how to make these measurements. Always measure the migration distance from the well, not from the previous band. Fill in the table above with your results.
2. Carefully examine the banding pattern in each well. Are there any banding patterns that are similar or the same? If so, which ones?
3. What does it indicate when a known and unknown DNA fingerprint have the same banding pattern?
4. Based on the results you analyzed on the gel, what conclusions can you make about the Midwest virus?
5. Based on the conclusion you made above about the Midwest virus, do you think a lock-down quarantine is necessary to contain the outbreak? Explain your answer.
6. Why is a restriction enzyme is used during DNA fingerprinting?
7. Why is it necessary to compare a DNA fingerprint of an unknown sample to DNA fingerprints of known samples?
8. Why is it that DNA from different sources will produce different banding patterns from each other when you use the same restriction enzyme? Use the following in your answer: restriction enzyme, base sequence, recognition site, fragment size, fragment number
9. How does gel electrophoresis separate DNA based on size? In your answer, be sure to mention: DNA's charge, electrophoresis current, agarose gel
10. Fill in the blank: Larger DNA fragments will produce bands _______ to/from the wells.
11. Fill in the blank: Smaller DNA fragments will produce bands _______ to/from the wells.
12. Below are illustrations of DNA that has been digested by a restriction enzyme in the locations shown with the dotted lines. The bands produced by sample A has been placed on the gel for you. Draw in bands in lanes B through E the correct positions we would expect to see the bands based on the DNA fragment sizes and fragment numbers shown and relative to the sizes and positions of the bands already in the first lane for sample A. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.32%3A_DNA_Fingerprinting.txt |
Learning Objectives
• Describe and explain Griffith's experiment originating bacterial transformation.
• Define and properly use the following terms: transformation, recombinant DNA, transgenic, competent cells, biotechnology, vector, genetic engineering, plasmid, horizontal gene transfer, selectable marker, GFP.
• Tell at least two applications for bacterial transformation.
• Explain how bacterial transformation works.
• Successfully conduct a bacterial transformation.
• Interpret results from bacterial transformation.
The Origins of Bacterial Transformation
British bacteriologist Frederick Griffith reported the first demonstration of bacterial transformation—a process in which external DNA is taken up by a cell, thereby changing its morphology and physiology. Griffith conducted his experiments with Streptococcus pneumoniae, a bacterium that causes pneumonia. Griffith worked with two strains of this bacterium called rough (R) and smooth (S). The two cell types were called “rough” (R) and “smooth” (S) after the appearance of their colonies grown on a nutrient agar plate.
The R strain is non-pathogenic (does not cause disease). The S strain is pathogenic (disease-causing), and has a capsule outside its cell wall. The capsule allows the cell to escape the immune responses of the host mouse.
When Griffith injected the living S strain into mice, they died from pneumonia. In contrast, when Griffith injected the live R strain into mice, they survived. In another experiment, when he injected mice with the heat-killed S strain, they also survived. This experiment showed that the capsule alone was not the cause of death. In a third set of experiments, a mixture of live R strain and heat-killed S strain were injected into mice, and—to his surprise—the mice died. Upon isolating the live bacteria from the dead mouse, only the S strain of bacteria was recovered. When this isolated S strain was injected into fresh mice, the mice died. Griffith concluded that something had passed from the heat-killed S strain into the live R strain and transformed it into the pathogenic S strain. He called this the transforming principle. These experiments are now known as Griffith's transformation experiments.
What occurred in Griffith's experiments in 1928 could not be fully explained at that time. Scientists then were unaware of the role of DNA blueprints for a living thing. Further, with such limited knowledge about DNA and what it does, scientists then were also unaware that bacteria are capable of "picking up" DNA from other bacterial cells or picking up DNA from their environment.
In Griffith's experiment, DNA from the dead S strain were transferred into the live R strain. This DNA provided the R strain with instructions for building the S strain's capsule. As a result, the live R strain cells began to produce the S strain's capsule and thereby developed the ability to evade the host's immune system. The result is the R strain acted in the same way as the S strain, became pathogenic, and caused death in the mice. In essence, what happened in these circumstances is bacterial transformation. Bacterial transformation is a process where bacteria absorb DNA from their environment resulting in new characteristics in the bacteria. The new characteristics that transformed bacteria (also called transgenic bacteria) exhibit is dependent on what types of genes are present in the DNA that they took up from their environment.
Definition
transgenic: describes a living thing that has DNA introduced into it through artificial means; the DNA added to the transgenic organism originates from a different species
Why Transform Bacteria?
Using living systems to benefit humankind is called biotechnology. Technically speaking, the domestication of plants and animals through farming and breeding practices is a type of biotechnology, however in a contemporary sense, we associate biotechnology with the direct alteration of an organism’s genetics to achieve desirable traits through the process of genetic engineering.
Genetic engineering involves the use of recombinant DNA technology, the process by which a DNA sequence is manipulated in vitro, thus creating recombinant DNA molecules that have new combinations of genetic material. The recombinant DNA is then introduced into a host organism. If the DNA that is introduced comes from a different species, the host organism is considered to be transgenic.
One example of a transgenic microorganism is the bacterial strain that produces human insulin. The insulin gene from humans was inserted into a plasmid. This recombinant DNA plasmid was then inserted into bacteria. As a result, these transgenic microbes have the DNA necessary to produce and secrete human insulin.
Many prokaryotes have the ability to acquire foreign DNA and incorporate functional genes into their own genome through “mating” with other cells (conjugation), viral infection (transduction), and taking up DNA from the environment (transformation). These mechanisms of DNA transfers are examples of horizontal gene transfer—the transfer of genetic material between cells of the same generation.
Beyond applications that generate useful products for humans, transformation is incredibly useful in studying genetics. Transformation has enabled scientists to expand understanding of the functions of specific genes and regulation of gene expression (which genes are turned on/off in what circumstances, and how those genes are regulated).
How does Bacterial Transformation Work?
Plasmids
A gene of interest (a segment of DNA that codes for a gene to be inserted into a living thing) are commonly inserted into plasmids. Plasmids are small pieces of typically circular, double-stranded DNA that replicate independently of the bacterial chromosome. In recombinant DNA technology, plasmids are often used as vectors - they are used for transferring DNA. Plasmids can be used to carry DNA fragments from one organism to another.
Plasmids used as vectors can be genetically engineered by researchers and scientific supply companies to have specialized properties. Some plasmid vectors contain genes that confer a selectable marker to help researchers separate bacteria carrying the plasmid from bacteria not carrying the plasmid. One of the most common selectable markers used is antibiotic resistance genes. For this, the plasmid is engineered with an antibiotic resistance gene so that all bacteria carrying the plasmid are resistant to that antibiotic. If the bacteria are then plated on petri plates containing the antibiotic in the medium, then only those bacteria that carry the plasmid with its antibiotic resistance gene will grow. Bacteria without the plasmid will be killed by the presence of the antibiotic in the medium since they lack the antibiotic resistance gene. The colonies that grow in the presence of the antibiotic can therefore be used in the ongoing research knowing that they carry the plasmid.
Bacterial Transformation
The most commonly used mechanism for introducing engineered plasmids into a bacterial cell is transformation, a process in which bacteria take up free DNA from their surroundings. In nature, free DNA typically comes from other lysed bacterial cells. In the laboratory, free DNA in the form of recombinant plasmids is introduced to the cell’s surroundings.
Some bacteria, such as Bacillus spp., are naturally competent, meaning they are able to take up foreign DNA. However, not all bacteria are naturally competent. In most cases, bacteria must be made artificially competent in the laboratory by increasing the permeability of the cell membrane. This can be achieved through chemical treatments that neutralize charges on the cell membrane or by exposing the bacteria to an electric field that creates microscopic pores in the cell membrane. These methods yield chemically competent or electrocompetent bacteria, respectively.
GFP
GFP stands for green fluorescent protein. This protein is coded in a gene that originated in the jellyfish species Aequorea victoria. GFP is commonly used in genetics research to visualize expression of a gene. GFP glows fluorescent green under ultraviolet light.
Laboratory Instructions
Your instructor will provide instructions for the transformation protocol you will use in this laboratory.
Questions
1. Define the following:
• transformation
• Recombinant DNA
• Genetic engineering
• Transgenic
• Biotechnology
• Vector
• Selectable marker
• Plasmid
• GFP
• Competent cells
2. Cells are made competent using one of two different approaches. What are these?
3. Explain how antibiotic resistance can be used as a selectable marker in genetic engineering.
4. Why are selectable markers used in genetic engineering?
5. Give at least two reasons it is useful to transform bacteria with certain genes of interest.
6. What is the purpose of the heat-shock step during bacterial transformation?
7. What would happen if you forgot to add the plasmid before the heat shock step? Would this change the results from this experiment? Explain.
8. What was added into the medium in the petri plates to insure that only bacteria carrying the plasmid would grow? | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.33%3A_Bacterial_Transformation.txt |
Learning Objectives
• Identify and name the following protozoan parasites in microscopic samples: Plasmodium sp., Trypanosoma cruzi, and Trichomonas vaginalis
• Name the diseases caused by the following protozoan parasites: Plasmodium sp., Trypanosoma cruzi, and Trichomonas vaginalis
• Explain how the following protozoan parasites are transmitted to humans: Plasmodium sp., Trypanosoma cruzi, and Trichomonas vaginalis
• Interpret the life cycles of the following protozoan parasites: Plasmodium sp., Trypanosoma cruzi, and Trichomonas vaginalis
• Examine the following protozoan parasites using a microscope and illustrate and label the specimens: Plasmodium sp., Trypanosoma cruzi, and Trichomonas vaginalis
Introduction to Protozoan Parasites
All protozoans are found in a life stage called trophozoites. The “troph” stage is actively feeding, mostly motile and responsible for symptoms in a host. Some protozoans can form a cyst stage – a resting, inactive stage that protects the organism because of a thick wall that is produced. These cysts enable the protozoan to survive harsh environmental conditions outside of a host and ensure the organism will be passed to other hosts. Ingestion of the cyst in contaminated food or water is a common method of acquiring many protozoan infections.
Many protozoan parasites require vectors for transmission to occur. A vector is a transport mechanism. With parasites requiring a vector, the vector is a living thing, often an arthropod species (e.g. mosquito).
The life cycles of parasites are very specific. Parasites require transmission to specific species of organisms (the hosts) in a specific order to continue spreading and reproducing. At times, a parasite may be transmitted to a host that is not in their life cycle. This accidental host can experience symptoms of parasite infection, but the parasite is unable to continue its life cycle and continue reproduction.
In additional to an accidental host, host species can also be classified as either the definitive host or the intermediate host:
• definitive host: a host species that can harbor either the adult form of a parasite or the sexual stage of a parasite
• intermediate host: a host species that can harbor an immature form of a parasite in a non-sexual stage
Protozoan parasites can undergo sexual reproduction, asexual reproduction, or both sexual and asexual reproduction depending on the species. Asexual reproduction will typically involve cell divisions to produce new individuals, whereas sexual reproduction will involve production and fusion of gametes ("male" and "female" cells that have half the DNA of the other cells and can fuse to produce genetically unique individuals).
Plasmodium sp. (Cause of Malaria)
Introduction to Plasmodium sp.
Plasmodium (plaz-mo’dee-um) causes malaria, one of the common causes of hemolytic anemia worldwide. There are four species of PlasmodiumP. vivax (most common), P. ovale, P. malariae, and P. falciparum (most lethal). Typical symptoms are fever, chills, headache, muscle pain, and sweating. According to WHO, there were an estimated 229 million cases of malaria per year worldwide, mostly (94%) in the African Region. Cases of malaria diagnosed in the United States were mostly seen in travelers and immigrants returning to the U.S. from countries where malaria is endemic.
Malaria is the number one cause of death by parasites in the world. To continue existing, it must alternate sexual and asexual cycles. Interruption of either life cycle will control the disease. Measures taken to interrupt the life cycle include attempts to eliminate the Anopheles mosquito, to protect the host from being bitten using chemical repellants and mosquito netting, to prophylactically treating travelers in high risk areas, to cure active cases with various antiparasitic drugs. The occurrence of drug resistant strains worldwide has dramatically increased in recent years. Contact with CDC will aid in determining the best prophylactic drug and drug of choice for treatment depending on the patient’s health and the area in which malaria was acquired.
Clinical Presentation of Malaria
The symptoms of uncomplicated malaria can be rather non-specific and the diagnosis can be missed if health providers are not alert to the possibility of this disease. Since untreated malaria can progress to severe forms that may be rapidly (<24 hours) fatal, malaria should always be considered in patients who have a history of exposure (mostly: past travel or residence in disease-endemic areas). The most frequent symptoms include fever and chills, which can be accompanied by headache, myalgias, arthralgias, weakness, vomiting, and diarrhea. Other clinical features include splenomegaly, anemia, thrombocytopenia, hypoglycemia, pulmonary or renal dysfunction, and neurologic changes. The clinical presentation can vary substantially depending on the infecting species, the level of parasitemia, and the immune status of the patient. Infections caused by P. falciparum are the most likely to progress to severe, potentially fatal forms with central nervous system involvement (cerebral malaria), acute renal failure, severe anemia, or acute respiratory distress syndrome. Other species can also have severe manifestations. Complications of P. vivax malaria include splenomegaly (with, rarely, splenic rupture), and those of P. malariae include nephrotic syndrome.
Asexual Stage of Plasmodium sp. in Humans
One becomes infected with Plasmodium through the bite of the infected female Anopheles mosquito. The mosquito transmits Plasmodium sporozoites via her saliva when she inserts her proboscis into human skin to obtain a blood meal. The blood provides nourishment for the eggs she will lay. Sporozoites injected into the blood stream leave the blood vascular system within a period of forty minutes and invade the parenchymal cells of the liver. In liver cells, the sporozoites undergo asexual multiplication. They are then liberated and invade red blood cells, initiating the blood stream phase of the infection.
An asexual cycle, known as schizogony, takes place within the red cells of the infected host. This process results in the formation of four to thirty-six new parasites in each red cell. Immature trophozoites, called a “ring” forms, develop which then enlarge to become mature trophozoites, filling most of the parasitized red blood cells. Asexual multiplication occurs when the trophozoites’ nuclear material and cytoplasm split. At the end of the schizogonic cycle the infected blood cells rupture, liberating merozoites, which, in turn, infect new red blood cells. Lysis of the red cells liberates products of metabolism of the parasites and the red cells. These toxic materials cause the symptoms of malaria – chills, fever, nausea, vomiting and headache. The fever spikes occur at varying intervals of 24, 48, or 72 hours depending on the species of Plasmodium present. The febrile period may last several hours, ending with a profuse sweating stage. This cycle is repeated many times.
Sexual Stage of Plasmodium sp. in Mosquitos
Sexual stage male and female gametocytes may also appear in the red blood cells. When the mosquito bites an infected person, she draws blood into her stomach which may contain male and female gametocytes. In the mosquito’s gut, the male gametocytes form spermatozoa and the female forms an ovum. Fertilization takes place. The resulting zygote can invade the gut wall and produce numerous sporozoites. The sporozoites migrate through the tissue of the mosquito to the salivary glands where they will be injected into the next human host when the mosquito takes another blood meal. The asexual cycle then proceeds in the new host.
Plasmodium sp. Life Cycle
Blood parasites of the genus Plasmodium. There are approximately 156 named species of Plasmodium which infect various species of vertebrates. Four species are considered true parasites of humans, as they utilize humans almost exclusively as a natural intermediate host: P. falciparum, P. vivax, P. ovale and P. malariae.
During a blood meal, a malaria-infected female Anopheles mosquito inoculates sporozoites into the human host.
Human Liver Stages (exo-erythrocytic schizogony)
Sporozoites infect liver cells
…and mature into schizonts
… which rupture and release merozoites.
(Of note, in P. vivax and P. ovale a dormant stage [hypnozoites] can persist in the liver and cause relapses by invading the bloodstream weeks, or even years later.)
Human Blood Stages (erythrocytic schizogony)
The parasites undergo asexual multiplication in the erythrocytes (red blood cells).
Merozoites infect red blood cells.
The ring stage trophozoites mature into schizonts, which rupture releasing merozoites.
Some parasites differentiate into sexual erythrocytic stages (gametocytes).
Blood stage parasites are responsible for the clinical manifestations of the disease.
The gametocytes, male (microgametocytes) and female (macrogametocytes), are ingested by an Anopheles mosquito during a blood meal.
Mosquito Stages. The parasites’ multiplication in the mosquito is known as the sporogonic cycle.
While in the mosquito's stomach, the microgametes penetrate the macrogametes generating zygotes.
The zygotes in turn become motile and elongated (ookinetes)…
… which invade the midgut wall of the mosquito where they develop into oocysts.
The oocysts grow, rupture, and release sporozoites…
…which make their way to the mosquito's salivary glands. Inoculation of the sporozoites into a new human host perpetuates the malaria life cycle.
Laboratory Instructions: Plasmodium sp.
1. Examine a blood smear infected with Plasmodium sp. and identify the ring-like immature trophozoites (will require 400x or 1000x magnification).
2. Carefully illustrate the sample as you see it in the microscope. Label "red blood cells" and "Plasmodium sp. ring stage" in your illustration.
Results & Questions: Plasmodium sp.
1. Carefully illustrate the sample as you see it in the microscope. Label "red blood cells" and "Plasmodium sp. ring stage" in your illustration.
2. What stage of the Plasmodium sp. life cycle did you examine (human liver stage, human blood stage, or mosquito stage)?
3. What disease does Plasmodium sp. cause in humans?
4. What human tissues are inhabited by the Plasmodium sp. parasite in humans?
5. How is Plasmodium sp. transmitted to humans?
6. What species is the definitive host of Plasmodium sp.? How do you know?
7. What species is the intermediate host of Plasmodium sp.? How do you know?
8. What are the four Plasmodium species that cause malaria in humans?
9. Where in the world is malaria present?
10. How can people in the United States present with a malaria infection?
11. Give at least four symptoms of malaria.
Trypanosoma cruzi (Cause of Chagas Disease)
Introduction to Trypanosoma cruzi
Trypanosoma cruzi (trip-an’o-so’muh/kroo’zye) is the protozoan responsible for causing Chagas disease. T. cruzi is limited to the western hemisphere, including California, the southern United States, Central and South America.It is most often seen in rural areas in Latin America where poverty is widespread. The vector for T. cruzi is reduviid bug species known as kissing bugs. The most common genera responsible for transmission of the disease are Triatoma, Rhodnius, and Panstrongylus. Infection usually occurs after bugs defecate on the bite site and are rubbed into the wound by the host scratching. Humans contract Chagas disease (South American Trypanosomiasis) when an infected bug bites the human skin and subsequently defecates in the wound. Infection may be mild or asymptomatic. There may be fever or swelling around the bug bite. Parasites may also be found in the circulating blood. Chagas disease occurs immediately after infection and may last a few weeks or months. In 20-30% of infected people, acute infection may result in severe inflammation of the heart muscle or the brain and lining around the brain.
Clinical Presentation of Chagas Disease
Chagas disease has an acute phase and chronic phase. The acute phase is usually asymptomatic, but can present with nonspecific somatic symptoms. Rarely, the acute phase may be more severe with potential cardiac or neurologic symptoms and signs. Nodular lesions or furuncles, usually called chagomas, may develop around the vector’s feeding site. Chagomas occurring on the on the eyelids are commonly referred to as palpebral and periocular firm swelling. Most acute cases resolve over a period of a few weeks or months into a subclinical chronic form of the disease (“indeterminate form”). Reactivation of Chagas disease from this asymptomatic form may occur in patients with HIV or those receiving immunosuppressive drugs.
The symptomatic chronic form (“determinate form”) may not occur for years or even decades after initial infection. This may include cardiac or gastrointestinal involvement, which occasionally occur together. The many complications of chronic Chagas disease can be fatal. Amastigote invasion of smooth muscle can lead to megaesophagus, megacolon, and dilated cardiomyopathy.
Life Cycle of Trypanosoma cruzi
An infected triatomine insect vector (or “kissing” bug) takes a blood meal and releases trypomastigotes in its feces near the site of the bite wound. Trypomastigotes enter the host through the bite wound or intact mucosal membranes, such as the conjunctiva.
Inside the host, the trypomastigotes invade cells near the site of inoculation, where they differentiate into intracellular amastigotes.
The amastigotes multiply by binary fission…
… and differentiate into trypomastigotes, and then are released into the circulation as bloodstream trypomastigotes.
Trypomastigotes infect cells from a variety of tissues and transform into intracellular amastigotes in new infection sites. Clinical manifestations can result from this infective cycle. The bloodstream trypomastigotes do not replicate (different from the African trypanosomes). Replication resumes only when the parasites enter another cell or are ingested by another vector.
The “kissing” bug becomes infected by feeding on human or animal blood that contains circulating parasites.
The ingested trypomastigotes transform into epimastigotes in the vector’s midgut.
The parasites multiply and differentiate in the midgut…
… and differentiate into infective metacyclic trypomastigotes in the hindgut.
Other less common routes of transmission include blood transfusions, organ transplantation, transplacental transmission, and foodborne transmission (via food/drink contaminated with the vector and/or its feces).
Laboratory Instructions: Trypanosoma cruzi
1. Examine a blood smear infected with Trypanosoma cruzi and identify the T. cruzi trophozoites (will require 400x or 1000x magnification).
2. Carefully illustrate the sample as you see it in the microscope. Label the following in your illustration:
• "red blood cells"
• "Trypanosoma cruzi trypomastigote"
• "flagellum"
• "undulating membrane"
• "nucleus"
Results & Questions: Trypanosoma cruzi
1. Carefully illustrate the sample as you see it in the microscope. Label the following in your illustration:
• "red blood cells"
• "Trypanosoma cruzi trypomastigote"
• "flagellum"
• "undulating membrane"
• "nucleus"
2. Explain how humans become infected with T. cruzi.
3. What disease is caused by T. cruzi infection in humans?
4. What geographic regions of the world is T. cruzi infection possible?
5. Give at least three symptoms of Chagas disease.
6. Can Chagas disease be asymptomatic? Explain your answer.
7. Carefully examine the life cycle of T. cruzi. Inside of the human host, is T. cruzi an intracellular parasites (lives inside host cells) or an intercellular parasite (lives outside of host cells)? Explain your answer.
Trichomonas vaginalis (Cause of Trichomoniasis)
Introduction to Trichomonas vaginalis
Trichomoniasis a common, treatable, sexually transmitted disease (STD). Most people who have trichomoniasis do not have any symptoms.
Trichomonas vaginalis (trick’o-mo’nas/vadj-i-nay’lis) has no cyst stage so it cannot survive outside its host very long. It is transmitted by intimate contact between individuals. It is the cause of trichomoniasis, commonly called “ping-pong vaginitis” because it may be passed back and forth between sexual partners. In the United States, an estimated 2 million people have the infection, but only about 30% develop any symptoms of trichomoniasis. While most infections are asymptomatic, it can cause prostate and epididymis infections in men. Females report frequent urination, itching, burning, and a vaginal discharge. Diagnosis often occurs when people seek medical help for what they believe is a urinary tract infection. Flagyl is a medication used to treat infections. Diagnosis is usually made by microscopically identifying the tear-dropped shaped trophozoites in a wet preparation of a vaginal or urethral discharge. Three to five flagella may be visible as a tuft at the anterior end of the cell. T. vaginalis will exhibit a quick, jerky, darting motility as it zooms around the field of vision.
In the United States, CDC estimates that there were more than two million trichomoniasis infections in 2018. However, only about 30% develop any symptoms of trich. Infection is more common in women than in men. Older women are more likely than younger women to have the infection.
Clinical Presentation of Trichomoniasis
About 70% of people with the infection do not have any signs or symptoms. When trich does cause symptoms, they can range from mild irritation to severe inflammation. Some people get symptoms within 5 to 28 days after getting the infection. Others do not develop symptoms until much later. Symptoms can come and go.
Men with trich may notice:
• Itching or irritation inside the penis;
• Burning after peeing or ejaculating; and
• Discharge from the penis.
Women with trich may notice:
• Itching, burning, redness or soreness of the genitals;
• Discomfort when peeing; and
• A clear, white, yellowish, or greenish vaginal discharge (i.e., thin discharge or increased volume) with a fishy smell.
Having trich can make sex feel unpleasant. Without treatment, the infection can last for months or even years.
Pregnant people with trich are more likely to have their babies early. Also, their babies are more likely to have a low birth weight (less than 5.5 pounds).
Laboratory Instructions: Trichomonas vaginalis
1. Examine the sample of T. vaginalis and identify the T. vaginalis trophozoites (use 400x or 1000x magnification).
2. Carefully illustrate the sample as you see it in the microscope. Label the following in your illustration:
• "Trichomonas vaginalis"
• "flagella"
• "undulating membrane"
• "nucleus"
• "axostyle"
• "posterior axostyle"
Results & Questions: Trichomonas vaginalis
1. Carefully illustrate the sample as you see it in the microscope. Label the following in your illustration:
• "Trichomonas vaginalis"
• "flagella"
• "undulating membrane"
• "nucleus"
• "axostyle"
• "posterior axostyle"
2. What disease is caused by T. vaginalis?
3. Give at least two common symptoms of trichomoniasis in men.
4. Give at least two common symptoms of trichomoniasis in women.
5. How frequently is trichomoniasis asymptomatic?
6. How is trichomoniasis spread?
7. How many host species does T. vaginalis have?
8. How is trichomoniasis diagnosed? | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.34%3A_Protozoan_Parasites.txt |
Learning Objectives
• Identify and name the following helminth parasites in microscopic samples: Enterobius vermicularis, Dipylidium caninum, and Schistosoma sp.
• Name the diseases caused by the following helminth parasites: Enterobius vermicularis, Dipylidium caninum, and Schistosoma sp.
• Explain how the following helminth parasites are transmitted to humans (or dogs/cats for D. caninum): Enterobius vermicularis, Dipylidium caninum, and Schistosoma sp.
• Interpret the life cycles of the following helminth parasites: Enterobius vermicularis, Dipylidium caninum, and Schistosoma sp.
• Examine the following helminth parasites using a microscope and illustrate and label the specimens: Enterobius vermicularis, Dipylidium caninum, and Schistosoma sp.
Enterobius vermicularis (Causes Enterobiasis)
Introduction to Enterobiasis
The pinworm, Enterobius vermicularis (en’tur-o’bee-us/vur-mick-yoo-lair’is), is thought to be the most common helminth parasite in temperate regions with a high level of sanitation. While children are more commonly infected than adults, pinworm infestations are found in all ages and socioeconomic groups. Humans are the only host of Enterobius vermicularis.
The life cycle of Enterobius vermicularis is 4-6 weeks. Adults worms inhabit the cecum (part of the large intestine/colon) and adjacent portions of the large and small intestine of the host. The male pinworm is inconspicuous, about 2 to 5 mm in length, and no more than 0.2 mm in width. The female may reach a length of 13 mm (1/2 inch) and a maximum width of 0.5 mm The female is distinguished by a long, thin, sharply pointed tail, which gives rise to the common name “pinworm”. The gravid females, containing 11,000 to 15,000 eggs, migrate down the intestinal tract to the anus where they deposit their eggs. Enterobius ova, having a sticky albumin coating, adhere to the perianal region. The eggs mature quickly and are infective within a few hours after they are deposited.
Recovery of eggs from the perianal region provides the most efficient method of diagnosis of pinworm infection. Repeated examinations on consecutive days are necessary because of the irregular migrations of the gravid female worms. Since pinworm eggs are generally deposited at night, the examination should be made in the early morning hours before the patient has washed or defecated. Typically, Graham’s Scotch Tape Method is employed. The sticky side of cellophane tape is applied to the anal area of the patient. The tape is then pressed firmly on a microscope slide, sticky side down. The slide is examined under the low power of a microscope for the presence of pinworm eggs. The eggs are identified by their flattened oval (asymmetrical) shape and possibly the presence of a well-developed embryo.
Migration of the female worm may cause pruritus ani (anal itching). The local itching may cause insomnia and restlessness in children or adults who are infected, as the worms migrate from the anus during the resting hours. In about one-third of infected children, eggs may be obtained from beneath the fingernails. Reinfection of the patient by hand-to-mouth transmission (from scratching the perianal area or handling contaminated fomites) is common and makes control of the parasite difficult. Enterobius ova can survive for 2-3 weeks outside the body in high humidity and moderate temperatures. If towels or linens are shared, these eggs can be passed to others. It is recommended that all members of the infected household be treated simultaneously. Infection of others through contaminated bedding, clothing, bath tubs, toilet seats, dry dust, and air-borne eggs may serve to infect persons at some distance. The prescription drug, mebendazole, is used to treat pinworm. There are over-the-counter drugs available for treatment of pinworms also. Vermox, antiminth, and Povan (pyrvinium pamoate) are acceptable treatments. Patients using Povan should be forewarned that it colors the stool a bright red color!
E. vermicularis occurs worldwide, with infections occurring most frequently in school- or preschool-children and in crowded conditions.
Clinical Presentation of Enterobiasis
Enterobiasis is frequently asymptomatic. The most typical symptom is perianal pruritus, especially at night, which may lead to excoriations and bacterial superinfection. Occasionally, invasion of the female genital tract with vulvovaginitis and pelvic or peritoneal granulomas can occur. Other symptoms include, teeth grinding, enuresia, insomnia, anorexia, irritability, and abdominal pain, which can mimic appendicitis. E. vermicularis larvae are often found within the appendix on appendectomy, but the role of this nematode in appendicitis remains controversial. Very rare instances of eosinophilic colitis associated with E. vermicularis larvae have been reported.
Life Cycle of Enterobius vermicularis
Gravid adult female Enterobius vermicularis deposit eggs on perianal folds.
Infection occurs via self-inoculation (transferring eggs to the mouth with hands that have scratched the perianal area) or through exposure to eggs in the environment (e.g. contaminated surfaces, clothes, bed linens, etc.).
Following ingestion of infective eggs, the larvae hatch in the small intestine...
...and the adults establish themselves in the colon, usually in the cecum.
The time interval from ingestion of infective eggs to oviposition by the adult females is about one month. At full maturity adult females measure 8 to 13 mm, and adult males 2 to 5 mm; the adult life span is about two months.
Gravid females migrate nocturnally outside the anus and oviposit while crawling on the skin of the perianal area.
The larvae contained inside the eggs develop (the eggs become infective) in 4 to 6 hours under optimal conditions.
Rarely, eggs may become airborne and be inhaled and swallowed. Retroinfection, or the migration of newly hatched larvae from the anal skin back into the rectum, may occur but the frequency with which this happens is unknown.
Laboratory Instructions: Enterobius vermicularis
1. Examine eggs of E. vermicularis (use 400x or 1000x magnification).
2. Carefully illustrate the egg sample as you see it in the microscope.
3. Examine an adult worm of E. vermicularis.
4. Carefully illustrate the adult worm sample as you see it in the microscope. Label the following in your illustration:
• "male" or "female" (as appropriate)
• "mouth"
• "cephalic expansion"
• "pharynx"
• "end bulb"
• "intestine"
• if the worm is a male:
• "testis"
• "spicule"
• "cloaca"
• if the worm is a female:
• "vulva"
• "posterior uterus"
Results & Questions: Enterobius vermicularis
E. vermicularis eggs
E. vermicularis adult helminth
1. Carefully illustrate the egg sample as you see it in the microscope.
2. Carefully illustrate the adult worm sample as you see it in the microscope. Label the following in your illustration:
• "male" or "female" (as appropriate)
• "mouth"
• "cephalic expansion"
• "pharynx"
• "end bulb"
• "intestine"
• if the worm is a male:
• "testis"
• "spicule"
• "cloaca"
• if the worm is a female:
• "vulva"
• "posterior uterus"
3. What disease is caused by E. vermicularis?
4. Where does E. vermicularis live inside its human host?
5. How can infection with E. vermicularis be diagnosed?
6. Give at least three symptoms of enterobiasis.
7. Can enterobiasis be asymptomatic?
8. Where geographically in the world does E. vermicularis infection occur?
9. What are three similarities of the male and female E. vermicularis worms?
10. What are four differences between the male and female E. vermicularis worms?
Dipylidium caninum (Dog Tapeworm)
Introduction to Dipylidium caninum
Dipylidium caninum is a common tapeworm of dogs and cats, but is occasionally found in humans. It has many common names including the “flea tapeworm”, “cucumber tapeworm”, and “double-pored tapeworm”. Geographic distribution of D. caninum is worldwide. This tapeworm is ubiquitous and common among pet dogs and cats. Human infection is rare, but has been reported from every inhabited continent.
Tapeworms are flatworms in the phylum Platyhelminthes. These helminths have a scolex (its "head") that is equipped with suckers and hooks, but no mouth. Tapeworms absorb nutrients through their tegument (their "skin"). The scolex and its suckers and hooks are used for attachment to the host intestine. Behind the scolex, a tapeworm is composed of segments called proglottids. The proglottids closest to the scolex are the smallest in size and are considered immature proglottids, that is, they lack functional reproductive organs and they lack eggs. New proglottids are produced just behind the scolex and neck of the tapeworm. As new proglottids are formed, the older proglottids will be pushed further from the scolex. As proglottids age, they will ultimately become larger and produce mature proglottids that contain mature ovaries and testes. Yes, that is correct, a tapeworm's mature proglottids contain both male and female genitalia, and are therefore capable of conducting sexual reproduction alone (a second worm is not involved in sexual reproduction in this case). The largest proglottids, and those most distant from the scolex, are called gravid proglottids. These gravid proglottids are distinct because sexual reproduction has already occurred and these proglottids contain a multitude of eggs. Gravid proglottids on the end of the tapeworm can break off and be released from its host in feces to release the eggs and continue the life cycle.
Diagnosis is made by demonstrating the typical proglottids or egg packets in the stool or the environment. Although concentration methods are usually not necessary due to the large size of the proglottids, flotation methods may miss infections as egg packets are heavy and may fail to float.
Clinical Presentation of the Dog Tapeworm
Most infections with Dipylidium caninum are asymptomatic. Pets may exhibit behavior to relieve anal pruritis (such as scraping anal region across grass or carpeting). Mild gastrointestinal disturbances may occur. The most striking feature in animals and children consists of the passage of proglottids. These can be found in the perianal region, in the feces, on diapers, and occasionally on floor covering and furniture. The proglottids are motile when freshly passed and may be mistaken for maggots or fly larvae.
Life Cycle of Dipylidium caninum
Gravid proglottids are passed intact in the feces or emerge from the perianal region of the host.
In the environment, the proglottids disintegrate and release egg packets, which are also occasionally found free in the feces.
The intermediate host (most often larval stages of the dog or cat flea Ctenocephalides spp.) ingests egg packets, and the oncosphere within is released into the larval flea’s intestine. The oncosphere penetrates the intestinal wall, invades the insect’s hemocoel (body cavity), and develops into a cysticercoid.
The cysticercoid remains in the flea as it matures from a larva into an adult.
The vertebrate host becomes infected by ingesting the adult flea containing the cysticercoid.
In the small intestine of the vertebrate host, the cysticercoid develops into the adult tapeworm after about one month. The adult tapeworms (measuring up to 60 cm in length and 3 mm in width) reside in the small intestine of the host, where they each attach by their scolex.
Gravid, double-pored proglottids detach from the strobila (body) and are shed in the feces.
Humans also acquire infection by ingesting the cysticercoid contaminated flea. Children are most frequently infected, possibly due to close contact with flea-infested pets.
Laboratory Instructions: Dipylidium caninum
1. Examine eggs of D. caninum (use 400x or 1000x magnification).
2. Carefully illustrate the egg sample as you see it in the microscope.
3. Examine an adult worm of D. caninum. The specimen will be separated into the following segments: scolex with neck and immature proglottids, mature proglottids, and gravid proglottids.
4. Carefully illustrate the three segments of the adult worm sample as you see it in the microscope. Label the following in your illustration:
• the smallest sample (scolex, neck, and immature proglottids):
• "scolex"
• "suckers"
• "hooks"
• "neck"
• "immature proglottids"
• intermediate-sized sample (mature proglottids)
• "mature proglottid"
• "genital pore"
• "ovary"
• "testes"
• "uterus"
• largest sample (gravid proglottids)
• "gravid proglottid"
• "eggs"
Results & Questions: Dipylidium caninum
D. caninum eggs
D. caninum adult helminth: scolex & immature proglottids
D. caninum adult helminth: mature proglottids
D. caninum adult helminth: gravid proglottids
1. Carefully illustrate the egg sample as you see it in the microscope.
2. Carefully illustrate the three segments of the adult worm sample as you see it in the microscope. Label the following in your illustration:
• the smallest sample (scolex, neck, and immature proglottids):
• "scolex"
• "suckers"
• "hooks"
• "neck"
• "immature proglottids"
• intermediate-sized sample (mature proglottids)
• "mature proglottid"
• "genital pore"
• "ovary"
• "testes"
• "uterus"
• largest sample (gravid proglottids)
• "gravid proglottid"
• "eggs"
3. Explain how immature proglottids become mature proglottids.
4. Explain how mature proglottids become gravid proglottids.
5. Explain how eggs are released from the host body to continue the life cycle.
6. What is the function of the scolex?
7. How does a tapeworm get its nutrients?
8. How does a tapeworm conduct sexual reproduction?
9. Give three signs/symptoms of D. caninum infection.
10. What species is/are the definitive host(s) of D. caninum?
11. What species is/are the intermediate host(s) of D. caninum?
Schistosoma sp. (Cause of Schistosomiasis)
Introduction to Schistosoma sp. (shis’to-so’muh)
There are several species of Schistosoma that cause disease in humans. Each species is unique to a particular area in the world. Since the United States does not have the specific host snails needed for its life cycle, infections are not contracted directly, yet many immigrants and travelers to endemic areas are infected. An estimated 236 million people require preventative drug treatment per year, out of which 105 million people were reported to have been treated. Preventative treatment, which should be repeated for a number of years, will reduce and prevent morbidity.
Infection occurs when your skin comes in contact with contaminated freshwater in which certain types of snails that carry schistosomes are living. Individuals infected with Schistosoma excrete the ova in the feces. Upon contact with water, the ova (which look like a cartoon talk bubble) develop into miracidia which enters the snail. Maturation in the snail results in the production of the free swimming cercaria. When the cercaria contacts the skin of a person who is in the water, it secretes enzymes that enable it to burrow through unbroken skin. The Schistosoma are then carried in the bloodstream to the liver or urinary bladder where they mature into an adult. Adults can live up to seven years. Schistosoma is dioecious – the male and female are separate animals. If both male and female are present, fertilization occurs, and ova are produced. The cycle then continues in a new host. Schistosoma infection may result in destruction of the liver, lungs, and/or urinary system. This is second most devastating parasitic disease. (Malaria is the most common). Praziquantel is an effective treatment.
Birds in the United States suffer from a Schistosoma infection. The Schistosoma parasite that infects birds has an intermediate snail host that is predominantly found in water in the Great Lakes and on the east coast. Sometimes when the free swimming cercaria of this bird parasite encounters a human swimming in the water, it will attempt to burrow through the skin of the human host instead of the normal bird host. Since humans are not the correct host for this parasite, the cercaria are unable to penetrate the human skin and instead, become embedded in the skin. This causes a painful, itchy inflammatory dermatitis called “swimmer’s itch”.
Geographic regions where schistosomiasis occurs include:
• Africa: contact with any freshwater in southern and sub-Saharan Africa–including the great lakes and rivers as well as smaller bodies of water– should be considered a risk for schistosomiasis transmission. Transmission also occurs in the Mahgreb region of North Africa and the Nile River valley in Egypt and Sudan .
• South America: Brazil, Suriname, Venezuela
• Caribbean: Dominican Republic, Guadeloupe, Martinique, Saint Lucia (risk in Caribbean is very low)
• The Middle East: Iran, Iraq, Saudi Arabia, Yemen
• Southern China
• Parts of Southeast Asia and the Philippines, Laos
• A recent focus of ongoing transmission has been identified in Corsica.
Clinical Presentation of the Schistosomiasis
Within days after becoming infected, you may develop a rash or itchy skin. Fever, chills, cough, and muscle aches can begin within 1-2 months of infection. Most people have no symptoms at this early phase of infection.
When adult worms are present, the eggs that are produced usually travel to the intestine, liver or bladder, causing inflammation or scarring. Children who are repeatedly infected can develop anemia, malnutrition, and learning difficulties. After years of infection, the parasite can also damage the liver, intestine, lungs, and bladder. Rarely, eggs are found in the brain or spinal cord and can cause seizures, paralysis, or spinal cord inflammation.
Symptoms of schistosomiasis are caused by the body’s reaction to the eggs produced by worms, not by the worms themselves.
Life Cycle of Schistosoma sp.
Schistosoma eggs are eliminated with feces or urine, depending on species.
Under appropriate conditions the eggs hatch and release miracidia...
...which swim and penetrate specific snail intermediate hosts.
The stages in the snail include two generations of sporocysts...
...and the production of cercariae.
Upon release from the snail, the infective cercariae swim, penetrate the skin of the human host...
...and shed their forked tails, becoming schistosomulae.
The schistosomulae migrate via venous circulation to lungs, then to the heart, and then develop in the liver, exiting the liver via the portal vein system when mature,
Male and female adult worms copulate and reside in the mesenteric venules, the location of which varies by species (with some exceptions).
For instance, S. japonicum is more frequently found in the superior mesenteric veins draining the small intestine...
...and S. mansoni occurs more often in the inferior mesenteric veins draining the large intestine.
However, both species can occupy either location and are capable of moving between sites. S. intercalatum and S. guineensis also inhabit the inferior mesenteric plexus but lower in the bowel than S. mansoni. S. haematobium most often inhabits in the vesicular and pelvic venous plexus of the bladder...
...but it can also be found in the rectal venules. The females (size ranges from 7–28 mm, depending on species) deposit eggs in the small venules of the portal and perivesical systems. The eggs are moved progressively toward the lumen of the intestine (S. mansoni,S. japonicum, S. mekongi, S. intercalatum/guineensis) and of the bladder and ureters (S. haematobium), and are eliminated with feces or urine, respectively
Laboratory Instructions: Schistosoma sp.
1. Examine eggs of Schistosoma sp. (use 400x or 1000x magnification).
2. Carefully illustrate the egg sample as you see it in the microscope.
3. Examine an adult worm of Schistosoma sp.
4. Carefully illustrate the adult worm sample as you see it in the microscope.
Results & Questions: Schistosoma sp.
Schistosoma sp. eggs
Schistosoma sp. adult helminth
1. Carefully illustrate the egg sample as you see it in the microscope.
2. Carefully illustrate the adult worm sample as you see it in the microscope.
3. What disease does Schistosoma sp. cause?
4. How do people become infected with Schistosoma sp.?
5. What is the definitive host species of Schistosoma sp.?
6. What is the intermediate host species of Schistosoma sp.?
7. List four species of Schistosoma that causes schistosomiasis in humans.
8. Give at least five symptoms of schistosomiasis.
9. Where in the human body to adult worms live?
10. How does Schistosoma sp. leave a human to continue its life cycle?
11. What areas of the world can humans contract schistosomiasis? | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.35%3A_Helminth_Parasites.txt |
Learning Objectives
• Define and differentiate between yeast and mold.
• Define, identify, and apply the following terms: budding, hyphae, mycelium, septate, coenocytic, sporangium, sporangiophore, sporangiospore, conidia, conidiophore, vesicle
• Describe how Aspergillus can cause infection in humans, the risk factors for aspergillosis, and the symptoms of infection.
• Describe how Coccidioides can cause infection in humans, the risk factors for infection, and the symptoms of infection.
• Give both the medical and common names for Coccidioides infections.
• Tell that antibiotics cannot be used to treat fungal infections and that they must be treated with antifungal medications.
Introduction to Fungi
Mycology is the study of fungi. Fungi are eukaryotic organisms which grow as either yeast or mold. However, there are some fungi that are dimorphic, meaning they can grow as yeast under certain environmental conditions (such as the warm moist lungs in the body) and mold under other conditions (such as in soil in the environment). For example, Coccidioides immitis, the fungus responsible for San Joaquin Valley Fever, is an example of a dimorphic fungus.
Fungi grow slower than bacteria and at a lower temperature and lower pH than most bacteria prefer. Sabouraud’s agar is selective media for fungi because it incorporates simple nutrients (glucose and peptone) at a pH of 4.5-5.6 which inhibits bacterial growth. Although some yeasts can grow at 36°C, we incubate all fungal cultures at 25 to 30°C for at least one week. Some fungi take three weeks or more to grow.
Yeast
The colonial appearance of most yeast is moist, creamy and white in color and similar in appearance to Staphylococcus colonies. Microscopically, yeast cells are unicellular and round to oval, whereas bacteria cells vary in shape (cocci, rods, spirals). Yeast cells are usually five to ten times larger than bacteria and can be visualized at 400X total magnification. Yeast reproduce asexually by budding and the newly produced cell, called a bud or blastospore, protrudes from the periphery of the parent cell. The blastospore may break off from the parent cell or stay attached. Successive blastospores remaining attached to the original cell result in the formation of pseudohyphae.
Most yeast have similar macroscopic and microscopic appearances. Biochemical tests, such as carbohydrate assimilation tests, must be performed to identify yeast species. Examples of yeast include Candida albicans which is an opportunistic pathogen (thrush, diaper rash, vaginal yeast infections) and Saccharomyces cerevisiae which is used to make bread, beer, and wine.
Mold
Fungi that grow as mold produce multicellular filaments called hyphae (plural) or hypha (singular). Most species of mold hyphal filaments are separated by a cross wall (septum) and are called septate hyphae. In mold species where the hyphal filaments not separated by cross walls are called aseptate or coencocytic hyphae. Hyphal filaments intertwined into a mass, known as mycelia (plural) or mycelium (singular), can be seen macroscopically as fuzzy or hairy, colorful colonies. Some of the hyphae, called vegetative hyphae, grow on or down into the agar surface to extract nutrients from the medium. Other hyphae, called aerial hyphae, grow above the agar surface and produce asexual reproductive spores.
The two types of asexual spores produced by molds are called sporangiospores and conidiospores. Sporangiospores are produced at the end of aerial hyphae called sporangiophores in a saclike structure called a sporangium. The sporangia of specific molds have characteristic shapes which can be used to identify the mold species. An example of a mold that produces sporangiospores is the bread mold, Rhizopus. Conidiospores are formed on aerial hyphae called conidiophores. Conidia may be one-celled (microconidia) or multicelled (macroconidia). Examples of fungi that produce conidiospores are Penicillium and Aspergillus.
Molds are usually identified in the laboratory by their characteristic macroscopic appearance, hyphal structure (septate or nonseptate) and type of asexual sporulation. Since spore formation is an important identification criterion, slide cultures are performed to observe sporulation without disturbing the hyphal structures.
Aspergillus
Introduction to Aspergillus (Cause of Aspergillosis)
Aspergillosis is an infection caused by Aspergillus, a common mold (a type of fungus) that lives indoors and outdoors. Most people breathe in Aspergillus spores every day without getting sick. However, people with weakened immune systems or lung diseases are at a higher risk of developing health problems due to Aspergillus. The types of health problems caused by Aspergillus include allergic reactions, lung infections, and infections in other organs.
There are approximately 180 species of Aspergillus, but fewer than 40 of them are known to cause infections in humans. Aspergillus fumigatus is the most common cause of human Aspergillus infections. Other common species include A. flavus, A. terreus, and A. niger.
Healthcare providers consider your medical history, risk factors, symptoms, physical examinations, and lab tests when diagnosing aspergillosis. Imaging tests such as a chest x-ray or a CT scan a patient's lungs or other parts of the body depending on the location of the suspected infection. If a healthcare provider suspects an Aspergillus infection in the lungs, they might collect a sample of fluid from your respiratory tract to send to a laboratory. Healthcare providers may also perform a tissue biopsy, in which a small sample of affected tissue is analyzed in a laboratory for evidence of Aspergillus under a microscope or in a fungal culture. A blood test can help diagnose invasive aspergillosis early in people who have severely weakened immune systems.
Types of Aspergillosis
• Allergic bronchopulmonary aspergillosis (ABPA): Occurs when Aspergillus causes inflammation in the lungs and allergy symptoms such as coughing and wheezing, but doesn’t cause an infection.2
• Allergic Aspergillus sinusitis: Occurs when Aspergillus causes inflammation in the sinuses and symptoms of a sinus infection (drainage, stuffiness, headache) but doesn’t cause an infection.3
• Azole-Resistant Aspergillus fumigatus: Occurs when one species of Aspergillus, A. fumigatus, becomes resistant to certain medicines used to treat it. Patients with resistant infections might not get better with treatment.
• Aspergilloma: Occurs when a ball of Aspergillus grows in the lungs or sinuses, but usually does not spread to other parts of the body.4 Aspergilloma is also called a “fungus ball.”
• Chronic pulmonary aspergillosis: Occurs when Aspergillus infection causes cavities in the lungs, and can be a long-term (3 months or more) condition. One or more fungal balls (aspergillomas) may also be present in the lungs.5
• Invasive aspergillosis: Occurs when Aspergillus causes a serious infection, and usually affects people who have weakened immune systems, such as people who have had an organ transplant or a stem cell transplant. Invasive aspergillosis most commonly affects the lungs, but it can also spread to other parts of the body.
• Cutaneous (skin) aspergillosis: Occurs when Aspergillus enters the body through a break in the skin (for example, after surgery or a burn wound) and causes infection, usually in people who have weakened immune systems. Cutaneous aspergillosis can also occur if invasive aspergillosis spreads to the skin from somewhere else in the body, such as the lungs.6
Clinical Presentation: Aspergillus
The different types of aspergillosis can cause different symptoms.1
The symptoms of allergic bronchopulmonary aspergillosis (ABPA) are similar to asthma symptoms, including:
• Wheezing
• Shortness of breath
• Cough
• Fever (in rare cases)
Symptoms of allergic Aspergillus sinusitis2 include:
• Stuffiness
• Runny nose
• Headache
• Reduced ability to smell
Symptoms of an aspergilloma (“fungus ball”)3 include:
• Cough
• Coughing up blood
• Shortness of breath
Symptoms of chronic pulmonary aspergillosis4,5 include:
• Weight loss
• Cough
• Coughing up blood
• Fatigue
• Shortness of breath
Invasive aspergillosis1 usually occurs in people who are already sick from other medical conditions, so it can be difficult to know which symptoms are related to an Aspergillus infection. However, the symptoms of invasive aspergillosis in the lungs include:
• Fever
• Chest pain
• Cough
• Coughing up blood
• Shortness of breath
• Other symptoms can develop if the infection spreads from the lungs to other parts of the body.
Laboratory Instructions: Aspergillus
1. Examine Aspergillus using a microscope. View the sample at 400x or 1000x magnification.
2. Carefully illustrate the Aspergillus sample as you see it in the microscope. Label the following in your illustration:
• "hyphae" or "hypha"
• "conidiophore"
• "vesicle"
• "conidia (spores)" or "conidiospores"
Results & Questions: Aspergillus
1. Carefully illustrate the Aspergillus sample as you see it in the microscope. Label the following in your illustration:
• "hyphae" or "hypha"
• "conidiophore"
• "vesicle"
• "conidia (spores)" or "conidiospores"
2. What areas of the human body can become infected with Aspergillus?
3. Where does Aspergillus live and how are humans exposed to Aspergillus?
4. Who populations are most at risk of developing aspergillosis?
5. What is the difference between a mold and yeast?
6. Is Aspergillus a mold or a yeast?
7. Name the Aspergillus structures that branch from the hyphae to produce asexual spores?
8. What are the Aspergillus asexual spores called?
9. Can aspergillosis be treated with antibiotics? Explain your answer.
Coccidioides (Causes Coccidioidomycosis aka Valley Fever)
Introduction to Coccidioides (Cause of Coccidioidomycosis aka Valley Fever)
Valley fever is an infection caused by a fungus that lives in the soil. About 20,000 cases are reported in the United States each year, mostly from Arizona and California, and the number of cases is increasing. Valley fever can be misdiagnosed because its symptoms are similar to those of other respiratory illnesses. Here are some important things to know about Valley fever, also called coccidioidomycosis.
The fungus that causes Valley fever, Coccidioides, is found in soil in the southwestern United States, parts of Mexico and Central America, and parts of South America. It has also been found in south-central Washington State. The fungus might also live in similar areas with hot, dry climates. People can get Valley fever by breathing in the microscopic fungus from the air in these areas. Valley fever does not spread from person to person.
In areas where Valley fever is common, it’s difficult to completely avoid exposure to the fungus because it is in the environment. There is no vaccine to prevent infection. That’s why knowing about Valley fever is one of the most important ways to avoid delays in diagnosis and treatment. People who have symptoms of Valley fever and live in, work in, or have visited an area where the fungus is common should ask their doctor to test them for Valley fever. Healthcare providers should be aware that Valley fever symptoms are similar to those of other respiratory illnesses and should consider testing for Valley fever in patients with pneumonia symptoms who live in or have traveled to an area where Coccidioides lives.
The Financial Cost of Valley Fever
Valley fever is a serious, costly illness:
• Nearly 75% of people with Valley fever miss work or school
• As many as 40% of people who get Valley fever are hospitalized
• The average cost of a hospital stay for a person with Valley fever is almost \$50,000
• About 60–80% of patients with Valley fever are given one or more rounds of antibiotics before receiving a correct diagnosis and appropriate treatment (antifungal medication is required for treatment)
Clinical Presentation of Coccidioides (Cause of Coccidioidomycosis aka Valley Fever)
Many people who are exposed to the fungus never have symptoms. Other people may have symptoms that include:
• Fatigue (tiredness)
• Cough
• Fever
• Shortness of breath
• Headache
• Night sweats
• Muscle aches or joint pain
• Rash on upper body or legs
The symptoms of valley fever can be similar to those of other respiratory illnesses, which may cause delays in diagnosis and treatment. For many people, symptoms go away within weeks or months without any treatment. But healthcare providers may prescribe antifungal medicine for some people to reduce symptoms or prevent the infection from getting worse. People who have severe lung infections or infections that have spread to other parts of the body always need antifungal treatment and may need to stay in the hospital.
Anyone can get Valley fever if they live in, work in, or travel to an area where the fungus lives in the environment. Valley fever can affect people of any age, but it’s most common in adults aged 60 and older. Also, certain groups of people may be at higher risk for developing the severe forms of valley fever, such as:
• People who have weakened immune systems, which may include people who:
• Have HIV
• Have had an organ transplant
• Are taking medications such as corticosteroids or tumor necrosis factor (TNF) inhibitors
• Pregnant people
• People who have diabetes
• People who are Black or Filipino
Laboratory Instructions: Coccidioides (Cause of Coccidioidomycosis aka Valley Fever)
1. Carefully examine the microscopic image of Coccidioides in the image above.
Results & Questions: Coccidioides (Cause of Coccidioidomycosis aka Valley Fever)
1. Based in careful examination of the microscopic image of Coccidioides, is this fungus growing as yeast or mold?
2. Label the image above to show the location of "hyphae."
3. Are the hyphae of Coccidioides septate or coenocytic? How can you tell?
4. How do people become exposed to Coccidioides?
5. Give the common name and the medical term for infection with Coccidioides.
6. What populations are at greater risk of infection by Coccidioides?
7. What are common symptoms of infection by Coccidioides?
8. Where geographically can patients become develop coccidioidomycosis?
9. What percentage of people with coccidioidomycosis become hospitalized?
10. Do patients with a Coccidioides infection struggle to get a correct diagnosis?
11. When should a healthcare professional consider coccidioidomycosis as a possible diagnosis?
12. Can Coccidioides infection be treated with antibiotics? Explain your answer. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.36%3A_Fungal_Parasites.txt |
Learning Objectives
• Name the following virus types and virus structures: helical capsid, icosahedral capsid, enveloped virus, complex virus, nucleic acid (DNA or RNA)/viral genome, capsid, protein, spike proteins, envelope, sheath, tail fibers
• Tell that viruses are not cells, but are particles that are almost always smaller than cells.
• Participate in viral epidemic simulation.
• Use data from viral epidemic simulation to determine the original carrier of the "virus."
• Calculate incidence rate and prevalence rate.
• Define, use, and recognize and name examples of the following: epidemiology, etiology, morbidity, morbidity rate, prevalence, incidence, mortality, sporadic diseases, endemic diseases, epidemic diseases, pandemic diseases, causative agent, reservoirs, passive carriers, active carriers, asymptomatic carriers, direct contact transmission, droplet transmission, indirect contact transmission, vehicle transmission, mechanical transmission, mechanical vector, quarantine, nosocomial infections, healthcare-associated infections
Introduction to Viruses
Viruses are noncellular parasitic entities that cannot be classified within any living kingdom. They can infect organisms as diverse as bacteria, plants, and animals. In fact, viruses exist in a sort of netherworld between a living organism and a nonliving entity. Living things grow, metabolize, and reproduce. In contrast, viruses are not cellular, do not have a metabolism or grow, and cannot divide by cell division. Viruses can copy, or replicate themselves; however, they are entirely dependent on resources derived from their host cells to produce progeny viruses—which are assembled in their mature form. No one knows exactly when or how viruses evolved or from what ancestral source because viruses have not left a fossil record. Some virologists contend that modern viruses are a mosaic of bits and pieces of nucleic acids picked up from various sources along their respective evolutionary paths.
Viruses are diverse entities: They vary in structure, methods of replication, and the hosts they infect. Nearly all forms of life—from prokaryotic bacteria and archaeans, to eukaryotes such as plants, animals, and fungi—have viruses that infect them. While most biological diversity can be understood through evolutionary history (such as how species have adapted to changing environmental conditions and how different species are related to one another through common descent), much about virus origins and evolution remains unknown.
Viruses were first discovered after the development of a porcelain filter—the Chamberland-Pasteur filter—that could remove all bacteria visible in the microscope from any liquid sample. In 1886, Adolph Meyer demonstrated that a disease of tobacco plants—tobacco mosaic disease—could be transferred from a diseased plant to a healthy one via liquid plant extracts. In 1892, Dmitri Ivanowski showed that this disease could be transmitted in this way even after the Chamberland-Pasteur filter had removed all viable bacteria from the extract. Still, it was many years before it was proved that these “filterable” infectious agents were not simply very small bacteria but were a new type of very small, disease-causing particle.
Virus Structures
Most virions, or single virus particles, are very small, about 20 to 250 nanometers in diameter. However, some recently discovered viruses from amoebae range up to 1000 nm in diameter. With the exception of large virions, like the poxvirus and other large DNA viruses, viruses cannot be seen with a light microscope. It was not until the development of the electron microscope in the late 1930s that scientists got their first good view of the structure of the tobacco mosaic virus, discussed above, and other viruses. The surface structure of virions can be observed by both scanning and transmission electron microscopy, whereas the internal structures of the virus can only be observed in images from a transmission electron microscope. The use of electron microscopy and other technologies has allowed for the discovery of many viruses of all types of living organisms.
Use this interactive tool to get a sense of how big viruses are in comparison to cells and familiar objects.*
*viruses in this interactive tool include: measles virus, hiv, phage, influenza virus, hepatitis virus, and rhinovirus
Viruses are noncellular, meaning they are biological entities that do not have a cellular structure. They therefore lack most of the components of cells, such as organelles, ribosomes, and the plasma membrane. A virion consists of a nucleic acid core (DNA or RNA), an outer protein coating or capsid, and sometimes an outer envelope made of protein and phospholipid membranes derived from the host cell. Viruses may also contain additional proteins, such as enzymes, within the capsid or attached to the viral genome. The most obvious difference between members of different viral families is the variation in their morphology, which is quite diverse. An interesting feature of viral complexity is that the complexity of the host does not necessarily correlate with the complexity of the virion. In fact, some of the most complex virion structures are found in the bacteriophages—viruses that infect the simplest living organisms, bacteria.
Viruses come in many shapes and sizes, but these features are consistent for each viral family. As we have seen, all virions have a nucleic acid genome covered by a protective capsid. The proteins of the capsid are encoded in the viral genome, and are called capsomeres. Some viral capsids are simple helices or polyhedral “spheres,” whereas others are quite complex in structure.
In general, viruses structures can be classified as: helical, icosahedral, enveloped, and complex, also known as head-and-tail. Helical capsids are long and cylindrical. Many plant viruses are helical, including TMV. Icosahedral viruses have shapes that are roughly spherical, such as those of poliovirus or herpesviruses. Enveloped viruses have membranes derived from the host cell that surrounds the capsids. Animal viruses, such as HIV, are frequently enveloped. Head-and-tail viruses infect bacteria and have a head that is similar to icosahedral viruses and a tail shaped like helical viruses.
Viral Reproduction
All viruses depend on cells for reproduction and metabolic processes. By themselves, viruses do not encode for all of the enzymes necessary for viral replication. But within a host cell, a virus can commandeer cellular machinery to produce more viral particles. Bacteriophages replicate only in the cytoplasm, since prokaryotic cells do not have a nucleus or organelles. In eukaryotic cells, most DNA viruses can replicate inside the nucleus, with an exception observed in the large DNA viruses, such as the poxviruses, that can replicate in the cytoplasm. With a few exceptions, RNA viruses that infect animal cells replicate in the cytoplasm. An important exception that will be highlighted later is Influenza virus.
The life cycle of bacteriophages has been a good model for understanding how viruses affect the cells they infect, since similar processes have been observed for eukaryotic viruses, which can cause immediate death of the cell or establish a latent or chronic infection.
The Lytic Cycle
During the lytic cycle of virulent phage, the bacteriophage takes over the cell, reproduces new phages, and destroys the cell. T-even phage is a good example of a well-characterized class of virulent phages. There are five stages in the bacteriophage lytic cycle. Attachment is the first stage in the infection process in which the phage interacts with specific bacterial surface receptors (e.g., lipopolysaccharides and OmpC protein on host surfaces). Most phages have a narrow host range and may infect one species of bacteria or one strain within a species. This unique recognition can be exploited for targeted treatment of bacterial infection by phage therapy or for phage typing to identify unique bacterial subspecies or strains. The second stage of infection is entry or penetration. This occurs through contraction of the tail sheath, which acts like a hypodermic needle to inject the viral genome through the cell wall and membrane. The phage head and remaining components remain outside the bacteria.
The third stage of infection is biosynthesis of new viral components. After entering the host cell, the virus synthesizes virus-encoded endonucleases to degrade the bacterial chromosome. It then hijacks the host cell to replicate, transcribe, and translate the necessary viral components (capsomeres, sheath, base plates, tail fibers, and viral enzymes) for the assembly of new viruses. Polymerase genes are usually expressed early in the cycle, while capsid and tail proteins are expressed later. During the maturation phase, new virions are created. To liberate free phages, the bacterial cell wall is disrupted by phage proteins such as holin or lysozyme. The final stage is release. Mature viruses burst out of the host cell in a process called lysis and the progeny viruses are liberated into the environment to infect new cells.
The Lysogenic Cycle
In a lysogenic cycle, the phage genome also enters the cell through attachment and penetration. A prime example of a phage with this type of life cycle is the lambda phage. During the lysogenic cycle, instead of killing the host, the phage genome integrates into the bacterial chromosome and becomes part of the host. The integrated phage genome is called a prophage. A bacterial host with a prophage is called a lysogen. The process in which a bacterium is infected by a temperate phage is called lysogeny. It is typical of temperate phages to be latent or inactive within the cell. As the bacterium replicates its chromosome, it also replicates the phage’s DNA and passes it on to new daughter cells during reproduction. The presence of the phage may alter the phenotype of the bacterium, since it can bring in extra genes (e.g., toxin genes that can increase bacterial virulence). This change in the host phenotype is called lysogenic conversion or phage conversion. Some bacteria, such as Vibrio cholerae and Clostridium botulinum, are less virulent in the absence of the prophage. The phages infecting these bacteria carry the toxin genes in their genome and enhance the virulence of the host when the toxin genes are expressed. In the case of V. cholera, phage encoded toxin can cause severe diarrhea; in C. botulinum, the toxin can cause paralysis. During lysogeny, the prophage will persist in the host chromosome until induction, which results in the excision of the viral genome from the host chromosome. After induction has occurred the temperate phage can proceed through a lytic cycle and then undergo lysogeny in a newly infected cell.
Epidemiology
In the United States and other developed nations, public health is a key function of government. A healthy citizenry is more productive, content, and prosperous; high rates of death and disease, on the other hand, can severely hamper economic productivity and foster social and political instability. The burden of disease makes it difficult for citizens to work consistently, maintain employment, and accumulate wealth to better their lives and support a growing economy.
Epidemiology is the science that underlies public health by examining the incidence, spread, transmission, and control of diseases in society. Epidemiology studies how disease originates and spreads throughout a population, with the goal of preventing outbreaks and containing them when they do occur. Over the past two centuries, discoveries in epidemiology have led to public health policies that have transformed life in developed nations, leading to the eradication (or near eradication) of many diseases (e.g. polio, smallpox, measles) that were once causes of great human suffering and premature death. However, the work of epidemiologists is far from finished. Numerous diseases continue to plague humanity, and new diseases are always emerging. Moreover, in the developing world, lack of infrastructure continues to pose many challenges to efforts to contain disease.
The field of epidemiology concerns the geographical distribution and timing of infectious disease occurrences and how they are transmitted and maintained in nature, with the goal of recognizing and controlling outbreaks. The science of epidemiology includes etiology (the study of the causes of disease) and investigation of disease transmission (mechanisms by which a disease is spread).
Analyzing Disease in a Population
Epidemiological analyses are always carried out with reference to a population, which is the group of individuals that are at risk for the disease or condition. The population can be defined geographically, but if only a portion of the individuals in that area are susceptible, additional criteria may be required. Susceptible individuals may be defined by particular behaviors, such as intravenous drug use, owning particular pets, or membership in an institution, such as a college. Being able to define the population is important because most measures of interest in epidemiology are made with reference to the size of the population.
The state of being diseased is called morbidity. Morbidity in a population can be expressed in a few different ways. Morbidity or total morbidity is expressed in numbers of individuals without reference to the size of the population. The morbidity rate can be expressed as the number of diseased individuals out of a standard number of individuals in the population, such as 100,000, or as a percent of the population.
There are two aspects of morbidity that are relevant to an epidemiologist: a disease’s prevalence and its incidence. Prevalence is the number, or proportion, of individuals with a particular illness in a given population at a point in time. For example, the Centers for Disease Control and Prevention (CDC) estimated that in 2012, there were about 1.2 million people 13 years and older with an active human immunodeficiency virus (HIV) infection. Expressed as a proportion, or rate, this is a prevalence of 467 infected persons per 100,000 in the population. On the other hand, incidence is the number or proportion of new cases in a period of time. For the same year and population, the CDC estimates that there were 43,165 newly diagnosed cases of HIV infection, which is an incidence of 13.7 new cases per 100,000 in the population. The relationship between incidence and prevalence can be seen in Figure 5. For a chronic disease like HIV infection, prevalence will generally be higher than incidence because it represents the cumulative number of new cases over many years minus the number of cases that are no longer active (e.g., because the patient died or was cured).
In addition to morbidity rates, the incidence and prevalence of mortality (death) may also be reported. A mortality rate can be expressed as the percentage of the population that has died from a disease or as the number of deaths per 100,000 persons (or other suitable standard number).
Patterns of Incidence
Diseases that are seen only occasionally, and usually without geographic concentration, are called sporadic diseases. Examples of sporadic diseases include tetanus, rabies, and plague. In the United States, Clostridium tetani, the bacterium that causes tetanus, is ubiquitous in the soil environment, but incidences of infection occur only rarely and in scattered locations because most individuals are vaccinated, clean wounds appropriately, or are only rarely in a situation that would cause infection. Likewise in the United States there are a few scattered cases of plague each year, usually contracted from rodents in rural areas in the western states.
Diseases that are constantly present (often at a low level) in a population within a particular geographic region are called endemic diseases. For example, malaria is endemic to some regions of Brazil, but is not endemic to the United States.
Diseases for which a larger than expected number of cases occurs in a short time within a geographic region are called epidemic diseases. Influenza is a good example of a commonly epidemic disease. Incidence patterns of influenza tend to rise each winter in the northern hemisphere. These seasonal increases are expected, so it would not be accurate to say that influenza is epidemic every winter; however, some winters have an usually large number of seasonal influenza cases in particular regions, and such situations would qualify as epidemics (Figure 6 and Figure 7).
An epidemic disease signals the breakdown of an equilibrium in disease frequency, often resulting from some change in environmental conditions or in the population. In the case of influenza, the disruption can be due to antigenic shift or drift (see Virulence Factors of Bacterial and Viral Pathogens), which allows influenza virus strains to circumvent the acquired immunity of their human hosts.
An epidemic that occurs on a worldwide scale is called a pandemic disease. For example, HIV/AIDS is a pandemic disease and novel influenza virus strains often become pandemic.
Etiology
When studying an epidemic, an epidemiologist’s first task is to determinate the cause of the disease, called the etiologic agent or causative agent. Connecting a disease to a specific pathogen can be challenging because of the extra effort typically required to demonstrate direct causation as opposed to a simple association. It is not enough to observe an association between a disease and a suspected pathogen; controlled experiments are needed to eliminate other possible causes. In addition, pathogens are typically difficult to detect when there is no immediate clue as to what is causing the outbreak. Signs and symptoms of disease are also commonly nonspecific, meaning that many different agents can give rise to the same set of signs and symptoms. This complicates diagnosis even when a causative agent is familiar to scientists.
Robert Koch was the first scientist to specifically demonstrate the causative agent of a disease (anthrax) in the late 1800s. Koch developed four criteria, now known as Koch’s postulates, which had to be met in order to positively link a disease with a pathogenic microbe. Without Koch’s postulates, the Golden Age of Microbiology would not have occurred. Between 1876 and 1905, many common diseases were linked with their etiologic agents, including cholera, diphtheria, gonorrhea, meningitis, plague, syphilis, tetanus, and tuberculosis. Today, we use the molecular Koch’s postulates, a variation of Koch’s original postulates that can be used to establish a link between the disease state and virulence traits unique to a pathogenic strain of a microbe.
Koch’s Postulates
1. The suspected pathogen must be found in every case of disease and not be found in healthy individuals.
2. The suspected pathogen can be isolated and grown in pure culture.
3. A healthy test subject infected with the suspected pathogen must develop the same signs and symptoms of disease as seen in postulate 1.
4. The pathogen must be re-isolated from the new host and must be identical to the pathogen from postulate 2.
Spread of Pathogens
Understanding how infectious pathogens spread is critical to preventing infectious disease. Many pathogens require a living host to survive, while others may be able to persist in a dormant state outside of a living host. But having infected one host, all pathogens must also have a mechanism of transfer from one host to another or they will die when their host dies. Pathogens often have elaborate adaptations to exploit host biology, behavior, and ecology to live in and move between hosts. Hosts have evolved defenses against pathogens, but because their rates of evolution are typically slower than their pathogens (because their generation times are longer), hosts are usually at an evolutionary disadvantage. This section will explore where pathogens survive—both inside and outside hosts—and some of the many ways they move from one host to another.
Reservoirs and Carriers
For pathogens to persist over long periods of time they require reservoirs where they normally reside. Reservoirs can be living organisms or nonliving locations. Nonliving reservoirs can include soil and water in the environment. These may naturally harbor the organism because it may grow in that environment. These environments may also become contaminated with pathogens in human feces, pathogens shed by intermediate hosts, or pathogens contained in the remains of intermediate hosts.
Pathogens may have mechanisms of dormancy or resilience that allow them to survive (but typically not to reproduce) for varying periods of time in nonliving environments. For example, Clostridium tetani survives in the soil and in the presence of oxygen as a resistant endospore. Although many viruses are soon destroyed once in contact with air, water, or other non-physiological conditions, certain types are capable of persisting outside of a living cell for varying amounts of time. For example, a study that looked at the ability of influenza viruses to infect a cell culture after varying amounts of time on a banknote showed survival times from 48 hours to 17 days, depending on how they were deposited on the banknote. On the other hand, cold-causing rhinoviruses are somewhat fragile, typically surviving less than a day outside of physiological fluids.
A human acting as a reservoir of a pathogen may or may not be capable of transmitting the pathogen, depending on the stage of infection and the pathogen. To help prevent the spread of disease among school children, the CDC has developed guidelines based on the risk of transmission during the course of the disease. For example, children with chickenpox are considered contagious for five days from the start of the rash, whereas children with most gastrointestinal illnesses should be kept home for 24 hours after the symptoms disappear.
An individual capable of transmitting a pathogen without displaying symptoms is referred to as a carrier. A passive carrier is contaminated with the pathogen and can mechanically transmit it to another host; however, a passive carrier is not infected. For example, a health-care professional who fails to wash their hands after seeing a patient harboring an infectious agent could become a passive carrier, transmitting the pathogen to another patient who becomes infected.
By contrast, an active carrier is an infected individual who can transmit the disease to others. An active carrier may or may not exhibit signs or symptoms of infection. For example, active carriers may transmit the disease during the incubation period (before they show signs and symptoms) or the period of convalescence (after symptoms have subsided). Active carriers who do not present signs or symptoms of disease despite infection are called asymptomatic carriers. Pathogens such as hepatitis B virus, herpes simplex virus, and HIV are frequently transmitted by asymptomatic carriers.
Mary Mallon, better known as Typhoid Mary, is a famous historical example of an asymptomatic carrier. An Irish immigrant, Mallon worked as a cook for households in and around New York City between 1900 and 1915. In each household, the residents developed typhoid fever (caused by Salmonella typhi) a few weeks after Mallon started working. Later investigations determined that Mallon was responsible for at least 122 cases of typhoid fever, five of which were fatal. See Eye on Ethics: Typhoid Mary for more about the Mallon case.
A pathogen may have more than one living reservoir. In zoonotic diseases, animals act as reservoirs of human disease and transmit the infectious agent to humans through direct or indirect contact. In some cases, the disease also affects the animal, but in other cases the animal is asymptomatic.
In parasitic infections, the parasite’s preferred host is called the definitive host. In parasites with complex life cycles, the definitive host is the host in which the parasite reaches sexual maturity. Some parasites may also infect one or more intermediate hosts in which the parasite goes through several immature life cycle stages or reproduces asexually.
Transmission
Regardless of the reservoir, transmission must occur for an infection to spread. First, transmission from the reservoir to the individual must occur. Then, the individual must transmit the infectious agent to other susceptible individuals, either directly or indirectly. Pathogenic microorganisms employ diverse transmission mechanisms.
Contact Transmission
Contact transmission includes direct contact or indirect contact. Person-to-person transmission is a form of direct contact transmission. Here the agent is transmitted by physical contact between two individuals (Figure 9) through actions such as touching, kissing, sexual intercourse, or droplet sprays. Direct contact can be categorized as vertical, horizontal, or droplet transmission. Vertical direct contact transmission occurs when pathogens are transmitted from mother to child during pregnancy, birth, or breastfeeding. Other kinds of direct contact transmission are called horizontal direct contact transmission. Often, contact between mucous membranes is required for entry of the pathogen into the new host, although skin-to-skin contact can lead to mucous membrane contact if the new host subsequently touches a mucous membrane. Contact transmission may also be site-specific; for example, some diseases can be transmitted by sexual contact but not by other forms of contact.
When an individual coughs or sneezes, small droplets of mucus that may contain pathogens are ejected. This leads to direct droplet transmission, which refers to droplet transmission of a pathogen to a new host over distances of one meter or less. A wide variety of diseases are transmitted by droplets, including influenza and many forms of pneumonia. Transmission over distances greater than one meter is called airborne transmission.
Indirect Contact Transmission
Indirect contact transmission involves inanimate objects called fomites that become contaminated by pathogens from an infected individual or reservoir (Figure 10). For example, an individual with the common cold may sneeze, causing droplets to land on a fomite such as a tablecloth or carpet, or the individual may wipe her nose and then transfer mucus to a fomite such as a doorknob or towel. Transmission occurs indirectly when a new susceptible host later touches the fomite and transfers the contaminated material to a susceptible portal of entry. Fomites can also include objects used in clinical settings that are not properly sterilized, such as syringes, needles, catheters, and surgical equipment. Pathogens transmitted indirectly via such fomites are a major cause of healthcare-associated infections (see Controlling Microbial Growth).
Vehicle Transmission
The term vehicle transmission refers to the transmission of pathogens through vehicles such as water, food, and air. Water contamination through poor sanitation methods leads to waterborne transmission of disease. Waterborne disease remains a serious problem in many regions throughout the world. The World Health Organization (WHO) estimates that contaminated drinking water is responsible for more than 500,000 deaths each year. Similarly, food contaminated through poor handling or storage can lead to foodborne transmission of disease (Figure 11).
Dust and fine particles known as aerosols, which can float in the air, can carry pathogens and facilitate the airborne transmission of disease. For example, dust particles are the dominant mode of transmission of hantavirus to humans. Hantavirus is found in mouse feces, urine, and saliva, but when these substances dry, they can disintegrate into fine particles that can become airborne when disturbed; inhalation of these particles can lead to a serious and sometimes fatal respiratory infection.
Although droplet transmission over short distances is considered contact transmission as discussed above, longer distance transmission of droplets through the air is considered vehicle transmission. Unlike larger particles that drop quickly out of the air column, fine mucus droplets produced by coughs or sneezes can remain suspended for long periods of time, traveling considerable distances. In certain conditions, droplets desiccate quickly to produce a droplet nucleus that is capable of transmitting pathogens; air temperature and humidity can have an impact on effectiveness of airborne transmission.
Tuberculosis is often transmitted via airborne transmission when the causative agent, Mycobacterium tuberculosis, is released in small particles with coughs. Because tuberculosis requires as few as 10 microbes to initiate a new infection, patients with tuberculosis must be treated in rooms equipped with special ventilation, and anyone entering the room should wear a mask.
Vector Transmission
Diseases can also be transmitted by a mechanical or biological vector, an animal (typically an arthropod) that carries the disease from one host to another. Mechanical transmission is facilitated by a mechanical vector, an animal that carries a pathogen from one host to another without being infected itself. For example, a fly may land on fecal matter and later transmit bacteria from the feces to food that it lands on; a human eating the food may then become infected by the bacteria, resulting in a case of diarrhea or dysentery (Figure 12).
Biological transmission occurs when the pathogen reproduces within a biological vector that transmits the pathogen from one host to another (Figure 12). Arthropods are the main vectors responsible for biological transmission (Figure 13). Most arthropod vectors transmit the pathogen by biting the host, creating a wound that serves as a portal of entry. The pathogen may go through part of its reproductive cycle in the gut or salivary glands of the arthropod to facilitate its transmission through the bite. For example, hemipterans (called “kissing bugs” or “assassin bugs”) transmit Chagas disease to humans by defecating when they bite, after which the human scratches or rubs the infected feces into a mucous membrane or break in the skin.
Biological insect vectors include mosquitoes, which transmit malaria and other diseases, and lice, which transmit typhus. Other arthropod vectors can include arachnids, primarily ticks, which transmit Lyme disease and other diseases, and mites, which transmit scrub typhus and rickettsial pox. Biological transmission, because it involves survival and reproduction within a parasitized vector, complicates the biology of the pathogen and its transmission. There are also important non-arthropod vectors of disease, including mammals and birds. Various species of mammals can transmit rabies to humans, usually by means of a bite that transmits the rabies virus. Chickens and other domestic poultry can transmit avian influenza to humans through direct or indirect contact with avian influenza virus A shed in the birds’ saliva, mucous, and feces.
Quarantining
Individuals suspected or known to have been exposed to certain contagious pathogens may be quarantined, or isolated to prevent transmission of the disease to others. Hospitals and other health-care facilities generally set up special wards to isolate patients with particularly hazardous diseases such as tuberculosis or Ebola (Figure 16.15). Depending on the setting, these wards may be equipped with special air-handling methods, and personnel may implement special protocols to limit the risk of transmission, such as personal protective equipment or the use of chemical disinfectant sprays upon entry and exit of medical personnel.
The duration of the quarantine depends on factors such as the incubation period of the disease and the evidence suggestive of an infection. The patient may be released if signs and symptoms fail to materialize when expected or if preventive treatment can be administered in order to limit the risk of transmission. If the infection is confirmed, the patient may be compelled to remain in isolation until the disease is no longer considered contagious.
In the United States, public health authorities may only quarantine patients for certain diseases, such as cholera, diphtheria, infectious tuberculosis, and strains of influenza capable of causing a pandemic. Individuals entering the United States or moving between states may be quarantined by the CDC if they are suspected of having been exposed to one of these diseases. Although the CDC routinely monitors entry points to the United States for crew or passengers displaying illness, quarantine is rarely implemented.
Healthcare-Associated (Nosocomial) Infections
Hospitals, retirement homes, and prisons attract the attention of epidemiologists because these settings are associated with increased incidence of certain diseases. Higher rates of transmission may be caused by characteristics of the environment itself, characteristics of the population, or both. Consequently, special efforts must be taken to limit the risks of infection in these settings.
Infections acquired in health-care facilities, including hospitals, are called nosocomial infections or healthcare-associated infections (HAI). HAIs are often connected with surgery or other invasive procedures that provide the pathogen with access to the portal of infection. For an infection to be classified as an HAI, the patient must have been admitted to the health-care facility for a reason other than the infection. In these settings, patients suffering from primary disease are often afflicted with compromised immunity and are more susceptible to secondary infection and opportunistic pathogens.
In 2011, more than 720,000 HAIs occurred in hospitals in the United States, according to the CDC. About 22% of these HAIs occurred at a surgical site, and cases of pneumonia accounted for another 22%; urinary tract infections accounted for an additional 13%, and primary bloodstream infections 10%. Such HAIs often occur when pathogens are introduced to patients’ bodies through contaminated surgical or medical equipment, such as catheters and respiratory ventilators. Health-care facilities seek to limit nosocomial infections through training and hygiene protocols such as those described in Control of Microbial Growth.
Laboratory Instructions
Viral Epidemic Scenario
One of the most rapid ways for a pathogen to spread is when people are asymptomatic carriers or when people are in the incubation period before symptoms occur. Since these people are not experiencing symptoms, they are unaware that they are carrying a pathogen and able to spread that pathogen. This is particularly true when the incubation period is lengthy (length of the incubation period is different for different pathogens).
You are among a population who is at risk of catching a virus at the start of an epidemic. Everyone in the population begins either as an uninfected person, except one person who is an asymptomatic carrier of the virus. No one knows who the infected person is including that person.
This virus passes when two people are in close proximity and the carrier exhales respiratory droplets into the air. The uninfected person inhales respiratory droplets from the nearby carrier and infection then occurs.
As you go about normal day to day life in this scenario, you will come into contact with different individuals in the classroom each "day." Each "day" you will interact with one classmate and breath each others respiratory droplets. You will not know until the end whether you were the original infected person, or if you become infected and transmit the infection to others.
Epidemic Day 1
A well plate will be used to track fluid samples from each participant.
1. Choose/be assigned a vial number.
2. Collect the vial with your number and a transfer pipet.
3. Write your vial number down in the Results & Questions section below.
4. Put 5 drops from your vial into the well on the well plate corresponding with your vial number and epidemic day 1.
Epidemic Day 2 (Transfer #1)
1. Names will be randomly chosen using a group randomizer.
2. Find the person you are paired with and write their vial number in the table for epidemic day 2 in the Results & Questions section below.
3. Use your transfer pipet to collect fluid from your vial. Your partner will do the same.
4. Drop 5 drops from your transfer pipet into your partner's vial (this represents you transferring your respiratory droplets to your partner).
5. Your partner will drop 5 drops from their transfer pipet to your vial (this represents your partner transferring their respiratory droplets to you).
6. Put any liquid remaining in the transfer pipet back into your own vial.
7. Close your vial securely and turn the vial upside-down 10 times to mix the vial well.
8. Collect liquid from your vial with the transfer pipet.
9. Put 5 drops from your vial into the well on the well plate corresponding with your vial number and epidemic day 2.
10. Put any liquid remaining in the transfer pipet back into your own vial.
Epidemic Day 3 (Transfer #2)
1. Names will be randomly chosen using a group randomizer.
2. Find the person you are paired with and write their vial number in the table for epidemic day 3 in the Results & Questions section below.
3. Use your transfer pipet to collect fluid from your vial. Your partner will do the same.
4. Drop 5 drops from your transfer pipet into your partner's vial (this represents you transferring your respiratory droplets to your partner).
5. Your partner will drop 5 drops from their transfer pipet to your vial (this represents your partner transferring their respiratory droplets to you).
6. Put any liquid remaining in the transfer pipet back into your own vial.
7. Close your vial securely and turn the vial upside-down 10 times to mix the vial well.
8. Collect liquid from your vial with the transfer pipet.
9. Put 5 drops from your vial into the well on the well plate corresponding with your vial number and epidemic day 3.
10. Put any liquid remaining in the transfer pipet back into your own vial.
Epidemic Day 4 (Transfer #3)
1. Names will be randomly chosen using a group randomizer.
2. Find the person you are paired with and write their vial number in the table for epidemic day 4 in the Results & Questions section below.
3. Use your transfer pipet to collect fluid from your vial. Your partner will do the same.
4. Drop 5 drops from your transfer pipet into your partner's vial (this represents you transferring your respiratory droplets to your partner).
5. Your partner will drop 5 drops from their transfer pipet to your vial (this represents your partner transferring their respiratory droplets to you).
6. Put any liquid remaining in the transfer pipet back into your own vial.
7. Close your vial securely and turn the vial upside-down 10 times to mix the vial well.
8. Collect liquid from your vial with the transfer pipet.
9. Put 5 drops from your vial into the well on the well plate corresponding with your vial number and epidemic day 4.
10. Put any liquid remaining in the transfer pipet back into your own vial.
Revealing the Viral Infections & Spread of Virus in the Population
1. After epidemic day 4, your instructor will place a single drop of a solution into each person's vial. If the vial turns red, you are "infected" with the virus.
2. If you are infected, fill out the table on the board to indicate your vial number and the vial numbers you exchanged "respiratory drops" with.
3. Your instructor will also use the well plate to determine the number of "infected" people in the population each day if the epidemic. They will share this information with the class so you can calculate the prevalence rates and incidence rates.
Results & Questions
My vial number is: _______
Epidemic Day Vial # Respiratory Drops Exchanged With
2
3
4
Vial # Mark with 'X' if infected on Epidemic Day 4 Vials that Exchanged Respiratory Droplets with this Vial
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
1. Fill out the table above for each of the epidemic days.
2. Fill out the table above to indicate which of the vials were positive for the virus and which vials the virus-positive vials exchanged respiratory droplets with.
3. Put your epidemiologist hat on. Use the information above to determine which vial was the original one that was infected. Which vial contained virus on epidemic day 1? How can you tell?
4. How many chances did people in this scenario have to become "infected?"
5. At the beginning of this viral epidemic scenario, only one person was "infected" with the virus. How many people were "infected" with the virus by epidemic day 4?
6. Explain why there were more people who were "infected" at the end of the scenario than the number of fluid exchanges.
7. What type or types of transmission occurred in this scenario (direct contact transmission, droplet transmission, indirect contact transmission, vehicle transmission, mechanical transmission)
8. When a person is asymptomatic, are they aware that they are spreading a pathogen?
9. Calculate the prevalence rate and incidence rate of the virus in the population for each epidemic day.
• Epidemic Day Prevalence Rate Incidence Rate
1
2
3
4
10. Define the following terms:
• epidemiology:
• etiology:
• morbidity:
• morbidity rate:
• prevalence:
• incidence:
• mortality:
• sporadic diseases:
• endemic diseases:
• epidemic diseases:
• pandemic diseases:
• causative agent:
• reservoirs:
• passive carriers:
• active carriers:
• asymptomatic carriers:
• direct contact transmission:
• droplet transmission:
• indirect contact transmission:
• vehicle transmission:
• mechanical transmission:
• mechanical vector:
• quarantine:
• nosocomial infections:
• healthcare-associated infections: | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.37%3A_Viruses_and_Viral_Epidemic_Simulation.txt |
Learning Objectives
• Describe the structures of viruses, including their sizes.
• Identify examples of different virus morphologies (e.g. helical, icosohedral, etc.)
• Explain how tobacco mosaic virus infects cells, spreads, and replicates.
• Tell that specific viruses will only infect specific species.
• Describe how TMV has significant impacts on agriculture.
• Extract TMV from tobacco.
• Conduct a bioassay using TMV, analyze results of the experiment by t-test, and state conclusions of the experiment.
Introduction to Viruses
Viruses are noncellular parasitic entities that cannot be classified within any living kingdom. They can infect organisms as diverse as bacteria, plants, and animals. In fact, viruses exist in a sort of netherworld between a living organism and a nonliving entity. Living things grow, metabolize, and reproduce. In contrast, viruses are not cellular, do not have a metabolism or grow, and cannot divide by cell division. Viruses can copy, or replicate themselves; however, they are entirely dependent on resources derived from their host cells to produce progeny viruses—which are assembled in their mature form. No one knows exactly when or how viruses evolved or from what ancestral source because viruses have not left a fossil record. Some virologists contend that modern viruses are a mosaic of bits and pieces of nucleic acids picked up from various sources along their respective evolutionary paths.
Viruses are diverse entities: They vary in structure, methods of replication, and the hosts they infect. Nearly all forms of life—from prokaryotic bacteria and archaeans, to eukaryotes such as plants, animals, and fungi—have viruses that infect them. While most biological diversity can be understood through evolutionary history (such as how species have adapted to changing environmental conditions and how different species are related to one another through common descent), much about virus origins and evolution remains unknown.
Viruses were first discovered after the development of a porcelain filter—the Chamberland-Pasteur filter—that could remove all bacteria visible in the microscope from any liquid sample. In 1886, Adolph Meyer demonstrated that a disease of tobacco plants—tobacco mosaic disease—could be transferred from a diseased plant to a healthy one via liquid plant extracts. In 1892, Dmitri Ivanowski showed that this disease could be transmitted in this way even after the Chamberland-Pasteur filter had removed all viable bacteria from the extract. Still, it was many years before it was proved that these “filterable” infectious agents were not simply very small bacteria but were a new type of very small, disease-causing particle.
Virus Structures
Most virions, or single virus particles, are very small, about 20 to 250 nanometers in diameter. However, some recently discovered viruses from amoebae range up to 1000 nm in diameter. With the exception of large virions, like the poxvirus and other large DNA viruses, viruses cannot be seen with a light microscope. It was not until the development of the electron microscope in the late 1930s that scientists got their first good view of the structure of the tobacco mosaic virus, discussed above, and other viruses. The surface structure of virions can be observed by both scanning and transmission electron microscopy, whereas the internal structures of the virus can only be observed in images from a transmission electron microscope. The use of electron microscopy and other technologies has allowed for the discovery of many viruses of all types of living organisms.
Use this interactive tool to get a sense of how big viruses are in comparison to cells and familiar objects.*
*viruses in this interactive tool include: measles virus, hiv, phage, influenza virus, hepatitis virus, and rhinovirus
V
iruses are noncellular, meaning they are biological entities that do not have a cellular structure. They therefore lack most of the components of cells, such as organelles, ribosomes, and the plasma membrane. A virion consists of a nucleic acid core (DNA or RNA), an outer protein coating or capsid, and sometimes an outer envelope made of protein and phospholipid membranes derived from the host cell. Viruses may also contain additional proteins, such as enzymes, within the capsid or attached to the viral genome. The most obvious difference between members of different viral families is the variation in their morphology, which is quite diverse. An interesting feature of viral complexity is that the complexity of the host does not necessarily correlate with the complexity of the virion. In fact, some of the most complex virion structures are found in the bacteriophages—viruses that infect the simplest living organisms, bacteria.
Viruses come in many shapes and sizes, but these features are consistent for each viral family. As we have seen, all virions have a nucleic acid genome covered by a protective capsid. The proteins of the capsid are encoded in the viral genome, and are called capsomeres. Some viral capsids are simple helices or polyhedral “spheres,” whereas others are quite complex in structure.
In general, viruses structures can be classified as: helical, icosahedral, enveloped, and complex, also known as head-and-tail. Helical capsids are long and cylindrical. Many plant viruses are helical, including TMV. Icosahedral viruses have shapes that are roughly spherical, such as those of poliovirus or herpesviruses. Enveloped viruses have membranes derived from the host cell that surrounds the capsids. Animal viruses, such as HIV, are frequently enveloped. Head-and-tail viruses infect bacteria and have a head that is similar to icosahedral viruses and a tail shaped like helical viruses.
Viral Reproduction
All viruses depend on cells for reproduction and metabolic processes. By themselves, viruses do not encode for all of the enzymes necessary for viral replication. But within a host cell, a virus can commandeer cellular machinery to produce more viral particles. Bacteriophages replicate only in the cytoplasm, since prokaryotic cells do not have a nucleus or organelles. In eukaryotic cells, most DNA viruses can replicate inside the nucleus, with an exception observed in the large DNA viruses, such as the poxviruses, that can replicate in the cytoplasm. With a few exceptions, RNA viruses that infect animal cells replicate in the cytoplasm. An important exception that will be highlighted later is Influenza virus.
The life cycle of bacteriophages has been a good model for understanding how viruses affect the cells they infect, since similar processes have been observed for eukaryotic viruses, which can cause immediate death of the cell or establish a latent or chronic infection.
The Lytic Cycle
During the lytic cycle of virulent phage, the bacteriophage takes over the cell, reproduces new phages, and destroys the cell. T-even phage is a good example of a well-characterized class of virulent phages. There are five stages in the bacteriophage lytic cycle. Attachment is the first stage in the infection process in which the phage interacts with specific bacterial surface receptors (e.g., lipopolysaccharides and OmpC protein on host surfaces). Most phages have a narrow host range and may infect one species of bacteria or one strain within a species. This unique recognition can be exploited for targeted treatment of bacterial infection by phage therapy or for phage typing to identify unique bacterial subspecies or strains. The second stage of infection is entry or penetration. This occurs through contraction of the tail sheath, which acts like a hypodermic needle to inject the viral genome through the cell wall and membrane. The phage head and remaining components remain outside the bacteria.
The third stage of infection is biosynthesis of new viral components. After entering the host cell, the virus synthesizes virus-encoded endonucleases to degrade the bacterial chromosome. It then hijacks the host cell to replicate, transcribe, and translate the necessary viral components (capsomeres, sheath, base plates, tail fibers, and viral enzymes) for the assembly of new viruses. Polymerase genes are usually expressed early in the cycle, while capsid and tail proteins are expressed later. During the maturation phase, new virions are created. To liberate free phages, the bacterial cell wall is disrupted by phage proteins such as holin or lysozyme. The final stage is release. Mature viruses burst out of the host cell in a process called lysis and the progeny viruses are liberated into the environment to infect new cells.
The Lysogenic Cycle
In a lysogenic cycle, the phage genome also enters the cell through attachment and penetration. A prime example of a phage with this type of life cycle is the lambda phage. During the lysogenic cycle, instead of killing the host, the phage genome integrates into the bacterial chromosome and becomes part of the host. The integrated phage genome is called a prophage. A bacterial host with a prophage is called a lysogen. The process in which a bacterium is infected by a temperate phage is called lysogeny. It is typical of temperate phages to be latent or inactive within the cell. As the bacterium replicates its chromosome, it also replicates the phage’s DNA and passes it on to new daughter cells during reproduction. The presence of the phage may alter the phenotype of the bacterium, since it can bring in extra genes (e.g., toxin genes that can increase bacterial virulence). This change in the host phenotype is called lysogenic conversion or phage conversion. Some bacteria, such as Vibrio cholerae and Clostridium botulinum, are less virulent in the absence of the prophage. The phages infecting these bacteria carry the toxin genes in their genome and enhance the virulence of the host when the toxin genes are expressed. In the case of V. cholera, phage encoded toxin can cause severe diarrhea; in C. botulinum, the toxin can cause paralysis. During lysogeny, the prophage will persist in the host chromosome until induction, which results in the excision of the viral genome from the host chromosome. After induction has occurred the temperate phage can proceed through a lytic cycle and then undergo lysogeny in a newly infected cell.
Introduction to Plant Viruses
Most plant viruses, like the tobacco mosaic virus, have single-stranded (+) RNA genomes. However, there are also plant viruses in most other virus categories. Unlike bacteriophages, plant viruses do not have active mechanisms for delivering the viral genome across the protective cell wall. For a plant virus to enter a new host plant, some type of mechanical damage must occur. This damage is often caused by weather, insects, animals, fire, or human activities like farming or landscaping. Movement from cell to cell within a plant can be facilitated by viral modification of plasmodesmata (cytoplasmic threads that pass from one plant cell to the next). Additionally, plant offspring may inherit viral diseases from parent plants. Plant viruses can be transmitted by a variety of vectors, through contact with an infected plant’s sap, by living organisms such as insects and nematodes, and through pollen. The transfer of a virus from one plant to another is known as horizontal transmission, whereas the inheritance of a virus from a parent is called vertical transmission.
Symptoms of viral diseases vary according to the virus and its host. One common symptom is hyperplasia, the abnormal proliferation of cells that causes the appearance of plant tumors known as galls. Other viruses induce hypoplasia, or decreased cell growth, in the leaves of plants, causing thin, yellow areas to appear. Still other viruses affect the plant by directly killing plant cells, a process known as cell necrosis. Other symptoms of plant viruses include malformed leaves; black streaks on the stems of the plants; altered growth of stems, leaves, or fruits; and ring spots, which are circular or linear areas of discoloration found in a leaf.
Table 1: Some Common Symptoms of Plant Viral Diseases
Symptom Appears as
Hyperplasia Galls (tumors)
Hypoplasia Thinned, yellow splotches on leaves
Cell necrosis Dead, blackened stems, leaves, or fruit
Abnormal growth patterns Malformed stems, leaves, or fruit
Discoloration Yellow, red, or black lines, or rings in stems, leaves, or fruit
Plant viruses can seriously disrupt crop growth and development, significantly affecting our food supply. They are responsible for poor crop quality and quantity globally, and can bring about huge economic losses annually. Others viruses may damage plants used in landscaping. Some viruses that infect agricultural food plants include the name of the plant they infect, such as tomato spotted wilt virus, bean common mosaic virus, and cucumber mosaic virus. In plants used for landscaping, two of the most common viruses are peony ring spot and rose mosaic virus. There are far too many plant viruses to discuss each in detail, but symptoms of bean common mosaic virus result in lowered bean production and stunted, unproductive plants. In the ornamental rose, the rose mosaic disease causes wavy yellow lines and colored splotches on the leaves of the plant.
Tobacco Mosaic Virus
The tobacco mosaic virus (TMV) is a plant pathogen that causes tobacco mosaic disease. TMV infects tobacco plants, tomato plants, bean plants, and other plant species within the plant family Solanaceae (sometimes called "nightshades") (Scholthof, 2000). Tobacco mosaic disease has been reported as early as the early 1800s in Colombia where tobacco was an important agricultural crop (Scholthof, 2008). This virus impacted the quality of the tobacco by causing it to taste incredibly bitter, and therefore could devastate the value of tobacco crops (Scholthof, 2008).
TMV is an RNA virus with a helical capsid. When TMV infects a tobacco plant, it can cause abnormal growth patterns (e.g. curling leaves), cell necrosis, and discoloration - see Figure 5 above (Scholthof, 2000). The impacts of TMV on tomato plant today are more profound (~20% crop loss) than on tobacco crops, since TMV-resistant tobacco crops are typically selected and grown agriculturally (~1% crop loss by TMV) (Scholthof, 2000).
As mentioned above in the Introduction to Plant Viruses section, plant cells have a thick cell wall that viruses cannot penetrate, and TMV is no different. A plant cell must be injured in order for TMV to cause infection (Scholthof, 2000). Transfer of the TMV particles involves mechanical transmission from a farm tool carrying the virus, a farm worker's hand that is carrying the virus from another plant or from tobacco they have used, or from an infected leaf to non-infected leaf when they make contact with each other (Scholthof, 2000). After TMV enters a plant cell, the protein capsid disassembles to release the RNA genome of the virus (Scholthof, 2000). Within minutes of a plant cell being infected with TMV, the released viral RNA starts to be used by the host cell's ribosomes to begin making viral proteins (Scholthof, 2000). In summary, the TMV viral proteins replicate the viral RNA genome (to make more virus particles), form subunits for the capsid (to make more virus particles), and facilitate transfer of the virus between adjacent plant cells through cell-to-cell plant communication junctions called plasmodesmata (Scholthof, 2000). Newly synthesized virus particles can not only travel through plasmodesmata, they can also enter the plant's phloem (passages that connect the entire plant), and therefore infect the entire plant (Scholthof, 2000). If an infected cell has its cell wall damaged, virus particles can be transmitted from that cell to infect other plants (Scholthof, 2000).
Laboratory Instructions
Grow Plants to Infect
1. Prepare a plant pot with soil.
2. Plant 5-6 tomato or bean seeds as instructed on the seed package.
3. Water and provide light for the plants for 4-6 weeks as instructed on the seed package.
What is a "Bioassay?"
An assay is a type of scientific test used to determine the composition of a substance. In this case, we will assay tobacco to determine:
1. Does this tobacco contain TMV?
2. How much infective TMV is in the tobacco?
3. Does milk have an impact on the ability of TMV to infect plant cells?
"Bio-" relates to life. In this case, "bio-" refers to the use of a plant used in this assay. Viruses cannot be grown on their own - they require cells. In this case, a living organism (a plant) is used to enable us to experiment with this virus.
Bioassay Instructions
Important
Wash your hands with soap anytime they may have touched anything that might contain tobacco mosaic virus. You may need to wash your hands multiple times throughout this laboratory protocol and be sure to wash your hands at the end of the laboratory so you do not carry and spread the virus into the environment.
This will prevent spread of the virus and prevent cross-contamination in the experiment.
Important
Thoroughly clean any laboratory materials (test tubes, stir rods, etc.) that could have come into contact with the tobacco mosaic virus with disinfectant.
This will prevent spread of the virus and prevent cross-contamination in the experiment.
Create a Virus Extract from Tobacco
1. Mix tobacco from 2-3 different sources (different brands of tobacco).
2. Use a mortar and pestle to produce finely ground tobacco. This will damage the cell walls of the tobacco to release the tobacco mosaic virus from infected cells.
3. Weigh 1 g of tobacco.
4. Measure 15 mL of diatomaceous earth dissolved in phosphate buffer and pour into a small beaker. The diatomaceous earth will be important for creating friction and causing damage to plant leaves to enable infection.
5. Add the 1 g of tobacco to the 15 mL of phosphate buffer with diatomaceous earth.
6. Use a stir rod to further mash the tobacco and stir it into the liquid. This will release tobacco mosaic virus into the phosphate buffer.
7. Put a funnel into a test tube.
8. Line the inside of the funnel with 3-4 layers of cheesecloth.
9. Pour the tobacco-phosphate buffer-diatomaceous earth mixture through the cheesecloth. This will remove tobacco particles, but allow the diatomaceous earth and buffer carrying the tobacco mosaic virus into the test tube.
10. The liquid in this test tube will hereafter be referred to as the "virus extract."
11. Wash your hands thoroughly.
Note
The "virus extract" contains tobacco mosaic virus. Any solution that this virus extract is added to should also contain the virus. Any solution that does not contain this virus extract should not contain the virus.
Prepare Three Test Samples & Apply to Plant Leaves
Prepare Plant for Sample Application
1. Choose three different branches of the plant that have healthy-looking leaves.
2. Use masking tape to create a "flag" on each branch, each labeled as:
1. Control (No Virus)
2. Virus in Water
3. Virus in Milk
Control (No Virus)
1. Label a clean test tube as "Control (No Virus)."
2. Measure 1.5 mL of the phosphate buffer containing diatomaceous earth and pour into the test tube.
3. Measure 1.5 mL of deionized water and pour into the test tube.
4. Gently mix the test tube by lightly swirling of lightly flicking the bottom of the test tube with your finger.
5. Dip a sterile cotton swab into the Control (No Virus) solution.
6. Gently swab the top surface of one leaf on the plant branch you labeled "Control (No Virus)"
7. Repeat steps 5-6 until all leaves on the "Control (No Virus)" branch have been swabbed. If any of the leaves on this branch already have discoloration, pull the leaf off the plant instead of swabbing it.
Virus in Water
1. Label a clean test tube as "Virus in Water."
2. Measure 1.5 mL of the virus extract solution and pour into the test tube.
3. Measure 1.5 mL of deionized water and pour into the test tube.
4. Gently mix the test tube by lightly swirling of lightly flicking the bottom of the test tube with your finger.
5. Dip a sterile cotton swab into the Virus in Water solution.
6. Gently swab the top surface of one leaf on the plant branch you labeled "Virus in Water" being careful not to touch any other leaves on the plant.
7. Repeat steps 5-6 until all leaves on the "Virus in Water" branch have been swabbed. If any of the leaves on this branch already have discoloration, pull the leaf off the plant instead of swabbing it.
8. Wash your hands well after this step to prevent spreading the virus to tools, bench tops, etc.
Virus with Milk
1. Label a clean test tube as "Virus with Milk."
2. Measure 1.5 mL of the virus extract solution and pour into the test tube.
3. Measure 1.5 mL of milk and pour into the test tube.
4. Gently mix the test tube by lightly swirling of lightly flicking the bottom of the test tube with your finger.
5. Dip a sterile cotton swab into the Virus with Milk solution.
6. Gently swab the top surface of one leaf on the plant branch you labeled "Virus with Milk" being careful not to touch any other leaves on the plant.
7. Repeat steps 5-6 until all leaves on the "Virus with Milk" branch have been swabbed. If any of the leaves on this branch already have discoloration, pull the leaf off the plant instead of swabbing it.
8. Wash your hands well after this step to prevent spreading the virus to tools, bench tops, etc.
Post-Inoculation Plant Care
1. Continue caring for plants as you did before Inoculation (light and water as indicated on the seed package). Take care that the leaves of the plants do not touch each other.
2. Wait 2-3 weeks before examining plant leaves and collecting data.
Results & Questions
# of Lesions on Leaf 1 # of Lesions on Leaf 2 # of Lesions on Leaf 3 # of Lesions on Leaf 4 # of Lesions on Leaf 5 # of Lesions on Leaf 6 Average # of Lesions
Control (No Virus)
Virus in Water
Virus with Milk
Class Average # of Lesions t-test Range
Control (No Virus)
Virus in Water
Virus with Milk
1. Fill in the table above with data from the tobacco mosaic bioassay. Depending on the number of leaves on the plant, you may or may not need every box in the table above.
2. Share the average number of lesions per leaf for Control (No Virus), Virus in Water, and Virus with Milk with your instructor. They will calculate the class average for each and t-test ranges. Write these data in the table above.
3. Examine the class average of lesions and the t-test ranges and interpret the t-test statistic. Write conclusions about whether or not these data are statistically significantly different from each other (see the t-test Statistical Analysis section in the Control of Microbial Growth lab for guidance):
• Control vs. Virus in Water:
• Control vs. Virus with Milk:
• Virus in Water vs. Virus with Milk:
4. Explain how TMV infects plant cells.
5. Why was diatomaceous earth used in this experiment (think about how TMV infects plant cells)?
6. How can TMV spread in a plant?
7. What plant species does TMV infect?
8. Can TMV infect humans? Explain your answer by discussing the specificity of viruses.
9. Define bioassay. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.38%3A_Virus_Bioassay.txt |
Learning Objectives
• Define the following terms: fomites, sterilization, asepsis, sepsis, commercial sterilization, disinfection, sanitization, antisepsis, degerming
• Give at least five examples of the categories of physical controls for microbes and what they involve.
• Give at least three examples of categories of chemical controls for microbes and what they involve.
• Successfully conduct an experiment comparing soap, disinfectant, and untreated surfaces (fomites) and the microbial load present.
• Graph and analyze results from control of microbial growth experiment.
• Interpret t-test results and state conclusions based on the interpretation.
Clean Enough?
How clean is clean? People wash their cars and vacuum the carpets, but most would not want to eat from these surfaces. Similarly, we might eat with silverware cleaned in a dishwasher, but we could not use the same dishwasher to clean surgical instruments. As these examples illustrate, “clean” is a relative term. Car washing, vacuuming, and dishwashing all reduce the microbial load on the items treated, thus making them “cleaner.” But whether they are “clean enough” depends on their intended use. Because people do not normally eat from cars or carpets, these items do not require the same level of cleanliness that silverware does. Likewise, because silverware is not used for invasive surgery, these utensils do not require the same level of cleanliness as surgical equipment, which requires sterilization to prevent infection.
Why not play it safe and sterilize everything? Sterilizing everything we come in contact with is impractical, as well as potentially dangerous. As this chapter will demonstrate, sterilization protocols often require time- and labor-intensive treatments that may degrade the quality of the item being treated or have toxic effects on users. Therefore, the user must consider the item’s intended application when choosing a cleaning method to ensure that it is “clean enough.”
Fomites
To prevent the spread of human disease, it is necessary to control the growth and abundance of microbes in or on various items frequently used by humans. Inanimate items, such as doorknobs, toys, or towels, which may harbor microbes and aid in disease transmission, are called fomites. Two factors heavily influence the level of cleanliness required for a particular fomite and, hence, the protocol chosen to achieve this level. The first factor is the application for which the item will be used. For example, invasive applications that require insertion into the human body require a much higher level of cleanliness than applications that do not. The second factor is the level of resistance to antimicrobial treatment by potential pathogens. For example, foods preserved by canning often become contaminated with the bacterium Clostridium botulinum, which produces the neurotoxin that causes botulism. Because C. botulinum can produce endospores that can survive harsh conditions, extreme temperatures and pressures must be used to eliminate the endospores. Other organisms may not require such extreme measures and can be controlled by a procedure such as washing clothes in a laundry machine.
Control of Microbial Growth: Sterilization
The most extreme protocols for microbial control aim to achieve sterilization: the complete removal or killing of all vegetative cells, endospores, and viruses from the targeted item or environment. Sterilization protocols are generally reserved for laboratory, medical, manufacturing, and food industry settings, where it may be imperative for certain items to be completely free of potentially infectious agents. Sterilization can be accomplished through either physical means, such as exposure to high heat, pressure, or filtration through an appropriate filter, or by chemical means. Chemicals that can be used to achieve sterilization are called sterilants. Sterilants effectively kill all microbes and viruses, and, with appropriate exposure time, can also kill endospores.
For many clinical purposes, aseptic technique is necessary to prevent contamination of sterile surfaces. Aseptic technique involves a combination of protocols that collectively maintain sterility, or asepsis, thus preventing contamination of the patient with microbes and infectious agents. Failure to practice aseptic technique during many types of clinical procedures may introduce microbes to the patient’s body and put the patient at risk for sepsis, a systemic inflammatory response to an infection that results in high fever, increased heart and respiratory rates, shock, and, possibly, death. Medical procedures that carry risk of contamination must be performed in a sterile field, a designated area that is kept free of all vegetative microbes, endospores, and viruses. Sterile fields are created according to protocols requiring the use of sterilized materials, such as packaging and drapings, and strict procedures for washing and application of sterilants. Other protocols are followed to maintain the sterile field while the medical procedure is being performed.
One food sterilization protocol, commercial sterilization, uses heat at a temperature low enough to preserve food quality but high enough to destroy common pathogens responsible for food poisoning, such as C. botulinum. Because C. botulinum and its endospores are commonly found in soil, they may easily contaminate crops during harvesting, and these endospores can later germinate within the anaerobic environment once foods are canned. Metal cans of food contaminated with C. botulinum will bulge due to the microbe’s production of gases; contaminated jars of food typically bulge at the metal lid. To eliminate the risk for C. botulinum contamination, commercial food-canning protocols are designed with a large margin of error. They assume an impossibly large population of endospores (1012 per can) and aim to reduce this population to 1 endospore per can to ensure the safety of canned foods. For example, low- and medium-acid foods are heated to 121 °C for a minimum of 2.52 minutes, which is the time it would take to reduce a population of 1012 endospores per can down to 1 endospore at this temperature. Even so, commercial sterilization does not eliminate the presence of all microbes; rather, it targets those pathogens that cause spoilage and foodborne diseases, while allowing many nonpathogenic organisms to survive. Therefore, “sterilization” is somewhat of a misnomer in this context, and commercial sterilization may be more accurately described as “quasi-sterilization.”
Control of Microbial Growth: Non-Sterilizing Approaches
Sterilization protocols require procedures that are not practical, or necessary, in many settings. Various other methods are used in clinical and nonclinical settings to reduce the microbial load on items. Although the terms for these methods are often used interchangeably, there are important distinctions.
The type of protocol required to achieve the desired level of cleanliness depends on the particular item to be cleaned. For example, those used clinically are categorized as critical, semicritical, and noncritical. Critical items must be sterile because they will be used inside the body, often penetrating sterile tissues or the bloodstream; examples of critical items include surgical instruments, catheters, and intravenous fluids. Gastrointestinal endoscopes and various types of equipment for respiratory therapies are examples of semicritical items; they may contact mucous membranes or nonintact skin but do not penetrate tissues. Semicritical items do not typically need to be sterilized but do require a high level of disinfection. Items that may contact but not penetrate intact skin are noncritical items; examples are bed linens, furniture, crutches, stethoscopes, and blood pressure cuffs. These articles need to be clean but not highly disinfected.
Figure 1 summarizes common protocols, definitions, applications, and agents used to control microbial growth.
Disinfection
The process of disinfection inactivates most microbes on the surface of a fomite by using antimicrobial chemicals or heat. Because some microbes remain, the disinfected item is not considered sterile. Ideally, disinfectants should be fast acting, stable, easy to prepare, inexpensive, and easy to use. An example of a natural disinfectant is vinegar; its acidity kills most microbes. Chemical disinfectants, such as chlorine bleach or products containing chlorine, are used to clean nonliving surfaces such as laboratory benches, clinical surfaces, and bathroom sinks. Typical disinfection does not lead to sterilization because endospores tend to survive even when all vegetative cells have been killed.
Antisepsis
Unlike disinfectants, antiseptics are antimicrobial chemicals safe for use on living skin or tissues. Examples of antiseptics include hydrogen peroxide and isopropyl alcohol. The process of applying an antiseptic is called antisepsis. In addition to the characteristics of a good disinfectant, antiseptics must also be selectively effective against microorganisms and able to penetrate tissue deeply without causing tissue damage.
Degerming
The act of handwashing is an example of degerming, in which microbial numbers are significantly reduced by gently scrubbing living tissue, most commonly skin, with a mild chemical (e.g., soap) to avoid the transmission of pathogenic microbes. Wiping the skin with an alcohol swab at an injection site is another example of degerming. These degerming methods remove most (but not all) microbes from the skin’s surface.
Sanitization
The term sanitization refers to the cleansing of fomites to remove enough microbes to achieve levels deemed safe for public health. For example, commercial dishwashers used in the food service industry typically use very hot water and air for washing and drying; the high temperatures kill most microbes, sanitizing the dishes. Surfaces in hospital rooms are commonly sanitized using a chemical disinfectant to prevent disease transmission between patients.
Chemical Approaches for Controlling Microbial Growth
Table 1: Chemical disinfectants can be used to control microbes. These types of chemicals fall into different chemical categories: phenolics, metals, halogens, alcohols, surfactants, bisbiguanides, alkylating agents, peroxygens, supercritical gases, chemical food preservatives, and natural food preservatives.
Chemical Disinfectants
Chemical Mode of Action Example Uses
Phenolics
Cresols
o-Phenylphenol
Hexachlorophene
Triclosan
Denature proteins and disrupt membranes Disinfectant in Lysol
Prevent contamination of crops (citrus)
Antibacterial soap
pHisoHex for handwashing in hospitals
Metals
Mercury
Silver
Copper
Nickel
Zinc
Bind to proteins and inhibit enzyme activity Topical antiseptic
Treatment of wounds and burns
Prevention of eye infections in newborns
Antibacterial in catheters and bandages
Mouthwash
Algicide for pools and fish tanks
Containers for long-term water storage
Halogens
Iodine
Chlorine
Fluorine
Oxidation and destabilization of cellular macromolecules Topical antiseptic
Hand scrub for medical personnel
Water disinfectant
Water treatment plants
Household bleach
Food processing
Prevention of dental carries
Alcohols
Ethanol
Isopropanol
Denature proteins and disrupt membranes Disinfectant
Antiseptic
Surfactants
Quaternary ammonium salts Lowers surface tension of water to help with washing away of microbes, and disruption of cell membranes Soaps and detergent
Disinfectant
Antiseptic
Mouthwash
Bisbiguanides
Chlorhexidine
Alexidine
Disruption of cell membranes Oral rinse
Hand scrub for medical personnel
Alkylating Agents
Formaldehyde
Glutaraldehyde
o-Phthalaldehyde
Ethylene oxide
β-Propionolactone
Inactivation of enzymes and nucleic acid Disinfectant
Tissue specimen storage
Embalming
Sterilization of medical equipment
Vaccine component for sterility
Peroxygens
Hydrogen peroxide
Peracetic acid
Benzoyl peroxide
Carbamide peroxide
Ozone gas
Oxidation and destabilization of cellular macromolecules Antiseptic
Disinfectant
Acne medication
Toothpaste ingredient
Supercritical Gases
Carbon dioxide Penetrates cells, forms carbonic acid, lowers intracellular pH Food preservation
Disinfection of medical devices
Disinfection of transplant tissues
Chemical Food Preservatives
Sorbic acid
Benzoic acid
Propionic acid
Potassium sorbate
Sodium benzoate
Calcium propionate
Sulfur dioxide
Nitrites
Decrease pH and inhibit enzymatic function Preservation of food products
Natural Food Preservatives
Nisin
Natamycin
Inhibition of cell wall synthesis (Nisin) Preservation of dairy products, meats, and beverages
t-test Statistical Analysis
The t-test is a statistical analysis that enables scientists to objectively determine, based on their data and not their opinions, whether the results from one experimental treatment are statistically different from another experimental treatment. For example, in this experiment, we will be able to determine if the soap-cleaned surfaces produced a statistically significantly different number of microbial colonies on petri plates than the uncleaned surfaces. If the t-test indicates that there is not a statistical difference, regardless of what the means (i.e. averages) are an how different those means may appear, they are statistically considered not different from each other.
To determine if a pair of experimental treatments are statistically different from each other, consider the means (i.e. averages) and the t-test ranges:
• If the mean of one treatment falls into the t-test range of the other treatment, and the mean of the other treatment falls into the t-test range of the first treatment, these results indicate that these treatments are not statistically significantly different from each other.
• If the mean of one treatment falls outside of the t-test range of the other treatment, and the mean of the other treatment falls outside of the t-test range of the first treatment, these results indicate these treatments are statistically significantly different from each other.
Example 38.1
uncleaned surface soap cleaned surface
mean number of colonies on the petri plate 98 12
t-test range 54 - 142 0 - 24
Solution
To determine if the microbial load on the uncleaned surface is statistically significantly different than the soap cleaned surface as the following questions:
1. Does the mean of the uncleaned surface fall into the t-test range of the soap cleaned surface? Answer No. 98 does not fall between 0 and 24.
2. Does the mean of the soap cleaned surface fall into the t-test range of the uncleaned surface? Answer: No. 12 does not fall between 54 and 142.
3. Check with the bullet points above this example. According to those, since the means do not fit into the t-test ranges of the other experimental groups, they are statistically significantly different.
Conclusion Statement (Option 1)
The soap cleaned surface had statistically significantly lower microbial load than the uncleaned surface.
Conclusion Statement (Option 2)
The uncleaned surface had a statistically significantly higher microbial load than the soap cleaned surface.
Example 38.2
disinfectant cleaned surface soap cleaned surface
mean number of colonies on the petri plate 9 12
t-test range 2 - 16 0 - 24
Solution
To determine if the microbial load on the disinfectant cleaned surface is statistically significantly different than the soap cleaned surface as the following questions:
1. Does the mean of the disinfectant cleaned surface fall into the t-test range of the soap cleaned surface? Answer Yes. 9 falls between 0 and 24.
2. Does the mean of the soap cleaned surface fall into the t-test range of the disinfectant cleaned surface? Answer: Yes. 12 falls between 2 and 16.
3. Check with the bullet points above this example. According to those, since the means fall into the t-test ranges of the other experimental groups, they are not statistically significantly different.
Conclusion Statement (Option 1)
The soap cleaned surface did not have a statistically significantly different microbial load than the disinfectant cleaned surface.
Conclusion Statement (Option 2)
The disinfectant cleaned surface did not have a statistically significantly different microbial load than the soap cleaned surface.
Laboratory Instructions
Purpose
The goal of this experiment is to swab various surfaces for microbes and to compare the number of microorganisms that grow on a Petri plate from the same surface in three different conditions:
1. The surface as it is, untreated
2. The surface that has been cleaned with soap
3. The surface that has been cleaned with disinfectant
Each person will choose a surface. Work as a group so that the surfaces you choose will be diverse and different. Surfaces may be anything that it will be safe to use soap and disinfectant on. Examples include:
• Door handles
• Pens/pencils
• Cell phones (being mindful of not getting sensitive parts wet when cleaning)
• The floor
• A shoe
• A lab bench
• Lab equipment
• A sink in the lab or the bathroom
• Other parts of the bathroom
• A computer mouse (being mindful of not getting sensitive parts wet when cleaning)
• Keys
• Anything else you can think of
Instructions
1. Choose a surface you would like to examine for microbes.
2. Label three petri plates with:
1. Your name
2. Your group number
3. The sample being swabbed
4. Label one of each as:
1. uncleaned
2. soap cleaned
3. disinfectant cleaned
3. Dip a sterile cotton swab into dH2O and then swab the surface of interest and streak across the Petri plate labeled "uncleaned."
4. Clean a separate spot of that same surface with soap and wipe dry. Dip a sterile cotton swab into dH2O and then swab the surface you cleaned with soap and streak across the Petri plate labeled "soap cleaned."
5. Clean a separate spot of that surface with disinfectant and wipe dry. Dip a sterile cotton swab into dH2O and then swab the surface you cleaned with disinfectant and streak the Petri plate labeled "disinfectant cleaned."
6. Invert all plates and place in the class bin (these will be put in the incubator until next class).
Results & Questions
1. The surface I am examining in this experiment is…
2. I predict the following will have the most bacterial growth (circle one):
uncleaned soap cleaned disinfectant cleaned
1. I predict the following will have the least bacterial growth (circle one):
uncleaned soap cleaned disinfectant cleaned
1. Record your individual results (colony number) in the table below:
uncleaned
soap cleaned
disinfectant cleaned
1. Record class results (average colony number) in the table below:
uncleaned
soap cleaned
disinfectant cleaned
mean (i.e. average) colony number
t-test range
1. Discuss your results.
• Which treatment produced the most microbial growth?
• Which treatment produced the least microbial growth?
• See questions 2. and 3. above. Were your predictions for the control of bacterial growth correct? Explain your answer.
2. What type of control of microbes did we employ in this laboratory – physical or chemical?
3. Use your individual data to create a bar graph (you may use a graphing software or graph paper – handwritten graphs should be done neatly with a ruler). Your bar graph should have 3 bars, one for each Petri plate. The y-axis should be the number of colonies. Remember to label all axes and give your graph a title.
4. Use your class data to create a bar graph (you may use a graphing software or graph paper – handwritten graphs should be done neatly with a ruler). Your bar graph should have 3 bars, one for each Petri plate. The y-axis should be the average number of colonies. Remember to label all axes and give your graph a title. Standard error should be shown as error bars above and below the average colony numbers on each bar of the bar graph.
• How to analyze data using t-test:
5. Interpret results of the t-test analysis. Give a proper conclusion statement for these results indicating that you conducted a statistical analysis (i.e. use the phrase "statistically signigicant" or statistically significantly"). | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.39%3A_Control_of_Microbial_Growth.txt |
Learning Objectives
• Define "antibiotic" and tell where these chemicals come from in nature (for non-synthetic or non-semisynthetic).
• Differentiate between broad spectrum antibiotics and narrow spectrum antibiotics.
• Tell that use of broad spectrum antibiotics contributes to increased antibiotic resistance in bacteria.
• Tell the purpose of the Kirby-Bauer test.
• Explain how the Kirby-Bauer test works including how diffusion of the antibiotics is important in the creation zones of inhibition.
• Define "zone of inhibition."
• Successfully conduct a Kirby-Bauer test (modified) and interpret the results.
• Explain why the Kirby-Bauer test is important for reducing antibiotic resistance.
• Describe why antibiotic resistance is a threat for successful treatment of bacterial infections.
Introduction to Antibiotics
Antibiotics are chemicals produced by some bacteria and fungi that, in small quantities, can inhibit the growth of bacteria. Much of the success we have achieved in treating infections since World War II is due to the discovery of antibiotics. Utilizing the information gleaned from studying these natural chemicals, scientists have artificially synthesized other useful antimicrobial chemicals in the laboratory. Sometimes these antimicrobials are completely synthesized in the lab (synthetic) and sometimes they are partly produced in nature and partly synthesized in the lab (semisynthetic).
Many microbes can produce antibiotics, but four genera produce most of the antibiotics used for treating human and animal infections. Bacillus and Streptomyces are bacteria. Penicillium and Cephalosporium are fungi. It is a constant challenge to develop new antibiotics to replace those antibiotics for which microbes have developed resistance.
The range of bacteria killed by an antibiotic determines its “spectrum of activity”. Antibiotics that are only effective against Gram-positive or only effective against Gram-negative bacteria have a narrow spectrum of activity. Antibiotics that are effective against many different types of bacteria are called broad spectrum antibiotics. Broad spectrum antibiotics are probably contributing to the escalating drug resistance we are seeing in microorganisms. Broad spectrum antibiotics often wipe out a person’s normal microbiome as well as the pathogen they are intended to kill, resulting in superinfections from organisms such as Candida albicans and Clostridium difficile that grow out of control when they do not have to compete with microbes in the normal microbiomes.
Empiric therapy takes place when an antimicrobial agent is given to the patient without performing a culture or other diagnostic test to determine the specific cause of the disease. Empiric therapy is prescribed in instances where the causative pathogen is likely and where diagnostic tests will not change the treatment. The selection of which drug to use is based solely on experience, observation and relevant clinical information including current resistance patterns in suspected pathogens. These antibiotics are typically broad-spectrum, in that they treat a wide variety of possible microorganisms. Examples of this include antibiotics prescribed for strep throat, pneumonia, urinary tract infections, and suspected bacterial meningitis in newborns aged 0 to 6 months.
Physicians are beginning to target infections with narrow spectrum antibiotics, or synergistically treat infections with small doses of multiple antibiotics to try to prevent antibiotic resistance.
The laboratory can aid the physician in selecting which antimicrobial agent is likely to kill the pathogen that is causing an infection in a patient. There are several methods that are used by clinical microbiologists in this determination, including the Kirby-Bauer Test.
Kirby-Bauer Test
The results of the Kirby-Bauer Test provide an accurate prediction of which antibiotics are likely to be effective against the pathogen. Because the Kirby-Bauer test is relatively simple to perform and is inexpensive, it has been extensively used in medical practice. The results are reported as S (sensitive), I (intermediate), and R (resistant) to an antibiotic. A sensitive result indicates the bacteria will die when it is exposed to the antibiotic. An intermediate result indicates the antibiotic must be used in combination with another antibiotic to clear the infection. A resistant result indicates the antibiotic does not kill the bacteria.
Petri plates with nutrient agar are covered with the bacteria that is being tested for its antibiotic sensitivity. After spreading the bacteria over the entire surface of the petri plates, small disks containing different antibiotics are placed at a distance from each other (or alone) on the petri plate. Each antibiotic will diffuse outward from the antibiotic disk creating a concentration gradient of the antibiotic in a circle surrounding the disk. Closest to the antibiotic disk will be the highest concentration of antibiotic. Further from the antibiotic disk will be the lowest concentration of antibiotic. Based on how sensitive the bacterial species/strain is to each antibiotic, bacterial will begin to grow at one of the antibiotic concentrations. Bacteria that can grow at higher concentrations of an antibiotic (closer to the disk) will have more resistance to that antibiotic. Bacteria that can only grow at lower concentrations of an antibiotic (further from the disk) will be more susceptible to that antibiotic. As a result, the size of the zone of inhibition (the region of the petri plate surrounding the antibiotic disk that does not have bacterial growth) will determine if the bacterial species/strain is sensitive, intermediate, or resistance to that antibiotic.
The Kirby-Bauer test, known as agar disk diffusion, must be strictly regulated for the results to be interpreted correctly. Such characteristics as the stability of the antibiotic, the rate of diffusion of the antibiotic, the bacteria being tested, the pH of the culture medium, the depth of the culture medium, the inoculum density, the incubation time, the incubation temperature, and the concentration of the antibiotic can affect the results. Nevertheless, when the Kirby-Bauer test is performed under standardized conditions (on Mueller-Hinton agar inoculated with a pure culture of microbes that is the correct turbidity to match McFarland 0.5 standard, etc.) and the results are interpreted according to the Interpretative Zone Standards published by the National Committee for Clinical Laboratory Standards. In this laboratory, this is a simplified version of the Kirby-Bauer Test in lab that is not standardized, but will allow you to learn the general principles involved in this procedure.
Laboratory Instructions
Kirby-Bauer Test for Antibiotic Sensitivity (Modified for Simplicity)
1. Label 3 TSA plates with your group name and "Kirby-Bauer."
2. Dip a sterile cotton swab into a Staphylococcus aureus TSB culture and completely coat the surface of the TSA plate with S. aureus. Cotton swabs should go into an antimicrobial solution and be left there for at least 10 minutes before disposal.
3. Repeat step 2 for all the TSA plates.
4. Allow the plates to dry for 5 – 10 minutes.
5. Gently place one penicillin G disk (disk is labeled as P) in the center of one of the petri plates. DO NOT press the disk into the agar and do not move the disk once placed on the agar.
6. Repeat step 5. for an erythromycin disk (disk is labeled as E) on a different petri plate.
7. On the third petri plate, place a streptomycin disk (disk is labeled as S) centrally on one side of the petri plate and a tetracycline disk (labeled as T or TE) centrally on the other side of the same petri plate making sure they are spread out from each other.
8. DO NOT INVERT THE PLATES! Place the petri plates in the incubator.
9. After given time for growth (24-48 hours), measure the zones of inhibition in mm and record results in the results table.
10. Identify which of the size ranges that each zone of inhibition falls into in the interpretation table. This will determine if this bacterial strain is susceptible, intermediate, or resistant to each antibiotic.
Results & Questions
antibiotic disk abbreviation
antibiotic name
antibiotic disk concentration
strain is resistant for this size of zone of inhibition (mm)
strain is intermediate for this size of zone of inhibition (mm)
strain is susceptible for this size of zone of inhibition (mm)
E
erythromycin
15 µg
13 or less
14 – 22
23 or more
P
penicillin G
10 units
28 or less
29 or more
S
streptomycin
10 µg
6 or less
7 – 9
10 or more
T (TE)
tetracycline
30 µg
14 or less
15 – 18
19 or more
antibiotic disk
diameter of the zone of inhibition (mm)
this S. aureus strain is... (resistant, intermediate or susceptible)... to this antibiotic
E (erythromycin)
P (penicillin G)
S (streptomycin)
T (TE) (tetracycline)
1. Complete the table above using measurements of the diameter of the zones of inhibition and the interpretation table above to determine if S. aureus is resistant, intermediate, or susceptible to each antibiotic.
2. If you had a patient with an infection of this strain of S. aureus, which antibiotic(s) might be a good choice for treatment? Explain your answer.
3. Define "zone of inhibition."
4. Explain how the size of the zone of inhibition relates to the concentration of the antibiotic.
5. Explain how the size of the zone of inhibition relates to the susceptibility or resistance to an antibiotic.
6. What is "antibiotic resistance?"
7. Why is using this Kirby-Bauer approach helpful to prevent bacterial strains from becoming resistant to antibiotics?
8. True or False. It is easy for scientists to develop new antibiotics.
9. What are the treatment options for a bacterial infection where the bacterial species is resistant to all types of antibiotics?
10. Why is it important to take steps to prevent bacterial strains from becoming antibiotic resistant? | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.40%3A_Bacterial_Susceptibility_to_Antibiotics_%28Kirby-Bauer_Test%29.txt |
Learning Objectives
• Define the terms microbial flora, microbiota, and microbiome.
• List locations in the human body where it is normal and healthy to have microbes growing.
• Explain how microorganisms keep humans healthy and give at least three ways they are beneficial to our health.
• Give at least three ways humans are not cultivating as healthy a microbiome as we could.
• Tell that the microbial communities are distinct in different areas of the human body.
• Tell that the microbial communities are distinct in different individuals.
• Grow microbes from your own skin microbiome.
• Characterize microbes grown from your own skin microbiome and tell the likely species they are.
Introduction to the Human Microbiome
The importance of the normal microbial flora (a.k.a. microbiota or microbiome) of the human body has been an area of increasing interest in both research and the popular media. One frequently cited statistic is that there are 10-100 times more bacterial than human cells in the body. More recent calculations, however, result in a ratio closer to 1:1, with an estimated 1013 human cells and 1013 – 1015 bacterial cells. The cellular contribution of microbes to the human body, however, is small compared to the genetic contribution. The human genome contains approximately 20,000 genes, but there are 3.3 million unique bacterial genes in the gut microbiota alone. That is a 150-fold difference between the human and bacterial genetic contribution. No matter the exact proportion of bacteria in the human body, the impact of the microbiota on our physiology is substantial.
It has been known for decades that animals raised without normal flora display a variety of health effects across many body systems. Not surprisingly, neither the digestive system nor the immune system develops properly. The cecum (part of the large intestine/colon) tends to be enlarged and other gastrointestinal (GI) abnormalities appear. The immune system is underdeveloped. More recently it has been shown that the central nervous system, including the brain, does not develop properly in these animals. Because bacteria produce vitamins necessary for animal nutrition (most notably vitamin K), animals without normal flora suffer from vitamin deficiencies. Lack of normal flora also makes animals more susceptible to infection with a variety of pathogens, particularly those that infect the GI tract. Although lack of normal flora generally has negative effects, it does also result in an absence of dental caries (cavities) and lower body fat.
Normal flora is found in all areas of the human body exposed to the environment (one exception is the lungs), but internal organs and body fluids are considered sterile in a healthy individual. This is generally true, although bacteria are sometimes found in these “sterile” tissues even in healthy people. For instance, a person’s blood will become bacteremic (contain bacteria) for up to three hours after brushing their teeth.
Each area of the human body contains a characteristic population of microbes, although the exact composition of each person’s flora is unique. The diversity of the bacteria populating the human gut alone is enormous, with an estimated 40,000 species. An increasing number of studies associates such shifts in the gut microbiota with outcomes such as susceptibility to infection, immune disorders, metabolic changes, and altered mood and behavior. Each of these physiological effects can be linked directly to chemical communication within the microbiota and between the microbiota and human.
Flora of the Skin
The exact microbial population on the skin depends on the specific body area. Moist areas, such as axilla (armpits) and groin, tend to have more (and different) bacterial growth compared to drier areas. The most common bacteria of the skin flora are the Gram-positive, catalase positive cocci of the genera Staphylococcus and Micrococcus. Although Staphylococcus aureus can occasionally be found on the skin, it is more commonly found in the nose in those people that carry it in their normal flora.
Flora of the Mouth and Upper Respiratory Tract
The flora of the mouth and upper respiratory tract is typically associated with a more diverse set of microbes. Streptococci, specifically, alpha-hemolytic Streptococci often referred to collectively as the “viridans Streptococci”are very prominent in the mouth. These include S. mutans, S. sanguis, and S. mitis. S. mutans in particular plays a critical role in the formation of plaque and dental caries (cavities). Although both Staphylococci and Streptococci are Gram-positive cocci, unlike the Staphylococci the Streptococci are catalase-negative, consistent with the low-oxygen environment of the mouth.
Other inhabitants of the mouth and upper respiratory tract include bacteria in the genera Neisseria and Haemophilus. As mentioned above, Staphylococcus aureus is most often found in the nose of those individuals who carry it in their normal flora. The fungal genus Candida is also common in the mouth and upper respiratory tract.
Flora of the Intestines
The most studied population of normal flora in the microbes living in the intestines. This population of microbes is commonly referred to as the gut microbiota or gut microbiome. Although the bacterial species most commonly associated with the intestines is Escherichia coli, it is actually not the most numerous in the intestine. Bacteria in the phylum Bacteroidetes form a large proportion of bacteria in the gut. The Gram-positive Firmicutes (such as Lactobacillus and Clostridium) and Actinobacteria (including Bifidobacterium) can be equally numerous. In healthy individuals, Proteobacteria (including E. coli and other Enterobacteriaceae) are the least abundant of the major bacterial groups in the intestines. There are many other groups of microbes found in the intestines, including fungi such as Candida. It is shifts in the proportions of these groups of microbes that are typically studied when investigating the role of normal flora on human health.
Laboratory Instructions
In this experiment, a few locations of the skin will be swabbed in order to examine microbes present in different locations.
Prepare Petri Plates and Swab Skin
1. Prepare 2 TSA plates per person.
2. Label the 2 TSA plates with your name.
3. Choose the 2 skin locations you would like to swab and label one petri plate with each location you chose.
4. Moisten a sterile cotton swab with DI or distilled water.
5. Swab the first skin location you chose and then do a zig-zag streak on the TSA plate you labeled with that skin location.
6. Repeat steps 4 and 5 for the other skin location you chose.
7. Invert the petri plates and incubate for 24 – 48 hours.
Examine Results
1. Examine the TSA plates for colony morphologies. Choose one isolated colony from each petri plate. Choose colonies that have different morphologies. Describe the colony morphologies in the results & questions section below.
2. Create two bacterial smears from the two isolated colonies you chose from each plate. You can use a single slide with one smear on each side. Label the slide so you do not get the two smears mixed up.
3. Heat fix the two bacterial smears. Fix each individually (heat-fix one smear, let the slide cool off, then heat-fix the other smear).
4. Conduct a Gram stain on the two bacterial smears (can be done at the same time.
5. Examine the Gram-stained cells with a microscope.
6. Record the cell shape, cell arrangement, and Gram for each of the colonies you chose.
7. Use the table below to "identify" each species found on your skin. Write the species you found for each skin location.
Skin Organism
Gram
Microscopic Morphology
Microscopic Arrangement
Colony Appearance on TSA
Staphylococcus epidermidis*
Positive
Cocci
Clusters
Large, round, white, glistening
Staphylococcus aureus
Positive
Cocci
Clusters
Large, round glistening, typically yellow
Corynebacterium spp.*
Positive
Pleomorphic rods
Club-shaped, bipolar
Dry and wrinkled
Yeasts
Positive
Large budding cells
Single
Large, round, moist
Bacillus spp.*
Positive
Bacilli
Single, chains
Round or irregular, surface dull becoming thick and opaque
Streptococcus viridans*
Positive
Cocci
Chains
Small, dome-shaped colony surrounded by an area of discoloration.
Micrococcus luteus
Positive
Cocci
Single, tetrads
Large, round, yellow, glistening colony
* These are the most commonly isolated microorganisms from healthy skin.
Results & Questions
Skin location #1: ______________________
Results from colony on skin location #1
Skin location #2: ______________________
Results from colony on skin location #2
colony color
colony appearance (shiny, dull, dry, moist, glistening, etc.)
colony form
Gram
cell shape
cell arrangement
likely species (based on the table above)
1. Complete the table above to summarize results of the two isolated colonies you chose.
2. Examining the results from the skin swabs, answer the following:
• How many distinct types of microbes can you identify on the petri plates (each distinct type would have a different colony morphology)?
• Which of the skin locations you swabbed had the most microbes present?
3. True or false. The microbes growing on your skin, under normal circumstances, are dangerous to your health.
4. Explain why microbes growing on your skin may actually be beneficial to your health.
5. List at least 3 ways microbes in your "gut" are important for keeping you healthy.
6. What happens to your microbiome when you take antibiotics?
7. What circumstances prevent humans from getting enough exposure to microbes to build up a healthy microbiome? Give at least three.
8. True or false. All microbial communities living on the skin have the same composition.
9. True or false. All humans have the same species composition in their gut microbiome. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.41%3A_Human_Microbiome.txt |
Learning Objectives
• Apply microbiological tools to isolate and identify bacterial species of unknown identities.
• Carefully document results of microbiological tests.
• Effectively collaborate with a classmate.
• Successfully identify the unknown bacterial species.
Introduction
In this project you will experience the type of process that microbiologists have traditionally used to identify a bacterial species. This will involve:
1. Isolating bacteria (one species per culture - must begin with an isolated colony to insure that there is only one species) - if bacteria are not isolated, you cannot rely on the results of any of the other tests you do.
2. Conducting biochemical tests to narrow down the possible species of the unknown bacteria
3. Collaborating with a partner and contributing equally to the work
4. Writing a scientifically-written report detailing the project, its experiments, and its results
Important
In scientific research and throughout the professional world, collaboration occurs often and is an essential skill. Employers want you to be able to collaborate effectively with others. You are building your job and career skills here!
Important
In scientific research and throughout the professional world, being able to clearly communicate through writing is an essential skill. Empolyers want you to be able to communicate effectively with others. You are building your job and career skills here!
Activity Log
For this project you will work in a pair. You are BOTH responsible for contributing equally to identifying the unknown bacteria.
Keep the following activity log to show your contributions to identifying your unknown bacteria. This insures that this group project is fair and all members of the group are contributing equally to the project. You will submit your activity log in your report. If your contributions to the project don't demonstrate equal contributions, deductions will be made to the report.
Date
Task
Student(s) In the Group Working On This Task
Instructor Signature
Instructions for Identifying Unknown Bacteria
1. Obtain a culture of unknown bacteria from your instructor. Your unknown culture will have a letter or a number. What letter or number is your unknown bacterial culture?
2. Your culture of unknown bacteria will have two different species in it. Follow the steps below carefully to identify these bacterial species.
Step 1: Isolate the Two Bacterial Species & Do Initial Gram Stain
1. Conduct eight (8) streak plates on TSA in order to obtain isolated colonies (each partner will conduct four). By doing eight streak plates, this increases the chances that you and your partner will obtain isolated colonies of both your bacterial species.
2. Make sure your names, unknown number/letter, and the date are on the Petri plates, invert the Petri plates, tape together, and put into the bin to be incubated.
3. Do a Gram stain to determine:
• Whether the mixture of bacterial species contains a Gram positive and a Gram negative species, both Gram positive, or both Gram negative.
• The cell shape of each bacterial species (i.e. coccus, bacillus, vibrio, spirillum, or spirochete).
• The cell arrangement of each bacteria species (i.e. single, pairs, chains (strepto-), or bunches (staphylo-).
4. Record results in the table below. Refer back to this table in Step 2 to make sure that the bacteria you isolate match the bacteria you saw in this Gram stain.
Unknown Bacterial Species #1
Unknown Bacterial Species #2
Gram
cell shape
cell arrangement
Note
Taking photos of results at every stage of the project will make a stand-out report! Include these photos in your report to provide visuals and evidence of your results. Make sure each photo is accompanied by a caption telling what the photo shows. If the photo is of a microscopic sample, make sure to indicate the magnification used when the photo was taken.
Step 2: Characterize Bacterial Colonies, Create Stock Cultures of Isolated Bacteria, & Do Gram Stain on Isolates
Note
Taking photos of results at every stage of the project will make a stand-out report! Include these photos in your report to provide visuals and evidence of your results. Make sure each photo is accompanied by a caption telling what the photo shows. If the photo is of a microscopic sample, make sure to indicate the magnification used when the photo was taken.
1. Carefully examine streak plates. Identify two bacterial colonies that have differences in appearance.
2. For the two different bacterial colonies you find, describe differences in the colony forms in the tables below. Do the best you can keeping your Petri plate closed to prevent contamination. It is essential that these bacterial colonies do not become contaminated with other bacteria or microbes floating in the air.
3. Label two TSA slants. Give each Unknown bacterial species a name (you can use names "A" and "B" or "1" and "2" or "Yessica" and "Yoli" - choose any names you and your partner would like - you will just need to keep straight which colony is which).
4. Transfer one third (1/3) of one of the isolated colonies to one of the TSA slants.
5. Transfer one third (1/3) of the other isolated colony to one of the TSA slants.
6. Use one third (1/3) of each of the colonies to make separate bacterial smears on a slide and conduct a Gram stain.
7.
8. Compare results from the Gram stains. Did you obtain the same types of bacteria and separate them successfully? If you see Gram positive and Gram negative cells together, they are not isolated. If you see bacilli and cocci together, they are not isolated.
1. If one or both bacterial species are not isolated (they are mixed with another species), re-examine the petri plates to see if there is another colony that appears different and create a new TSA slant, and create a new bacterial smear on a slide. Do the Gram stain on this if there is still time. If there is not time, save your bacterial smear for next class to see if the bacteria are now isolated.
2. If you do not have better isolated colonies or continue to find that you have a mixture, conduct more streak plates to isolate bacterial colonies again.
9. When you have successfully isolated your bacteria, record results from the Gram stain in the tables below.
Step 3: Follow the Flow Chart to Identify Bacterial Species
Note
Taking photos of results at every stage of the project will make a stand-out report! Include these photos in your report to provide visuals and evidence of your results. Make sure each photo is accompanied by a caption telling what the photo shows. If the photo is of a microscopic sample, make sure to indicate the magnification used when the photo was taken.
Unknown Bacterial Species ___________________
(put name you chose in the blank space above)
Gram
cell shape
cell arrangement
colony form
colony elevation
colony margin
colony color
colony diameter (mm)
test 1:
test 2:
test 3:
bacterial species
Unknown Bacterial Species ___________________
(put name you chose in the blank space above)
Gram
cell shape
cell arrangement
colony form
colony elevation
colony margin
colony color
colony diameter (mm)
test 1:
test 2:
test 3:
bacterial species
1. After your bacteria have been isolated and you have good results from your Gram stain, begin to follow the flow chart below.
• For example, if your Bacteria "A" is Gram-positive, use your TSA stock slant to inoculate a starch plate to do a starch hydrolysis test.
• For example, if your Bacteria "B" is Gram-negative, use your TSA stock slant to to inoculate a SIM deep culture to test for H2S production.
• Use the results (positive or negative) from each test to determine the next test you should do on the flow chart.
• Return to the appropriate chapters in this lab manual to assist with conducting each test: | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.42%3A_Unknown_Bacteria_Identification_Project.txt |
Learning Objectives
• Write an organized and well formatted scientific report detailing the Unknown Identification Project.
• Understand expectations of the Unknown Identification Project Report and grading guidelines.
Instructions for Writing Unknown Bacterial Species Identification Project Report
Originality
TurnItIn
The words in your report must be 100% your own. To make sure that the report is 100% your own, you will submit this report using TurnItIn. TurnItIn compares your report with websites, books, publications, and the work of other students who have submitted their documents to TurnItIn. For your report to be acceptable, the similarity rating between your work and other works will need to be below 20%.
Citing Sources
You may use outside resources to provide information that will help you to write the report (e.g. information about Gram stains, oxidase test, starch hydrolysis test, etc.), but you must cite the sources of information you used. The text must also be fully in your own words. Do not just change a couple words around. TurnItIn is smarter than this and will flag the text.
Example 42.1
Example of scientific writing using information from an external source:
The initial Gram stain was unsuccessful the first time since all of the cells appeared purple, indicating that the decolorization step was too short (Pakpour and Horgan, 2021). As a result, the initial Gram stain was repeated using a decolorization step that was 10 seconds longer than in the previous attempt. This produced a Gram stain where Gram negative rods and Gram positive cocci were apparent.
More Information about this Example
In this example, the authors' last names are Pakpour and Horgan and the work is from 2021. A full reference would then be provided at the end of the report in a "Works Cited" section.
Works Cited Section
A works cited section is found at the end of a scientific report. It lists the full references for all external resources cited throughout the entire report. There are many different ways to format a Works Cited section. What matters most is:
• you use the same professional formatting for your references (i.e. consistency)
• references are complete (with title, year, author name(s), publisher/journal/book name, URL (if applicable), page numbers/name (if applicable), etc.)
• references are listed in alphabetical order
Example 42.2
Works Cited
Hartline, 2022. Starch Hydrolysis. Microbiology Laboratory Manual. LibreText. Accessed on 10/30/2022 from:
Pakpour and Horgan, 2021. Lab 3: Simple, Negative, and Gram Stain. General Microbiology Lab Manual (Pakpour & Horgan). LibreText. Accessed on
Quoting Sources
Quoting another author in the sciences is rare. As a result, in this report, quoting another author is not allowed. Take information from texts and put it into your own words and cite the source. Don't just change one or two words (Turn It In will flag this). You need to write your own sentence using the ideas you read. Or even better, synthesize your own ideas and results together with ideas from another source and then cite the source.
This type of synthesis is shown in the example above discussing the Gram stain. Events and results from the laboratory were synthesized together into the same sentence with the idea in the cited source that the decolorization step was too short since all the cells appeared purple.
Working with Your Partner
Since you and your partner worked together and will have done the same steps and will have the same results, it may be tempting to share parts of your reports with each other. TurnItIn will recognize the similarities between your reports and flag it as plagiarism.
You must write your own report without sharing text with your partner. However, you are encouraged to get your partner to proofread your report and give feedback and comments to help improve the report before you submit it. The images in your reports may be the same since you will share the same results and will want to choose the best photos for the report.
Organization of the Report
The report should be broken into the following sections. Each section should have a bold header with larger font that the text in each section. Do you see how this document is separated into sections with bold headers? This is how your report should look too, except name each section as follows:
• Introduction
• Methods
• Results & Discussion
• Conclusions
• Works Cited
• Activity Log
Example 42.3
Introduction
Introduction text / subsections here.
Methods
Methods subsections and text here.
Results & Discussion
Results and discussion subsections and text here.
Conclusions
Conclusion text here.
Works Cited
Full references here in alphabetical order.
Activity Log
Photos of activity log pages here.
Introduction
An introduction gives important background information to the reader. Don't write this report assuming that your instructor knows all of this information already and therefore you shouldn't include it. Instead, write the report as if you were writing it for a biology student who hasn't taken microbiology yet. Therefore, you will need to:
• Address the reasons why identifying bacterial species is important.
• Describe how (generally speaking) identifying bacterial species is done.
• Describe each of the microbiological tests and microbiological approaches that you used in this project including:
• how each works
• what each test tells about the bacteria
• how you determine the results of a test
Example 42.4
For example, if you identified your bacteria as Bacillus subtilis and Escherichia coli, you will have different paragraphs or sub-sections in the Introduction on the following topics:
• Streak plate
• Gram stain
• Starch hydrolysis
• H2S production
• Oxidase test
• Lactose fermentation
In the paragraph introducing the Gram stain (for example), you will need to discuss:
• How the crystal violet stain will remain inside a thick layer of peptidoglycan
• How the crystal violet stain will be decolorized from a thin layer of peptidoglycan
• How cells with the thin peptidoglycan layer are stained with a different color
• The structure of the cell walls of Gram-positive bacteria
• The structure of the cell walls of Gram-negative bacteria
• The color Gram positive bacteria appear in the microscope after the Gram stain
• The color Gram negative bacteria appear in the microscope after the Gram stain
Important
End the introduction with a paragraph that describes the basic premise and approach of this project and how this approach either resulted in the successful identification of both unknown bacteria, one identification was successful and the other unsuccessful, or that both were not correctly identified. Be sure to name the species you had and what species you identified them as.
It may seem like this is spoiling the ending of the paper - and it is! This is common among scientific publications to end the Introduction with a paragraph that tells the reader what they can expect to read about in the rest of it.
Methods
Methods will tell the approaches you used to conduct each test. The Methods section does not discuss the concepts of each test, the meaning of each test, or the results of the tests. The Methods section describes what you did with your bacteria in the laboratory for each test and how you did the tests. The Methods is written in paragraph form (do no bulletpoint or number steps - do not copy from laboratory protocals). Separate the Methods section into subsections.
Example 42.5
Continuing the example from above where you identified your bacteria as Bacillus subtilis and Escherichia coli...
Methods
Streak Plate
Text here about what you did to make your streak plates (e.g. using aseptic technique, using a Bunsen burner and loop, where bacteria were collected from [e.g. original Unknown culture, stock TSA slant, etc.], how the petri plate was streaked in quadrants while flaming loop in between, plate inverted and incubated at 37°C for 48 hours).
Gram Stain
Text here about what you did to make bacterial smears and conduct Gram stains.
Starch Hydrolysis Test
Text here about what you did to do the starch hydrolysis test.
H2S Production Test
Text here about what you did to do the H2S production test.
Oxidase Test
Text here about what you did to do the oxidase test.
Lactose Fermentation Test
Text here about what you did to do the lactose fermentation test.
Since you are only describing how you did a test (the steps you took in the laboratory to do the tests), each subsection in the Methds may be relatively short. The Methods section should not include any results and is not the place to discuss the theory of each test. It should simply state how each test/component was done.
Methods
H2S Production
To test whether Unknown Bacteria A produces H2S, Unknown Bacteria A was aseptically transferred from the stock TSA slant into a SIM deep using an inoculation needle. This culture was incubated at 37 °C for 48 hours before it was examined to determine test results.
Results & Discussion
Separate the Results & Discussion section into subsections.
Example 42.7
Continuing the example from above where you identified your bacteria as Bacillus subtilis and Escherichia coli...
Results & Discussion
Initial Gram Stain of Mixed Culture
Text & figures here about streak plate results and discussion
Quadrant Streak Plate
Text & figures here about streak plate results and discussion
Gram Stain
Text & figures here about Gram stain results and discussion
Starch Hydrolysis Test
Text & figures here about starch hydrolysis results and discussion
H2S Production Test
Text & figures here about H2S production test results and discussion
Oxidase Test
Text & figures here about oxidase test results and discussion
Lactose Fermentation Test
Text & figures here about lactose fermentation test results and discussion
Important
Each Results & Discussion subsection should contain both results and discussion:
• Results: simply tells the observation(s) from each test.
• Discussion: interprets observations and the meanings of the results of each test.
Example 42.8
Result for Gram stain Section: "The initial Gram stain showed a mixture of purple-colored cocci arranged in chains and pink-colored bacilli."
Discussion for Gram stain Section: "These results indicate that Unknown Culture #4 contains Gram-positive cocci, possibly Streptococci, and Gram-negative rods. The bacteria staining Gram-positive have a thick layer of peptidoglycan in their cell walls and lack an outer membrane. The Gram-negative bacterial species have cell walls with a thin layer of peptidoglycan and an outer membrane."
The Results and the Discussion should be synthesized together in paragraph form in each section.
Example 42.9
Example of a Gram Stain subsection of the Results & Discussion:
"The initial Gram stain showed a mixture of purple-colored cocci arranged in chains and pink colored rods. This indicates that Unknown Culture #4 contains Gram-positive cocci, likely Streptococci, and Gram-negative rods. The bacteria staining Gram-positive have a thick layer of peptidoglycan in their cell walls and lack an outer membrane. The Gram-negative bacterial species have cell walls with a thin layer of peptidoglycan and an outer membrane.
"After these bacteria were isolated using the quadrant streak-plate technique, the subsequent Gram stains showed one isolate appeared as purple cocci and the other isolate were pink bacilli. There was no evidence of pink bacilli with the purple cocci or purple cocci with the pink rods. This indicates that the two species that were examined in the initial Gram stain were successfully isolated and separated into their own cultures. The purple-staining cocci are a Gram positive bacterial species and will hereafter be referred to as "Unknown Bacteria A." The pink-staining rods are a Gram negative bacterial species and designated as "Unknown Bacteria B" in the remainder of this report."
In the example text above, the description would be further supported with images of the initial Gram stain, the Gram stain of Unknown Bacteria A, and the Gram stain of Unknown Bacteria B. These images will require captions to indicate what the image is. Images can be placed as separate figures, each with their own caption, or arranged together as a single figure with a single caption.
Example 42.10
This is an example of a single photo showing results with its caption:
Figure 1: Results from initial Gram stain of the original Unknown Bacterial Culture showing both species mixed together in this culture.
Example 42.11
This is an example of multiple photos that have been put together as a single figure with one caption:
Conclusions
In the conclusions section of the report, including the following:
• Name the two bacterial species that you identified the species as from the laboratory tests you conducted.
• Tell if the bacterial species identified were in fact the species that you had in the unknown culture. If they were not what was identified, name the correct species.
• If there was a misidentification, tell what species you did have and reflect on and discuss sources of error or events during the project that might have led to misidentification.
• If you successfully identified the bacterial species, reflect on and discuss what likely contributed to your successful identification of the bacterial species.
Activity Log
Take clear photos of the activity log or scan these pages and include them in this section of the report.
Writing Style
Write in Third Person
Write in the third person. This means that you act as a narrator as if you were outside of the experiment and not involved in the experiment.
• First person (don't write this way): I examined my Gram stain and saw that the cells were pink.
• First person (don't write this way): We examined our Gram stain and saw that the cells were pink.
• Second person (don't write this way): When you examined the Gram stain, you saw that the cells were pink.
• Third person (write this way): Upon examining the Gram stain, pink cells were observed.
Write in Past Tense
Write in the past tense.
• Future tense (don't write this way): A Gram stain will be done.
• Present tense (don't write this way): A Gram stain is being done.
• Past tense (write this way): A Gram stain was done.
Proofread
Use Grammar and Spelling Checkers
Check all of your text for proper grammar and spelling. Make adjustments were necessary.
Write Early & Read Multiple Times Before Submitting
Complete your report at least a few days before submitting it. Read it a couple times after writing it (including on separate days). Make edits on each reading.
Have a Classmate Proofread and Give Feedback
Complete your report early, print out a copy or email it to a classmate and ask them to read it over and make notes to help you to improve your report. Make edits that you deem will improve your report as suggested by your classmate.
Instructions for Submitting the Report
Upload a file with your Unknown Identification Project Report in the PROJECT: Unknown Identification Project Report assignment in Canvas. This upload will use TurnItIn.
TurnItIn will:
• Electronically analyze and review the text in your document to insure your work is original.
• Compare your work to its student paper repository.
• Compare your work to current and archived web site content.
• Compare your work to periodicals, journals, and publications.
• Create an originality report of your work.
• Both you and your instructor will be able to see the originality report.
See how to upload your work to this assignment in this video guide:
Grading
Low Participation Activity Log Deductions
• Reports with activity logs that show 20% less work than your partner will receive a 10% deduction.
• Reports with activity logs that show 40% less work than your partner will receive a 20% deduction.
• Reports with activity logs that show 50%-70% less work than your partner will receive a 50% deduction.
• Reports with activity logs that show 75%-100% less work than your partner will receive an 80% deduction.
Grading Rubric
Five Stars (100%)
Four Stars (85%)
Three Stars (70%)
Two Stars (50%)
One Star (20%)
Zero Stars (0%)
Introduction
(40%)
The Introduction section thoroughly and accurately addresses all expected components for the introduction. Excellent writing quality and style. Citations are included as appropriate.
The Introduction section addresses most of the expected components and most or all of the components are accurate. Writing style demonstrates quality and thoughtful development. Citations are included as appropriate.
The Introduction section addresses some of the expected components and most/some of the components are accurate. Writing and writing style may be good or may requires some improvement. Citations may or may not be included.
The Introduction section addresses a few of the expected components and some of the components are accurate. Writing and writing style requires additional development. Citations may or may not be included.
The Introduction section only slightly addresses the expected components. Writing and writing style requires additional development. Citations may or may not be included.
The Introduction section is absent or addresses almost none of the expected components. Writing and writing style requires a lot more development. Citations may or may not be included.
Methods
(10%)
The Methods section thoroughly and accurately addresses all expected components without including text better suited to the introduction or results & discussion sections. Excellent writing quality and style.
The Methods section addresses most of the expected components and most or all of the components are accurate without including text better suited to the introduction or results & discussion sections. Writing style demonstrates quality and thoughtful development.
The Methods section addresses some of the expected components and most/some of the components are accurate and may include text that would be better suited to the introduction or results & discussion sections. Writing and writing style requires additional development.
The Methods section addresses a few of the expected components and some of the components are accurate and may include text that would be better suited to the introduction or results & discussion sections. Writing and writing style requires additional development.
The Methods section addresses a few of the expected components and some of the components are accurate and may include text that would be better suited to the introduction or results & discussion sections. Writing and writing style requires additional development.
The Methods section is absent or addresses almost none of the expected components and may include text that would be better suited to the introduction or results & discussion sections. Writing and writing style requires a lot more development.
Results and Discussion
(40%)
The Results and Discussion section thoroughly and accurately addresses all expected components. Quality figures are provided to support results and have captions. Excellent writing quality and style. Citations are included as appropriate.
The Results and Discussion section addresses most of the expected components and most or all of the components are accurate. Figures are provided to support results and may or may not have captions. Writing style demonstrates quality and thoughtful development. Citations are included as appropriate.
The Results and Discussion section addresses some of the expected components and most/some of the components are accurate. Figures are provided to support results and may or may not have captions. Writing and writing style requires additional development. Citations may or may not be included.
The Results and Discussion section addresses a few of the expected components and some of the components are accurate. Figures may or may not be provided to support results and may or may not have captions. Writing and writing style requires additional development.
The Results and Discussion section does not address most of the expected components. Figures may or may not be provided to support results and may or may not have captions. Writing and writing style requires additional development.
The Results and Discussion section is absent or addresses almost none of the expected components. Writing and writing style requires a lot more development. Citations may or may not be included.
Conclusions
(5%)
Report conclusions are reflective, clearly states what species were identified through the project and whether or not these identifications were accurate, offers multiple thorough and thoughtful plausible explanations for each successful and unsuccessful identifications.
Writing is excellent.
Report conclusions are reflective, clearly states what species were identified through the project and whether or not these identifications were accurate, and provides at least one thoughtful and plausible explanation for each successful and unsuccessful identifications.
Writing style is good to excellent.
Report conclusions are reflective, clearly states what species were identified through the project and whether or not these identifications were accurate, and provides one explanation for each successful and unsuccessful identifications.
Writing style may be good or may require additional development.
Report conclusions states what species the unknown species actually were, and may provide one explanation successful and unsuccessful identifications.
Writing style requires additional development.
Report conclusions states what species the unknown species actually were, and does not provide any explanations for successful and unsuccessful identifications.
Writing style requires lots of additional development.
Conclusions section is absent or fails to address appropriate content for this section.
Works Cited
(5%)
The Works Cited section has consistent formatting for all references. References are provided for each citation in the text. Information included is thorough. References are listed in alphabetical order.
The Works Cited section has consistent formatting for all references. References are provided for each citation in the text. Information included is mostly thorough, but some important information is missing. References are listed in alphabetical order.
The Works Cited section has mostly consistent formatting for all references. References are provided for most of the citations in the text. Information included is mostly thorough, but some important information is missing. References order may or may not be alphabetical.
The Works Cited section has inconsistent formatting. References are provided for most/some of the citations in the text. Information included is missing components. References order may or may not be alphabetical.
The Works Cited section lacks formatting. References are missing or most of the information required for full references are missing. References order may or may not be alphabetical.
Works Cited section is either missing or is just a list of titles or websites used without any attempt at formatting or organization.
Sample Unknown Identification Report
To help you better grasp how all of these guidelines look in a cohesive report, a sample report has been developed for your reference. This report is to help you better understand:
• The overall formatting for the report
• The writing style for this type of report
• The type of content that is appropriate for each section of the report
• How citations may appear throughout the text
• Appropriate formatting for figures and captions
***This sample report is NOT provided for you to copy even a single sentence. Even if you change some of the words in a sentence you copy, this is still plagiarism. The ideas can be the same in the reports, but the way they are worded MUST be completely your own way of writing.*** | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/01%3A_Labs/1.43%3A_Unknown_Bacteria_Identification_Project_Report.txt |
Answer
6 g ("six grams")
How this Answer is Calculated
1. Calculation setup: 400 mL x [15 g /1000 mL]
2. Calculate parentheses first: 400 mL x 0.015 g/mL
3. Multiply (mL units cancel out): 6 g
2.02: Exercise 2.2
13 g ("thirteen grams")
1. Calculation setup: 650 mL x [20 g /1000 mL]
2. Calculate parentheses first: 650 mL x 0.02 g/mL
3. Multiply (mL units cancel out): 13 g
2.03: Exercise 2.3
6 g ("six grams")
1. Calculation setup: 200 mL x [30 g /1000 mL]
2. Calculate parentheses first: 200 mL x 0.03 g/mL
3. Multiply (mL units cancel out): 6 g
2.04: Exercise 2.4
13.5 g ("thirteen point five grams")
1. Calculation setup: 300 mL x [45 g /1000 mL]
2. Calculate parentheses first: 300 mL x 0.045 g/mL
3. Multiply (mL units cancel out): 13.5 g
2.05: Exercise 2.5
5.25 g ("five point two five grams")
1. Calculation setup: 150 mL x [35 g /1000 mL]
2. Calculate parentheses first: 150 mL x 0.035 g/mL
3. Multiply (mL units cancel out): 5.25 g
2.06: Exercise 4.1
Answer
0.0025 m ("zero point zero zero two five meters")
How this Answer is Calculated
1. Calculation setup: 2,500 μm x 10-6
2. Calculate the exponent first: 2,500 μm x 0.000001
3. Multiply: 0.0025 m
2.07: Exercise 4.2
Answer
0.000012 m ("zero point zero zero zero zero one two meters")
How this Answer is Calculated
1. Calculation setup: 12,000 nm x 10-9
2. Calculate the exponent first: 12,000 nm x 0.000000001
3. Multiply: 0.000012 m
2.08: Exercise 4.3
Answer
0.035 m ("zero point zero three five meters")
How this Answer is Calculated
1. Calculation setup: 35 mm x 10-3
2. Calculate the exponent first: 35 mm x 0.001
3. Multiply: 0.035 m
2.09: Exercise 4.4
Answer
4,000 μm ("four thousand micrometers")
How this Answer is Calculated
1. Calculation setup: 0.004 m ÷ 10-6
2. Calculate the exponent first: 0.004 m ÷ 0.000001
3. Divide: 4,000 μm
2.10: Exercise 4.5
Answer
300 mm ("three hundred millimeters")
How this Answer is Calculated
1. Calculation setup: 0.3 m ÷ 10-3
2. Calculate the exponent first: 0.3 m ÷ 0.001
3. Divide: 300 mm
2.11: Exercise 4.6
Answer
850 nm ("eight hundred and fifty nanometers")
How this Answer is Calculated
1. Calculation setup: 0.00000085 m ÷ 10-9
2. Calculate the exponent first: 0.00000085 m ÷ 0.000000001
3. Divide: 850 nm
2.12: Exercise 5.1
40X
Calculation
1. Formula: total magnification = (objective lens magnification) x (ocular lens magnification)
2. Plug numbers into the formula: total magnification = (4X) x (10X)
3. Multiply: 40X
2.13: Exercise 5.2
100X
Calculation
1. Formula: total magnification = (objective lens magnification) x (ocular lens magnification)
2. Plug numbers into the formula: total magnification = (10X) x (10X)
3. Multiply: 100X
2.14: Exercise 5.3
400X
Calculation
1. Formula: total magnification = (objective lens magnification) x (ocular lens magnification)
2. Plug numbers into the formula: total magnification = (40X) x (10X)
3. Multiply: 400X
2.15: Exercise 5.4
1000X
Calculation
1. Formula: total magnification = (objective lens magnification) x (ocular lens magnification)
2. Plug numbers into the formula: total magnification = (100X) x (10X)
3. Multiply: 1000X
2.16: Exercise 5.5-A
Answer
eyepiece
Note
the eyepiece contains the ocular lens (usually 10X magnification)
2.17: Exercise 5.5-B
Answer
revolving nosepiece
2.18: Exercise 5.5-C
Answer
objective lens
Note
There are multiple objective lenses. Revolving the nosepiece will change the objective lens in use to increase or decrease magnification of the sample.
2.19: Exercise 5.5-D
stage
2.20: Exercise 5.5-E
stage clip
2.21: Exercise 5.5-F
Answer
light source / illuminator
2.22: Exercise 5.5-G
base
2.23: Exercise 5.5-H
diaphragm
2.24: Exercise 5.5-I
stage controls
2.25: Exercise 5.5-J
fine focus
2.26: Exercise 5.5-K
course focus
2.27: Exercise 5.5-L
arm
2.28: Exercise 5.6-ocular lens
Answer
E. A magnifying lens located inside the microscope part where a person looks into the microscope.
2.29: Exercise 5.6-revolving nosepiece
Answer
D. A structure capable of rotating to change the objective lens being used to magnify the sample.
2.30: Exercise 5.6-arm
Answer
L. A structural component that serves to support the eyepiece, revolving nosepiece, and stage.
2.31: Exercise 5.6-stage control
Answer
A. Adjusts the position of a slide on the stage.
2.32: Exercise 5.6-base
Answer
H. A structural component that serves to support the weight of the microscope from underneath.
2.33: Exercise 5.6-course focus
Answer
B. Adjusts the focus of the microscope by moving the stage up and down in large increments.
Note
The course focus is used with the 4X objective lens and the 10X objective lens only and should never be used with the 40X or 100X objective lenses (it could crack a slide or damage the lenses).
2.34: Exercise 5.6-fine focus
Answer
K. Adjusts the focus of the microscope by moving the stage up and down in smaller increments.
Note
The fine focus is mostly used with the 40X objective and 100X objective.
2.35: Exercise 5.6-light source illuminator
Answer
F. Provides light that shines on the sample and carries the image of the specimen through the magnifying lenses. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/02%3A_Exercise_Answers/2.01%3A_Exercise_2.1.txt |
Answer
M. A magnifying lens which increases magnification of the specimen that can easily be changed by rotating between lenses of different magnifications.
2.37: Exercise 5.6-stage
Answer
C. A surface where a slide is positioned under the objective lens and over the light source.
2.38: Exercise 5.6-stage clip
Answer
I. Secures a slide in place on the microscope.
2.39: Exercise 5.6-diaphragm
Answer
G. A structure capable of changing the amount of light passing through the specimen.
2.40: Exercise 5.6-eyepiece
Answer
J. Where the microscope user looks into the microscope to view a magnified image of the specimen.
2.41: Exercise 6.1-1
organic molecule
Explanation
This molecule contains a carbon atom (C) and hydrogen atoms (H). Since it has both C and H, it is an organic molecule.
2.42: Exercise 6.1-2
organic molecule
Explanation
This molecule contains carbon atoms (C) and hydrogen atoms (H). Since it has both C and H, it is an organic molecule.
2.43: Exercise 6.1-3
Answer
inorganic molecule
Explanation
This molecule does not contain carbon atoms (C) or hydrogen atoms (H). Since it does not have both C and H, it is an inorganic molecule.
Note
Don't be thrown off by the "C" in NaCl. This is not a carbon atom. "Cl" is the element chlorine.
2.44: Exercise 6.1-4
Answer
inorganic molecule
Explanation
This molecule does not contain carbon atoms (C) or hydrogen atoms (H). Since it does not have both C and H, it is an inorganic molecule.
Note
Don't be thrown off by the "C's" in CaCl2. These are not a carbon atoms. "Ca" is the element calcium. "Cl" is the element chlorine.
2.45: Exercise 6.1-5
Answer
inorganic molecule
Explanation
Even though this molecule does contain hydrogen atoms (H), it does not contain any carbon atoms (C). Since it does not have both C and H, it is an inorganic molecule.
2.46: Exercise 6.1-6
organic molecule
Explanation
This molecule contains carbon atoms (C) and hydrogen atoms (H). Since it has both C and H, it is an organic molecule.
2.47: Exercise 6.1-7
organic molecule
Explanation
This molecule contains carbon atoms (C) and hydrogen atoms (H). Since it has both C and H, it is an organic molecule.
2.48: Exercise 6.1-8
Answer
inorganic molecule
Explanation
This molecule does not contain any hydrogen atoms (H). Since it does not have both C and H, it is an inorganic molecule.
Note
Most inorganic molecules do not contain carbon atoms, but they can. Note that this molecule does not contain any hydrogen atoms (H) with the carbon atom (C), thereby making it inorganic.
2.49: Exercise 10.1
Pink cells after the Gram stain (assuming the Gram stain was completed successfully) means that this is a species that is considered Gram-negative.
A species that is Gram-negative has a thin layer of peptidoglycan in its cell wall and an outer membrane containing lipopolysaccharide (LPS).
2.50: Exercise 10.2
Purple cells after the Gram stain (assuming the Gram stain was completed successfully) means that this is a species that is considered Gram-positive.
A species that is Gram-positive has a thick layer of peptidoglycan in its cell wall and does not have an outer membrane.
2.51: Exercise 10.3
Purple colored cells and pink colored cells after the Gram stain (assuming the Gram stain was completed successfully) means that there are at least two species of bacteria in the sample and one species is Gram-positive and one species is Gram-negative.
The species that is Gram-positive has a thick layer of peptidoglycan in its cell wall and does not have an outer membrane.
The species that is Gram-negative has a thin layer of peptidoglycan in its cell wall and an outer membrane containing lipopolysaccharide (LPS). | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/02%3A_Exercise_Answers/2.36%3A_Exercise_5.6-objective_lens.txt |
1. Two different species of bacteria (Look for different cell shapes and different Gram results).
2. One species is Gram-positive (purple) and one species is Gram-negative (pink).
3. The Gram-positive cells are cocci and the Gram-negative cells are bacilli.
Note
While only two species of bacteria can be distinguished, it is possible that there could be more than two. There are many species of Gram-positive cocci and many species of Gram-negative bacilli.
2.53: Exercise 10.5
1. One species of bacteria (look for different cell shapes and different Gram results - all cells appear as pink rods).
2. Gram-negative (pink)
3. bacilli
Note
Just because all cells appear as Gram-negative bacilli, that doesn't mean that it is for sure only one species of bacteria. There are many different species of Gram-negative bacilli.
2.54: Exercise 10.6
1. Two different species of bacteria (look for different cell shapes and different Gram results).
2. One species is Gram-positive (purple) and one species is Gram-negative (pink).
3. The Gram-positive cells are bacilli and the Gram-negative cells are cocci.
Note
While only two species of bacteria can be distinguished, it is possible that there could be more than two. There are many species of Gram-positive bacilli and many species of Gram-negative cocci.
2.55: Exercise 15.1
Answer
211,000,000,000 cells/mL in original sample or 2.11 x 1011 cells/mL in original sample
Solution
• # of colonies counted = 211
• amount of diluted sample added to the petri plate in mL = 100 μL = 0.1 mL
• dilution of the petri plate counted = 10-8
Step 1: Determine the concentration of cells in the diluted sample:
(# of colonies counted on the petri plate) ÷ (amount of diluted sample added to the petri plate in mL) = CFU in diluted sample (cells/mL)
(211 colonies) ÷ (0.1 mL diluted sample added to petri plate) = 2,110 cells/mL in the diluted sample
Step 2: Determine the concentration of cells in the original sample:
(CFU in diluted sample) ÷ (dilution of the petri plate counted) = CFU in original sample (cells/mL)
(2,110 cells/mL in diluted sample) ÷ (10-8 dilution of the petri plate counted) = 211,000,000,000 cells/mL in original sample = 2.11 x 1011 cells/mL in original sample
CFU = 211,000,000,000 cells/mL in original sample or 2.11 x 1011 cells/mL in original sample
2.56: Exercise 15.2
Answer
32,400,000 cells/mL in original sample or 3.24 x 107 cells/mL in original sample
Solution
• # of colonies counted = 162
• amount of diluted sample added to the petri plate in mL = 50 μL = 0.05 mL
• dilution of the petri plate counted = 10-4
Step 1: Determine the concentration of cells in the diluted sample:
(# of colonies counted on the petri plate) ÷ (amount of diluted sample added to the petri plate in mL) = CFU in diluted sample (cells/mL)
(162 colonies) ÷ (0.05 mL diluted sample added to petri plate) = 3,240 cells/mL in the diluted sample
Step 2: Determine the concentration of cells in the original sample:
(CFU in diluted sample) ÷ (dilution of the petri plate counted) = CFU in original sample (cells/mL)
(3,240 cells/mL in diluted sample) ÷ (10-4 dilution of the petri plate counted) = 32,400,000 cells/mL in original sample or 3.24 x 107 cells/mL in original sample
CFU = 32,400,000 cells/mL in original sample or 3.24 x 107 cells/mL in original sample
2.57: Exercise 15.3
Answer
356,000,000 cells/mL in original sample or 3.56 x 108 cells/mL in original sample
Solution
• # of colonies counted = 89
• amount of diluted sample added to the petri plate in mL = 250 μL = 0.25 mL
• dilution of the petri plate counted = 10-6
Step 1: Determine the concentration of cells in the diluted sample:
(# of colonies counted on the petri plate) ÷ (amount of diluted sample added to the petri plate in mL) = CFU in diluted sample (cells/mL)
(89 colonies) ÷ (0.25 mL diluted sample added to petri plate) = 356 cells/mL in the diluted sample
Step 2: Determine the concentration of cells in the original sample:
(CFU in diluted sample) ÷ (dilution of the petri plate counted) = CFU in original sample (cells/mL)
(356 cells/mL in diluted sample) ÷ (10-6 dilution of the petri plate counted) = 356,000,000 cells/mL in original sample or 3.56 x 108 cells/mL in original sample
CFU = 356,000,000 cells/mL in original sample or 3.56 x 108 cells/mL in original sample
2.58: Exercise 15.4
Answer
approximately 9 x 106 cells/mL
Solution
1. Find the number corresponding to the absorbance on the graph's axis (in this case 0.04 on the x-axis).
2. On the graph, draw a straight line along the absorbance reading on the axis until it reaches the standard line. In this case, draw straight up from 0.04 until it hits the dotted standard line.
3. On the graph, draw a straight line from the point identified in step 2. to the other axis (in this case away toward the y-axis).
4. Where you intersect the other axis, determine the value of this point on the graph. This will be the approximate CFU at this absorbance. At 0.04 absorbance, the standard line is about at about 9, but the unit given on the graph indicates these numbers should be multiplied by 106. This is how 9 x 106 cells/mL was determined as the CFU at this absorbance.
2.59: Exercise 15.5
Answer
70.1 x 106 cells/mL or 7.0 x 107 cells/mL
Solution
• Equation for standard line: y = 226.21x
• The absorbance (which is x): 0.31
• Set up the equation: y = (226.21) x (0.31)
• Calculate y: 70.125
• Recall that the graph axis indicates y is x 106, so the final answer is: 70.1 x 106 cells/mL or 7.0 x 107 cells/mL
2.60: Exercise 15.6
Answer
40.7 x 106 cells/mL or 4.1 x 107 cells/mL
Solution
• Equation for standard line: y = 226.21x
• The absorbance (which is x): 0.18
• Set up the equation: y = (226.21) x (0.18)
• Calculate y: 40.718
• Recall that the graph axis indicates y is x 106, so the final answer is: 40.7 x 106 cells/mL or 4.1 x 107 cells/mL
2.61: Exercise 22.1
Answers
• Did this bacterial species produce gas? Yes
• Did this bacterial species produce acid? Yes
• Did fermentation occur? Yes
Solution
• Gas was produced. The upside-down tiny tube inside the test tube (the Durham tube) is not completely filled with liquid medium. It has some gas in it. This indicates the bacterial species produced gas during fermentation and it was trapped inside of the Durham tube.
• Acid was produced. The medium is yellow. When the medium was inoculated it was red. When the pH of this medium becomes acidic, a pH indicator will change from red to yellow. Since the medium is yellow, it indicates that acid was produced to reduce the pH.
• Fermentation occurred. If either acid or gas is produced or if acid and gas are produced then fermentation has occurred.
2.62: Exercise 22.2
Answers
• Did this bacterial species produce gas? No
• Did this bacterial species produce acid? No
• Did fermentation occur? No
Solution
• Gas was not produced. The upside-down tiny tube inside the test tube (the Durham tube) is completely filled with liquid medium. The Durham tube therefore does not have any gas in it (no bubble). If the bacterial species produced gas during fermentation it would have been trapped inside of the Durham tube.
• Acid was not produced. The medium is red. When the medium was inoculated it was red. Since the medium is still red, no acid was produced. In this medium, when acid is produced, a pH indicator changes the medium to yellow.
• Fermentation did not occur. If neither acid or gas are produced, this indicates fermentation did not occur. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/02%3A_Exercise_Answers/2.52%3A_Exercise_10.4.txt |
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Identification of two Bacterial Species in a Mixed Culture Using Traditional Microbiological Identification Approaches
Written By: Rosanna Hartline
Introduction
Bacterial Diversity & Importance of Species Identification
Bacteria are among the most diverse types of life on Earth (Locey and Lennon, 2016). There are billions, perhaps trillions, of bacterial species occupying habitats that are living, such as animals, and non-living habitats with a vast variety of temperatures, pH, and salinities (Parker et al., 2022). The metabolic and physical and adaptations enabling bacterial survival is wide-ranging, particularly given that these microscopic organisms are only a single cell big (Parker et al., 2022). The impacts bacteria have on other living things and the environment are extensive. Bacteria are responsible for critical nutrient cycling activities in environmental systems that would not occur without them (Parker et al., 2022). Without bacteria, nutrient availability on Earth would be highly limited and greatly reduce the amount of life that could exist on this planet (Parker et al., 2022).
Bacteria live symbiotically with other living things in parasitic, commensal, and mutualistic ways (Parker et al., 2022). In fact, living in and on a single human, there are more than 100 trillion bacterial cells (Locey and Lennon, 2016). To understand the interactions of each bacterial species and their impacts on the environment and other living things, bacterial species must be identified and characterized. The implications of the types of bacterial species living in different habitats and environments ranges from destructive and lethal effects of some bacterial species to bacterial roles and activities that are crucial for the existence of life. In this study, the goal was to apply traditional microbiological approaches to successfully identify bacterial species of unknown identifies in a mixed culture. To accomplish this, the following approaches were used: quadrant streak plate, Gram stain, starch hydrolysis test, H2S production test, oxidase test, and lactose fermentation test.
Quadrant Streak Plate
To identify bacterial species using traditional microbiological methods, isolating a single bacterial species from other microbes is crucial. Subsequent microbiological tests will have indeterminant results if there are multiple microbial species mixed, since there is no way of knowing which species is producing any positive test result. To isolate bacterial species, a commonly used approach is the quadrant streak plate technique (Raymond et al., 2022). This approach utilizes a petri plate with nutrient-rich media and four streak zones (Raymond et al., 2022). The first zone will have the densest growth since it is streaked directly from the source of the microbes being examined. The following zones are pulled from the streak immediately before, and the microbes present on the inoculating loop are killed by flaming before each subsequent streak. The result is dilution of the number of microbes in each streak, until the fourth quadrant produces locations on the petri plate where only single cells are deposited on the petri plate agar (Hartline, 2022). A bacterial colony will develop from a single cell as the cells divide by binary fission, and thereby produce an isolated colony that will contain only one bacterial species (Klamm, 2019). These isolated colonies can then be characterized, aiding in species identification, and then may be transferred to a separate culture to create a monoculture for subsequent identification tests. Qualities such as colony color, form, size, and appearance are useful information to distinguish between different species of bacteria. These can be determined by careful examination of isolated colonies on a petri plate.
Gram Stain
The diversity of bacterial species in the world is subdivided into two major groups based on their cell wall structures and are designated as either Gram-positive or Gram-negative based on the results of the Gram stain (Raymond et al., 2022). The Gram stain will stain Gram-positive bacteria purple since the purple-colored crystal violet stain is not removed from the Gram-positive cell wall during the decolorization step of the Gram stain due to the thick peptidoglycan layer in the cell wall outside of the plasma membrane (Parker et al., 2022). The Gram-positive bacterial cell wall also contains teichoic acid and does not have an outer membrane (Hartline, 2022). For Gram-negative bacteria, the purple-colored crystal violet stain is readily removed from its thin peptidoglycan layer during the decolorization step of the Gram stain and will appear pink due to the counterstain safranin used after the decolorization step (Raymond et al., 2022). Most species that stain Gram-negative have a thin peptidoglycan layer outside the of plasma membrane with an outer membrane outside of the peptidoglycan layer that has embedded lipopolysaccharide (also known as endotoxin) (Hartline, 2022; Parker et al., 2022).
Starch Hydrolysis Test
One of the many microbiological tests used to characterize bacterial species and aid in species identification is the starch hydrolysis test. Starch is a polysaccharide, and therefore is composed of many sugars that bacteria can use as an energy source and a carbon source (Parker et al., 2022). If a bacterial species has the amylase gene, it can produce the enzyme amylase enabling it to hydrolyze (break down) starch and utilize this rich energy and carbon source (Hartline, 2022). To determine if bacteria hydrolyze starch, bacteria are grown on a starch plate and following incubation, iodine is added to the plate. Iodine reacts with starch to produce a blue-black color enabling visualization of starch and where it is and is not present in the petri plate agar (Pakpour and Horgan 2021). If starch has been broken down by the bacteria, the agar will lack the blue-black color around the bacterial growth indicating that starch has been hydrolyzed, since starch is absent near the bacteria (Pakpour and Horgan 2021).
H2S Production
Use of SIM deep medium can be used to determine multiple characteristics of a bacterial species in a single test tube, including determining if bacteria can produce H2S (Pakpour and Horgan 2021). Production of H2S can indicate that either a bacterial species can convert cysteine (an amino acid) to pyruvate or that a bacterial species conducts a form of anaerobic respiration where H2S is produced when a sulfur-containing compound is used as a final electron acceptor (Hartline, 2022). The SIM medium contains ferrous sulfide and if bacteria produce H2S, the ferrous sulfide will react with H2S to produce a black precipitate, indicating the bacterial species is H2S-positive (Pakpour and Horgan 2021). The absence of black precipitate characterizes the bacterial species as H2S-negative, since it does not produce H2S gas.
Oxidase Test
Cytochrome c oxidase is an enzyme that some bacteria use as part of their electron transport chain in aerobic respiration (Raymond et al., 2022). Only some bacterial species have the cytochrome c oxidase gene and therefore produce this enzyme, making it a useful characteristic for differentiating bacterial species in the identification process (Raymond et al., 2022). Some bacterial species do not have the cytochrome c oxidase enzyme, but they can still conduct aerobic respiration using different enzymes (Hartline, 2022). Testing for the presence or absence of this enzyme is not an indicator of whether a species conducts aerobic respiration or not, just whether the species uses the cytochrome c oxidase as the enzyme that transfers electrons from the electron transport chain to oxygen, the final electron acceptor in this process (Hartline, 2022). The oxidase test determines whether a bacterial species has the cytochrome c oxidase enzyme. This test uses a chemical called tetramethyl-p-phenylenediamine and if cytochrome c oxidase is present in the bacteria, it will remove electrons from this chemical and the product molecule is a blueish or purplish color (Klamm, 2019). Therefore, oxidase positive bacteria produce a blueish or purplish color in the first 30 seconds of mixing the chemical with live bacteria and oxidase negative bacteria do not produce this color change.
Lactose Fermentation Test
Some bacterial species can use fermentation as a method of producing ATP and some bacterial species are not. Further, the types of sugars that different bacterial species can ferment also differs based on the genes (and therefore enzymes) that the species have or do not have (Hartline, 2022). The lactose fermentation test determines whether bacterial species are capable of utilizing lactose, a type of sugar, to conduct fermentation and will indicate whether a bacterial species will make acid, gas, or both as byproducts of the fermentation process (Lee 2021). Bacterial species that metabolize lactose have the gene for the enzyme beta-galactosidase enabling the breakdown lactose, a disaccharide, into monosaccharides that can enter catabolic pathways such as fermentation (Hartline 2022). To conduct this test, a lactose-containing medium with a pH indicator and Durham tube is inoculated with the bacteria being tested. If acid is produced by the fermentation process, the medium will change from red to yellow (Lee, 2021). If gas is produced by the fermentation process, a pocket of gas will form inside of the Durham tube. If no lactose fermentation occurs, no acid or gas will be produced.
Experimental Overview
Bacterial identification is an important skill for people in a vast range of professions where bacteria can impact their work including healthcare, food safety, and water safety. This experiment aimed to separate the bacterial species in a mixed culture and utilized traditional microbiological identification approaches to identify the bacterial species present in that culture. These techniques resulted in successful identification of Bacillus subtilis as one of the species in that mixed culture. The other species in the culture was Escherichia coli but was misidentified in this process as Serratia marcescens.
Methods
Quadrant Streak Plate
From a TSB culture containing a mixture of two unknown bacterial species, a loopful of culture was aseptically transferred to a single streak along one side of a petri plate. The loop was flamed and cooled before an edge of the initial streak was dragged into a second streak on the petri plate (Hartline, 2022). The loop was again flamed, cooled, and part of the second streak was dragged into the third streak (Hartline, 2022). This was repeated one more time to complete a fourth streak (Hartline, 2022). This process was repeated eight times to produce a total of eight quadrant streak plates. The plates were inverted and incubated at 30 °C for 48 hours.
Gram Stain
Bacterial smears were prepared by either by aseptically transferring several loops of liquid culture to a microscope slide or aseptically transferring a bacterial colony using a loop from a petri plate to a drop of saline on a microscope slide. The slide was placed onto a slide warmer until dry and then heat-fixed by passing the slide through a Bunsen burner flame three times. Crystal violet was applied to stain the smear for one minute, rinsed with deionized water, covered with Gram’s iodine for one minute, rinsed with deionized water, decolorized with ethanol for five to fifteen seconds, rinsed with deionized water, counterstained with safranin for one minute, and rinsed with deionized water (Hartline, 2022). Following this staining procedure, the slide was blotted with bibulous paper and examined with a light microscope.
Starch Hydrolysis Test
A loop was used to aseptically transfer isolated bacteria from a TSA slant culture to create a single-line streak on the surface of a petri plate with starch agar medium. The plate was inverted and incubated at 37 °C for 48 hours. The plate was then flooded with iodine to visualize presence and absence of starch in the agar.
H2S Production
An inoculation needle was used to aseptically transfer bacteria from a TSA slant to a SIM deep culture and incubated at 37 °C for 48 hours.
Oxidase Test
A sterile plastic loop was used to aseptically transfer bacteria from the SIM deep culture to a moistened oxidase test strip. Results of the test were interpreted within the first 30 seconds. This test was repeated a total of three times to ensure that results were accurate.
Lactose Fermentation Test
Bacteria were aseptically transferred from a TSA slant to a test tube with phenol red lactose medium containing a Durham tube. This culture was incubated at 37 °C for 12 days.
Results & Discussion
Initial Gram Stain of Mixed Bacterial Culture
A Gram stain of the mixed culture of unknown bacteria designated as ‘H’ revealed two distinctly different bacterial species since one stained purple and one stained pink. Both species exhibited rod-shaped cells and as a result are designated as bacilli.
Pink-colored cells indicated these cells were a Gram-negative species and therefore likely have a cell wall containing a thin layer of peptidoglycan and an outer membrane with embedded lipopolysaccharide (Parker et al., 2022). The crystal violet primary stain was removed from the Gram-negative cells during the decolorization step of the Gram stain due to the thin layer of peptidoglycan being unable to retain the stain. These cells appeared pink due to the counterstain safranin.
The purple-colored cells suggested these were a Gram-positive species that have a thick layer of peptidoglycan in its cell walls resulting in the crystal violet stain being retained during the decolorization step of the Gram stain (Parker et al., 2022).
The initial Gram stain of the mixed bacterial culture ‘H’ revealed that the two unknown bacterial species were a Gram-positive bacillus species and a Gram-negative bacillus species.
Isolation of Bacterial Species
As a result of the quadrant streak plate procedure, isolated colonies were successfully produced. There were two distinctly different types of colonies that were differentiated by shape, color, and size. These differences in colony characteristics suggest that these are different microbial species (Figure 2).
One of the colony types, hereafter designed as bacterial specie ‘alpha,’ were a bright ivory color and shiny (Hartline, 2022). The colonies measured between 1 mm and 3 mm in diameter and had a circular form with sharp edges and raised elevation.
The other colony type, designated as ‘beta,’ produced dry and dull looking colonies with a milky-yellow coloration, an irregular form, flat elevation, and measured between 3 mm and 5 mm in diameter (Hartline, 2022). These colonies had distinct color variations that occurred as irregular, wavy rings within the colonies.
Identification of Bacterial Species Alpha
Gram Stain
A Gram stain conducted on the unknown isolated bacterial species Alpha revealed that this species has rod-shaped cells, or bacilli, and the cells appeared pink after the Gram stain (Figure 3).
Since unknown bacterial species Alpha produced pink-colored cells in the Gram stain, this indicates it is a Gram-negative species (Hartline, 2022). This species likely has a cell wall with a thin layer of peptidoglycan outside of the plasma membrane and an outer membrane containing lipopolysaccharide (Hartline, 2022; Parker et al., 2022).
H2S Test
Since unknown bacterial species Alpha was Gram-negative, the next indicated test to conduct was to assess whether this species produces H2S gas or not. After inoculation and incubation of a SIM deep, the medium did not produce a black coloration (Figure 4).
Since the SIM deep did not produce a black coloration, this means that no H2S was produced in the culture to interact with the ferrous sulfide in the medium (Pakpour and Horgan 2021). Therefore, unknown bacterial species Alpha does not produce H2S and is H2S-negative. This result reveals that unknown bacterial species Alpha does not produce pyruvate from cysteine and does not conduct the form of anaerobic respiration that produces H2S gas as a byproduct (Hartline, 2022).
Although not directly a part of this identification protocol, the presence or absence of motility can be determined using this SIM deep culture (Pakpour and Horgan 2021). Growth migrated away from the stab line in the SIM deep suggesting unknown bacterial species Alpha is motility positive and therefore has one or more flagella enabling movement in this bacterial species (Hartline, 2022; Pakpour and Horgan 2021).
Oxidase Test
Due to the H2S negative test result, the identification protocol next indicates the oxidase test. The oxidase test did not produce a purple or blue color in the first 30 seconds (Figure 5). The test was repeated a total of three times to ensure the validity of this result and the results did not change.
The lack of purple or blue color in under 30 seconds means that unknown bacterial species Alpha is oxidase negative. These results indicate that unknown bacterial species Alpha does not have the cytochrome c oxidase enzyme or the gene that codes for cytochrome c oxidase (Raymond et al., 2022). While this result does not indicate whether this bacterial species can conduct aerobic respiration or not, it does indicate that it does not use the cytochrome c oxidase enzyme to transfer electrons from the electron transport chain to O2 (Hartline, 2022). If this species does conduct aerobic respiration, it uses a different enzyme for this process at the end of the electron transport chain (Hartline, 2022).
Lactose Fermentation Test
Since the oxidase test produced a negative result for unknown bacterial species Alpha, the next indicated identification test in the protocol was the lactose fermentation test. After inoculation and incubation, the phenol red lactose medium maintained a red color and the Durham tube was still filled with the liquid medium and lacked a gas pocket inside of it (Figure 6).
Since the phenol red lactose medium did not become yellow, this indicates that acid was not produced by the bacteria. No air pocket in the Durham tube in this culture indicates that no gas was produced by the bacteria in this culture. Since neither gas nor acid were produced, this indicates that bacterial species Alpha is lactose fermentation negative. This result suggests this bacterial species either does not conduct fermentation, does not have the beta-galactosidase gene for breaking down lactose, or both (Hartline, 2022).
Based on this result, unknown bacterial species Alpha was identified using this laboratory protocol as Serratia marcescens.
Identification of Bacterial Species Beta
Gram Stain
The isolated bacterial species designated as Beta was Gram-stained and results show the cells appear as purple rods (Figure 7).
The purple coloration of this bacterial species after the Gram stain indicates this species is Gram-positive. This result indicates this species has a thick layer of peptidoglycan in its cell wall that retains the purple coloration of the crystal violet stain during the Gram stain protocol (Parker et al., 2022). The rod-shaped cells indicate this speices have cells that are Gram-positive bacilli.
Starch Hydrolysis Test
Since unknown bacterial species Beta stained Gram-positive, the next indicated test in the identification protocol was the starch hydrolysis test. Following incubation of the unknown bacteria on a starch plate and staining the starch plate with iodine, a clear zone surrounded the bacterial growth on the petri plate (Figure 8).
The starch hydrolysis test showed a clear zone surrounding the bacterial growth indicating that starch was absent in the agar surrounding unknown bacterial species Beta (Pakpour and Horgan 2021). This result reveals that Beta is starch hydrolysis positive and therefore must have the amylase gene to produce the amylase enzyme that breaks down starch (Hartline, 2022).
Since the starch hydrolysis test was positive for unknown bacterial species Beta, the identification protocol indicates that this species is Bacillus subtilis.
Conclusion
Unknown bacterial species Alpha was identified as Serratia marcescens using this identification protocol using traditional microbiological techniques. In fact, this species was not S. marcescens, but was Escherichia coli indicating one or more errors occurred resulting in incorrect identification of this bacterial species. There are two plausible explanations for this misidentification. First, it is possible when unknown bacterial species Alpha was aseptically collected from the TSA slant that the loop was too hot, and any bacteria collected from the slant were killed by the heat of the loop. As a result, the lactose fermentation tube may not have been successfully inoculated, resulting in the lactose fermentation negative result. The other possible explanation for incorrect identification is that a contaminant colony on the quadrat streak plate was collected and tested throughout this identification process instead of the actual unknown bacterial species that was targeted.
Unknown bacterial species Beta was identified as Bacillus subtilis in this experiment. The Gram-positive bacterial species in unknown culture ‘H’ was indeed B. subtilis and therefore identification of this species was successful. This result was likely due to several factors including careful aseptic technique to prevent contamination, thoughtful examination and documentation of each laboratory result throughout the process, and careful execution of all the laboratory protocols to ensure accurate experimental results.
Works Cited
Hartline R. 2022. Microbiology Laboratory Manual. LibreText. Retrieved from: https://bio.libretexts.org/Courses/West_Hills_College_-_Lemoore/Microbiology_Laboratory_Manual
Klamm LS. 2019. Klamm’s Microbiology Laboratory Manual. MOspace Institutional Repository. Retrieved from: https://mospace.umsystem.edu/xmlui/handle/10355/69341
Lee, 2021. MB352 General Microbiology Laboratory 2021 (Lee). LibreText. Retrieved from: https://bio.libretexts.org/Courses/North_Carolina_State_University/MB352_General_Microbiology_Laboratory_2021_(Lee)
Locey KJ, Lennon JT. 2016. Scaling laws predict global microbial diversity. Proceedings of the National Academy of Sciences of the United States of America. 113: 5970-5975.
Pakpour N, Horgan S. 2021. General Microbiology Lab Manual (Pakpour & Horgan). LibreText. Retrieved from: https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/Microbiology_Labs/Book%3A_General_Microbiology_Lab_Manual_(Pakpour_and_Horgan)
Parker N, Schneegurt M, Thi Tu A-H, Lister P, Forster BM, Allen S, Auman A, Brelles-Mariño G, Alhadeff Feldman M, Flowers P, Pinchuk G, Rowley B, Sutherland M, Franklund C, Paterson A. 2022. Microbiology. OpenStax. Retrieved from: https://openstax.org/details/books/microbiology
Raymond J, Boorse G, Mason A. 2022. Red Mountain Microbiology. Maricopa Community Colleges. Retrieve from: https://open.maricopa.edu/redmountainmicro/
Attributions*
*Attributions are not a part of the sample Unknown Identification Project Report and are not to be included in student reports. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/02%3A_Exercise_Answers/2.63%3A_Sample_Project_Report.txt |
Materials
Student Preparation of TSB Medium
• TSB medium powder
• stir plates (1 per group)
• magnetic stir bars (1 per group)
• 50 or 100 mL graduated cylinders (1 per group)
• 250 mL or 300 mL beakers for making TSB medium (1 per group)
• balances
• weigh papers or boats
• weigh utensils
• distilled or deionized water
Re-creation of Spallanzani's Experiment
• test tube racks (1 per group)
• test tubes (2 per group)
• test tube caps (1 per group)
• 5 mL or 10 mL pipet (1 per group)
• pipet bulbs or pipet pumps (1 per group)
• 10-20 mL student-prepared TSB (before autoclaving)
• labeling tape and markers
Re-creation of Pasteur's Experiment
• 125 mL Erlenmeyer flasks (2 per group)
• neoprene stoppers with one hole, size 5 (2 per group)
• 5 mm flint glass tubing, prepped as straight glass tubes (1 per group)
• 5 mm flint glass tubing, prepped as swan-neck glass tubes (1 per group)
• 50 mL or 100 mL graduated cylinder (1 per group)
• 100 mL student-prepared TSB (before autoclaving)
• labeling tape and markers
Preparations
Re-creation of Pasteur's Experiment
Straight Glass Tubes
1. Wear safety goggles.
2. Use a file to score around the flint glass tubing to cut lengths of glass tubing about 6-8 cm in length.
3. Put on protective gloves (not lab gloves; use thicker gloves such as gardening gloves or autoclave gloves).
4. Snap the tubing at the score site.
5. Repeat until you have enough straight-glass tubes for class (1 per group).
Swan-Neck Tubes
1. Wear safety goggles.
2. Use a file to score around the flint glass tubing to cut lengths of glass tubing about 20-25 cm in length.
3. Put on protective gloves (not lab gloves; use thicker gloves such as gardening gloves or autoclave gloves).
4. Snap the tubing at the score site.
5. Use a Bunsen burner to heat the glass tube to bend the tube into a swan-neck shape. This will require you to heat the glass tube at two different locations where one site is bent downward and the other site is bent upward. Make sure that there is enough of a straight section before the swan-neck curves so the straight section can pass through the one-hole neoprene stopper.
6. Repeat until you have enough swan-neck glass tubes for class (1 per group).
Put Glass Tubes through Stoppers
1. Carefully push a straight glass tube through the hole in one of the neoprene stoppers.
2. Carefully push the straight region of the swan-neck class tube through the hole in one of the neoprene stoppers.
3. Repeat for each group.
After Lab Class
1. Autoclave the test tubes and flasks prepared by students for the Spallanzani and Pasteur experiment re-creations.
2. Incubate the test tubes and flasks prepared by students for the Spallanzani and Pasteur experiment re-creations in a 30°C or 37°C incubator or leave out on a bench.
3. When growth is observed in the uncovered test tube from the Spallanzani experiment have students examine results from the Spallanzani experiment re-creation.
4. When growth is observed in the flask with the straight glass tube from the Pasteur experiment have students examine results from the Pasteur experiment re-creation.
3.02: Get to Know the Microscope and Microbes
Materials
• light microscopes (1 per student or 1 per pair of students)
• slides: letter 'e'
• prepared slides of your choosing:
• a helminth species
• a fungal species
• a protozoan species
• bacterial species | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/03%3A_Instructor_Setup/3.01%3A_Myth_of_Spontaneous_Generation.txt |
Burn Test
Note
There are multiple ways this test can be run with a class:
Option 1: Have all student groups test all samples.
Option 2: Have all student groups test one sample (recommended sample: sugar) and have the other samples pre-prepared and students interpret the pre-prepared results.
Option 3: Instructor demonstrates one sample (recommended sample: sugar) and all other samples are pre-prepared and students interpret the results.
Option 4: All samples are pre-prepared and students interpret the results.
Consider which option you are choosing when determining the quantities of materials you will need below.
Preparation
• If you choose one of Options 2, 3, or 4 above, prepare the burn test as follows:
• Place a small amount of each substance in its own separate test tube (baking soda, hair, salt, starch, sugar, vegetable oil, water), preferably a screw-top test tube that can be re-used semester after semester.
• Wear safety goggles. Without a cap or top on the test tube, place the bottom of the test tube in the flame of a Bunsen burner while the opening of the test tube is facing away from you (you only need to do this for the organic samples since the inorganic samples will not burn). You may want to do this in a fume hood.
• Continue burning until the organic samples turn black (this can take a while for oil - just persist).
• Once cool, add caps or tops to the test tubes
Materials
• If you choose option 1 or 2, make sure to have the following on hand:
• test tubes (enough for each sample students will test per group)
• Bunsen burners
• test tube holders
• safety goggles or safety glasses
• the substances that will be tested: baking soda, hair, salt, starch, sugar, vegetable oil, water
• droppers to transfer liquid samples
• spoons or other utensils to transfer solid samples
• test tube racks (enough for 1 per group)
Building Monomers of Biological Molecules
Materials
• organic molecular modeling sets (enough for each student to have one or to share with another student)
Foods & their Biological Molecules
Preparation
The day of or the night before the lab, create food item suspensions as follows:
1. add pieces of green banana to a blender and add DI or distilled water
2. puree until homogenous
3. transfer solution to storage container
4. clean blender
5. repeat steps 2-5 using black banana, chicken meat (muscle), and egg
Materials
• blended suspensions of green banana, black banana, chicken meat, egg
• five test tubes per group
• labeling tape
• labeling markers
• safety goggles or safety glasses (1 per student)
• test tube rack (1 per group)
• droppers (2-3 per food suspension)
• DI or distilled water
• biuret reagent in dropper bottles (1 for every two groups - groups can share)
• Benedict's solution in dropper bottles (1 for every two groups - groups can share)
• hot plates with beakers containing boiling water or almost boiling water (1 for every two groups - groups can share)
• iodine in dropper bottles (1 for every two groups - groups can share)
• brown paper or brown paper towels
• chemical waste container (biuret and Benedict's waste - can be disposed of in the same container)
3.04: Aseptic Technique
Preparation
• 2 test tubes with sterile TSB per group
Materials
• 2 test tubes with sterile TSB per group
• test tube rack (1 per group)
• Bunsen burner (1 per group)
• inoculating loop (1 per group)
• labeling tape
• marker
After Lab Class
1. Incubate test tubes of (hopefully still) sterile TSB at 37°C until the next lab class.
2. Next lab class have students examine test tubes for growth and discuss possible sources of contamination (particularly for groups that had TSB showing growth).
3.05: Plating on Petri Plates for Isolation
Preparation
• sterile TSB test tubes (2 to grow cultures and another 3, 6, 9, or 12 [depending on how many cultures you want groups to share])
• 3 TSA Petri plates per group
• Inoculate TSB test tube with Escherichia coli and incubate at 37°C 24 hours before lab class
• Inoculate TSB test tube with pigmented strain of Serratia marcescens and incubate at 30°C 24 hours before lab class
• Just before lab class, aseptically transfer 200 μm of Escherichia coli culture to sterile TSB test tube(s) - add to 1-4 sterile TSB test tubes (E. coli cultures for students to use)
• Just before lab class, aseptically transfer 200 μm of Serratia marcescens culture to sterile TSB test tube(s) - add to 1-4 sterile TSB test tubes (S. marcescens cultures for students to use)
• Just before lab class, aseptically transfer 100-200 μm of Escherichia coli culture to sterile TSB test tube(s) and 100-200 μm of Serratia marcescens culture to the same test tube(s) - add to 1-4 sterile TSB test tubes (E. coli + S. marcescens cultures for students to use)
Materials
• 3 TSA plates per group
• test tube racks (1 per group)
• Bunsen burner (1 per group)
• inoculating loop (1 per group)
• markers
• culture of E. coli
• culture of S. marcescens (pigmented strain)
• culture of E. coli + S. marcescens (pigmented strain)
After Lab Class
1. Incubate quadrant streak plates at 30°C for 24-48 hours.
2. Refrigerate streak plates to prevent overgrowth if the next lab class occurs after 24-48 hours.
3. Have students examine streak plates and characterize colonies next lab class. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/03%3A_Instructor_Setup/3.03%3A_Molecules_of_Life.txt |
Preparation
1. Have test tubes with sterile TSB on hand.
2. 24 hours before the laboratory, inoculate test tubes of sterile TSB with a bacterial species of your choice (Escherichia coli or Staphylococcus aureus are good options) and incubate at the appropriate temperature for that species (37 °C for the E. coli and S. aureus).
Remember that you will have multiple groups needing these cultures so make enough cultures so each group has their own or enough so each group only shares the culture with another group.
Note
You can use multiple bacterial species to have students compare different cell morphologies and arrangements. The Results & Questions section of this lab will require editing to provide questions for multiple species.
Materials
• 24-hour bacterial cultures of choice in TSB - see Preparation section above
• plain microscope slides (at least 1 per group)
• Bunsen burners (1 per group)
• inoculating loops (1 per group)
• (if using bacteria on solid medium rather than TSB) droppers with saline, DI water, or distilled water
• slide warmer
• test tube racks (1 per group plus one or more racks to hold cultures)
• wood test tube clamps/holders (1 per group)
• small chemical waste containers (1 per group)
• methylene blue in a dropper bottle (1 per group or enough for two groups to share one bottle)
• distilled or DI water in squirt bottles (1 per group)
• bibulous paper (enough for each groups to blot their slides)
• light microscopes (1 per group)
• colored pencils
• (optional, but recommended) Wax pencils (1 per group)
• (if using oil immersion) immersion oil dropper bottles
• (if using oil immersion) lens paper
3.07: Gram Stain
Preparation
1. Have test tubes with sterile TSB on hand.
2. 12-18 hours before the laboratory, inoculate test tubes of sterile TSB with a mixture of Escherichia coli and Staphylococcus aureus and incubate at 37 °C .
Note
Remember that you will have multiple groups needing these cultures so make enough cultures so each group has their own or enough so each group only shares the culture with another group.
Materials
• 12-18 hour cultures of E. coli and S. aureus mixture - see Preparation section above
• plain microscope slides (at least 1 per group)
• Bunsen burners (1 per group)
• inoculating loops (1 per group)
• (if using bacteria on solid medium rather than TSB) droppers with saline, DI water, or distilled water
• slide warmer
• test tube racks (1 per group plus one or more racks to hold cultures)
• wood test tube clamps/holders (1 per group)
• small chemical waste containers (1 per group)
• crystal violet in a dropper bottle (1 per group or enough for two groups to share one bottle)
• Gram's iodine in a dropper bottle (1 per group or enough for two groups to share one bottle)
• 95% ethanol in a dropper bottle (1 per group or enough for two groups to share one bottle)
• safranin in a dropper bottle (1 per group or enough for two groups to share one bottle)
• distilled or DI water in squirt bottles (1 per group)
• bibulous paper (enough for each groups to blot their slides)
• light microscopes (1 per group)
• colored pencils
• (optional, but recommended) Wax pencils (1 per group)
• (if using oil immersion) immersion oil dropper bottles
• (if using oil immersion) lens paper | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/03%3A_Instructor_Setup/3.06%3A_Simple_Stain.txt |
Preparation
1. Have test tubes with sterile TSB on hand.
2. 36-48 hours before the laboratory, inoculate test tubes of sterile TSB with Bacillus subtilis and incubate at 37 °C .
Note
Remember that you will have multiple groups needing these cultures so make enough cultures so each group has their own or enough so each group only shares the culture with another group.
Materials
• 36-48 hour cultures of B. subtilis - see Preparation section above
• plain microscope slides (at least 1 per group)
• Bunsen burners (1 per group)
• inoculating loops (1 per group)
• (if using bacteria on solid medium rather than TSB) droppers with saline, DI water, or distilled water
• slide warmer
• test tube racks (1 per group plus one or more racks to hold cultures)
• wood test tube clamps/holders (1 per group)
• small chemical waste containers (1 per group)
• malachite green in a dropper bottle (1 per group or enough for two groups to share one bottle)
• safranin in a dropper bottle (1 per group or enough for two groups to share one bottle)
• distilled or DI water in squirt bottles (1 per group)
• hot plates (1 per group or enough for two groups to share)
• large beakers - 1000 mL+ (1 per group or enough for two groups to share)
• wire racks (1 per group or enough for two groups to share)
• paper towels
• bibulous paper (enough for each groups to blot their slides)
• light microscopes (1 per group)
• colored pencils
• (optional, but recommended) wax pencils (1 per group)
• (if using oil immersion) immersion oil dropper bottles
• (if using oil immersion) lens paper
3.09: Capsule Stain
Materials
• 12-18 hour bacterial culture in skim milk broth culture (Enterobacter aerogenes or Serratia marcescens)
• plain microscope slides (at least 1 per group)
• Bunsen burners (1 per group)
• inoculating loops (1 per group)
• test tube racks (1 per group plus one or more racks to hold cultures)
• wood test tube clamps/holders (1 per group)
• small chemical waste containers (1 per group)
• 1% crystal violet
• 20% copper sulfate
• light microscopes (1 per group)
• colored pencils
• (if using oil immersion) immersion oil dropper bottles
• (if using oil immersion) lens paper
3.10: Acid-Fast Stain
Preparation
1. Have test tubes with sterile TSB on hand.
2. 12-18 hours before the laboratory, inoculate test tubes of sterile TSB with a mixture of Staphylococcus epidermidis and Mycobacterium chelonae and incubate at 37 °C .
Materials
• 12-18 hour cultures (enough for groups to share)
• plain microscope slides (at least 1 per group)
• Bunsen burners (1 per group)
• inoculating loops (1 per group)
• (if using bacteria on solid medium rather than TSB) droppers with saline, DI water, or distilled water
• slide warmer
• test tube racks (1 per group plus one or more racks to hold cultures)
• 400-500 mL beakers with ~200 mL of water each (1 per group)
• hot plate (1 per group)
• slide rack hat fits over beaker (1 per group)
• paper towels
• carbol fuchsin in dropper bottles (1 per group)
• acid-alcohol in dropper bottles (not ethanol from the Gram stain kit) (1 per group)
• methylene blue in dropper bottles (1 per group)
• wood test tube clamps/holders (1 per group)
• small chemical waste containers (1 per group)
• distilled or DI water in squirt bottles (1 per group)
• bibulous paper (enough for each groups to blot their slides)
• light microscopes (1 per group)
• colored pencils
• (optional, but recommended) Wax pencils (1 per group)
• (if using oil immersion) immersion oil dropper bottles
• (if using oil immersion) lens paper | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/03%3A_Instructor_Setup/3.08%3A_Endospore_Stain.txt |
Plate Counts
Preparation
• sterile TSB in test tubes on hand for E. coli cultures (1 per group)
• TSA plates (4 per group)
• Sterilize:
• microcentrifuge tubes (7 per group)
• tips for pipettes (for pipettes that can dispense 990 μL and 100 μL)
• inoculate sterile TSB in test tubes with E. coli 8-16 hours before lab and incubate at 37 °C (1 per group)
Materials
• sterile microcentrifuge tubes (7 per group)
• sterile tips for pipettes (for pipettes that can dispense 990 μL and 100 μL)
• pipettes for dispensing 990 μL and 100 μL (enough for each group or for two groups to share)
• containers for used pipette tips
• 8-16 hour E. coli cultures in TSB (1 per group)
• TSA plates (4 per group)
• vortex (enough of them for groups to share)
• labeling marker (1 per group)
• spreader tools (1 per group)
• ethanol in squirt bottles
• empty petri plates (1/2 per group)
• Bunsen burners (1 per group)
• strikers (1 per group)
Absorbance Measurements
Preparation
• test tubes with 5 mL sterile TSB (4 per group)
• inoculate sterile TSB in test tubes with E. coli 8-16 hours before lab and incubate at 37 °C (1 per group) - if the plate counts are being done with the absorbance measurements, these are the same cultures used for the plate counts
Materials
• test tubes with 5 mL sterile TSB (4 per group)
• 8-16 hour E. coli cultures in TSB (1 per group)
• pipettes for transferring 5 mL (enough for each group to complete four transfers
• transfer pipettes (5 per group)
• cuvettes
• waste container for E. coli samples emptied from cuvettes
• spectrophotometer set to 600 nm (enough for two groups to share)
• labeling tape (1 per group)
• labeling marker (1 per group)
Creating a Standard Line between Plate Counts and Absorbance
Materials
• graph paper or computers with graphing software (1 per student)
• rulers (1 per student if using graph paper)
3.12: Eukaryotic Cells
Materials
• eukaryotic cell models (1 per group)
• light microscopes (1 per student or 1 per pair of students)
• colored pencils
• prepared slides of your choosing:
• a protozoan species
• an algal species
• a fungal species
• a helminth species
3.13: Starch Hydrolysis
Preparation
• 24 hour cultures of E. coli, B. subtilis, and P. vulgaris (enough for groups/individual students to share)
• starch agar plates (one per student or one per group)
Materials
• 24 hour cultures of E. coli, B. subtilis, and P. vulgaris (enough for groups/individual students to share)
• starch agar plates (one per student or one per group)
• test tube racks (1 per group/student)
• inoculating loops (1 per group/student)
• strikers (1 per group/student)
• Bunsen burners (1 per group/student)
• iodine (enough dropper bottles for groups or individual students to share)
• labeling markers (1 per group)
3.14: Catalase Test
Preparation
• 24 hour cultures of Staphylococcus aureus and Streptococcus pyogenes (enough for groups to share)
Materials
• 24 hour cultures of Staphylococcus aureus and Streptococcus pyogenes (enough for groups to share)
• new microscope slides (one per group)
• inoculating loops (one per group)
• hydrogen peroxide (enough for groups to share)
• transfer pipettes or droppers
Note
The hydrogen peroxide cannot be old or have been exposed to light for prolonged periods of time. If this is the case, the reaction may not work. Be sure to use new or newer hydrogen peroxide in a light-proof container. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/03%3A_Instructor_Setup/3.11%3A_Determination_of_Bacterial_Numbers.txt |
Preparation
• 24 hour petri plate cultures of Pseudomonas aeruginosa or Alcaligenes faecalis (oxidase positive) and Escherichia coli or Proteus mirabilis (oxidase negative) (enough for groups to share)
Materials
• 24 hour petri plate cultures of Pseudomonas aeruginosa or Alcaligenes faecalis (oxidase positive) and Escherichia coli or Proteus mirabilis (oxidase negative) (enough for groups to share)
• empty petri plates (enough for each group to have 1/2 of an empty petri plate)
• oxidase test strips (enough for each group to have 2)
• DI or distilled water in squirt bottles
• sterile transfer pipettes or sterile transfer swabs (enough for each group to have 2)
3.16: Citrate Test
Preparation
• 24 hour cultures of citrate positive species (Klebsiella, Enterobacter, Proteus, Serratia, Pseudomonas, or Salmonella) and citrate negative species (Escherichia coli or Shigella) (enough for groups/individual students to share)
• Simmons' citrate agar slants (two per group)
Materials
• 24 hour cultures of citrate positive species (Klebsiella, Enterobacter, Proteus, Serratia, Pseudomonas, or Salmonella) and citrate negative species (Escherichia coli or Shigella) (enough for groups/individual students to share)
• Simmons' citrate agar slants (two per group)
• test tube racks (1 per group)
• inoculating loops (1 per group)
• strikers (1 per group)
• Bunsen burners (1 per group)
• labeling tape (1 per group)
• labeling markers (1 per group)
3.17: Bacterial Oxygen Requirements
Preparation
• 24 hour cultures of Pseudomonas aeruginosa, Escherichia coli, and a Clostridium species of choice (if using) - Clostridium species are obligate anaerobes so cultivation will require strict anaerobic conditions (enough for groups to share)
• thioglycollate tubes (enough for each group to have 2-3, depending on the number of species being tested)
• TSA plates (2 per group)
Materials
• 24 hour cultures of Pseudomonas aeruginosa, Escherichia coli, and a Clostridium species of choice (if using) - Clostridium species are obligate anaerobes so cultivation will require strict anaerobic conditions (enough for groups to share)
• thioglycollate tubes (enough for each group to have 2-3, depending on the number of species being tested)
• labeling tape (1 per group)
• labeling markers (1 per group)
• test tube racks (1 per group)
• inoculating loops (1 per group)
• strikers (1 per group)
• Bunsen burners (1 per group)
• TSA plates (2 per group)
• anaerobic jar or bags with heat sealer
• GasPak anaerobic generators
• anaerobic indicators
3.18: Fermentation
Preparation
• 24 hour cultures of Escherichia coli, Bacillus subtilis, and Proteus vulgaris (enough for groups to share)
• phenol red glucose medium in test tubes with Durham tubes (3 per group)
• phenol red lactose medium in test tubes with Durham tubes (3 per group)
Materials
• 24 hour cultures of Escherichia coli, Bacillus subtilis, and Proteus vulgaris (enough for groups to share)
• phenol red glucose medium in test tubes with Durham tubes (3 per group)
• phenol red lactose medium in test tubes with Durham tubes (3 per group)
• test tube racks (1 per group)
• inoculating loops (1 per group)
• strikers (1 per group)
• Bunsen burners (1 per group)
• labeling tape (1 per group)
• labeling markers (1 per group)
3.19: SIM Deep Tests
Preparation
• 24 hour cultures of Escherichia coli, Proteus vulgaris, and Staphylococcus aureus (enough for groups to share)
• SIM deeps (3 per group)
Materials
• 24 hour cultures of Escherichia coli, Proteus vulgaris, and Staphylococcus aureus (enough for groups to share)
• SIM deeps (3 per group)
• test tube racks (1 per group)
• inoculating needles (1 per group)
• strikers (1 per group)
• Bunsen burners (1 per group)
• labeling tape (1 per group)
• labeling markers (1 per group)
• Kovac's reagent (enough for groups to share)
Expected Test Results
H2S production indole production motility
Escherichia coli - + +
Proteus vulgaris + - +
Staphylococcus aureus - - -
3.20: Coagulase Test
Preparation
• inoculate petri plates with Staphylococcus aureus and Staphylococcus epidermidis 24 hours before class (enough from groups to share)
Materials
• petri plates with Staphylococcus aureus and Staphylococcus epidermidis 24 hours before class (enough from groups to share)
• transfer pipet
• rabbit plasma (suitable for coagulase test)
• slides (slide test)
• test tubes (test tube test)
• sterile plastic inoculation loops
• tape
• markers
3.21: Gelatin Hydrolysis
Preparation
• 24 hour cultures of one gelatin hydrolysis positive species and one gelatin hydrolysis negative species (enough for groups to share):
• gelatin hydrolysis positive species: Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Serratia marcescens
• gelatin hydrolysis negative species: Staphylococcus epidermidis, Escherichia coli
• gelatin hydrolysis medium in test tubes (2 per group)
Materials
• 24 hour cultures (enough for groups to share)
• gelatin hydrolysis medium in test tubes (2 per group)
• test tube racks (1 per group)
• inoculating needles (1 per group)
• strikers (1 per group)
• Bunsen burners (1 per group)
• labeling tape (1 per group)
• labeling markers (1 per group) | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/03%3A_Instructor_Setup/3.15%3A_Cytochrome_C_Oxidase.txt |
Preparation
• 24 hour cultures of Escherichia coli, Alcaligenes faecalis, and Pseudomonas aeruginosa (enough for groups to share)
• nitrate broth medium in test tubes with Durham tubes (3 per group)
Materials
• 24 hour cultures of Escherichia coli, Alcaligenes faecalis, and Pseudomonas aeruginosa (enough for groups to share)
• nitrate broth medium in test tubes with Durham tubes (3 per group)
• 0.8% sulfanilic acid in dropper bottles (enough for groups to share)
• 0.6% N, N-Dimethyl-alpha-naphthylamine (enough for groups to share)
• zinc powder (enough for groups to share)
• wooden applicator sticks for zinc powder
• test tube racks (1 per group)
• inoculating loops (1 per group)
• strikers (1 per group)
• Bunsen burners (1 per group)
• labeling tape (1 per group)
• labeling markers (1 per group)
3.23: MR-VP Tests
Preparation
• 24 hour cultures of Escherichia coli and Enterobacter aerogenes
• MR-VP broth in test tubes (two per group)
Materials
• 24 hour cultures of of Escherichia coli and Enterobacter aerogenes
• MR-VP broth in test tubes (two per group)
• test tube racks (1 per group)
• inoculating loops (1 per group)
• strikers (1 per group)
• Bunsen burners (1 per group)
• labeling tape (1 per group)
• labeling markers (1 per group)
• empty test tubes (two per group)
• transfer pipets (2 per group)
• methyl red reagent in dropper bottles (enough for groups to share)
• Barritt's reagent A in dropper bottles (enough for groups to share)
• Barritt's reagent B in dropper bottles (enough for groups to share)
3.24: EMB Agar
Preparation
• 24 hour cultures of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus (enough for groups/individual students to share)
• EMB agar plates (three per student or three per group)
Materials
• 24 hour cultures of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus (enough for groups/individual students to share)
• EMB agar plates (three per student or three per group)
• test tube racks (1 per group/student)
• inoculating loops (1 per group/student)
• strikers (1 per group/student)
• Bunsen burners (1 per group/student)
• labeling markers (1 per group)
3.25: Mannitol Salt Agar
Preparation
• 24 hour cultures of Staphylococcus aureus and Staphylococcus epidermidis (enough for groups/individual students to share)
• mannitol salts agar plates (one per student or one per group)
Materials
• 24 hour cultures of Staphylococcus aureus and Staphylococcus epidermidis (enough for groups/individual students to share)
• mannitol salts agar plates (one per student or one per group)
• test tube racks (1 per group/student)
• inoculating loops (1 per group/student)
• strikers (1 per group/student)
• Bunsen burners (1 per group/student)
• labeling markers (1 per group)
3.26: DNA RNA and DNA Replication
Instruction Instructions
This activity works best when students are stepped through the activity by their instructor. Walk through the instructions one step at a time and keep students together on that same step. Walk around to make sure students are following instructions accurately. Correct students who deviate from instructions or who skip ahead.
3.27: PCR
Preparation & Materials
It is recommended to choose a PCR kit provided by a science education supplier. Follow the materials and preparation instructions for the kit you choose. You will also need to provide students with detailed laboratory instructions since kit instructions differ. Here are a couple examples of kits:
3.28: DNA Fingerprinting
Preparation
• 1X TAE buffer (enough for the electrophoresis chambers you are using and for making the gels)
• electrophoresis gels (enough for electrophoresis chambers you are using - one for the class or one for each group)
• 1% agarose heated in 1X TAE buffer
• at least 3 wells are needed per group (4 if you are using a ladder)
Materials
• 1X TAE buffer (enough for the electrophoresis chambers you are using and for making the gels)
• electrophoresis gels (enough for electrophoresis chambers you are using - one for the class or one for each group)
• power supplies (enough for the electrophoresis chambers you are using)
• DNA samples:
• option 1: have students load loading dye into the wells, but have loading dye in different microcentrifuge tubes labeled as "East coast virus," "West coast virus," and "Midwest virus." Use this option if you don't have enough class time for letting the gel fully run and stain. If you are using this option, print out copies of one of the gel images below (1 per student) so students can analyze the results
• option 2: purchase a virus DNA fingerprinting kit through a biology education supplier that has a quick-stain approach. You may wish to include in this option a DNA ladder.
• sterile micropipette tips to dispense 20 μL
• micropipettes to dispense 20 μL
• rulers
Gel Result Printouts
Choose one of the gel images below if you are using option 1 described above. Print out enough of the gel of your choice for each student to have one printout.
3.29: Bacterial Transformation
Materials
• bacterial transformation kit(s) (enough for entire class) - examples of kits are linked below
• bacterial transformation protocol handouts (1 per student)
• additional materials may be required by the kit you choose
Preparation
• follow the specific preparation instructions in the bacterial transformation kit you choose
3.30: Protozoan Parasites
Materials
• prepared microscope slides of Plasmodium sp. (enough for the class to share)
• prepared microscope slides of Trypanosoma cruzi (enough for the class to share)
• prepared microscope slides of Trichomonas vaginalis (enough for the class to share)
• (if using 1000x) immersion oil (enough for the class to share)
• light microscopes (1 per student or 1 per pair of students)
• colored pencils | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/03%3A_Instructor_Setup/3.22%3A_Nitrate_Reduction.txt |
Materials
• prepared microscope slides of Enterobius vermicularis eggs and adult worms (enough for the class to share)
• prepared microscope slides of Dipylidium caninum eggs and adult worm compsite (enough for the class to share)
• prepared microscope slides of Schistosoma sp. eggs and adult worms (enough for the class to share)
• (if using 1000x) immersion oil (enough for the class to share)
• light microscopes (1 per student or 1 per pair of students)
• colored pencils
3.32: Fungal Parasites
Materials
• Aspergillus slides (1 per student or 1 per pair of students)
• microscopes (1 per student or 1 per pair of students)
• colored pencils
3.33: Viral Epidemic
Setup Instructions
• Follow setup instructions in the kit.
Activity Instructions
• There will need to be an even number of people in the "population," so if your class has an odd number of students, the instructor can join the simulation to make the numbers even.
• Watch students carefully when they add their sample into the well plates to make sure they are putting them in the correct locations.
• Use a random group generator to randomize student pairs (otherwise, groups of students who are in the same social circle will end up sharing their drops with each other and you will be less likely to get good results). You can find a free random group generator at ClassTools.Net
3.34: Virus Bioassay
Materials
• plant grow lights
• tomato or bean seeds
• rolling tobacco (3 different brands mixed together) (1 gram per group)
• Tobacco Mosaic Virus Inoculation Kit (1 per class)*
• 50 mL beakers (1 per group)
• mortar and pestle (enough for groups to share)
• balances (enough for groups to share)
• weigh papers/boats (1 per group)
Note
*I have found the beans they provide in this kit difficult to grow. Unless you are good at growing these types of beans, recommend to use seed packets for tomato or bean plants and follow the instructions on the packet.
3.35: Control of Microbial Growth
Preparation
• prepare 3 TSA petri plates per student
• spreadsheet with formulas preprared to calculate t-test results for the class results
Materials
• 3 TSA petri plates per student
• sterile cotton swabs (3 per student)
• sterile distilled or DI water in beakers, containers or cups (1 per group)
• soap in squeeze bottles (1 per group)
• disinfectant spray (1 per group)
• markers
• graph paper and rulers or computers for graphing (1 per student)
• spreadsheet prepared to calculate t-test results for the class results
3.36: Bacterial Susceptibility to Antibiotics (Kirby-Bauer Test)
Preparation
• TSA petri plates (enough for 3 per group)
• 18-24 hour TSB cultures of Staphylococcus aureus (one per group or enough for groups to share)
Materials
• TSA petri plates (enough for 3 per group)
• 18-24 hour TSB cultures of Staphylococcus aureus (one per group or enough for groups to share)
• test tube racks (1 per group)
• Bunsen burners (1 per group)
• strikers (1 per group)
• sterile cotton swabs (3 per group)
• waste containers with disinfectant for used cotton swabs (1 per group)
• tweezers (1 per antibiotic type - label tweezers so they do not get mixed up)
• antibiotic disks (one of each type per group):
• erythromycin (15 µg)
• penicillin G (10 units)
• streptomycin (10 µg)
• tetracycline (30 µg)
3.37: Human Microbiome
Preparation
• 2 TSA plates per student
Materials
• 2 TSA plates per student
• sterile cotton swabs (2 per student)
• DI or distilled water
• markers
• materials for Gram staining
• plain microscope slides (at least 1 per group)
• Bunsen burners (1 per group)
• inoculating loops (1 per group)
• droppers with saline, DI water, or distilled water
• slide warmer
• wood test tube clamps/holders (1 per group)
• small chemical waste containers (1 per group)
• crystal violet in a dropper bottle (1 per group or enough for two groups to share one bottle)
• Gram's iodine in a dropper bottle (1 per group or enough for two groups to share one bottle)
• 95% ethanol in a dropper bottle (1 per group or enough for two groups to share one bottle)
• safranin in a dropper bottle (1 per group or enough for two groups to share one bottle)
• distilled or DI water in squirt bottles (1 per group)
• bibulous paper (enough for each groups to blot their slides)
• light microscopes (1 per group)
• (optional, but recommended) Wax pencils (1 per group)
• (if using oil immersion) immersion oil dropper bottles
• (if using oil immersion) lens paper
3.38: Unknown Bacteria Identification Project
Preparation
• 24 hour TSB cultures, each with one Gram-negative species listed on the project flow chart and one Gram-positive species listed on the project flow chart (enough for one per pair of students)
• See Instructor Setup sections for the following chapters:
• Plating on Petri Plates for Isolation
• Gram Stain
• Starch Hydrolysis
• SIM Deep Tests
• Cytochrome c Oxidase
• Coagulase Test
• Fermentation Test
• An assignment in your learning management system (such as Canvas) where students can upload their project reports and have them checked with TurnItIn. Be sure to include your grading rubric in TurnItIn and Canvas. | textbooks/bio/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/03%3A_Instructor_Setup/3.31%3A_Helminth_Parasites.txt |
An Introduction to Microbiomes
Microorganisms represent the fundamentals of life and interact with almost every facet of it. Yet, for the majority of their existence they have been largely ignored, or rather unseen by humans. The unraveling of the complex interactions between all life forms is an endless duty and seems to generate more questions than answers. However, the cracks in knowledge produced by peering into the unknown offers insight into our life and the world around us. These minute creatures have shaped Earth’s evolution since their dawn almost three-and-a-half billion years ago, and continuously affect our environment and health. The importance of the planet’s collection of microbes is realized more and more each day, and our unceasing investigation of them will surely unlock secrets of life we could never imagine.
The study of microbiomes is fairly novel in the context of understanding and applying their communal existence to ourselves and surroundings. Though scientists have recognized symbiotic relationships and traditionally focused on individual microbes and their interactions with human health, environmental impact, industrial applications, etc., their respective communities and influence as a whole in these areas have only just begun to be elucidated. That is in no small part due to the daunting task of cataloging the immense and complex interplay between the multitude of different microorganisms in a given environment, though rapid advances in technology have begun to ease analysis.
What is a microbiome?
A microbiome can be best described as a collective polymicrobial community, or ‘microbiota’, and its associated activity with genetic and physio-chemical constituents in a defined spaciotemporal habitat (Figure 1). These members of the microbiota include bacteria, archaea, algae, protozoa, fungi, and viruses (though the latter is somewhat debated since viruses and their derivatives aren’t technically living). Within this symbiotic context with a particular eukaryotic host, the entire entity is termed a ‘holobiont’ and the aggregate of genetic material termed the ‘hologenome’. Interactions between these partners may have long occurred, shaping the evolution of each, whereas others may be novel or transient, sometimes resulting in prompt change and infectious diseases. The change in the normal microbiota, or dysbiosis, can result in a variety of different diseases. As so, their study has been especially important in the fields of life sciences, human health, and medicine.
Quick Quiz
Query \(1\)
Our fascination with microorganisms begun before we even fully understood them or were even able to see them. Their implications concerning human health were primarily explored during the ‘golden age of microbiology’ with the work of Louis Pasteur and Robert Koch. Their experiments and discoveries shed light on not only the ubiquitous nature of microbes, but their importance in our everyday lives. Other significant milestones and historical microbiological development can be viewed in Figure 2. Human health and infectious diseases were central to the field of microbiology, though food microbiology, industrial applications, and microbial ecology became increasingly explored. Over the last couple centuries, a microbial catalog of knowledge has slowly grown, but much of these findings were limited to those organisms that could be cultured and measured. The advent of sequencing and ‘multi-omics’ technologies has since allowed researchers to document microorganisms that were previously missed or ignored with traditional techniques, and with further advances, larger microbial communities and symbioses can be better understood. The ‘microbiome’ was first defined in the late 1980s when a group of microbial ecologists were studying the rhizosphere, which provided context to better describe these polymicrobial communities (Whipps et al., 1988). Many other similar definitions have been published since then with varying specifics on genetic expression, symbioses, and ecological interactions (Lederberg & McCray, 2001, Marchesi & Ravel, 2015, Berg et al., 2020). The ‘holobiont’ concept stems from Adolf Meyer-Abich’s ‘theory of holobiosis’ proposed in 1943 and was independently conceived and popularized in the early 1990s by Lynn Margulis, though it only described the host and a single symbiont (Margulis, 1991, Baedke et al., 2020). Since then has been expanded to include the entire microbiota in multiple symbiotic contexts (Simon et al., 2019). In recent decades there has been a steady increase in microbiome publications as the subject has grown in popularity. Along with that, there has been more analytical breakdown as certain microbiomes are being described with emphasis on specific members, such as the ‘bacteriome’, ‘archaeome’, ‘mycobiome’, ‘protistome’, and ‘virome’, and these terms are best used to refer to the distinct contribution of those particular microbes within the entire microbiome context. In general, though, most microbiomes are delineated by their specific host or type of environment, with the human microbiome being the most popular example.
The Human Microbiome
It was evident that the human microbiome and its involvement in a micro and macro scale needed to be characterized. The Human Microbiome Project (HMP) set out in 2007 with this as one of its primary goals (Turnbaugh et al., 2007). The program also set out with initiatives to develop a set of microbial genome sequences, explain the relationship between disease and microbiome changes and evaluate the data with multi-omics approaches, develop new tools and technology for computational analysis, establish a data analysis and coordinating center and research repositories, as well as address ethical, social, and legal implications of HMP research (Human Microbiome Project). The second phase of the HMP launched in 2014, called the Integrative Human Microbiome Project (iHMP), having the main mission to completely characterize the human microbiota with a key focus on human health and disease using three projects: pregnancy and preterm birth, onset of inflammatory bowel disease (IBD), and onset of type 2 diabetes (NIH Human Microbiome Project, The Integrative HMP (iHMP) Research Network Consortium, 2019). Aside from these, the human microbiome and disruption of the microbiota has been linked to several other important conditions and diseases including multiple sclerosis, diabetes (types 1 and 2), allergies, asthma, autism, and cancer (Backhed et al., 2012, Hsiao et al., 2013, Petersen and Round, 2014, Trompette et al., 2014, Garrett, 2015, Lloyd-Price et al., 2016).
It makes sense that the human microbiome can have such an impact on human health and behavior if you consider that we are essentially a collection of organisms forming a living entity. In a way, our symbionts may even actually define more of who we are than just our own unique biological makeup. For instance, the ratio of microbial cells associated with a human body could equal, if not exceed (traditional estimates were tenfold), the number of human cells (Sender et al., 2016). Even more interesting is viewing our genetic makeup; the human genome contains about 20,000 genes, but its hologenome contains > 33 million genes brought by its microbiota (Huttenhower et al., 2012, Lloyd-Price et al., 2016, Simon et al., 2019). Furthermore, the composition and rate of change of each person’s microbiota is distinctive from one individual to another since it is influenced by variables like age, lifestyle, diet, antibiotics, occupation, environment, etc. (Gilbert et al., 2018). The genetic wealth and member diversity contributed from the microbiota has roles in adaptation, survival, development, growth, and reproduction of the holobiont and can affect fitness in the short term as well as have long lasting effects concerning the evolution of both partners (Rosenberg, and Zilber-Rosenberg, 2011).
Co-evolution of the host-microbiota symbiosis can be considered even more unique when viewing the microbial consortium at different locations or organs in the host, as their makeup is governed by and reflects specific physiological processes in those areas. For example, the bacteria found in the human gut microbiota are primarily from the phyla Bacteroides and Firmicutes, whereas Actinobacteria and Proteobacteria command the skin microbiome, though there is some overlap and it is important to note that there are differences depending on exact location (e.g. dry vs. moist areas of the skin) (Grice and Segre, 2011, Jandhyala et al. 2015). Though there are differences between various microbiota within a holobiont, they can still influence each other to some degree. In the case of the gut and skin microbiotas in humans, deemed the ‘gut-skin axis’, there are indications that both the health of the gastrointestinal (GI) tract and skin, as well as their response to stressors, are correlated (Levcovich et al., 2013, O’Neill et al., 2016, Salem et al. 2018). Even more interesting is the effects certain microbiota can have on germ-free organs like the brain. Studies on the ‘gut-brain axis’ show that the microbiota in the GI tract, and in some cases disruption of it, are associated with many mental illnesses and neurodegenerative disorders including depression, anxiety, autism, schizophrenia, Parkinson’s disease, and Alzheimer’s disease (Clapp et al. 2017, Foster et al. 2017, Cryan et al. 2019). A variety of different ‘axes’ which demonstrate interplay between microbiota, organs, and locations have been identified in the human body and much of what is known about their connections is novel and early in its research.
Environmental Microbiomes
Not only can our microbiome regulate who we are, but those communities in the surrounding environment can affect us, our microbiome, and others. Environmental microbiomes can directly or indirectly affect our health through ecological interactions. For example, soil microbiomes in the rhizosphere of plant roots and plant microbiomes of economically important crops have implications in agriculture, human health, and ecology (Saleem et al., 2019, Hirt, 2020). Plant growth, health, soil nutrient cycling and availability, and defense against potential pathogens are dictated by their own symbionts as well as their microbial neighbors in the ground, which include a variety of bacteria, protists, viruses, and network of fungi known as mycorrhizae (Busby et al., 2017, Hannula et al., 2017, Pratama and van Elsas, 2018, Zhong et al., 2019,). By better understanding the functional interactions of all the players in these environmental microbiomes, it may be possible to address problems like agricultural soil fertility, plant disease, and pollution. Thus, harnessing better microbiomes either by specifically engineering a microbial consortia for a targeted area/specimen or transplanting a natural community could improve sustainable agricultural practices which are desperately needed to feed the expanding population of humans while protecting the environment (Elhady et al., 2018, Arif et al., 2020, Hernandez-Alvarez et al., 2022).
Studying animal-microbiome systems can also give us information about our environment. For instance, how anthropogenic-induced changes have impacted things like climate-change and approaches to maintain homeostasis with various environmental factors. A promising reservoir of research is the microbiota of marine animals since most of the planet is covered in water. Corals, sponges, various fish, and marine mammals have all been investigated to document the response of their microbial symbionts to changing environmental factors (Apprill, 2017). Other animal microbiome models are being used in an analogous means to understand how the microbiota could potentially influence human health and fitness. The microbiomes of classic biomedical models such as the fruit fly, zebrafish, and nematode worm are being investigated since these organisms are well known and readily available to work with in many labs (Douglas, 2019). Though, it is important to consider a variety of animal host-microbiome models as each could contribute unique insights to beneficial microbial interactions within the human holobiont. For example, the gut microbiome of honeybees, the skin microbiome of freshwater polyps, and the individual interaction of Vibro fischeri and the Hawaiian bob-tailed squid have provided valuable information to understanding host-microbiome relationships (Douglas, 2019). Further research of these and other systems are needed for the pursuit of technological and medical advances or cultural/societal changes necessary for overall human and planetary benefit.
Microbiome Analysis
Traditionally, it has been difficult to characterize complete microbial communities, as most projects require substantial time and resources. Though, advancements in NGS and ‘omics’ technologies have begun to allow researchers to tackle microbiome analytics using a variety of approaches including metagenomics, metatranscriptomics, proteomics, metabolomics, culturomics, etc.(Integrative HMP (iHMP) Research Network Consortium, 2014, Bashiardes et al., 2016, Daliri et al., 2017, Janson and Hofmockel, 2018, Lin et al., 2019, Diakite et al., 2020, ). Each technique is selected and applied depending on the experimental setup and what questions are being addressed. For instance, does the research care about the identification of members of the microbiome, how they are interacting with the host or each other, what macromolecules are present, what genes are being expressed, what is the functional potential, etc. These different types of investigations can then be integrated together in network analyses to establish linkages and correlations within the microbiome datasets, though statistical models and analytical tools must be carefully selected to avoid false outcomes and shortcomings (Jiang et al., 2019). Due to the particular limitations of these approaches and the unavoidably large datasets produced by microbiomic studies, it is paramount to continually develop novel technologies to unearth the knowledge buried in these microbiomes.
Considering the abundance of microbiomes that no doubt play role in human health, the extent to which microorganisms sway our lives is almost impossible to foresee, and the opportunities they present for improvement to industry, agriculture, environmental and human health is potentially unlimited.
Check Your Understanding
• What is the difference between a microbiome and microbiota?
• In what ways does a microbiome influence a holobiont?
• How can an environmental microbiome indirectly impact human health? | textbooks/bio/Microbiology/Microbiomes_-_Health_and_the_Environment_(Parks)/01%3A_An_Introduction_to_Microbiomes/1.01%3A_An_Introduction_to_Microbiomes.txt |
Analyzing Microbiomes
Choosing the correct approach when characterizing a microbiome is important when considering what questions are trying to be answered. Who are the players? What is the function? What genes are being expressed? How do they interact?
Even though when assessing a microbiome all members of the microbiota should be considered, some insight can be gained by viewing individual groups, such as the prokaryotic component, which can be further compartmentalized into the bacteriome and archaeome. In some cases, it is difficult to completely characterize all of the members due to inherent biological differences between these communities, and in other situations, certain groups may be absent or irrelevant to the study. In so, analyzing these individual pieces could help reveal their distinct importance and contribution to the overall big picture.
The type of analysis also depends on the research question. If the sole interest is to identify all the bacteria in a sample, then a metagenomic approach may work best. Whereas, if an interest lies in what the microorganisms are doing, then a metatranscriptomics or metaproteomics approach would be more appropriate. If there is an interest in one or a few particular members of the microbiome, culture-based techniques (culturomics) could be implemented, which can often catch rare microbes that are missed using sequencing techniques because of data filtering protocols (Lagier et al., 2012, Allaband et al., 2019). However, it is usually difficult to characterize an entire microbiome based on just a few microbes and achieving multiple isolations is extremely difficult as a ‘one-size-fits-all’ type media does not exist.
Sample Collection
Depending on the type of research questions and the particular microbiome of interest, there are different ways to collect microbial communities that will be subjected to analysis. That is, the approach to collecting microbes from the human gut is much different than getting a soil sample. Really, there is no perfect method for any one microbiome, though some may be more difficult to obtain than others, and will depend on a variety of factors such as feasibility, cost, patient acceptance, and downstream analytical methods (Allaband et al., 2019). It can also be very difficult to capture all microbes within a microbiome sample, as inevitably some will be missed or cannot be collected by a particular technique. For example, when analyzing the gut microbiome, stool collection is preferred due to its ease and frequent accessibility, but this method often misses microbes in the small intestine and others which are mucosally adherent as opposed to excreted (Eckburg et al., 2005, de Carcer et al., 2010, Allaband et al., 2019). Additionally, collection and storage materials, storage time, and transport options should be considered as these can compromise sample integrity and create problems for various analytical approaches (Costello et al., 2009, Caporaso et al., 2011, McDonald et al., 2018).
Approaches to Characterization
What is the Difference between Culture-Dependent and Independent Methods of Microbial Community Characterization?
-Excerpt taken from Findley and Grice, 2014 available under the Creative Commons CC0 public domain dedication:
Up until the 1980s, microbiologists routinely relied on culture-dependent methods for microbial isolation, identification, and characterization. Colony morphology, stains (i.e., Gram stain), biochemical characteristics (i.e., coagulase test), motility tests, antibiotic resistance profiles, and other characteristics guided bacterial and/or fungal identification and taxonomy. However, this approach has several limitations, including an inability to mimic in vivo conditions and selection against slow-growing and/or fastidious organisms. With recent advances in sequencing technologies and development of bioinformatics tools and reference databases, researchers are now better equipped to capture microbial diversity without the biases of culture-based approaches.
Culture-independent methods of microbial identification rely on a targeted amplicon strategy, which employs highly conserved microbe-specific molecular markers and does not rely on growing isolates in pure culture. The 16S ribosomal RNA (rRNA) gene is used for bacterial identification, while fungi and other microeukaryotes are identified using either the 18S rRNA gene or the Internal Transcribed Spacer (ITS) region. A complementary approach to amplicon-based surveys is whole genome shotgun metagenomics. With this approach, one can identify the microbiota present and gain insight into the functional potential of the microbiota in an untargeted manner.
Within the culture-independent targeted approach, PCR amplification techniques, like quantitative PCR (qPCR) and reverse transcriptase PCR (RT-PCR) can be implemented to detect and quantify specific organisms, genes, or expression values within the microbiome sample. Untargeted approaches typically give whole community characterization and include metagenomics, metatranscriptomics, metaproteomics, and metabolomics which have a variety of applications depending on the type of study.
Culturomics
Cultivating all the microorganisms within a certain microbiome can be quite challenging and time-consuming, but nonetheless can provide important information that would otherwise be missed with other approaches. Creating media that mimics the original environment can help to capture organisms that exist at lower concentrations than that of the detectable threshold of molecular tools (Lagier et al., 2012). The application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has enhanced culture-dependent studies by allowing fast and reliable identification of bacterial taxa in a cost-effective manner (Seng et al., 2009, Lagier et al., 2012). Even though there have been advances in culturomics, the approach still has certain biases based on the types of microbes that can be easily grown (e.g. aerobic microbes are much easier to grow than anaerobic ones in most cases), as well as the selection for those that grow more rapidly and outcompete the others in a sample (Allaband et al., 2019). Thusly, it is imperative to close the gap between microbial richness observed in nature versus what is cultivable in the lab in order to better understand in vivo microbial functioning within a microbiome (Sarhan et al., 2019).
Metagenomics and DNA sequencing
Next-generation sequencing (NGS), also known as high-throughput sequencing (HTS), allows the generation of thousands of sequencing reads in a cost-effective manner (Nkrumah-Elie, et al., 2018). These reads can then be bioinformatically processed and annotated to identify microbial taxa, genes, potential functions, etc. depending on the sample type and experimental conditions (Rivera-Pinto et al., 2018). However, differences in wet lab work, sequencing platforms, processing pipelines, and statistical approaches can affect data output, which make reproducibility come into question (Gloor et al., 2016). Usually, information about microbiome compositions are provided as relative abundances instead of the true abundances due to sequencing platform imitations. This data is considered an arbitrary sum and is referred to as compositional data (Jiang et al., 2019). As sequence generation and pipelines improve and become standardized, the development and accessibility for analytical tools can help to streamline and produce consistent and more accurate results while aggregating data for microbiome characterization and meta-analysis without advanced expertise (Markowitz et al., 2007, Dhariwal et al., 2017, Gonzalez et al., 2018, Chong et al., 2020, Mitchell et al., 2020, Bharti and Grimm, 2021, Chen et al., 2021).
Metatranscriptomics and RNA sequencing
While metagenomic studies have been instrumental in identifying various microbes and genes in a given sample, it does not differentiate between viable cells or when those genes are being expressed (Gosalbes, et al., 2011, Bashiardes et al., 2016). Metatranscriptomic strategies of microbiome analysis allow researchers to capture gene activity which can delineate the functional dynamics of a particular community and it’s interactions with its host or environment. These RNA-based approaches can complement metagenomics, as the extent of expression of those annotated genes can be measured (Franzosa et al., 2014). This is considerably important concerning certain disease states because the presence of a pathogen does not always produce deleterious conditions, but rather it is the functional activity of certain organisms.
RNA-seq experiments usually begin with isolation of total RNA in a sample, then selection is based upon whether it is prokaryotic or eukaryotic in origin, and mRNA must be isolated from other RNA species (i.e. rRNA and tRNA) (Bashiardes et al., 2016). cDNA is then synthesized from the mRNA, adapters are ligated to create a library, amplification and sequencing follows, and the generated reads are mapped to a reference genome where expression can be measured (Giannoukos et al., 2012, Bashiardes et al., 2016). These experiments can be difficult and sensitive, as care must be taken with sample collection, and avoiding contamination and degradation from ribonucleases is important to maintain integrity of the samples as well. Furthermore, metatranscriptomics may not always give the whole picture of community expression due to the high complexity of organisms, the delicateness of RNA, the wide range of transcript expression, and various limitations with current technology (Shakya et al., 2019).
Metaproteomics
Metaproteomics gives a snapshot of the entire protein content of a microbiome sample under various conditions, which can further provide insight into gene activity and metabolic functions. This strategy identifies peptides by matching tandem mass spectrometry (MS/MS) data to protein sequence databases, which can be further assigned to functional groups via annotation databases (Sajulga et al., 2020). This approach can be convoluted though, as many peptides match to similar proteins produced by different organisms, and proteins can be multi-functional making it difficult for assignment to any one group. Additionally, bioinformatic analysis of metaproteomes isn’t exactly standardized which makes reproducibility and comparison of studies problematic (Schiebenhoefer et al., 2019, Sajulga et al., 2020).
Metabolomics
Metabolomics analyzes the non-protein metabolites that are produced and/or regulated by a given microbiome, which adds another angle of characterization to community-host-environmental interaction and functional dynamics. By understanding how various microbes process metabolites like natural products, nutrients, and medications, we can better determine how they influence their ecosystem through molecular interactions. Researchers typically obtain these molecules through gas or liquid chromatography and subsequently analyze them by mass spectrometry (Allaband et al., 2019, Bauermeister et al., 2021). Metabolomics strategies can either be targeted or untargeted, where the former is more sensitive and compares metabolites to a pre-determined bank with reference standards, however, it cannot detect novel molecules. Since most metabolites produced by microbiomes are unknown, an untargeted approach using tandem mass spectrometry is useful in detecting many small molecules, though annotation of modified metabolites is challenging (Watrous et al., 2012, Wang et al., 2016, Allaband et al., 2019). As with other microbiome analytical approaches, various data analysis tools and pipelines of the metabolome need to be standardized to a degree to improve overall research context. Moreover, major advancements in the known inventory of microorganism-derived metabolites and their functions are required to enhance metabolomics studies (Lee-Sarwar et al., 2020, Bauermeister et al., 2021).
Conclusion and Future Directions
While various approaches to characterizing a microbiome each have their advantages and drawbacks, network analyses integrating all the -omics (i.e. metagenomics, metatranscriptomics, metaproteomics, and metabolomics) could be best to gain a holistic view of compositional and functional dynamics (Bashiardes et al., 2016, Jiang et al., 2019). Novel and improved approaches to collect and analyze various microbiomes will help to minimize loss of information and provide a better picture of how these communities interact with and influence their host or environment. Also, the establishment of more comprehensive publicly available databases, and protocols for streamlining computational procedures will allow more consistent and reproducible research for upon which knowledge can be accumulated.
Check your Understanding
• What is the difference between culture-dependent and independent methods of microbiome analysis?
• What is the importance of culturomics in the scope of microbiome analysis?
• How can inaccurate or inconsistent results be produced from different metagenomic approaches to microbiome analysis?
• When would a metatranscriptomic analysis be more appropriate than a metagenomic approach? How do these approaches complement each other?
• What are the benefits and limitations of metabolomic analysis?
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Environmental Metagenomics
Chapter taken from Hozzein, 2020 [1] available under the Creative Commons Attribution 3.0 License.
Introduction
Metagenomics can be defined as the techniques and procedures that are used for the culture-independent analysis of the total genomic content of microorganisms living in a certain environment [2]. It has many useful applications with very promising potential in both medical and environmental microbiology. The most common use of metagenomics in environmental microbiology is studying the diversity of microbial communities in particular environments through the analysis of rRNA genes and how these communities change in response to changes in physical and chemical properties of these environments [3].
Metagenomics also provides an opportunity to obtain and identify novel enzymes with industrial applications from extreme environments where unculturable extremophiles live. In such circumstances, functional metagenomics enables the isolation of genes coding for extremozymes, enzymes that are capable of being catalytically active in extreme conditions, or genes that will allow for better understanding of the mechanisms that make such organisms resistant to extreme environmental conditions [4].
Metagenomics has special importance when it comes to studying soil microbiology. It is estimated that the number of distinct microorganisms in 1 gram of soil exceeds the number of microbial species cultured so far [5]. Therefore, metagenomics seems to be the ideal culture-independent technique for unraveling the biodiversity of soil microorganisms and to study how this biodiversity is affected with continuously changing conditions.
Sequencing technologies and metagenomics
Recently, taxonomic profiling, characterization, and analysis of microbial communities are being mostly performed using different next-generation sequencing (NGS) platforms. Metagenomic samples are high-throughput, short-read sequences, and the cost is relatively decreasing. In addition, these platforms are advantageous, avoiding the need for cloning of DNA fragments [6].
Recent advances in NGS technologies were developed to suit various numbers of applications, cost, and capabilities [7]. The most commonly used platforms are the 454 Life Sciences (Roche) and Illumina systems (Solexa) [8]. The 454 sequencing technology, which was the first commercially available next-generation technology, is based on the pyrosequencing technique. It provides high throughput and relatively cheap analysis [9]. During the sequencing reaction in this technique, nucleotide incorporation into the growing chain is detected by the capture of the released pyrophosphate, which is converted into a light through an enzymatic reaction. Different nucleotides are sequentially added into each nucleotide incorporation event; therefore the light signal can be attributed to a specific nucleotide. Finally, the light signals are converted into sequence information. In the 454 pyrosequencer, the DNA fragments are amplified after being fixed on beads in a water-oil emulsion [10]. Pyrosequencing has been employed widely in the analysis of microbial diversity in many environments including marine environments [11] and different soil environments [12, 13].
Illumina sequencing technology relies on the use of fluorescently labeled reversible terminator nucleotides. Instead of being chemically modified to prevent further DNA synthesis (dideoxynucleotides) which is the case with Sanger sequencing, the terminator nucleotides are attached with blocking group that can be removed from the nitrogen base in a single step. DNA synthesis takes place on a chip where primers are attached. After each cycle, the dyes attached to each nucleotide are excited by laser followed by scanning of the incorporated bases. In order for the next synthesis cycle to proceed, the blocking group and the dye are first removed by a chemical reaction. Illumina sequencing platform was successfully used to study microbial diversity in many environments [14, 15, 16].
In addition to the abovementioned technologies, recently developed sequencing technologies are available and being employed in metagenomic studies. These include SOLiD 5500 W Series developed by Applied Biosystems, single-molecule real-time (SMRT) DNA sequencing from Pacific Biosciences, and Ion Torrent semiconductor sequencing [8]. More innovative technologies are being developed that could be of great use for metagenomic studies in the near future. Strand sequencing technologies, currently being developed by Oxford Nanopore technologies, enable the sequencing of intact DNA strand that passes through a protein nanopore [17]. Irys Technology, developed by BioNano Genomics, represents one of the very promising new technologies in genomics era [8].
The metagenomic approaches
Metagenomics research strategy starts with selecting a proper ecological or biological environment of interest that hosts a wide variety of microbial communities which may have potential biotechnological applications. Environments that attract metagenomic researchers are mainly those characterized with extreme conditions or unique environmental conditions. These include environments with highly acidic or alkaline pH; high metal concentrations, pressures, or radiation; and high salinity or extreme temperatures [4].
Metagenomic analysis starts with isolating genomic DNA that represents the whole community in the soil sample, constructing a DNA library from the isolated DNA, and screening the available library for a target gene. It is important here to select the DNA extraction method that will provide enough yield and DNA that represents the diversity of the whole microbial community in the target environment. This is still one of the most challenging steps of metagenomic analysis. The chemical and physical characteristics of soils are very wide and complex, depending on the type of the soil examined, that will make it difficult to develop a reference method for DNA extraction from soils. Besides, soils contain many substances that are co-extracted with genomic DNA and harbor inhibitory effects on the downstream processing of the extracted DNA. Examples include humic and fulvic acids [18]. Therefore, optimization and comparison between different extraction methods are usually required for each type of soil [19, 20, 21, 22].
A DNA library is then constructed from the genomic DNA isolated from the target environment. This is performed by fragmenting the isolated DNA into fragments with appropriate sizes that would allow for their cloning. This is performed by either using restriction enzyme digestion or mechanical shearing. DNA fragments obtained from such processes are cloned into the proper cloning vector. Plasmid vectors are used for small DNA fragments, and the libraries generated are called small-insert genomic libraries. Large inserts are cloning into cosmid or fosmid vectors which can hold inserts up to 40 kb in size or BAC vector which can carry inserts with sizes that exceed 40 Kb [23].
DNA libraries are usually constructed in a microorganism that is well-studied and is easy to manipulate inside the laboratory such as Escherichia coli. In case there is a need for expressing the genes within the DNA inserts in other microorganisms, shuttle vectors are used to transfer the libraries into a proper host [24].
Finally, a screening assay is applied to search for a gene of a particular function, and the gene product is functionally analyzed. There are two different metagenomic strategies that are commonly used in research. The first one is focused on the use of marker genes such as the ribosomal genes 16S rRNA [25] and 18S rRNA [26] to study the composition of the microbial community in a certain environment or specific protein-coding gene with medical or industrial importance [27, 28, 29]. Such a strategy is called targeted metagenomics. The second approach is the shotgun metagenomics, in which a wide coverage of genomic DNA sequences is achieved using high-throughput next-generation sequencing to assess the entire taxonomic structure or functional potential of microbial communities [30].
The most challenging aspect of the screening process in metagenomics is the analysis of a huge amount of sequence data that are generated from the constructed library. A wide range of bioinformatic tools has been developed over the years to help analyze the metagenomic data and compare it to available online databases.
Acknowledgments
This work was funded by the Deanship of Scientific Research at Princess Nourah bint Abdulrahman University, through the Research Groups Program Grant no. (RGP-1438-0006).
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21. Gupta, P., Manjula, A., Rajendhran, J., Gunasekaran, P., & Vakhlu, J. (2017). Comparison of Metagenomic DNA Extraction Methods for Soil Sediments of High Elevation Puga Hot Spring in Ladakh, India to Explore Bacterial Diversity. Geomicrobiology Journal, 34(4), 289–299. https://doi.org/10.1080/01490451.2015.1128995
22. Mazziotti, M., Henry, S., Laval-Gilly, P., Bonnefoy, A., & Falla, J. (2018). Comparison of two bacterial DNA extraction methods from non-polluted and polluted soils. Folia Microbiologica, 63(1), 85–92. https://doi.org/10.1007/s12223-017-0530-y
23. Simon C., Daniel R. (2010) Construction of Small-Insert and Large-Insert Metagenomic Libraries. In: Streit W., Daniel R. (eds) Metagenomics. Methods in Molecular Biology (Methods and Protocols), vol 668. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-823-2_2
24. Lam, K. N., Cheng, J., Engel, K., Neufeld, J. D., & Charles, T. C. (2015). Current and future resources for functional metagenomics. Frontiers in Microbiology, 6, 1196. https://www.frontiersin.org/article/10.3389/fmicb.2015.01196
25. Païssé, S., Valle, C., Servant, F., Courtney, M., Burcelin, R., Amar, J., & Lelouvier, B. (2016). Comprehensive description of blood microbiome from healthy donors assessed by 16S targeted metagenomic sequencing. Transfusion, 56(5), 1138–1147. https://doi.org/https://doi.org/10.1111/trf.13477
26. West, D. (2018). Use of an 18s rRNA metagenomics approach as a method of detection of multiple infections in field blood samples collected on FTA cards from cattle , MSc by research thesis, University of Salford.
27. Nurdiani, D., Ito, M., Maruyama, T., Terahara, T., Mori, T., Ugawa, S., & Takeyama, H. (2015). Analysis of bacterial xylose isomerase gene diversity using gene-targeted metagenomics. Journal of Bioscience and Bioengineering, 120(2), 174–180. https://doi.org/10.1016/j.jbiosc.2014.12.022
28. Ufarté, L., Laville, É., Duquesne, S., & Potocki-Veronese, G. (2015). Metagenomics for the discovery of pollutant degrading enzymes. Biotechnology Advances, 33(8), 1845–1854. https://doi.org/10.1016/j.biotechadv.2015.10.009
29. Lanza, V. F., Baquero, F., Martínez, J. L., Ramos-Ruíz, R., González-Zorn, B., Andremont, A., Sánchez-Valenzuela, A., Ehrlich, S. D., Kennedy, S., Ruppé, E., van Schaik, W., Willems, R. J., de la Cruz, F., & Coque, T. M. (2018). In-depth resistome analysis by targeted metagenomics. Microbiome, 6(1), 11. https://doi.org/10.1186/s40168-017-0387-y
30. Quince, C., Walker, A. W., Simpson, J. T., Loman, N. J., & Segata, N. (2017). Shotgun metagenomics, from sampling to analysis. Nature Biotechnology, 35(9), 833–844. https://doi.org/10.1038/nbt.3935 | textbooks/bio/Microbiology/Microbiomes_-_Health_and_the_Environment_(Parks)/02%3A_Analyzing_Microbiomes/2.02%3A_Environmental_Metagenomics.txt |
Human Health and Disease
The human microbiome plays a vital role in maintaining homeostasis of various organ systems and protecting against infectious agents. It can be subcategorized into local or regional microbiomes throughout the body, such as the gut, oral, skin, lung, and vaginal microbiomes. These manicured microecosystems are highly organized and complex, with each person having their own distinct makeup and distribution of various microorganisms. Though, individual microbiomes are unique, there is still capacity to understand key role players in the community and their potential for adaptation to improve human health in general.
Disruption, or dysbiosis, of the microbiome can cause serious diseases and allow for opportunistic infections to occur. This disturbance can be caused by a variety of factors including changes in diet, exercise, geographical location, age, habits, as well as medical intervention like antimicrobial chemotherapy. This change in microbial composition subsequently leads to the development and exacerbation of a number of diseases impacting essentially every aspect of human physiology including the digestive, respiratory, integumentary, central nervous, and reproductive systems. Alternatively, dysbiosis may be the result of a particular disease, and in other cases disease-dysbiosis may be bidirectional (Silverman et al., 2019). The human microbiome can be influenced by many of the above mentioned factors, but also others like life partners, pet ownership, and occupation, which do not necessarily correlate with or contribute to dysbiosis (Kiecolt-Glaser et al., 2019, Kates et al., 2020).
A major driving feature of many of these elements is the host’s genetics, which can also directly impact their microbiome (Tabrett and Horton, 2020). Part of the gut microbiome and even individual types of bacteria have been shown to be heritable (Kurilshikov et al., 2017), and it is likely that other areas of the human microbiome, like the skin microbiome, could be passed from parent to offspring as well (Si et al., 2015). The host genotype is intimately linked to its microbiome and has been shown to affect dysbiosis-induced diseases such as inflammatory bowel disease and atopic dermatitis (Knights et al., 2013, Dabrowska and Witkiewicz, 2016, Woo and Sibley, 2020). Host genetics and their microbiome can even indirectly affect one’s susceptibility to other diseases. For example, the attractiveness of mosquitos to particular individuals is an heritable trait which is influenced by the many factors, including the skin microbiome; therefore altering the potential of contracting a mosquito-borne pathogen, such as a Plasmodium parasite which causes malaria (Martinez et al., 2020). It seems there is a combination between environmental factors, host genetics, behavior, and others that continually build and adapt the human microbiome, however, is worthy to note that the former may play a larger role in shaping particular microbiomes, such as the gut microbiome (Rothschild et al., 2018).
References
1. Dąbrowska, K., & Witkiewicz, W. (2016). Correlations of Host Genetics and Gut Microbiome Composition. Frontiers in Microbiology, 7, 1357. https://www.frontiersin.org/article/10.3389/fmicb.2016.01357
2. Kates, A. E., Jarrett, O., Skarlupka, J. H., Sethi, A., Duster, M., Watson, L., Suen, G., Poulsen, K., & Safdar, N. (2020). Household Pet Ownership and the Microbial Diversity of the Human Gut Microbiota. Frontiers in Cellular and Infection Microbiology, 10, 73. https://www.frontiersin.org/article/10.3389/fcimb.2020.00073
3. Kiecolt-Glaser, J. K., Wilson, S. J., & Madison, A. (2019). Marriage and Gut (Microbiome) Feelings: Tracing Novel Dyadic Pathways to Accelerated Aging. Psychosomatic Medicine, 81(8), 704–710. https://doi.org/10.1097/PSY.0000000000000647
4. Knights, D., Lassen, K. G., & Xavier, R. J. (2013). Advances in inflammatory bowel disease pathogenesis: linking host genetics and the microbiome. Gut, 62(10), 1505. https://doi.org/10.1136/gutjnl-2012-303954
5. Kurilshikov, A., Wijmenga, C., Fu, J., & Zhernakova, A. (2017). Host Genetics and Gut Microbiome: Challenges and Perspectives. Trends in Immunology, 38(9), 633–647. https://doi.org/https://doi.org/10.1016/j.it.2017.06.003
6. Martinez J., Showering, A., Oke, C., Jones, R. T., & Logan, J. G. (2020) Differential attraction in mosquito–human interactions and implications for disease control. Phil. Trans. R. Soc. B. Biol. Sci, 376(1818), 20190811. https://doi.org/10.1098/rstb.2019.0811
7. Rothschild, D., Weissbrod, O., Barkan, E., Kurilshikov, A., Korem, T., Zeevi, D., Costea, P. I., Godneva, A., Kalka, I. N., Bar, N., Shilo, S., Lador, D., Vila, A. V., Zmora, N., Pevsner-Fischer, M., Israeli, D., Kosower, N., Malka, G., Wolf, B. C., … Segal, E. (2018). Environment dominates over host genetics in shaping human gut microbiota. Nature, 555(7695), 210–215. https://doi.org/10.1038/nature25973
8. Si, J., Lee, S., Park, J. M., Sung, J., & Ko, G. (2015). Genetic associations and shared environmental effects on the skin microbiome of Korean twins. BMC Genomics, 16(1), 992. https://doi.org/10.1186/s12864-015-2131-y
9. Silverman, G. J., Azzouz, D. F., & Alekseyenko, A. v. (2019). Systemic Lupus Erythematosus and dysbiosis in the microbiome: cause or effect or both? Current Opinion in Immunology, 61, 80–85. https://doi.org/10.1016/j.coi.2019.08.007
10. Tabrett, A., & Horton, M. W. (2020). The influence of host genetics on the microbiome. F1000Research, 9, F1000 Faculty Rev-84. https://doi.org/10.12688/f1000research.20835.1
11. Woo, T. E., & Sibley, C. D. (2020). The emerging utility of the cutaneous microbiome in the treatment of acne and atopic dermatitis. Journal of the American Academy of Dermatology, 82(1), 222–228. https://doi.org/10.1016/j.jaad.2019.08.078 | textbooks/bio/Microbiology/Microbiomes_-_Health_and_the_Environment_(Parks)/03%3A_Human_Health_and_Disease/3.01%3A_Human_Health_and_Disease.txt |
The Gut Microbiome
The microbiome associated with the human gastrointestinal tract, termed the gut microbiome, may arguably be the most important component of the collective human microbiome. It has been shown to affect numerous other regions of the body and serves a role in many types of diseases throughout it. This is because many of the microbial products are absorbed in the alimentary canal and distributed throughout the body via the cardiovascular system.
Sample procurement to characterize the gut microbiome usually comes in the form of fecal matter, which readily available and less invasive. However, there are novel attempts to characterize gut microbiome constituents by sampling the mucosal-luminal interface (Yan et al., 2020). DNA and other features of interest can then be extracted from the microbes to provide information regarding gut microbiome composition and function.
Composition
The gut microbiome is quite versatile and its composition can vary widely among individuals with different ethnicities, across geographic locations, and with age (Yatsunenko et al., 2012, Gaulke and Sharpton, 2018). The host’s genetics do play a role in the composition of the gut microbiome, as certain members are heritable, while other’s abundances are the causal result of congenital diseases (Xu et al., 2020). Though diet, which is closely related to the aforementioned variables, may be the single strongest influencing factor when it comes to structuring this digestive community and alterations are reflected in both short-term and long-term dietary interventions (David et al., 2014, Xu and Knight, 2015). Indeed, the type of diet, such as a high-fat diet, will have a direct impact on the gut microbiome. More specifically, the unique macromolecules within the gut can modify microbial abundance and predicted functions. For example, a certain oil in the diet could result in higher microbial richness, however, this diversity may not alone be a predictor of better health outcomes (Abulizi et al., 2019). Dietary vitamin content and even receptors required for processing them are in part modulated by the microbiome. For example, vitamin D deficiency and downregulation of its affiliated receptor is associated with pathogenesis of various diseases and their restoration promotes healthy host-microbe homeostasis (Jin et al., 2015). It is also interesting to note that diet usually changes throughout the year as certain food items become available or absent depending on the season. An increased gut prevalence of Bacteroidetes, which can digest complex plant carbohydrates, could be explained by a diet consisting heavily of produce during a harvest season (Davenport et al., 2014). The composition and diversity of the gut microbiome can even be linked to personality traits. Those individuals with larger social networks have a more diverse gut microbiome, and those affected by stress and anxiety show an altered composition with reduced diversity (Johnson, 2020). The connection between the gut microbiome and mental states and behaviors will be discussed more in the section “Mental Health”.
Dysbiosis and Disease
The gastrointestinal mucosal immune system modulates and responds to the gut microbiome, where the resident members aid in its development and transient pathogens cause dysfunction, leading to various diseases (Shi et al., 2017). Inflammatory diseases such as systemic lupus erythematosus, rheumatoid arthritis, IBD, and ankylosing spondylitis are implicated in the impaired interaction between the intestinal microbiota and mucosal immune system (Arbuckle et al., 2003, Mikuls et al., 2012, Costello et al., 2015). Microbial dysbiosis within these cases are marked by changes in composition and diversity of specific groups of organisms (Shi et al., 2017). So, the gut microbiome can not only serve as an indicator for such diseases, but may also eventually become a target for treatment, with maintaining proper homeostasis a major goal.
Gut microbiome dysbiosis, whether it causes a particular disease or manifests as a result of it, is quickly becoming the focus of many gastrointestinal illnesses. Specific members of the gut microbiome (e.g. bacteriome, mycobiome, virome) can even vary and be affected differently depending on the disease and its severity. For example, while much of the focus is usually on bacteria in the gut, it is important to not discount the contributions of other microorganisms like bacteriophages and fungi. The gut phageome can vary in diversity, complexion, and in so has been shown to contribute to diseases like IBD, malnutrition, and AIDS (Norman et al., 2015, Reyes et al., 2015, Monaco et al., 2016, Shkoporov and Hill, 2019). Similarly, the gut mycobiome has shown to have roles in IBD, colorectal cancer, and even neurological diseases (Forbes et al., 2019, Gu et al., 2019, Qin et al., 2021). Though, it is likely the complex interactions between all members of the gut microbiome with the host undoubtedly play a role in various degrees for the progression of gastrointestinal and other diseases.
Those with Inflammatory Bowel Disease (IBD) experience substantial fluctuations in the gut microbiome, which is implicated due to signs and symptoms of the disease state, diet, as well as increased medication during flare ups (Walters et al., 2014, Halfvarson et al., 2017). IBD is a blanket term for two disorders, ulcerative colitis and Crohn’s disease, which are characterized by chronic inflammation of the gastrointestinal tract and commonly results in frequent diarrhea, abdominal pain, bloody stool, weight loss, and fatigue. It is likely that the gut microbiome plays both a role in the development of these conditions and is affected by the induced changes. The gut virome component responds to disease-induced environmental change of IBD patients by shifting from virulent to temperate bacteriophage core, which subsequently affects the bacteriome and dysbiosis condition (Clooney et al., 2019). A familial study of patients with Crohn’s disease showed an increase in the number of pathogenic bacteria, and a decrease in beneficial bacteria. In particular, three potentially pathogenic biofilm-forming species from both the bacteriome (Serratia marcescens and Escherichia coli) and mycobiome (Candida tropicalis) interact with each other and impact the host immune system by increasing levels of proinflammatory cytokines and mucolytic enzymes which cause oxidative and tissue damage (Hoarau et al., 2016).
Diabetes mellitus is another disease in which its progression is partially in response to gut microbiome dysbiosis. While there are other factors that play into diabetes such as culture, genetics, age, lifestyle, etc., this can be interlinked with an individual’s microbiome. Studies over both type 1 and type 2 diabetes have shown that a change in the gastrointestinal microbial ecology is associated with diabetic subjects as compared with their healthy counterparts (Giongo et al., 2010, Larsen et al., 2010, Musso et al., 2011, Sohail et al., 2017). Type 1 diabetes stems from destruction of pancreatic beta cells, which results in decreased insulin production and elevated blood glucose levels. The gut microbiome may contribute to the disease by dysbiosis-associated immune regulation causing destruction of the beta cells and/or gut leakiness, endotoxemia, and chronic low-grade inflammation associated with certain enteric microbes (Cani et al., 2007, Lee et al., 2011, McDermott and Huffnagle, 2014, Sohail et al., 2017). Type 2 diabetes is characterized by the body’s improper regulation and secretion of insulin and is associated by hyperglycemia. Physiological changes in the GI tract could be induced by dysbiosis in the gut microbiome leading to gut permeability and insulin resistance (Everard and Cani, 2013). In general, the gut microbiota composition is less in terms of diversity and richness for those with type 2 diabetes, though an increase in abundance of certain groups like Bifidobacterium could improve conditions associated with pathogenesis (Cani et al., 2007, Sohail et al., 2017). It seems that an alteration of the microbial gut profile has considerable effects on host metabolism, gastrointestinal physiology, gut fermentation capabilities, and immunity which can have many other downstream implications (Boulange et al., 2016).
Obesity is commonly associated with type 2 diabetes as well as other comorbidities that are linked to gut microbiome dysbiosis. As mentioned earlier, diet strongly affects the composition and function of the gut microbial community and subsequently impacts the host’s metabolic capabilities. In fact, a high-fat/calorie or improper diet that results in dysbiosis is evident earlier than the signs of the host’s metabolic abnormalities, and so gut microbiome dysbiosis may be the principle ingredient responsible for the progression of obesity and type 2 diabetes (Nagpal et al., 2018). A high-sugar diet seems to promote an abundance of Mollicutes, a class within Firmicutes, which in turn suppresses Bacteroidetes (Turnbaugh et al., 2008), and this higher ratio of Firmicutes/Bacteroidetes has been proposed as a biomarker and hallmark indicator for obesity (de Bandt et al., 2011, Zou et al., 2020). However, it is important to consider other factors such as physical activity and medication that could cause a variation in diversity of the gut microbiome, as this ratio does not always definitively denote obesity (Magne et al., 2020). Though, there are similarities between many of the microbiome-linked contributing factors of pathogenesis progression for diet-induced diseases. For example, a high-fat diet induces increased gut permeability that allows exogenously produced bacterial compounds (e.g. lipopolysaccharides) to diffuse through the intestinal barrier, which then can interact with immune cells and lead to inflammation (Cani et al., 2007, Nagpal and Yadav, 2017). However, diet isn’t the sole factor that can promote gut leakiness, and it seems that obesity in general causes an altered gastrointestinal state (Nagpal et al., 2018).
Chemotherapeutic Intervention and C. diff
Medication and antimicrobial drugs can also have drastic effects on the gut microbiome that invoke risk of secondary infections, allergies, and other diseases like obesity (Becattini et al., 2016). Though many of these prescribed treatments are necessary to combat infectious diseases, the aftermath may have more serious consequences. Not only does antimicrobial therapy disrupt the resident microbiome, but misuse, suboptimal dosing, and patient noncompliance can create conditions conducive to fostering antimicrobial resistance through selective pressure.
Clostridioides difficile (commonly called C. diff) infections are directly associated with antibiotic-induced dysbiosis in the gastrointestinal tract. C. difficile is part of the normal microbiota, however, as an opportunistic pathogen it can invade or colonize empty niches brought about by dysbiosis and cause potentially fatal episodes of pseudomembranous colitis, which is associated with abdominal cramping, pain, sepsis, and bouts of diarrhea (Kho and Lal, 2018). Infection and transmission of this organism has been well known for its prevalence in hospital settings, primarily affecting the elderly and immunocompromised, however, community-associated infections have recently increased in what was once considered low-risk populations (Rouphael et al., 2008, Baker et al., 2010, Hensgens et al., 2012, Benson et al., 2015, Johanesen et al., 2015). It is also alarming that this organism has resistance mechanisms to many commonly prescribed antimicrobials, including β-lactams, aminoglycosides, lincomycin, tetracyclines, and erythromycin (George et al., 1978), and more recently ‘hypervirulent’ strains have developed resistance to fluoroquinolones (He et al., 2013, Johanesen et al., 2015). C. difficile infections have an enrichment of fungi that associate with the bacteriome and perhaps antifungal therapy could help improve treatment success if administered in conjunction with specific antibacterial drugs (Stewart et al., 2019). Though, these infections can have lingering impacts on the gut microbiome, as further antimicrobial therapy that is usually required can perpetuate the situation. The inflammation as a result of the disease induces the production of antimicrobial peptides by epithelial cells and neutrophils which inhibit the growth of the natural resident commensal microbes (Leber et al., 2015).
While many events of gut dysbiosis are directly linked to the chemotherapeutic effects on microbes since they are prescribed to target microbes responsible for the infection, some medications which are meant to address other diseases, like antidepressants for mental health, have undesired effects on the microbiome (Maier and Typas, 2017). In the cases of multi-drug combinations (e.g. non-steroidal anti-inflammatory drugs (NSAIDs), antidepressants, laxatives, proton-pump inhibitors (PPIs), etc.), it is not the number of drugs that affect gut microbiome diversity, but rather the types of drugs (Rogers and Aronoff, 2016). Though these scenarios become complicated as it is difficult to ascertain whether the alterations observed on the microbiome are from the drug’s mechanism of action, a downstream side effect, or originate from the condition that is being treated, and it is likely a complex combination of all factors for each disease and medication (Rogers and Aronoff, 2016, Maier and Typas, 2017, Jackson et al., 2018).
Fecal Microbiota Transplant
Although pharmaceutical drugs are of dire importance to treat various diseases, whether they are infectious in nature or not, other avenues must be pursued for those that may benefit from restoration of the gut microbiome. Probiotics and fecal microbiota transplant (FMT) can serve as viable options for the prevention and treatment of gut microbiome dysbiosis. Probiotics are considered foodstuffs with microorganisms, usually bacteria (many being lactic acid bacteria) and yeast, and their byproducts that have a beneficial effect on human health when introduced into the body. Many probiotics are commercially available to consumers in the forms of products like yogurt, kefir, buttermilk, sauerkraut, pickles, premade vitamin supplements and many others (more information about probiotics and fermented foods can be found in the section “Food and Fermentation: Your Microbiome is What you Eat”). Specifically, probiotics can be used for the treatment and prevention of many of the aforementioned gut microbiome dysbiosis-associated diseases, especially those induced by antibiotics (Kim et al., 2019). The beneficial microbes outcompete pathogens for resources or prevent them from establishing a niche in which to grow (Ouwehand et al., 1999).
In more extreme cases of gut microbiome dysfunction and disease, like those from C. difficile infection (CDI) in which antibiotics are ineffective and can potentially exacerbate the problem, other measures must be taken. Fecal microbiota transplant therapy takes a stool sample containing the gut microbiome from a healthy donor and relocates it into the infected patient’s colon. The introduced microbiota then helps move the gut microbiome towards homeostasis by restoring the structure of beneficial microbes and metabolites (Fujimoto et al., 2021). This procedure is usually reserved for those patients with recurrent CDI and has shown to be highly successful and is considered safer and more effective than prolonged antibiotic usage (Mattila et al., 2012, Cammarota et al., 2015), though is also being investigated for first-line treatment of CDI (Camacho-Ortiz et al., 2017). FMT has gained traction for its success and is being further considered as a therapeutic option in other treatment protocols, such as those for cancer patients undergoing cancer immunotherapy to help improve response or manage toxicity (McQuade et al., 2020), and individuals undergoing allogenic hematopoietic stem cell transplant for hematological disorders that experience graft-versus-host disease complications from it (Zhang et al., 2021). However, precautions must be taken for FMT, as the donor’s sample could potentially harbor other pathogenic microbes, like multi-drug resistant Escherichia coli, that can result in pathogenesis, further complications, and even death for the recipient (DeFilipp et al., 2019, Martinez-Gili et al., 2020). More comprehensive research and FMT trials must be performed in order to optimize this procedure to better match donors with recipients and to further understand the exact mechanisms of microbiome rehabilitation.
Conclusion
Gut microbiome intervention may be the key to future treatments of diseases associated with dysbiosis like IBD, diabetes, obesity, colorectal cancer, etc., and offers a viable alternative to many traditional pharmaceutical interventions. Though, the gut microbiome is plastic and continually changes with its host’s environment and lifestyle, so stabilization is constant work. Additionally, creating an ideal ‘cocktail’ of microbes that will maintain homeostasis when implemented can be challenge. While there are general members of the gut microbiome that exist at a constant level and show some correlation to normal health, there may not be a true ‘standard’ gut microbiome due to the vast differences between people across the world. So, this type of therapy may require a more unique and individualized approach that depends on the disease and characteristics of both the host and their microbiome.
Check Your Understanding
• What factors influence the composition of the gut microbiome?
• How is gut microbiome dysbiosis brought about?
• What diseases are associated with gut microbiome dysbiosis? Are features of pathogenesis always a cause or effect of these events? Explain.
• Explain the treatment options that are available for gut microbiome dysbiosis.
References
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13. David, L. A., Maurice, C. F., Carmody, R. N., Gootenberg, D. B., Button, J. E., Wolfe, B. E., Ling, A. v, Devlin, A. S., Varma, Y., Fischbach, M. A., Biddinger, S. B., Dutton, R. J., & Turnbaugh, P. J. (2014). Diet rapidly and reproducibly alters the human gut microbiome. Nature, 505(7484), 559–563. https://doi.org/10.1038/nature12820
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15. DeFilipp, Z., Bloom, P. P., Torres Soto, M., Mansour, M. K., Sater, M. R. A., Huntley, M. H., Turbett, S., Chung, R. T., Chen, Y.-B., & Hohmann, E. L. (2019). Drug-Resistant E. coli Bacteremia Transmitted by Fecal Microbiota Transplant. New England Journal of Medicine, 381(21), 2043–2050. https://doi.org/10.1056/NEJMoa1910437
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The Oral Microbiome
The oral microbiome as with other site-specific microbiomes on and inside the human body is very distinct for each individual and its makeup and function is reflective of a variety of factors. Even within the context of the oral cavity, there are several unique niches with their own microbial ecosystem, including saliva, tongue, teeth, gingiva, throat, tonsils, and others. Each of these habitats exhibit diverse and complex interactions between bacteria, archaea, fungi, viruses, and protozoa, where dysfunction can lead to a number diseases, both rare and common (Wade, 2013, Sampaio-Maia et al., 2016).
Factors Affecting Composition
The composition of the oral microbiome and its respective niches is inherently dependent on host genetics, but other factors and habits such diet and smoking have a substantial effect on diversity. Members of the oral cavity demonstrate more heritability than the gut microbiome, and higher abundances of certain heritable organisms, such as Prevotella pallens, are associated with a lack of dental caries, while on the other hand, Streptococcus mutans and Lactobacillus species are linked to cavities (Davenport, 2017, Xiao et al., 2018). Detection of the specific groups of microbes has traditionally been difficult since many are fastidious and cannot be cultivated. So other techniques like metagenomics, metatranscriptomics, and proteomics have been implemented to better characterize the microorganisms present and their respective roles (Parahitiyawa et al., 2010, Grassl et al., 2016). These studies have found that the oral microbiota is quite different even between healthy individuals, and so it may be the microbial functionality that is more important in the progression of various diseases, such as the role of biofilm formation, plaque buildup, and sugar metabolism in the development of dental caries (Segata et al., 2012, Takahashi et al., 2010, Duran-Pinedo and Frias-Lopez, 2015, Sato et al., 2015, Davenport, 2017).
Dysbiosis and Disease
The oral cavity is such a dynamic location that constantly experiences a variety of different foods, drinks, oral hygiene products, and other environmental stimuli with each composed of a multitude of macromolecules, compounds, and potentially other microorganisms, and so it makes sense that its microbiome often fluctuates in diversity (Parahitiyawa et al., 2010). Though there are differences in composition between individuals, specific sites, and over periods of time, species such as Streptococcus mitis and Granulicatella adiacens are conserved and generally present throughout the oral microbiome, while the presence of other specific microbes are associated with a particular disease (Aas et al., 2005). However, diseases like Periodontitis, which is the chronic inflammation of the gums and tooth supporting structures and one of the most common oral diseases, has many organisms associated with the condition, so discerning which ones are primarily responsible is a complex task. There are a variety of viruses that cause oral-related conditions, such as the Human papilloma virus (HPV) which is known for causing lesions and warts in the mouth, as well as head and neck squamous cell carcinoma (Kumaraswamy and Vidhya, 2011). An increase of the protozoa Entamoeba gingivalis and Trichomonas tenax are observed in patients with gingival disease, but are not the causative agents, rather just taking advantage of the increased food sources (bacteria and food debris) from poor hygiene (Wantland et al., 1958). There is also a variety of fungi present in the oral cavity with Candida species being the most common, and many of the members in the oral mycobiome are responsible for chronic diseases, however correlation isn’t exactly clear (Ghannoum et al., 2010). Archaea, primarily methanogens, are also present in the oral microbiome, and while there aren’t technically any known pathogens in this domain, there is an increase in abundance observed in patients with periodontitis (Lepp et al., 2004, Mattarazo et al., 2011, Wade, 2013, Willis and Gabaldon, 2020).
Generally, the oral microbiome is linked with aspects oral health and has primary implications in dental and periodontal diseases, however it can contribute to vitality and diseases in other parts of the body such as cardiovascular disease, stroke, Alzheimer’s disease, cystic fibrosis, rheumatoid arthritis, diabetes, pneumonia, and preterm birth (Seymour et al., 2007, Duran-Pinedo and Frias-Lopez, 2015, Kori et al., 2020, Willis and Gabaldon, 2020). Additionally, the oral microbiome is implicated in various forms of cancer including esophageal, pancreatic, gastric, liver, colorectal, and oral (Willis and Gabaldon, 2020, Bakhti et al., 2021, Mohammed et al., 2021). In many of these types of cancers the Gram-negative anaerobe, Fusobacterium nucleatum, is a primary culprit as it can promote cancer by activation of cell proliferation, promotion of cellular invasion, induction of chronic inflammation and immune evasion (Al-hebshi et al., 2017, McIlvanna et al., 2021). Another contributing factor to cancer development is the use of tobacco products which cause oral microbiome dysbiosis (Al-habshi et al., 2017, Gopinath et al., 2021, Sajid et al., 2021). As with other microbiomes, deviation from the normal composition can result in altered function and progression of disease.
Saliva
Saliva is an important component in maintaining homeostasis in the oral cavity, as it lubricates food, initiates the digestive process, and defends against bacteria. Disruption in secretion can lead to changes in the oral microbiome which promotes progression of oral and other diseases (Grassl et al., 2016). Since saliva contacts virtually all surfaces within the oral cavity it is involved with all other duties of the mouth, and adaptive and innate immune defense mechanisms can be considered the most important in terms of microbiological clinical relevance. The protein and gycoprotein content regulates the oral microbiome by promoting the colonization of commensal microbiota while helping eliminate pathogenic microbes (Cross and Ruhl, 2018). These macromolecules aid in bacterial adhesion and biofilm formation so that they aren’t dislodged by salivary flow and other oral physiological processes (Mandel, 1987). The establishment of beneficial microbes prevents pathogenic bacteria from gaining a foothold, and the agglutinins found in saliva aid in removal through binding and then swallowing (Scannapieco, 1994). The co-evolution of the microbiota and humans has cultivated the development of specific bacterial adhesins for colonization of the preferred microorganisms, thus establishing a mutualism, though pathogenic microbes are quick to adapt to changing binding motifs (Springer and Gagneux, 2013, Cross and Ruhl, 2018).
Saliva can harbor numerous microbes, with one milliliter containing approximately one hundred million microbial cells (Marsh et al., 2015) and over 600 different species (Dewhirst et al., 2020, Willis and Gabaldon, 2020). In one study, the prominent genera found across various types of saliva samples (i.e. spit, drool, and oral rinse) from healthy individuals were Streptococcus (17.5%), Prevotella (15.5%), Veillonella (15.3%), Neisseria (12.7%) and Haemophilus (10%) (Lim et al., 2017). Though it can be difficult to differentiate a core salivary microbiota from other specific oral niches since it coats the oral cavity.
For those suffering from various diseases, the relative abundance of certain microorganisms and general composition of the oral microbiota is altered as compared to healthy controls. For example, patients suffering from chronic obstructive pulmonary disease (COPD) and periodontitis have varying abundances of Veillonella, Rothia, Actinomyces, and Fusobacterium in saliva samples (Lin et al., 2020). Periodontitis and COPD are comorbid diseases that are commonly associated with other conditions like rheumatoid arthritis, diabetes mellitus, and cardiovascular diseases (Scher et al., 2014, Wang et al., 2014, Chrysanthakopoulos and Chrysanthakopoulos, 2014, Lin et al., 2020). Dysfunction of bacterial ecology in saliva is exacerbated by COPD and periodontitis, and so restoration of the salivary microbiota may treat or reduce the severity of these diseases and their comorbidities (Jeffcoat et al., 2014, Zhou et al., 2014, Lin et al., 2020).
Alterations of the salivary microbiome are also associated with certain human viral infections like the herpes virus, influenza, and SARS-CoV (Blostein et al., 2021, Miller et al., 2021). Saliva generally is beneficial to oral health, though changes in it’s makeup could be detrimental and further aggravate disease. For instance, the saliva microbiota and their byproducts could be responsible for increased susceptibility of infection of gum tissues with herpes simplex virus 1, especially in individuals with periodontitis lesions (Zuo et al., 2019). Eukaryotic viruses can also directly interact with oral bacteria and affect disease severity, as is the case with streptococci and influenza which results in an increased viral load (Kamio et al., 2015). Similarly, among patients with COVID-19 there are differences in the salivary bacterial community based on SARS-CoV-2 viral load, though the exact dynamics and repercussions of the interaction isn’t yet well understood. It is possible that COVID-19-induced inflammation could directly impact the oral microbiome and contribute to other diseases connected with dysbiosis (Miller et al., 2021).
Dysbiosis of the fungal component in the salivary microbiome also contributes to overall oral microbial community changes and detrimental effects to the human host. The mycobiome is an important constituent of the oral microbiome though its member’s abundance is much less than that of the bacteriome. There are two main genera in the salivary mycobiome: Candida and Malassezia, where the former is associated with dental plaque bacteria, carbohydrate-rich microbial communities, and acidic pH conditions which contribute to dental caries (Hong et al., 2020). Other interactions between the mycobiome and bacteriome in saliva has been observed in the chronic inflammatory disease oral lichen planus (OLP). OLP causes swelling, discoloration, and open sores of the mucosal membranes in the oral cavity, primarily affecting the buccal region (cheek), but the lips, gingiva, and tongue may also be affected. Similar to plaque buildup and cavity formation, this disease is characterized by an increased abundance of genera Candida, but also Aspergillus, as well as a decrease in biodiversity (Li et al., 2019).
While diversity and abundance of specific groups within the salivary microbiome are associated with various diseases, it is important to consider other factors like diet, lifestyle habits, and genetics that can influence the progression of any particular disease.
Teeth, Plaque, and Cavities
Plaque formation occurs when a multispecies biofilm builds layers on the surface of teeth over time. This structure not only contains a variety of microbes, some of which are pathogenic, but proteins, carbohydrates, minerals, antimicrobial peptides and other compounds that dictate its structure and activity (Amerongen and Veerman, 2002, Flemmig and Beikler, 2011, Zarco et al., 2011). For normal healthy individuals, plaque biofilms are important in maintaining oral homeostasis and good tooth conditions as they can trap pathogens or prevent them from thriving due to competitive inhibition. However, regular detachment of these biofilms through oral hygiene and salivary flow are necessary to prevent pathogen establishment and their escape from immune responses and antimicrobial therapy (Avila et al., 2009, Filoche et al., 2009, Van Essche et al., 2010, Flemmig and Beikler, 2011, Zarco et al., 2011).
The persistence of oral biofilms contributes directly to cavity formation as carbohydrate-fermenting microbes within produce acidic byproducts which lower oral pH and damage tooth enamel (Selwitz et al., 2007, Ling et al., 2010). Cavities form once the surface layers of the tooth wear away and lesions form in the dentin, resulting in oral pain, tooth decay and loss (Selwitz et al., 2007, Zarco et al., 2011). Dental caries are the most prevalent disease for children worldwide, and dental care is the most common unmet need among children in the United States (Loesche and Grenier, 1976, Acs et al., 1999, Low et al., 1999, Peterson et al., 2013).
The fastidious nature of several members of plaque polymicrobial communities have made it traditionally difficult to characterize an exact consortium responsible for dental caries, however, recent studies using NGS technologies have detailed a few signature genera. Streptococcus mutans and Lactobacilli spp. are the primary culprits, but other genera such as Fusobacterium, Bifidobacterium, and Actinomyces are found in high abundance in people with cavities (Munson et al., 2004, Chhour et al., 2005, Corby et al., 2005, Peterson et al., 2013). The fungal yeast, Candida albicans, is also found regularly in children with severe early childhood caries (S-ECC) (Xiao et al., 2018). Interestingly, other members of Streptococcus including S. parasanguinis, S. mitis, S. oralis, and S. sanguinis are associated with individuals exhibiting good dental health (Corby et al., 2005, Peterson et al., 2013). Overall, during the progression of caries there is a reduction in species diversity in these communities (Peterson et al., 2013).
Gingivae and Periodontitis
Aside from dental caries, periodontitis, a.k.a. ‘gum disease’, is the other most common oral disease in humans, and it also results from alterations in oral microbial ecology. This inflammatory condition affects the supporting structures surrounding the teeth where the microbial communities that inhabit the subgingival area serve to trigger its onset (Hajishengallis and Lamont, 2012, Hong et al., 2015). Though progression of the disease comes in episodes, the continual breakdown of periodontium tissue (gingiva, periodontal ligament, cementum, and alveolar bone) leads to alveolar bone breakdown, formation of pocket lesions, and tooth loss (Listgarten, 1986). This condition is very difficult treat as specific antibiotics must be administered, though they are often unsuccessful as pathogens can hide in plaque, develop resistance, and quickly recolonize through reserves in the mucous membranes of the oral cavity. Once pockets form in the periodontium, periodontitis becomes irreversible, as the tissues are unable to reattach to the bone and the pathogens within cannot be completely eradicated or removed (Pihlstrom et al., 2005, Horz and Conrads, 2007, Van Essche et al., 2011, Zarco et al., 2011).
Culture-based studies have shown that periodontitis is associated with varying levels of abundance of three bacterial species: Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola, which are collective referred to as the ‘red complex’ (Socransky et al., 1998, Rocas et al., 2001, Socransky and Hafajee, 2005, Teles et al., 2010). More recent genetic analysis has revealed other bacteria associated with the disease, including those from the classes Clostridia, Negativicutes, and Erysipelotrichia; the genera Synergistes, Prevotella, and Fusobacterium; and the species Filifactor alocis (Vartoukian et al, 2009, Griffen et al., 2012, Costalonga and Herzberg, 2014, Willis and Gabaldon, 2020). Some methanogenic archaeal species have even been implicated in the disease, and perhaps serve a metabolic role as a ‘hydrogen sink’ for secondary fermenters (Lepp et al., 2004, Matarazzo et al., 2011). Viruses, both eukaryotic and bacteriophages, are also thought to play a part in the etiology of periodontitis. While bacteriophages manage biofilm formation through bacterial predation, the role of eukaryotic viruses found in the oral cavity such as those in the Herpesvirus family, is more enigmatic (Martinez et al., 2021). The protist Trichomonas tenax is frequently found in patients with severe periodontitis, though it is not known if its presence is a cause or a result of the disease (Benabdelkader et al., 2019. It is generally thought that this condition arises from events of dysbiosis causing an increase in community diversity and richness which then alters antagonistic/synergistic relationships, metabolic functions, and the oral environment (Kolenbrander et al., 2006, Shi et al., 2015, Ng et al., 2021). Those bacteria that may be absent or inhibited during dysbiosis conditions, and are associated with good periodontal health include those from the phylum Proteobacteria and the Firmicutes, class Bacilli, and the genera Streptococcus, Actinomyces, and Granulicatella (Griffen et al., 2012, Liu et al., 2012, Willis and Gabaldon, 2020).
Periodontitis resulting from oral microbiome dysbiosis has been linked to other diseases like chronic kidney disease, as well as several types of cancers including oral, esophageal, gastric, lung, pancreatic, prostate, hematologic, and breast (Fitzpatrick and Katz, 2010, Ioanndiou and Swede, 2011, Michaud et al., 2017). These associations stemming from the oral cavity could be due to dysbiosis-induced microbiome changes and recruitment of disease causing pathogens, production of harmful microbial-derived byproducts, and/or modulation of the immune system causing an increase in proinflammatory cytokines (Abnet et al., 2005, Kurkivuori et al., 2007, Chalabi et al., 2008, Meurman, 2010, Willis and Gabaldon, 2020). In reality, there are likely a multitude of factors and interactions between microbial communities and the host that lead to the progression of these diseases, however, it is still important to determine certain organismal signatures or functions that could indicate a condition and perhaps aid in diagnosis or treatment.
Tongue
The tongue is another niche for a variety of microbial communities, and though it is a maneuverable oral centerpiece that comes into contact with the rest of the cavity, it has its own unique makeup of microbes. The tongue microbiome serves as an ideal model to analyze changing microbial consortia and understand microbial community dynamics. By employing a novel computational method known as oligotyping, which relies on identifying certain nucleotide content of genetic sequences, a better resolution of microbial taxonomic distribution can be achieved (Eren et al., 2014, Welch et al., 2014, Wilbert et al., 2020). As expected, the tongue experiences large fluctuations in relative abundance across taxa, but this isn’t completely explained by obvious human behavior such as oral hygiene and food/liquid intake. The complex microbial organization that is observed is likely a combination of many factors, like other microbiomes, such as host immunity, physiology influenced by circadian rhythm, epithelial cell renewal, other microbial (e.g. bacteriophage) interactions, as well as certain host behaviors and lifestyles (Welch et al., 2014, Wilbert et al., 2020).
The changing microbiome of the tongue can also be linked to various diseases and conditions in both the oral cavity and throughout the rest of the body. Viewing the tongue for diagnosis isn’t a novel approach either, as traditional Chinese medical practices have viewed tongue phenotypes to discern various illnesses for thousands of years, and currently physical aspects of the tongue are being connected with its microbiome composition to better diagnosis certain diseases like oral, liver, gastric, colorectal, and pancreatic head cancers (Jiang et al., 2012, Han et al., 2014, Mukherjee et al., 2017, Cui et al., 2019 Lu et al., 2019, Mohammed et al., 2021). It is also interesting that members of the tongue microbiome can help regulate blood pressure and cardiovascular health through metabolism of dietary nitrate, and oral hygiene (e.g. tongue cleaning) can help promote the growth of these beneficial organisms (Tribble et al., 2019). While good habits like proper oral hygiene can cultivate a healthy oral microbiome and improve overall health, bad habits like smoking negatively affect microbial communities which contributes to various diseases. For example, metagenomic analysis investigating bacterial species and their gene content showed significant differences of certain species, strains, and metabolic pathways within the tongue microbiome between smokers and never smokers (Sato et al., 2020). This further demonstrates the usefulness of tongue microbiome analysis as a reliable technique to explore and diagnosis certain diseases and conditions.
Conclusion
Though not as well characterized as the gut microbiome, the oral microbiome has a significant impact on human health. Within the oral cavity, specific niche microbiomes of saliva, teeth, gums, and tongue are each compositionally unique and implicated in various conditions and diseases. Moreover still, other oral niches like the buccal mucosa, palate, pharynx, and tonsils not detailed here are distinctive and have certain associated diseases initiated with respective microbiome dysbiosis (Gao et al., 2014, Fukui et al., 2018, van der Meulen et al., 2018). Areas within the oral microbiome may eventually become routine sites for medical observation since samples are easily acquired, especially saliva, and microbial analysis can serve as a fast and reliable option for diagnosis and potential treatment of oral disorders as well as other diseases.
As the oral cavity is the initial point of contact and entrance for foreign microbial loads into the body, oral disease and its associated microbial ecology appear to serve as a conduit for a multitude of other diseases. In a sense the oral microbiome can be considered a precursor to the gut and lung microbiomes, as microbes are undoubtedly mixed with food, drink, and air before being swallowed or inhaled and taken further into the respective system. It is therefore important to understand and better characterize the oral microbiome so that medical diagnosis and intervention can be improved. However, with vast differences in microbial community diversity and composition between humans across the word, this will be quite the challenge to determine normalized healthy consortia respective to people of various geographic regions, ages, lifestlyes, etc.
Check Your Understanding
• What aspects of the oral microbiome allow for the formation of unique niches?
• How does dysbiosis within the oral microbiome contribute to certain diseases? (What aspects of changes in microbial community diversity and richness could cause oral and other diseases?)
• Why may saliva sampling be considered a viable option for diagnosis of oral and other diseases?
• How do oral biofilms contribute to cavity formation and tooth decay?
• Why are microbial infections associated with periodontitis hard to treat?
• How do certain lifestyles influence various niches of the oral microbiome and lead to disease?
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The Skin Microbiome
The microorganisms that inhabit the largest human organ are important in a variety of ways in regard to host health. The skin microbiome defends against pathogens, educates the immune system, helps wound healing, and moderates progression of diseases, and in return receives nutrients and real estate for colonization, creating a symbiotic establishment (Byrd et al., 2018). As the primary external interface to the environment, the skin serves as the initial physical barrier against invasion of potential pathogens. The territory of the skin itself is harsh for most microbes; its cool, acidic, dry, and considered nutrient poor, and so only those organisms that have adapted to these conditions can successfully colonize it. A majority of these microbes rely on obtaining nutrients from sweat, sebum, and dead skin cells by using proteases and lipases to break apart various compounds to liberate usable resources (Byrd et al., 2018). Though, there are adverse conditions and a general lack of food for microbes on the skin, there is still quite a diverse community which is unique to certain individuals and body locations.
Better characterization of the skin microbiome and its site-specific diversity can allow for greater understanding of skin diseases such as atopic dermatitis, acne, rosacea, and psoriasis that are associated with dysbiosis (Grice and Segre, 2011). The role of resident and transient microorganisms are important in the onset and progression of these types of diseases, and their study may help in diagnosis and treatment protocols.
Composition and Stability
Within a single square centimeter on the skin there can be up to one billion microorganisms, and this mixed community of bacteria, viruses, protozoa, fungi, and mites can be both good and bad for host health (Grice and Segre, 2011, Weyrich et al., 2015). Overall, there are four main bacterial phyla that constitute the human skin microbiome: Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes (in descending order of abundance) (Grice et al., 2009). Microbial composition primarily depends on the specific location of skin site, and particularly whether it is dry, moist, or sebaceous (i.e. oily). Moist areas like the bend of the elbow or the feet harbor bacteria like Staphylococcus or Corynebacterium species and tend to have higher species diversity, while sebaceous locations promote the growth of Propionibacterium species and are lower in diversity (Grice et al., 2009, Oh et al., 2016, Byrd et al., 2018).
The composition of fungi on the skin also depends on location and physiological properties. Fungal members of the skin microbiome primarily consist of species from the genus Malassezia, with some from Penicillium and Aspergillus, and less of a few other genera. Specifically, the community of fungi on the feet have a high amount of diversity and it tends to change more over time as compared with other locations on the body. This may be due to environmental exposure, sock and shoe usage, or the fact that specific sites like plantar heels, toe webs and toenails are commonly infected by fungal pathogens, which can be difficult to treat. (Findley et al., 2013).
Small arthropods less than half a millimeter also colonize the skin and makeup part of the microbiota. Two species of mites from the genus Demodex utilize lipids produced in the sebaceous regions of the skin; the larger species D. folliculorum tends to cluster around hair follicles while the smaller D. brevis situated near the eyelid rim is more antisocial (Schommer and Gallo, 2013). These lipid-eating mites normally have a symbiotic relationship with humans, however, they could potentially serve as mechanical vectors for transport of pathogenic bacteria, and their population buildup and/or associated events of dysbiosis could promote inflammatory reactions (Lacey et al., 2009, Lacey et al., 2011).
Eukaryotic viruses on the skin are primarily transient, exhibit lower site-specific affinity, and the most different from person to person. This is likely due to the fact that they are obligate intracellular pathogens with special constraints. Also, maybe as expected, various bacteriophage abundance in certain sites is dependent on the corresponding bacterial genera (Oh et al., 2016).
Microbial communities on the skin remain stable despite the constantly changing external environment that humans bring about. Over both short- and long-term time intervals, sebaceous sites are the most stable, and moist areas like the feet are the least stable. Interestingly, dry sites with high environmental contact and disruption, like the palms of hands, exhibit community stability over time (Oh et al., 2016). Though, variability and stability may be dependent on individual host habits and lifestyles. At an early age, however, there are drastic changes in the skin microbiome until it becomes established. At birth, the baby transitions from an essentially sterile environment in the womb, to open air and constant microbial exposure where initial colonization of the skin begins. The method of delivery also affects initial community composition on the skin. When a baby is born conventionally through the vaginal canal, the members of the skin microbiome reflect the vaginal microbiome, and if born via cesarean section, the infant’s skin community more closely resembles that of the mother’s skin (Dominguez-Bello et al., 2010). In the initial few months, the skin community predominately consists of Streptococci and Staphylococci bacteria, but as aging ensues the abundance of these genera decrease and diversity and numbers of others begins to increase and level out (Capone et al., 2011). This has long-term effects on health, as the evolution of an infant’s skin microbiome helps to regulate and mature both the skin and immune system.
Immune System Interaction
Exposure to potentially pathogenic microorganisms at a young age can help educate the immune system. Immediately after childbirth, initial skin colonization by microorganisms is allowed without the classical inflammatory response, though shortly after this period, these microbes promote the development of distinct components of the immune system for future pathogen encounters (PrabhuDas et al., 2011, Naik et al., 2012, Naik et al., 2015, Belkaid and Harrison, 2017). This has been observed in infant’s skin microbiome and an early colonization of Staphylococcus aureus being associated with a lower risk of developing atopic dermatitis, as the immune system is prepared for this organism that can exacerbate and perpetuate the disease (Kennedy et al., 2017, Blicharz et al., 2019). S. aureus can further modulate the immune system through toxin production and colonization, which initiates leukocyte responses and stimulates the adaptive and innate immune systems (Niebuhr, et al., 2011, Nakamura et al., 2013, Nakatsuji et al., 2016). Skin colonization of Staphylococcus epidermidis elicits similar immune responses, and a pre-association with this microbe could help defend against certain fungal and parasitic skin infections (Naik et al., 2012, Naik et al., 2015). Further investigation of microbe and immune system interactions will help uncover exact molecular functions and relationships, which could have future implications in therapy and treatment of skin diseases (Byrd et al., 2018).
Pathogens, Dysbiosis, and Skin Diseases
The skin microbiome also contributes to human health and combating infectious diseases in a preventative measure. In addition to educating the immune system, the commensal microorganisms that call the epidermis home help to prevent pathogen invasion by physically taking up niches and secreting certain antimicrobial compounds. For example, S. epidermidis, which is a part of the normal microbial flora of the skin, produces antimicrobial peptides and proteases that selectively targets and inhibits growth of pathogens such as S. aureus (Cogen et al., 2010, Iwase et al., 2010, Schommer and Gallo, 2013).
Though many commensal microorganisms on the skin can help to prevent pathogen colonization, these microbes themselves may be opportunistic pathogens and can cause an infection when certain conditions arise. For example, S. epidermidis is a common cause of infections when transmitted from the skin into the body, usually through a medical procedure like catheterization (Otto, 2012). Staphylococci are known for their ability to form biofilms on medical devices, which make them more difficult to treat (Otto, 2009). The fungi, Candida albicans, is another normal skin resident that can cause opportunistic infections. It is though that disruption of the normal skin flora, such as antibiotic therapy, could induce virulence factor production by the yeast, resulting in penetration of epidermal tissue and a subsequent case of candidiasis (Kuhbacher et al., 2017). Other skin diseases associated with dysbiosis, or various skin pathogens, include atopic dermatitis, acne, rosacea, psoriasis, and chronic wound healing.
Atopic dermatitis (AD), the most common form of eczema, is a chronic inflammatory condition of the skin that is caused by a mutation in several genes, including the fillagrin gene responsible for encoding a protein that helps maintain epidermal health (Bierber, 2008). AD is characterized by itchy, dry rashes that become vulnerable to infection when the skin barrier is breached by constant scratching, and occurs more frequently in areas with low microbial diversity, such as the inside of the elbow or back of the knee (Weyrich et al., 2015). S. aureus colonization is directly related to disease severity of AD, as it produces virulence factors that disrupt the integrity of the skin barrier. There is also an increase in abundance of S. epidermidis, and Clostridium and Serratia species, and this increase of select species suggests that a lower amount of microbial diversity plays a role in the progression of AD (Kong et al., 2012, Oh et al., 2013, Williams and Gallo, 2015). Selectively targeting pathogens such as S. aureus for treatment of AD proves to be difficult as the normal microbiota also suffers resulting in dysbiosis.
Psoriasis is similar to AD in that the disease results in inflamed, scaly skin plaques that are itchy and painful. This condition is also associated with altered microbial diversity, and there is an association with the development of psoriasis and oral streptococcal infections, though the connection is not exactly known (Norlind, 1955, Owen et al., 2000). Within psoriatic lesions, there is an increase Proteobacteria and Firmicutes, a decrease in Actinobacteria, and specifically a decrease within the genera Propionibacterium (Gao et al., 2008, Fahlen et al., 2012, Statnikov et al., 2013). Though there is a decrease in general of microbial diversity in psoriatic lesions, there hasn’t been any specific microbial causative agent identified for the disease.
Rosacea is a common chronic dermatosis which primarily manifests as persisting erythema (redness), telangiectasia (dilated or broken blood vessels), bulging, swelling, and/or raised patches in superficial facial skin (Picardo and Ottaviani, 2014). Like other cutaneous diseases, development of rosacea is linked with skin microbiome composition, and how those communities influence skin immune responses. In particular, an increase in Demodex mite abundance and density is observed in those with rosacea. They potentially contribute to the disease state through immune system activation, damaging epithelial tissue, and/or the exposure of antigenic proteins of bacteria released from their digestive tract (Forton and Seys, 1993, Georgala et al., 2001, Lacey et al., 2007, Koller et al., 2011, Casas et al., 2012, Forton, 2012, ). The induced reactions from microbiome shifts, genetics, and environmental factors then likely invoke other inflammatory triggers, which includes overgrowth of certain bacteria like S. epidermidis (Schommer and Gallo, 2013).
Acne (acne vulgaris) is a skin condition that results from hair follicles and sebaceous glands that are clogged with oil, bacteria, and dead skin cells, which creates whiteheads and blackheads. Propionibacterium acnes is a primary etiological agent in acne; it’s secretion of lipases, proteases, and hyaluronidases damage skin pores and induce inflammatory responses (McKelvey et al., 2012). Although this species of microbe is part of the normal skin microbiota, there are differences in certain strains of P. acnes that may explain differential virulence. Some disease-associated strains also have genes for antibiotic resistance, making treatment options other than chemotherapy a necessity (Fitz-Gibbon et al., 2013). Other commensal microorganisms, like S. epidermidis, could interact with P. acnes and be implicated in acne formation also, further demonstrating that residents can become pathogenic when opportunistic conditions arise (Bek-Thomsen et al., 2008, Weyrich et al., 2015, Dreno et al., 2017).
Chronic skin wounds (duration longer than three months) and their capacity to heal are also affected by the skin microbiome, especially in those individuals who are elderly, obese, immunocompromised, or diabetic (Weyrich et al., 2015, Byrd et al., 2018). Though the lesions or ulcers may not be initially caused by a microorganism, their presence, infection, and polymicrobial biofilm formation can be deleterious to the healing process and cause further complications (McKelvey et al., 2012, Wolcott et al., 2013). Analysis of skin wound microbiomes have shown a compilation of a diverse array of genera, but that microbial diversity is lower as compared with healthy skin (Gardiner et al., 2017, Kalan et al., 2019). Perhaps a higher microbial diversity allows for easier elimination of potential pathogens from the wound and promotes faster healing. An increase in facultative anaerobes, specifically the genus Enterobacter, are significant indicators in the persistence of chronic wounds and their lack of healing, possibly due to their versatile metabolism (Verbanic et al., 2020). Antibiotic therapy is an option to eliminate certain bacterial pathogens for chronic wounds, however, the resulting changes in the microbiome and addressing fungal and viral constituents may necessitate multiple different treatment approaches (Price et al., 2009). Further studies are needed in order to see whether individual therapy and targeted therapeutic intervention would promote faster healing in cases with chronic wounds (Kong, 2011, Weyrich et al., 2015, Verbanic et al., 2020).
It is likely that changes in microbial community composition and the elicited immune response work in combination with host genetics and other environmental factors to cause various cutaneous disorders (Weyrich et al., 2015 ). This makes treatment of these complex conditions difficult, and targeting a few potential pathogens is rarely successful, especially if it is not known whether their presence is a cause or effect. So, future therapeutic efforts may focus on a similar approach to treating gut dysbiosis with FMT, and as so, use healthy skin microbiota transplants to repair and stabilize the skin microbiome for patients inflicted with skin diseases (Williams and Gallo, 2015).
Conclusion
The skin microbiome is incredibly complex, resilient, and has a strong influence on health and disease. Characterization and defining a normal skin microbiome could help in future diagnosis and treatment of disease, although factors like differences in host genetics, lifestyles, and particular skin locations must be accounted for. As research and medical technologies advance, it may be possible to utilize these microbial neighbors on the skin in efforts to promote better overall health.
Check Your Understanding
• What features of human skin affect the composition and stability of its microbiome?
• How is the skin microbiome influenced in early life, and what implications does this have in human health?
• What diseases are associated with dysbiosis of the skin microbiome? How do these come about and what particular microorganisms are associated with each?
• How do certain members of the skin microbiome impede the healing process of chronic wounds?
References
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The Respiratory Microbiome
The respiratory microbiome (specifically the lower portions) was once thought to be nonexistent, as many considered healthy lungs to be a sterile environment. Like other microbiomes though, the upper and lower respiratory system are environments rich with bacteria, fungi, archaea, and viruses, and because of the novelty of study many of the members in these groups have not been identified. However, it is known that many of such organisms are not only responsible for or exacerbate a number of pulmonary diseases, but that the respiratory microbiome can help to mitigate infection and influence treatment of certain illnesses (Unger and Bogaert, 2017, Watson et al., 2019).
The human respiratory system begins with the upper respiratory system; starting with the nose and nostrils, then proceeds to the nasal cavity, pharynx, and larynx. The lower respiratory system then starts with the trachea, moves to the bronchi and bronchioles, and ends with the lungs. Each of these structures and areas have their own microbial niche which serve various roles in maintaining health, but also have the potential to be compromised. The respiratory system is similar to the oral and digestive system, in that they interact intimately with external stimuli and bring in foreign matter and microbes during regular biological processes, like inhalation. These systems also have overlapping parts (e.g. oral cavity and pharynx), where their respective microbiomes serve similar roles such as resistance to disruptive environmental factors and host defense (Dickson et al., 2014, Zaura et al., 2014, Hakansson et al., 2018). The mucosal surfaces of the respiratory tract also have a natural healthy biofilm where the resident microbiota reside to maintain homeostatic functions, however, as an extension of dysbiosis, these biofilms can become altered and obtain pathogenic microbes which contribute to respiratory diseases (Hamilos, 2019).
Composition
Immediately after birth the respiratory microbiome becomes colonized, though the collection of microbes found here during infancy is not much different than other locations in the body, and generally reflect the mode of delivery (Dominguez-Bello et al., 2010). The respiratory microbiome, as well as others, become differentiated over time when the different species of microbes adapt and outcompete each other based on their niche, host genetics, and environmental factors (Bosch et al., 2016). Colonization additionally depends on microbial immigration, microbial elimination, and relative reproduction rates of its members (Figure 1) (Dickson and Huffnagle, 2015). With age, the respiratory microbiome of each particular individual reflects the aforementioned factors as well as other lifestyle habits, and so they can be vastly different between people. Although these respiratory microbiomes are unique, a core set of microbes can be defined, with most of them being aerobic or facultatively anaerobic (Stearns et al., 2015, Hakansson et al., 2018).
Microbial composition also depends on the specific structure or niche within the respiratory system. The nasal microbiome largely consists of Staphylococci, Corynebacteria, and Streptococci, which is most likely due to its similarity and proximity to the skin (Mika et al., 2015, Shilts et al., 2016). Communities within the paranasal sinuses are highly diverse and include lactic-acid producing bacteria such as Lactobacilli, Enterococcus, and Pediococcus, and once in the nasopharynx the collection of microbes becomes more complex and favors oxygen-utilizing groups (Abreu et al., 2012, Biesbroek et. al, 2014, Teo et al., 2015). In the lower respiratory system, the microbiome resembles a bit of a mix from both the nasal and oral cavities, with a majority of bacteria from the genera Streptococcus, Fusobacteria, Pseudomonas, Veillonella, and Prevotella (Cui et al., 2014, Beck et al., 2015, Dickson and Huffnagle, 2015, Hakansson et al., 2018).
While much focus of the respiratory microbiome has been on bacteria (like many other microbiomes), study of the mycobiome and virome components have revealed their importance in pulmonary health and disease. Fungal spores are commonly inhaled, especially in high numbers during peak seasons, and depending on the particular organism and host immune health, they can cause infection and respiratory complications (Pashley et al., 2012, Denning et al., 2014, Nguyen et al., 2015). For healthy people, the respiratory mycobiome is predominated by environmental fungi including Aspergillus, Cladosporium, and Penicillium species (Charlson et al., 2012, van Woerden et al., 2012, Underhill and Iliev, 2014). However, members from these genera and other more well-known fungal pathogens, like Candida albicans, can be implicated in respiratory illnesses due to microbial community changes (Nguyen et al., 2015). The respiratory virome can also modulate various pulmonary diseases through respective bacterial interactions and host immune response. For bacteriophage composition, there are observed differences between healthy individuals and patients suffering from diseases associated with respective bacterial infections (Wilner et al., 2009). There is a diverse range of eukaryotic viruses that make up the normal human and respiratory virome, with the majority consisting of adenoviruses, herpesviruses and human papillomaviruses (HPVs), though these may only be transient and quickly cleared by the immune system or via the mucociliary escalator (Wilner et al., 2009, Popgeorgiev et al., 2013). More research needs to be conducted over the normal respiratory virome to determine its functionality and importance, though usually more focus is catered to those viruses directly involved in pathogenesis and dysbiosis.
Dysbiosis and Disease
The normal matrix of organisms in the respiratory system maintain homeostasis through their complex interactive network with each other and their host. Pathogens like Streptococcus pneumoniae and Haemophilus influenzae are actually part of the resident respiratory microbiome and interact with commensal microbes, however, they can incite infection when they are not kept in check due to community perturbation (de Steenhuijsen et al., 2015). During events of dysbiosis, pathogens and opportunistic pathogens can contribute to and exacerbate several respiratory diseases including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, pneumonia, otitis media, and other acute infections like influenza.
Asthma is a chronic lung disease that usually develops at an early age, and its complex etiology makes it difficult to treat. People with asthma experience inflammation and tightening of their airways and a production of extra mucus impairs breathing, talking, and being active. The prevalence of asthma has increased in recent decades, and a decrease in microbe exposure during youth could be partially responsible (Ober and Yao, 2011, Prescott, 2013). Proper development of the immune system is reliant on contact with microbes, and those individuals who are regularly subjected to a diverse variety of microorganisms at an early age have a reduced risk of developing asthma (Ownby et al., 2002, Fujimura et al., 2010, Fall et al., 2015). Aside from grooming the immune system, the intake of microbes influences the composition of the mucosal microbiota that lines the air passageways, which could directly affect asthma development (Durack et al., 2016). Indeed epidemiologic studies have shown that the disruption of the microbiome, such as in the case of antibiotic use during childhood, create a predisposition for this allergic disease (Khalkhali et al., 2014). Specifically, the increased presence of genus Haemophilus and other Proteobacteria, as well as the fungus Aspergillus fumigatus in the respiratory system are signature of asthma patients (Hilty et al., 2010, Teo et al., 2015, Urb et al., 2015). These pathogens would normally be cleared by the immune system or stifled by the resident microbiota, however, early childhood dysbiosis resulting in hampered immune functions and/or a discontent microbial consortium in the respiratory system contribute to the development of this condition.
Chronic obstructive pulmonary disease (COPD) is another chronic inflammatory lung disease that causes obstructed airflow to the lungs and is exacerbated by microbial dysbiosis. Emphysema, a condition where the alveoli are damaged due to cigarette smoke and other irritants, and chronic bronchitis, a condition characterized by inflammation of the lining of the airways, are the most common contributors to COPD. An alteration of the lower respiratory microbiome causing reduced diversity or the presence of specific organisms can further aggravate COPD causing a worsening of symptoms (Huang et al., 2014). Viral infections, like those caused by the rhinovirus, are commonly detected (in about half the patients) during COPD exacerbations (Seemungal et al., 2001, Rohde et al., 2003, Mallia et al., 2011). Also during these bouts, there is an increase in pathogens from the bacterial genera Haemophilus, Pseudomonas, and Moraxella and a general shift towards the phylum Proteobacteria in the respiratory microbiome (Huang et al., 2014, Millares et al., 2014). Additionally, patients with COPD exhibit a decline or absence of certain normal lung organisms that could contribute to mucosal homeostasis and prevention of pathogen overgrowth, such as Firmicutes (Streptococcus spp.), as well as Bacteroidetes (Prevotella spp.) (Fukata and Arditi, 2014, Sze et al., 2015, Hakansson et al., 2018). These compositional changes are at least partially brought about by current COPD treatments like steroid and antibiotic usage, and so future therapy may need to take the respiratory microbiome into further consideration for various approaches (Wang et al., 2016).
Cystic fibrosis (CF) is a genetic disease that causes normal cellular secretion of mucus, sweat, and digestive juices to become thicker and viscous. In the respiratory system, the buildup of sticky mucus can clog airways, impair lung function, and make breathing difficult. Bacterial pathogens, like Staphylococcus aureus, Pseudomonas aeruginosa, and Haemophilus parainfluenzae, tend to grow in the sputum and can cause infections which worsening CF conditions (Keogh and Stanojevic, 2018). Their biofilm-forming capabilities also contribute to the excess mucus production, further causing complications as infectious agents can be resilient and persistent (Høiby et al, 2010, Orazi and O’Toole, 2017). While changes in the microbiome are not typically responsible for exacerbations of CF, antibiotic administration is the primary treatment option to eliminate these pathogens residing in the mucus, however, prolonged use can result in decreased microbiome diversity and dysbiosis in the respiratory system as well as other areas (Zhao et al., 2012, Carmody et al., 2013 Price et al., 2013, Dickson et al., 2014).
Pneumonia is a microbial infection of the lungs that can cause the alveoli to fill with pus or other liquids, and severe cases can be life-threatening, especially to children, the elderly, and those who are immunocompromised (Wardlaw et al., 2006, Chalmers et al., 2011, Valley et al., 2015). The intensity of the infection is directly correlated with the microbial load and diversity, so etiologic determination is important for proper treatment, as the onset of pneumonia can have bacterial, fungal, or viral origins (Iwai et al., 2014). Viral pneumonia is commonly caused by influenza or rhinovirus, however, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has now also become a major cause post 2019 (Jain et al., 2015, Zhou et al., 2020, Pettigrew et al., 2021). Respiratory viral infections are the most common cause acute respiratory illnesses, and often times, these viral infections then lead to secondary bacterial infections, like pneumonia, due to host immune reactions and induced dysbiosis in the respiratory tract and gastrointestinal system (Hanada et al., 2018). Streptococcus pneumoniae is the most common culprit for bacterial pneumonia, and while it regularly resides in the upper respiratory tract, under dysbiotic conditions this organism can proliferate and spread to the lower respiratory tract and cause illness (File, 2003, de Steenhuijsen Piters et al., 2014). In patients with pneumonia, there is a decrease in many Gram-negative anaerobic bacteria that are apart of the resident microbiota, some of which are associated with a reduced risk of hospital-acquired pneumonia and clearance of S. pneumoniae (Bousbia et al., 2012, de Steenhuijsen Piters et al., 2014, Krone et al., 2014). Although, changes in the respiratory microbiome are more apparent in bacterial pneumonia rather than viral, it is not known whether this alteration in microbial consortia is a cause or effect of the disease, though it is likely conditional of both circumstances (Ramos-Sevillano et al., 2019, Pettigrew et al., 2021).
Conclusion
The respiratory microbiome is an up-and-coming avenue for research, diagnosis, and treatment of several pulmonary illnesses. While much of its characterization is still in it’s infancy, proper identification of microorganisms is important for management of respiratory diseases (Nguyen et al., 2016). As antibiotic resistance continues to surge, novel approaches are necessary to mitigate not only respiratory illnesses, but to maintain general health. And similarly to other microbiome-related treatments, like fecal microbiota transplant for the gut, eventually oral or aerosol administered microbiota may be implemented to treat pulmonary conditions (Huang and Boushey, 2015). Continual research detailing both core and individual respiratory microbiomes will push the advancement of these ‘futuristic’ medical motions.
Drag and Drop Quiz
Drag each contributing factor of the respiratory microbiome composition into the appropriate category.
Check your Understanding
• How is the colonization of the respiratory microbiome influenced, and what factors affect its composition over time?
• What factors of the respiratory microbiome affect the development of asthma?
• Which microorganisms are implicated in COPD and periods of exacerbation? How does dysbiosis contribute to this chronic condition?
• Why do you think a greater change is observed in the respiratory microbiota in patients with bacterial pneumonia versus viral?
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The Vaginal Microbiome
The vaginal microbiome is a reproductive organ-specific niche that harbors a unique collection of microorganisms that are important in a variety of aspects to human health. This microbial community has significance in maintaining vaginal homeostasis, protecting against urogenital infections, host immunity, and reproductive capacities. In recent years, next generation sequencing (NGS) techniques have been employed to classify the vaginal microbiome into community state types (CSTs), or vaginotypes, based upon composition, which can enhance epidemiological studies to make better associations between the microbiome and host-vaginal health (France et al., 2020, Mancabelli et al., 2021).
Composition and Role
The vaginal microbiome is composed of over 200 different bacterial species, though it is primarily dominated by members from Lactobacillus genus (Ma et al., 2012, Auriemma et al., 2021). The Lactobacilli protect the vagina from potential pathogen invasion through their fermentation of vaginal epithelial cell-produced glycogen into lactic acid, which lowers vaginal pH, as well as their production of various antimicrobial compounds, and resource/space competitive inhibition (Boskey et al., 2001, Amabebe and Anumba, 2018, Chee et al., 2020, Jang et al., 2019). Though, the bacteriome is usually the emphasized feature of the vaginal microbiome, it also harbors protists, fungi, archaea and viruses, with each occupying their own niche in the normal healthy network (Belay et al., 1990, Bradford and Ravel, 2017, Happel et al., 2020, Chacra and Fenollar, 2021). The composition isn’t always fixed however, as factors like the menstrual cycle, hormone fluctuation, sexual partners, hygiene, genetics, age, the environment, drug use, and other aspects of lifestyle can affect the microbial makeup (Aagaard et al., 2012, Fettweis et al., 2014, Hyman et al., 2014, Zapata and Quagliarello, 2015, Martin and Marrazzo, 2016, Diop et al., 2019, Chacra and Fenollar, 2021).
As mentioned earlier, using 16S rRNA sequencing, the vaginal microbiome has been categorized into CSTs and allowed for deeper analysis, where comparisons of the consortia within these classifications can be used to make associations with host and vaginal health. There are 5 main CSTs each predominated by a particular species of Lactobacilli, except for group IV, which has been further dissected into additional subgroups: CST I—L. crispatus dominated, CST II—L. gasseri dominated, CST III—L. iners dominated, and CST V—L. jensenii dominated (Ravel et al., 2011, France et al., 2020, Chacra and Fenollar, 2021, Mancabelli et al., 2021). CST IV is not dominated by any particular species, and contains a mixture of both strict and facultative anaerobes including Gardnerella, Atopobium, Lactobacillus, Bifidobacterium, etc., where certain controversial subgroups (e.g. CST IV-A, CST IV-B, CST IV-C, CST IV-D, CST IV-G, etc.) have various combinations depending on the study (Gajer et al., 2012, Albert et al., 2015, France et al., 2020, Mancabelli et al., 2021). Each of the five groups has correlations with vaginal pH, microbial colonization and biodiversity, as well as host characteristics such as pregnancy, ethnicity, and age, though it is not always absolute (France et al., 2020, Mancabelli et al., 2021). With reproducible organization of the vaginal microbiome, connections can be made with medical conditions, which could benefit treatment and diagnosis of vaginal diseases and associated affairs.
Dysbiosis and Disease
As with other microbiomes associated with the human body, disturbance of the resident vaginal microbiome can result in complications of health. Many diseases of the urogenital tract such as bacterial vaginosis (BV), urinary tract infections (UTIs), yeast infections, and several sexually transmitted infections (STIs) are caused by pathogenic microbes associated with dysbiosis (Taha et al., 1998, Donders et al., 2000, Wiesenfeld et al., 2003; Lai et al., 2009, De Seta et al., 2019, Mancabelli et al., 2021). Normally, the local flora maintains homeostasis, however, changes in composition provide opportunistic pathogens a window to proliferate and invade.
Bacterial vaginosis, associated with CST IV, stems from a perturbance of the vaginal flora, specifically a decrease in Lactobacilli and an increase in other microbes like Gardnerlla vaginalis, Atopboium vaginae, Ureaplasma urealyticum, and others that are only usually found in low numbers (Gajer et al., 2012, Margolis and Fredricks, 2015, Onderdonk et al., 2016, Zozaya et al., 2016). This condition can be asymptomatic in up to half the women with BV, and in the others can be diagnosed by observed changes in vaginal discharge or by the Nugent scoring system which utilizes a Gram stain (Figure 1) (Nugent et al., 1991, Amsel et al., 1983, Schwebke, 2000). While not exactly considered a sexually transmitted disease, bacterial vaginosis is commonly associated with certain sexual practices, though other factors such as hygiene, nutrition, intrauterine devices, hormonal changes, and certain comorbidities can contribute to susceptibility (Avonts et al, 1990, Calzolari et al., 2000, Neggers et al., 2007, Verstraelen et al., 2010, Zabor et al, 2010, Margolis and Fredricks, 2015). Women with this disease also have a higher risk to contract sexually transmitted infections, and it can be linked to reproductive complications and poor infant health (Wiesenfeld et al., 2003, Prince et al., 2015, Chacra and Fenollar, 2021). Typically, antibiotics targeting anaerobic bacteria are administered to treat BV, however recurrence is common, likely due to the antimicrobial-resistant nature of biofilms formed by the pathogens and/or regular exposure to external reservoirs (Swidinski et al., 2008, Oduyebo et al., 2009, Marrazzo et al., 2012, Bradshaw and Sobel, 2016). Probiotics, specifically containing L. crispatus, may be the better option, and vaginal microbiota transplantation from a healthy donors is a promising treatment course of action (Hemmerling et al., 2010, Ma et al., 2019).
Urinary tract infections are a common problem, especially for females, which are disproportionately affected. This infection is most likely to occur in the urethra or bladder (though in severe cases the kidneys can be affected), which causes pain in the pelvic area and during urination, the frequent urge to urinate, and the presence of blood in urine (Lee and Neild, 2007, Sheerin, 2011, Hooton, 2012). The vaginal microbiome, or more specifically some normal residents, like Escherichia coli, can move to the urogenital tract and cause issues resulting in a UTI, which often occurs via sexual activity (Nicolle et al., 1982, Foxman, 2014, Stapleton, 2016, Lewis and Gilbert, 2020). Other fastidious microorganisms, like those abundant in women experiencing BV, contribute to a higher risk of contracting an infection in the urinary tract as compared with those not suffering from dysbiosis in the vagina (Hillebrand et al., 2002, Sumati and Saritha, 2009). In these cases, the vaginal opening may harbor potential uropathogens (Figure 2) and transient exposure to the urinary tract could prompt colonization or other reactions (e.g. immunomodulation) that results in a UTI (Lewis and Gilbert, 2020). Antibiotics are traditionally used to treat UTIs, however probiotics and estrogen administration could help to restore the Lactobacillus colonization and protect against complicated and recurrent infections (Raz and Stamm, 1993, Eriksen, 1999, Prais et al., 2003, Stapleton et al., 2011, Tan and Chlebicki, 2016, Lewis and Gilbert, 2020).
Yeast infection of the vaginal region caused by Candida species, also known as vulvovaginal candidiasis (VVC), is a common condition with severe symptoms and a high recurrence rate (Oerlemans et al., 2020). Those affected experience vaginal itchiness or soreness, dyspareunia (painful intercourse), abnormal vaginal discharge, redness, swelling, and thinning of the vaginal wall (Chew and Than, 2016, Oerlemans et al., 2020). While it is the second-most common infection of the vagina behind BV, this disease primarily affects premenopausal women with a low vaginal pH value under 4.5 (Kim and Park, 2017, Gupta et al., 2019). The exact cause of VVC isn’t exactly clear, though it is thought it can come as a result of microbiome dysbiosis induced by prolonged antibiotic usage, which allows various Candida species to overgrow and establish an infection (Goldacre et al., 1979, Mitchell, 2004, Peters et al., 2014, van de Wijgert and Verwijs, 2020). Traditionally, antifungal medication has been used to treat VVC, however administration of probiotic vaginal microbes may be more prudent as their mechanisms of pathogen colonization and biofilm formation inhibition are more effective to prevent disease recurrence (Petrova et al., 2016, Tachedjian et al., 2017, Allonsius et al., 2019, Oerlemans et al., 2020, van de Wijgert and Verwijs, 2020).
Individuals with vaginal microbiome dysbiosis characterized by a decrease in abundance of Lactobacilli species (i.e. BV) are at a higher risk of contracting sexually transmitted infections, such as those caused by Neisseria gonorrhoeae, Trichomonas vaginalis, Chlamydia trachomatis, and Mycoplasma genitalium (Martin et al., 1999, Cherpes et al., 2003, Peipert et al., 2008, Brotman et al., 2010, Molenaar et al., 2018, De Seta et al., 2022). Infections caused by these organisms usually result in symptoms similar to other genital infections such as vaginal itching, pain, unusual discharge, rash, etc. which like other conditions (e.g. vaginitis), result in decreased epithelial integrity and can exacerbate pathogen invasion (Miller and Shattock, 2003, Greenbaum et al., 2019). In those individuals with BV and non-inflamed tissue, the increased risk for STIs could be due to the negative effects dysbiosis-related bacteria have on the innate immune system (Murphy and Mitchell, 2016, Liebenberg et al., 2017). Similarly, contraction of sexually transmitted viral infections like human immunodeficiency virus (HIV), herpesviruses, and human papillomavirus (HPV) are frequently associated with vaginal dysbiosis, immunomodulation, and disruption of the epithelial barrier (Sewankambo et al., 1997, Borgdorff et al., 2016, Siqueira et al., 2019, Torcia, 2019, De Seta et al., 2022). While there is correlation between vaginal dysbiosis and STIs, the protective mechanisms of the resident vaginal microbiota are unknown and still need to be uncovered (De Seta et al., 2022).
Reproduction, Pregnancy, and Infant health
The vaginal microbiome has further implications in host immunity, fertility, pregnancy, spontaneous preterm birth, and infant health (Anahtar et al., 2015, Fettweis et al., 2019, Gupta et al., 2020, Xu et al., 2020). Indeed, there are a multitude of factors that affect reproduction like age, genetics, hormone levels, fallopian tube blockage, menstrual cycle, and vaginal pH, however only recently has the vaginal microbiome been studied for its association with various fertility factors (Xu et al., 2020, Fan et al., 2022).
Changes in the resident vaginal flora and infections by certain pathogens can cause complications for reproductive health and pregnancy. For example, infection by Group B Streptococcus (GBS) has been associated with a decline in ovarian function, pregnancy loss, preterm delivery, and is the leading cause of bacteremia and meningitis in newborns (Zaleznik et al., 2000, Phares et al., 2008, Kolter and Henneke, 2017, Tazi et al., 2019, Xu et al., 2020). The vaginal microbiome of pregnant women is less rich and diverse than non-pregnant individuals, likely caused by changes in sex hormone levels (Farage et al., 2010, Aagaard et al., 2012). These alterations can cause shifts in the vaginal microbiome that could then result in infection and a risk of preterm or spontaneous labor (Wylie et al., 2018, Fettweis et al., 2019, Feehily et al., 2020, Gupta et al., 2020).
Infants become introduced to the microbial world via their mother, and predominately right after birth where vaginal versus cesarean delivery has a great impact on composition (Chu et al., 2017). However, exposure may happen earlier in utero, as this environment may not be as sterile as once thought as some studies have shown the presence of microbes in the placenta and amniotic fluid (Aagaard et al., 2014, Collado et al., 2016, Kolter and Henneke, 2017). The establishment of a newborn’s microbiome has profound effects on the development of immunity and metabolism as well as the onset of diseases like atopic dermatitis and obesity in later life (Rautava et al., 2012, Collado et al., 2016, Ta et al., 2020).
Conclusion
The vaginal microbiome is a complex community which affects many facets of human health and disease including urogenital, reproductive, immune, and infant. Characterization of the vaginal flora has allowed categorization into community state types which can help to predict and diagnose disease states. Within so, these dynamic changes that occur under various conditions produce a unique fingerprint for the vaginal microbiome which can be analyzed and potentially treated in a specific manner (Ceccarani et al., 2019, Lagenaur et al., 2020, Abou Chacra and Fenollar, 2021). While antibiotic administration has its benefits, novel approaches using targeted application of probiotic microbiomes (e.g. a gel containing specific Lactobacilli species) could help in treating diseases associated with vaginal dysbiosis by restoring those disrupted communities, as well as alleviating certain negative consequences of chemotherapy (Pino et al., 2019, Lagenaur et al., 2020 Oerlemans et al., 2020). More in-depth and continued research of the vaginal microbiome is necessary to illuminate the interactions and connections of it members to human health.
Drag and Drop Quiz
Drag the species of bacteria that dominates each vaginal community state type.
Check your Understanding
• Explain vaginal microbiome CSTs. Why are they important?
• Which CST is associated with BV, and which genus is typically absent?
• How does BV contribute to the development of other conditions like UTIs, VVC, and STIs?
• How does the vaginal microbiome influence reproduction?
• Where does a newborn acquire its microbiome, and what factors affect the composition?
• What alternatives to antimicrobials are promising for the treatment of vaginal microbiome associated diseases?
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Mental Health and Multi-Microbiome Interactions
It may seem obvious that localized microbiomes are responsible for diseases related to their respective areas (e.g., IBD and the gut microbiome or AD and the skin microbiome), however, it is a fascinating phenomenon that various microbiomes can affect each other and influence health in different parts of the body. Even more captivating is the link between certain microbiomes and mental health conditions presuming the brain is devoid of microbes.
These links between microbiomes are referred to as axes, and initial connections began with the most well-studied microbiome, the gut. Since many gut-derived microbial-produced molecules and compounds are spread through the bloodstream, associations with various organs are formed, such as the gut-brain axis, gut-skin axis, gut-lung axis, or a combination of multiple; gut-brain-skin axis. There are likely overlapping connections between all microbial components that form the human holobiome, thus teasing apart the exact members and their functions is a dutiful task.
Gut Interaction with Other Microbiomes
As mentioned earlier, the dissemination of molecules and compounds from the gut to the rest of the body creates an interconnected highway affecting most, if not all, parts of the body. Certainly, juxtaposed regions such as the oral cavity and the proximal portions of the GI tract have a relationship between their microbiomes, which it termed the gut-oral axis (GOA). These linked microbiomes have been shown to have immunomodulatory roles in the development of rheumatoid arthritis (RA) and osteoarthritis (OA) under dysbiosis, where the abundance of oral Porphyromonas gingivalis and intestinal Prevotella copri could be responsible (Drago et al., 2019, du Teil Espina et al., 2019). It has also been proposed that dysbiosis of the gut-oral microbiome axis is implicated in cirrhosis of the liver through pathogen invasion, resultant systemic inflammation, and impaired immunity and liver function (Acharya et al., 2017). Even gastrointestinal conditions like IBD and cancer of the colon, liver, and pancreas are linked with both the gut and oral microbiome dysbiosis, further demonstrating this strong interorgan connection to human health (Park et al., 2021).
The gut and the skin microbiomes are also linked via the gut-skin axis (GSA), and as the primary interface to the environment, they have major roles in physiological health (Figure 1). Dysbiosis of the gut-skin microbiome axis has influence on both GI and cutaneous disorders such as IBD, celiac disease, atopic dermatitis, psoriasis, acne, and other dermatologic issues, where each of these diseases can be associated with the ‘partner disease’ (i.e. a disease in one system linked to a disease in another system) (O’Neill et al., 2016, Salem et al., 2018, De Pessemier et al., 2021). Though crosstalk is bidirectional, more research seems to have focused on skin disorders and homeostasis as a result of gut microbiome health, where diet, immunomodulation, intestinal permeability, and metabolite secretion are major contributing factors (De Pessemier et al., 2021). However, there is evidence that Mallassezia restricta, a fungal member of the skin microbiota, is associated with Crohn’s disease and can exacerbate colitis (Limon et al., 2019, Sinha et al., 2021).
The gut-lung axis (GLA) has roles in the regulation of immunity and the development of various respiratory diseases. (Frati et al., 2019). The respiratory system and GI tract are connected by the mesenteric lymphatic system, where intact or fragmented microbes and their metabolites enter systemic circulation after passing through the intestinal barrier and can migrate to the pulmonary system to modulate immune responses (Enaud et al., 2020). The pathophysiology of diseases such as atopy and asthma are complicated and can be contributed to a variety of factors, however, there is evidence that dysbiosis of the gut microbiome contributes to the development of asthma, particularly in youths who exhibit a decrease in Lachnospira and increase in Clostridium spp. (Penders et al., 2007, Watson et al.,2019). Gut dysbiosis is also involved in chronic obstructive pulmonary disease (COPD) exacerbation, where fiber deficiency in an individual’s diet can contribute to chronic inflammation. Metabolism of fiber by gut microbes produces anti-inflammatory short-chain fatty acids (SCFAs), which could reduce inflammation both systemically and in the lungs, and so targeted dietary intervention for these patients may be a viable treatment addition for COPD and other respiratory diseases associated with inflammation like COVID-19 (Li et al., 2018, Vaughan et al., 2019, Allali et al., 2021). Moreover, SCFAs also have an important role in the defense against secondary infections in those afflicted by viral respiratory infections, further demonstrating the importance of gut microbiome health in connection with the GLA (Sensio et al., 2020).
Like other axes, immunomodulation by the gut microbiome also has influence on the gut-vagina axis (GVA), though it is less extensively studied. One promising avenue of treating vaginal diseases, specifically cervical cancer associated with human papilloma virus (HPV), is the use of mucosal lactic-acid bacteria (LAB)-based vaccines to modulate the gut microbiome. The approach could work as a prophylactic or for direct therapy, and be more easily administered via an oral route instead of parenteral (Taghinezhad-S, et al., 2021). Endometriosis and infertility are also disorders associated with sex hormone levels and inflammation, which once again are influenced by the gut microbiome composition and state. Specifically, there are some members in the gut who can affect the levels of circulating estrogen through metabolic processes and an altered state could lead to increased risk or symptom severity of these disorders (Salliss et al., 2022). Other female reproductive-associated diseases such as polycystic ovarian syndrome (PCOS), also associated with gut dysbiosis, could be ameliorated by diet modification and restoration of gut homeostasis. Here, the use of flaxseed oil could increase diminished levels of SCFAs observed in those with PCOS and protect against inflammation characteristic of the disease (Wang et al., 2020).
The maternal microbiome, including gut, vagina, and breast milk, can greatly influence the colonization and health of the infant after birth, but also affect the fetus prior to delivery. Fetal immune development is most likely influenced in the womb from translocation of maternal gut microbes and/or their metabolites across the placental barrier or ingestion of amniotic fluids (Walker et al., 2017, Nyangahu and Jaspan, 2019). Post-reproduction, the infant gut microbiome is continually developed through initial diet, and more specifically vertical transmission of the contents in the mother’s breast milk which includes its own unique microbiome (Ojo-Okunola et al., 2018, Ojo-Okunola et al., 2019, Quin et al., 2020). The human milk microbiome promotes infant gut colonization of probiotic strains which have roles in programming the immune and metabolic systems, as well as anti-infective, anti-allergic, and anti-tumor properties (Heikkila and Saris, 2003, Olivares et al., 2006, Lara-Villoslada et al., 2007, Civardi et al., 2015, Hassan et al., 2015, Walker and Iyengar, 2015, Boix-Amoros et al., 2016, Ojo-Okunola et al., 2018).
Mental Health Axes
The thought of microorganisms controlling aspects of cognitive function in their host, especially humans, is fascinating to say the least. While connections between other microbiomes, organ systems, and diseases may be less surprising, the microbial link to psychiatric, neurodevelopmental, age-related, and neurodegenerative disorders is very much intriguing. The human microbiome can communicate with the brain in a variety of ways; through the immune system, metabolism, endocrine system, circulatory system, and the nervous system, where microbes, their induced immune response, and their metabolites such as short-chain fatty acids, branched chain amino acids, and peptidoglycans are involved (Figure 2) (Liang et al., 2018, Cryan et al., 2019, Olsen and Hicks, 2019, Hadian et al., 2020, Bear et al., 2021). Several local microbiomes each contribute affects to the mental state, and whether it is gut-, skin-, oral-, lung-, etc. derived can dictate roles in various disorders. This is not just a one-way street though, as microbes around the body can alter mental states but also be affected by governance of the brain in a bi-directional manner (Ma et al., 2019).
Gut-Brain
Similar to other types of microbiome and organ interactions, the gut’s role in mental health is central and most well studied, which makes sense as it is the largest repository of microorganisms associated with the human body. The gut-brain axis (GBA; and gut plus essentially every other microbiome and brain axis, e.g., gut-skin-brain) is implicated in a number of cognitive functions and disorders including autism, anxiety, depression, stress, pain sensitivity, learning capacities, memory loss, moods and emotions, behavior (dietary, social, and reproductive), schizophrenia, Parkinson’s disease, and Alzheimer’s disease (Desbonnet et al., 2013, Stumpf et al., 2013, Dash et al., 2015, Gareau, 2016, Luczynski et al., 2016, Hoban et al., 2017, Liang et al., 2018, Manderino et al., 2017, Nishida and Ochman, 2017, Vuong et al., 2017, Cowan et al., 2018, Cryan et al., 2019, Bear et al., 2021, Narengaowa et al., 2021). The gut microbiome actually develops in sync with the brain and psychology, and disturbances during different stages of growth can result in the onset of different diseases (Figure 3) (Borre et al., 2014, Gur et al., 2015, Sampson and Mazmanian, 2015, Dinan and Cryan, 2016, Luczynski et al., 2016, Sharon et al., 2016, Kundu et al., 2017, Vuong et al., 2017, Carlson et al., 2018, Liang et al., 2018).
Early perturbation of the gut microbiome during the post-natal period, and even within the womb, can especially increase susceptibility to developing mental disorders since these are critical stages for development of the gut-brain axis and mind (Borre et al., 2014, Gur et al., 2015, Diaz Heijtz, 2016, Mika et al., 2016, Slykerman et al., 2016, O’Mahony et al., 2017, Liang et al., 2018). Even up through senescence, an abnormal gut microbiota is linked with several mental disorders, though the good news is that they can be remedied or improved by returning the gut to a homeostatic state. As the prevalence of mental disorders and neurological diseases have been steadily increasing, healing mental health by exploring and implementing options such as fecal microbiota transplant, diet intervention, probiotics, prebiotics, and psychobiotics are a must (Cryan and Dinan, 2012, Dinan et al., 2013, Liang et al., 2015, Pirbaglou et al., 2016, He et al., 2017, Kang et al., 2017, Mika et al., 2017, Bruce-Keller et al., 2018, Liang et al., 2018, Yang et al., 2018, Kesika et al., 2021, Margolis et al., 2021).
There are several ideas as to why there has been an increase in mental and neurological disorders in relation to the human microbiome. The “Gut Microbiota” hypothesis suggests this escalation is a direct result from gut microbiome dysbiosis due to factors of modern society such as diet, antibiotics, and stress (Liang et al., 2018b). The “Old Friends” hypothesis proposes that the co-evolution of early humanity with its microbiota in a less civilized and more natural ecosystem promoted the development of a stronger immune system, but now changes in lifestyle and environment leads to weaker immunity and subsequent disorders (Strachan, 1989, Rook and Lowry, 2008, Kramer et al., 2013, Rook, 2013). Lastly, the “Leaky Gut” theory implies that damage to the mucosal barrier of the gut increases intestinal permeability, which allows biomolecules and microorganisms to gain access various parts of the body that they normally couldn’t, and thereafter cause disease (Leclercq et al., 2012, Smythies and Smythies, 2014, Kelly et al., 2015, Potgieter et al., 2015, Slyepchenko et al., 2017, Liang et al., 2018). All of theses propositions are clear in their indication that the gut microbiome and its part in immune development has a definite role in the functioning of the mind, though the exact extent and mechanisms remain to be discovered.
Other Microbiomes and Mental Health
The majority of human microbiome interaction with regards to human health usually includes the gut to some degree, though there are some questions as to whether certain microbiomes create their own axes with the brain.
The oral microbiome can potentially get direct or indirect access to the brain through the olfactory tract, or via the circulatory system where blood can transport microbes to the blood-brain barrier (BBB), perivascular spaces, and circumventricular organs (Figure 4) (Olsen and Singhrao, 2015, Ranjan et al., 2018, Olsen and Hicks, 2019). Microbial access to the bloodstream can commonly occur during dental procedures, and pathogen invasion of the brain can impact neuro-immune activity and inflammation (Olsen, 2008). These oral-brain axis-derived infections could contribute to mental disorders such as Alzheimer’s disease (AD), dementia, Down’s syndrome, bipolar disorder, and autism spectrum disorder (ASD; which can lead to oral dysbiosis) (Ilievski et al., 2018, Olsen and Hicks, 2019, Maitre et al., 2020). Oral dysbiosis can also promote the development of ASD by affecting the metabolome and thus can create a troublesome positive feedback loop (Mussap et al., 2016, Wang et al., 2016).
Concerning the skin-brain axis, dysbiosis of the skin microbiome and chronic wounds can elicit systemic effects and induce neural responses that eventually affect the central nervous system (Figure 5) (Hadian et al., 2020). Chronic wounds exhibit persistent inflammation and can have various etiologies including diabetic foot ulcers (DFU), venous ulcers, arterial ulcers, pressure ulcers, surgical wounds, and other traumatic wounds (Green et al., 2014, Renner and Erfurt-Berge, 2017, Bui et al., 2018, Pedras et al., 2018, Hadian et al., 2020). In these cases, a compromised skin barrier can allow pathogens and their derivatives to enter the bloodstream, and if the skin is in a dysbiotic state, then abundant pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa can amplify this effect by inducing epithelial permeability (Roy et al., 2014, Basler et al., 2017). These microbes, metabolites, pro-inflammatory mediators, and other constituents circulate within the blood, potentially induce permeability in the blood-brain barrier (BBB), and eventually reach the brain to cause disorders such as wound-associated depression, anxiety, and other cognitive disorders (Wang et al., 2011, Zhang et al., 2015, Hadian et al., 2020). The skin-brain axis acts in a bi-direction manner too, with studies showing connections between certain mental disorders like post-traumatic stress disorder, and skin diseases associated with dysbiosis like psoriasis, chronic urticaria (hives), and atopic dermatitis (Gupta et al., 2017, Beri, 2018).
The lung-brain axis connects pulmonary microbes to neurodegenerative disorders and behavior characteristics, and though not much research has focused on this specific link, there are indications that air pollution can be a trigger. Pollutants in the air come from a variety of sources including engine emissions, coal combustion, biomass burning, and secondary photochemical products, such as ground level ozone (O3) (Mumaw et al., 2016). These compounds can be a source of chronic neuroinflammation and persistently affect microglial cells in the central nervous system, which in turn can increase risk of diseases such as Alzheimer’s disease, Parkinson’s disease, and autism, as well as elicit a decline in cognitive function in the elderly (Power et al., 2011, Wellenius et al., 2012, Roberts et al., 2013, Volk et al., 2013, Heneka et al., 2014, Jung et al., 2015, Kirrane et al., 2015, Mumaw et al., 2016). It is likely that air pollution also negatively affects the lung microbiome, and dysbiosis could further aggravate immune responses and cognitive disruption (Mousavi et al., 2021, Whiteside et al., 2021).
Is there a brain microbiome?
That is, are there resident microbes residing with a living brain? With many neurodegenerative and neuropsychiatric diseases lacking a clear etiology, determining any and all potential associations could help in therapeutic efforts (Link, 2021). The healthy brain is an assumedly sterile environment, though the same was once thought about other organs like the lungs. Interestingly, one study found evidence for the existence of viable bacteria in the human brain. Here, researchers were interested to see whether microbial invasion accompanied damaged brains observed in HIV/AIDS. After sequencing total RNA from cerebral white matter, they found bacteria and phage sequences in both experimental and control brain samples, which was further validated by 16S rRNA gene target amplification and in situ staining (Branton et al., 2013, Link, 2021).
If there were resident microbes in the brain, they would certainly be at much lower abundances as compared with other regions such as the gut or the oral cavity. Though there has been much interest in characterizing the presence of pathogens in unhealthy brains for disorders like Alzheimer’s disease, which is an arduous feat in itself, finding residents in a healthy state could prove even more difficult (Zhan et al., 2016, Alonzo et al., 2018, Dominy et al., 2019, Link, 2021). That is, identification approaches could easily miss novel or fastidious microorganisms, and contamination is hard to avoid. Furthermore, a wide-range of controlled and unbiased studies are necessary to catalogue a possible brain microbiome, however, completing this task in humans brings about ethical implications and sampling limitations (Link, 2021). It is fascinating to consider the prospect of symbiotic brain microorganisms, and how they could influence our health or who we are.
Conclusion
Mental faculties contribute greatly to human health, and therefore characterizing the involvement of the human microbiome is of substantial importance. Many of these select microbiomes overlap, and the disorders associated with each are likely entangled in a complex array. Brain functions are extremely complex as is, and piecing together the microbial puzzle within their reach further complicates matters. However, there is great optimism in considering the human microbiome as a source or target for advanced therapy to resolve mental disorders and other diseases, which could drastically change the way we think about medicine.
Discussion Concept Mapping
With a partner or as a class, connect two or more microbiomes using a concept map. Explain how and/or why the various microbiomes are linked (e.g., disease cause vs. effect, direct vs. indirect, pathways, consortia translocation, factors affecting composition, etc.).
Check your Understanding
• In what ways does the gut microbiome connect with other microbiomes and regions of the body? How does it influence the development of diseases in these areas?
• How could early use of antibiotics influence the development of asthma in children?
• How could gut microbiome metabolites (SCFAs) contribute to healthy human states?
• In what ways does the human microbiome communicate with the brain?
• What options are available to treat mental disorders through the gut microbiome?
• Why have mental illnesses been increasing in prevalence and how could the gut microbiome be involved?
• How are microbiomes, other than the gut, implicated in mental health?
• What role does skin microbiome dysbiosis and chronic wounds have in the development of cognitive disorders? | textbooks/bio/Microbiology/Microbiomes_-_Health_and_the_Environment_(Parks)/03%3A_Human_Health_and_Disease/3.07%3A_Mental_Health_and_Multi-Microbiome_Interactions.txt |
Environmental Nutrient Cycling and Human Health
The importance of microorganisms is unquestionable in regard to how nutrients circulate throughout each ecosystem. There are direct and indirect links between Earth’s ecosystems and human health, though like other microbial networks, they are sometimes unfathomably complex.
Climate change may be the most obvious association between environmental and human health, however, the solution to balance is not one likely easily achieved. Each type of ecosystem is unique, and reflects different changes to anthropogenic activity. Global soil microbiomes and organic foliar litter have great impact on worldwide biogeochemical cycling, plant health, and bioremediation, where environmental changes can disrupt microbial taxonomic distribution and functional profiles (Albright et al., 2020, Naylor et al., 2020). The ocean microbiome is vast considering it is the largest ecosystem on the planet, and plays a tremendous part in biogeochemical cycling, ecosystem dynamics, and response to climate change(Moran, 2015, Acinas et al., 2019, Marz et al., 2021). Also, since much of the ocean microbiome is uncharted, it could also serve as a major untapped reservoir for novel and progressive biosynthetic products (Paoli et al., 2021). Other aquatic environments such rivers, lakes, wetlands, and freshwater systems and their interaction with sediments and plants greatly influence carbon and other nutrient cycles in their respective ecosystems (Amado and Roland, 2017, Avila et al., 2019, Trevathan-Tackett et al., 2021).
In a cyclic manner, climate change can also greatly affect microbiome dynamics of large ecosystems like glaciers, tundra, permafrost, and even dry deserts, which can further exacerbate disturbances in that region and beyond. (Hamilton et al., 2013, Tripathi et al., 2019, Vigneron et al., 2019, Hough et al., 2020, Ray et al., 2020). These environments harbor several dormant microorganisms that can produce greenhouse gases like carbon dioxide and methane, and if they become metabolically active en masse, this could drastically increase contribution to climate change (Feng et al., 2020). Not only can ecosystem biodiversity be affected, but these changes can impact human society and health. Thus, it is important to consider analytical strategies to better understand global change so future actions can be coordinated to mitigate any negative consequences, and evaluating microbiomes may be part of the solution.
Global Change and the Soil Microbiome: A Human-Health Perspective
Article by Ochoa-Hueso, 2017 licensed under the terms of the Creative Commons Attribution License (CC BY).
The importance of the gut and the soil microbiomes as determinants of human and ecosystem health, respectively, is gaining rapid acceptation in the medical and ecological literatures. This suggests that there is a wealth of highly transferable knowledge about the microbial ecology of human and non-human ecosystems that is currently being generated in parallel, but mostly in isolation from one another. I suggest that effectively sharing this knowledge could greatly help at more efficiently understanding and restoring human health and the functioning of ecosystems, which are currently under wide-spread pressure. I illustrate this by comparing the effects of nitrogen deposition on ecosystem carbon sequestration with unhealthy dietary habits and human disease. The deposition of N, a key nutrient for plant growth, may increase carbon sequestration (equivalent to obesity) through several mechanisms, including a reduction in the ability of soil microbes to process organic matter, which some argue could help mitigate climate change. However, this usually results in a degradation of ecosystem health and, thus, cannot represent a real solution. Similarly, human obesity is linked to an alteration of the composition and functioning of microbial communities inhabiting the gut, which is often attributed to unhealthy dietary habits, including ingesting high amounts of simple sugars and processed foods. Finally, I advocate for the explicit recognition of the many commonalities between the functioning of the gut and ecosystems and a broader multidisciplinary collaboration among experts in ecology and human health, including the engineering of soil microbial communities designed ad-hoc to restore ecosystem health.
Nitrogen Deposition and Carbon Sequestration in a Changing Climate
It has been widely proposed that atmospheric nitrogen (N) deposition could help mitigate climate change by increasing the rates of carbon (C) sequestration in terrestrial ecosystems (Knorr et al., 2005; Reich et al., 2006; Yue et al., 2016). Two commonly observed responses are typically proposed as mechanisms: first, a greater amount of N usually implies a higher capacity for plant growth, which would result in a greater amount of C retained within the system (Magnani et al., 2007; de Vries et al., 2009; Laubhann et al., 2009). Of course, for this to be true, it is necessary that the increase in the rates of C uptake and accumulation exceed the C emission rates, whatever the main route by which the latter happens, including plant and/or microbial respiration and changes in fire dynamics due to an excess of biomass accumulation (Dezi et al., 2010; Fenn et al., 2010). The second main mechanism is linked to a reduction in decomposition rates, particularly of recalcitrant organic matter, which would, therefore, accumulate within the system (Knorr et al., 2005; Waldrop and Zak, 2006). Otherwise, this accumulated C may be lost to the atmosphere in the form of CO2 after being respired by soil microorganisms (Janssens et al., 2010). Of course, the relative importance of these mechanisms depend on how plant communities and soil microorganisms respond, directly and indirectly, to the additional inputs of N which, in any case, usually ends up resulting in a disruption of the interaction between these two key components of the ecosystem (Liu et al., 2014).
The Need for a New Perspective
In this article, I will adopt a human health perspective, hardly used in the discipline of global change ecology, to substantiate why atmospheric N deposition cannot represent a positive (i.e., healthy) alternative to mitigate climate change. In the medical literature, it is now widely recognized that human beings are like ecosystems (in fact, some consider us as living ecosystems) in which the eukaryotic cells that form part of our bodies and the prokaryotic cells that live in and on us are deeply interconnected, whereas the enormous importance of our microbiome to human health is also increasingly gaining acceptation (Bengmark, 1998; Berendsen et al., 2012; Ha et al., 2014; Alivisatos et al., 2015; Tilg and Adolph, 2015; Blaser, 2016; Blaser et al., 2016). The fact that many modern diseases, including conditions of the nervous and circulatory systems, skin and heart and allergies (including atopic dermatitis and food allergies), are directly caused by alterations in the microbial communities that live in our interior and exterior is also gaining rapid acceptation (Ha et al., 2014; Tilg and Adolph, 2015; Chang et al., 2016; Tang and Lodge, 2016). In this sense, the word ecosystem is widely used in the current literature of integrative medicine and gastroenterology. However, the opposite does not frequently happen in ecology [i.e., (cautiously) comparing ecosystems with the human body], despite the wealth of knowledge in the medical and human health literature that we, as ecologists, could apply in, for example, issues related to understanding the functioning (i.e., metabolism) of ecosystems and plant-soil-microbe interactions subjected to human pressure (Berendsen et al., 2012; Blaser et al., 2016; Table 1; Figure 1). Therefore, I will finally defend the need to approach problems in ecology from a more multidisciplinary, fresher perspective.
Why Nitrogen Deposition Cannot be the Solution to Climate Change
The reason why I think that a temporary, N deposition-induced increase in the rate of C sequestration will not contribute to mitigating climate change in the long term is equivalent to the reason of those that argue that an increase in obesity rates in human populations derived from a diet rich in simple sugars and processed food and the consequent alteration of their microbiome will not successfully and permanently solve any public health problem of today’s societies. Ingesting large amounts of simple sugars, processed foods, sugary drinks and saturated fats is definitely better than starving, but that does not mean that it is a healthy practice. And the same happens with N deposition and C sequestration. In ecosystems where N is still a limiting nutrient, which is quite common worldwide (LeBauer and Treseder, 2008), an increase in the availability of N can increase ecosystem productivity to levels comparable to human obesity (Tian et al., 2016), but that does not mean that the ecosystem is healthier and, therefore, that this will result in a long-term benefit (Bobbink et al., 2010; Jones et al., 2014). In this sense, a healthy ecosystem may be defined here as a highly multifunctional ecosystem that can maintain an adequate supply of services, at least as compared to a previously defined reference state.
In medicine, the term dysbiosis refers to changes in the composition of the microbiome that are not beneficial to the individuals, including a loss of abundance and diversity of beneficial microorganisms and increased number of pathogens, and that result in the development of a condition (Ha et al., 2014; Tilg and Adolph, 2015). This term could also be used to describe ecosystems that are dysfunctional due to alterations of their microbial communities. In this sense, it has been repeatedly shown through experimental studies and meta-analyses that increased N deposition is typically associated with changes in soil microbial communities (usually related to a decrease in abundance and biodiversity; Treseder, 2004, 2008; Ramirez et al., 2010; Zeng et al., 2015), reduced ecosystem functionality (alterations of energy metabolism; Waldrop and Zak, 2006; Treseder, 2008; Liu et al., 2014) and short- to mid-term increases in C sequestration, especially in aboveground biomass, but also in the soil and roots (comparable to obesity, as previously mentioned; Xia and Wan, 2008; Yue et al., 2016). Given that metabolic disorders and obesity in humans are clearly associated with a deterioration in the health status of individuals that may even result in cases of fatality due to chronic diseases, sudden death or, quite commonly in the natural world, to increased sensitivity to other environmental stresses (Mathur and Barlow, 2015; Monteiro et al., 2015), I think that we would do well to be cautious when we consider, perhaps naively, the potential benefits of a N that, after all, is the result of the atmospheric pollution derived from our activities (Gruber and Galloway, 2008).
The “Deceptively Simple” Solution
The connections between human health, disease, and the microbiome, especially in the case of the gut, are becoming increasingly apparent and are attracting the public attention, especially because of the high social cost of unhealthy dietary habits and lifestyles and the “deceptively simple” solution of the problem (Mathur and Barlow, 2015; Tilg and Adolph, 2015; Blaser, 2016). In the case of both people and ecosystems, (i) ensuring a healthy supply of nutrients derived from the breakdown and cycling of unprocessed food/organic matter, (ii) minimizing the use of antibiotics (particularly those associated with the livestock industry in the case of ecosystems; Park and Choi, 2008) and chemicals (including herbicides and pesticides in the case of ecosystems) that destroy the microbiome, unless this is strictly necessary, and (iii) promoting practices that favor the system’s ability to self-regenerate, something that living systems do wonderfully well, and that increase its resilience against pathogens and extreme events could be part of the solution, if not all, of the problem.
Of course, there are opportunities to aid in the recovery of our damaged and degraded ecosystems as well as there are possibilities to recover the lost or damaged intestinal flora (Brudnak, 2002; Sheth et al., 2016). This can be achieved by the use of properly designed probiotics or fecal transplants or, in the case of ecosystems, inocula assembled in the lab from pure cultures or soil samples obtained in the field from healthy ecosystems (Bowker, 2007; Chiquoine et al., 2016; Wubs et al., 2016) in conjunction with a balanced nutrient supply (i.e., organic matter inputs, the equivalent to prebiotics; Mathur and Barlow, 2015; Sheth et al., 2016). In this sense, the concept of synbiotics (i.e., synchronous administration of probiotics and prebiotics) could represent a particularly promising benchmark borrowed from the human health literature to successfully restore degraded ecosystems (Tang and Lodge, 2016) and, thus, the human probiotics industry has an opportunity to play a key role in this development.
Concluding Remarks
Recognizing and understanding the similarities and deep connections between the gut and the belowground world, where roots are the equivalent to our gut and the rhizosphere is the gut microflora (Berendsen et al., 2012) can help us advance the understanding of ecosystems by leaps and bounds through the search of similar microbial indicators of disease (e.g., Bacteroidetes to Firmicutes ratio in humans; Mathur and Barlow, 2015) and, therefore, to implement quick and successful measures in ecosystem management rather than relying, perhaps naively, on that the very same thing that caused climate change (i.e., pollutant emissions to the atmosphere) will also be part of the solution. From here, I advocate for the development of a new field of research that specifically aims at recognizing and make practical use of the profound links between the functioning of the gut and the ecosystems that extend beyond our bodies and that benefits from a truly multidisciplinary collaboration among experts in the areas of global change ecology and human health.
Author Contributions
The author confirms being the sole contributor of this work and approved it for publication.
Conflict of Interest Statement
The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Acknowledgments
I am indebted to Dr. Lilia Serrano for her tirelessly encouragement to write this opinion article.
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• Which ecosystems have the greatest impact on global nutrient cycling?
• Which microbiome is an under-explored source for the discovery of biosynthetic products?
• How could global warming’s effect on permafrost and tundra further increase greenhouse gases?
• What are the pros and cons of nitrogen deposition to counteract climate change?
• What are some suggested solutions to improve environmental and human health concerning microbiomes?
References
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The Ocean Microbiome and Marine Life
The ocean is teeming with life, both macro and micro, where their adaptation and success are dependent on both a local and global scale. Oceanic microorganisms have major roles in nutrient cycling, ecological interactions, and influence and respond to environmental changes (Doney et al., 2012). Temperature is the strongest factor that determines microbial community composition, which parallels depth stratification, and these communities can serve as indicators for anthropogenically-induced climate changes (Sunagawa et al., 2015). It is predicted that carbon fixation by microbial primary producers will decrease which will have downstream effects on ocean microbiome structure and overall trophic level interactions (Moran, 2015).
While free-living marine microorganisms can provide relevant information and predictive models about the ocean, those host-associated microbes can also give environmental insight into the largest ecosystem on the planet.
Marine Animal Microbiomes: Toward Understanding Host–Microbiome Interactions in a Changing Ocean
Article by Apprill, 2017 licensed under the terms of the Creative Commons Attribution License (CC BY).
All animals on Earth form associations with microorganisms, including protists, bacteria, archaea, fungi, and viruses. In the ocean, animal–microbial relationships were historically explored in single host–symbiont systems. However, new explorations into the diversity of microorganisms associating with diverse marine animal hosts is moving the field into studies that address interactions between the animal host and a more multi-member microbiome. The potential for microbiomes to influence the health, physiology, behavior, and ecology of marine animals could alter current understandings of how marine animals adapt to change, and especially the growing climate-related and anthropogenic-induced changes already impacting the ocean environment. This review explores the nature of marine animal–microbiome relationships and interactions, and possible factors that may shift associations from symbiotic to dissociated states. I present a brief review of current microbiome research and opportunities, using examples of select marine animals that span diverse phyla within the Animalia, including systems that are more and less developed for symbiosis research, including two represented in my own research program. Lastly, I consider challenges and emerging solutions for moving these and other study systems into a more detailed understanding of host–microbiome interactions within a changing ocean.
Introduction
Marine animals are the icons of life in the oceans. They represent about two million species (Mora et al., 2011) and include a wide range of body designs, from the highly simplistic sponges lacking true tissues and organs to the complex vertebrates containing specialized tissues and organs, such as fish and marine mammals, with some iconic representatives presented in Figure 1. The bodies of marine animals span several orders of magnitude in size, from the abundant planktonic copepod (1–2 mm) to the highly mobile blue whale (30 m), the largest animal on Earth. Marine animals are key members of ocean ecosystems and serve as both prey of and predators for other animals within the complex ocean food web. In contrast to terrestrial animals, marine animals have developed strategies for osmoregulation as well as highly specialized approaches for maintaining homeostasis within diverse temperature, oxygen and pressure gradients of the ocean (Graham, 1990; Knoll and Carroll, 1999). Marine animals also possess sophisticated specializations and functions that promote their success on or within their benthic or pelagic habitats, including specializations for living or enduring depths (outlined in Figure 1) that vary widely in factors such as light availability, access to food and predator exposure.
Marine animals share the sea with a vast diversity of microorganisms, including protists, bacteria, archaea, fungi, and viruses which comprise millions of cells in each milliliter of the 1.3 billion km3 of water comprising the oceans (Eakins and Sharman, 2010). These microorganisms are several micrometers or smaller in size, but collectively their roles in oxygen production, nutrient cycling, and organic matter degradation provide critical functions to the oceans and Earth (Arrigo, 2005; Falkowski et al., 2008). Microorganisms that associate with marine animals are part of the animal’s microbiome, or collection of microorganisms that reside on or within the animal. Some of the microorganisms comprising the microbiomes of marine animals are thought to originate from this surrounding supply of seawater-associated cells (e.g., Nussbaumer et al., 2006), while other cells appear to have strict inheritance patterns, passed on through generations from the host (Sharp et al., 2007).
Over the past two decades, the widespread application of genomic and more integrative microbiological approaches have advanced our understanding of animal microbiomes (reviewed within McFall-Ngai et al., 2013). Symbiotic relationships between microorganisms and marine animals have been studied for decades, but technological advancements are providing new insights into the sheer diversity of microbial life in association with animals in the sea (Smith, 2001; Douglas, 2010). For example, reef-building corals are acknowledged as the icons of animal–microbial symbiosis in the sea, with corals hosting photosynthetic symbionts that make critical contributions to host nutrition (Muscatine et al., 1981). New reports of diverse protists, bacteria, archaea, and viruses in association with corals provide insights into the role of these cells for fulfilling diverse functional processes within the different niches of the coral host (reviewed within Thompson et al., 2015; Bourne et al., 2016). In fact, for many terrestrial animals, new reports of microbial symbioses provide insights into the variety of genetic and biochemical interactions and the ways that microorganisms contribute to animal health, behavior, and ecology (e.g., Ley, 2010; Cho and Blaser, 2012).
Understanding the microbiomes of marine animals is a growing research area within the field of marine science. Currently, the science is heavily focused on identifying consistent or “core” microbial members of the microbiome (Shade and Handelsman, 2012). After first gaining an understanding of “who’s there” generally using diversity-based surveys targeting the small subunit (SSU) ribosomal RNA (rRNA) gene, these microbiomes are then often examined as a whole or in smaller units to understand the function of the cells, the nature of the associations and ultimately gain insight into the role of the microbiome in animal health, physiology, ecology, and behavior (Ezenwa et al., 2012; McFall-Ngai et al., 2013). Additionally, the ocean environment is changing at unprecedented rates due to climate-related and anthropogenic-induced impacts (Halpern et al., 2008; Doney et al., 2012), and the microbiome is also being investigated for its possible role as a sentinel of a changing host (Ainsworth and Gates, 2016).
How environmental changes and animal life history events affect the microbiomes of marine animals is growing area of research, and there is an emerging focus on better understanding interactions between the animal, microbiome, and ocean environment, including the elements that may define their exchanges (e.g., Meron et al., 2011; Lesser et al., 2016; Webster et al., 2016). Therefore, this review considers the symbiosis and dissociated stages of animal–microbiome associations, and discusses factors and causes that may alter interactions between animals and their microbiome. Next, this review discusses current research examining animal–microbiome relationships and interactions, by focusing on select systems that represent diverse marine animal phyla and which span the range of being more to less developed for microbiome research. Two of these systems, corals and marine mammals, are represented in my own research program. Lastly, this review concludes with a discussion of challenges in marine animal–microbiome research and opportunities available to further advance knowledge of animal–microbiome interactions in the ocean.
Conceptual Model of Factors Contributing to Host-Microbiome Interactions
Host–microbiome dynamics are generally described as falling into two main categories: symbiosis, in which the organisms are involved in a normal metabolic and immune signaling interactions, and secondly dysbiosis, in which the relationship or interactions are heavily altered, possibly related to a major stress or infection event. While host–microbiome symbiosis and dysbiosis has been mostly considered in humans and humanized models (Hamdi et al., 2011; Nicholson et al., 2012; Scharschmidt and Fischbach, 2013), many of the same concepts are applicable to organisms in the sea (Egan and Gardiner, 2016), and are being explored in various systems (discussed below). The exact factors and mechanisms tipping the scale between symbiosis and dysbiosis will probably vary with complexity of the host anatomy and immune functioning (e.g., simplistic sponges and corals compared to more complex fish and sharks) as well as with the complexity of interactions that may occur between the members of the microbiome.
A normal animal–microbiome relationship in the ocean could be referred to as a “symbiotic” state, although the exact nature of the relationship may vary for each cell in the association. For example, cells residing on the surface or within the gut cavity of an animal are physically associated, yet do not share as intimate of an association as those microbes residing intracellularly with the host’s cells. This normal symbiotic state is subject to a variety of environmental fluctuations, which are generally defined by the characteristics of the habitat (Figure 1). For example, in the ocean’s upper photic zone, animals are exposed to variations in temperature and light, and host–symbiont interactions, especially in ectothermal animals, could alter on cycles such as seasons that generally control the temperature and light environment. Normal fluctuations in animal-specific patterns could also alter host–microbiome relationships. For example, changes in diet, possibly due to short-term prey availability, can alter gut microbiota and host–microbiome metabolic exchanges in other systems (e.g., David et al., 2014), and similar diet trends may also affect marine animals. Stress is another factor more complex animals encounter on a daily basis (e.g., squid, crabs, fish), which could be related to social/territorial encounters or chasing or fleeing from prey, and the short-term production of stress hormones such as cortisol can influence host–microbiome relationships (e.g., Moloney et al., 2014).
There are also normal animal life events that occur on longer time frames or that are more drastic in scope, such as animal development, aging, and reproduction. In non-marine animals, these factors have been shown to cause alterations in animal-microbial relationships (e.g., Heintz and Mair, 2014). These changes can be drastic enough to cause a state of “altered symbiosis” that could extend for short or longer term. For example, the gut microbiome of women generally becomes altered during pregnancy (Koren et al., 2012). Events resulting in normal animal stress may also lead to a more altered symbiotic state, for example if social conflict was more chronic, perhaps due to the pressures of a particular habitat. Data from humans and humanized models suggests that the microbial community and associated genes do fluctuate with the normal variations and animal life events, and both may be considered “healthy” fluctuations (Nicholson et al., 2012). However, how these fluctuations affect exchanges between the host and microbiome is much less understood.
If symbiosis and altered symbiosis are considered as normal host–microbiome variations throughout an organism’s life, dysbiosis is the breakdown in the relationship, generally related to one or more major stressors, and can greatly alter host health and lead to a disease state (Holmes et al., 2011). The stressor may come from an external source, such as a pollutant, infective agent, or a longer-term natural environmental change—and there are probably countless other factors that could fit this category (Figure 2). For example, one of the most visible signs of host–microbiome dysbiosis is with scleractinian corals, whose relationship with unicellular algae breaks down after long-term yet small increases in seawater temperature, causing the coral to become “bleached” (Brown, 1997). In humanized models, major stressors such as malnutrition are related to less physically visible changes in innate immunity, which are linked to microbial ecology (Hashimoto et al., 2012). Understanding the relationship between symbiosis, dysbiosis and host health and functioning are general topics of research in most host–microbiome studies, but the environmental changes occurring in the ocean environment have made this area of research more pressing for marine animals. Overall, the concepts behind the model presented in Figure 2, as well as variations of this model, are generally driving much of the current research examining animal-microbial relationships in the ocean.
Overview of Diverse and Emerging Animal-Microbiome Study Systems
The microbiomes of diverse marine animals are currently under study, from simplistic organisms including sponges (e.g., Webster et al., 2010) and ctenophores (Daniels and Breitbart, 2012) to more complex organisms such as sea squirts (Blasiak et al., 2014) and sharks (Givens et al., 2015). Below I present some of the current study systems that represent a diverse cross-section of marine animal phyla, and trends of research in these systems including focus on symbiosis and dysbiosis. The organisms are generally presented in order from increasing to decreasing knowledge about the host-microbiome relationship.
The relationship between the Hawaiian bobtail squid Euprymna scolopes (phylum Mollusca) and the bioluminescent bacterium Vibrio fisheri (also recognized as Aliivibrio fisheri) is one of the best studied symbiotic relationships in the sea and is a choice system for general symbiosis research (Figures 3A,B). The E. scolopes-V. fisheri relationship has provided insight into fundamental processes in animal-microbial symbioses, and especially biochemical interactions and signaling between the host and bacterium (McFall-Ngai, 2000, 2014). Much of this research focuses on establishment of the symbiosis, with less focus on dysbiosis. Additionally, because V. fisheri exists in the light organ, these studies have been primarily limited to this one isolated relationship, with the remainder of the squid’s microbiome virtually unstudied (but see Barbieri et al., 2001; Collins et al., 2012). The E. scolopes–V. fisheri system offers simplicity for the study of host–microbial interactions and numerous helpful developments in animal husbandry, genomic tools, and experimental design that could be applied to ask more comprehensive questions about squid–microbiome interactions, including the conditions leading to dysbiosis of relationships.
Similar to E. scolope, the gutless marine oligochaete worm Olavius algarvensis (phylum Annelida) is another relatively well-studied marine host to microbes. One major difference is that it has been studied within the context of a larger consortium of microorganisms compared to E. scolope. These 3 cm long worms reside within shallow marine sediments of the Mediterranean Sea. The worms do not contain a mouth or a digestive or excretory system, but are instead nourished with the help of a suite of extracellular bacterial endosymbionts that reside upon coordinated use of sulfur present in the environment (Dubilier et al., 2001). This system has benefited from some of the most sophisticated ‘omics and visualization tools (Woyke et al., 2006). For example, multi-labeled probing has improved visualization of the microbiome (Schimak et al., 2016) and transcriptomics and proteomics have been applied to examine host–microbiome interactions, including energy transfer between the host and microbes (Kleiner et al., 2012) and recognition of the consortia by the worm’s innate immune system (Wippler et al., 2016). The major strength of this system is that it does offer the ability to study host–microbiome interactions with a low diversity microbial consortium, and it also offers a number of host and microbial genomic resources (e.g., Woyke et al., 2006; Ruehland et al., 2008). Dysbiosis has not been heavily investigated in this system, and given the growing knowledge of host–microbial interactions, O. algarvensis could be an imperative animal for dysbiosis research.
As mentioned above, corals (phylum Cnidaria) (Figure 3C) are one of the most common examples of an animal host whose symbiosis with microalgae can turn to dysbiosis, and is visibly detected as bleaching. Coral microbiomes have been examined in a variety of studies, which demonstrate how variations in the ocean environment, most notably temperature, light, and inorganic nutrients, affect the abundance and performance of the microalgal symbionts, as well as calcification and physiology of the host (Dubinsky and Jokiel, 1994; Anthony et al., 2008). Studies have also suggested that resident bacteria, archaea, and fungi additionally contribute to nutrient and organic matter cycling within the coral, with viruses also possibly playing a role in structuring the composition of these members, thus providing one of the first glimpses at a multi-domain marine animal symbiosis (reviewed in Bourne et al., 2016). The gammaproteobacterium Endozoicomonas is emerging as a central member of the coral’s microbiome, with flexibility in its lifestyle (Figure 3D) (Neave et al., 2016, 2017). Ocean disturbances including elevated temperature and ocean acidification have been shown to disrupt the coral’s associated bacteria (Thurber et al., 2009; Meron et al., 2011), including relationships with Endozoicomonas (Morrow et al., 2015). However, some members of this microbiome appear to be stable across large environmental gradients (Hernandez-Agreda et al., 2016). In addition to nutrition, the microbiome plays a role in coral health and stress. Temperature and light stress to corals can result in overproduction of reactive oxygen species (ROS), which can be detrimental to Symbiodinium and result in bleaching, but the associated bacteria have also recently been shown to contribute extracellular ROS (Diaz et al., 2016; Zhang et al., 2016), which could play a signaling role with the host or within the microbiome. Given the recent mass bleaching occurring on reefs (Hughes et al., 2017), corals will likely continue to be a useful and popular system for symbiosis and dysbiosis research. There are number of resources available to further promote study of the coral microbiome, including integrated databases (Franklin et al., 2012; Madin et al., 2016), a growing number of host and microbial genomes (Shinzato et al., 2011; Bayer et al., 2012; Neave et al., 2017), and laboratory amendable “model” systems (Weis et al., 2008; Baumgarten et al., 2015).
Sponges (phylum Porifera) are common members of the ocean’s diverse benthic habitats and their abundance and ability to filter large volumes of seawater have led to the awareness that these organisms play critical roles in influencing benthic and pelagic processes in the ocean (Bell, 2008). They are one of the oldest lineages of animals, and have a relatively simple body plan that commonly associates with bacteria, archaea, algal protists, fungi, and viruses (reviewed within Webster and Thomas, 2016). Sponge microbiomes are composed of specialists and generalists, and complexity of their microbiome appears to be shaped by host phylogeny (Thomas et al., 2016). Studies have shown that the sponge microbiome contributes to nitrogen cycling in the oceans, especially through the oxidation of ammonia by archaea and bacteria (Bayer et al., 2008; Radax et al., 2012). Most recently, microbial symbionts of tropical sponges were shown to produce and store polyphosphate granules (Zhang et al., 2015), perhaps enabling the host to survive periods of phosphate depletion in oligotrophic marine environments (Colman, 2015). The microbiomes of some sponge species do appear to change in community structure in response to changing environmental conditions, including temperature (Simister et al., 2012a) and ocean acidification (Morrow et al., 2015; Ribes et al., 2016), as well as synergistic impacts (Lesser et al., 2016). Understanding the effect of these altered host–microbiome interactions on sponge growth and ecology are topics for further research. As such, there are a number of resources to support research on sponges including a curated database of sponge–microbial sequences (Simister et al., 2012b), cultivated microbial isolates and sponge cell cultures from some species (Taylor et al., 2007) to facilitate investigations.
Atlantic killifish, (Fundulus spp., Phylum Chordata) (Figure 3E) are one of the most abundant estuarine fishes in North America, and are related to other families with more global distributions in coastal areas (Fritz et al., 1975; Lotrich, 1975). The killifish have a broad North American geographic distribution yet limited subpopulation movement, and thus the Atlantic killifish have become a useful field-residing model species for examining biological and ecological responses to natural environment conditions (salinity, oxygen, pH, and temperature) as well as chemical pollutants (Burnett et al., 2007). While the killifish microbiome (Figure 3F) has not been extensively studied, there is work examining the influence of pollutants on the skin and mucus of the fish, which suggests that this skin microbial community is relatively resistant to change (Larsen et al., 2015). Populations of the fish offer a unique host genetic resistance to toxicity (Hahn et al., 2004), and it is possible that this resistance is also facilitated by features of the microbiome. The Atlantic killifish appear to be an ideal study species for microbiome investigations and especially the response of the host–microbiome symbiosis to changing ocean conditions. Specifically, the killifish can be maintained in laboratory aquaria, they are hardy and amendable to experimental manipulation, and spawning material can be acquired for developmental (Burnett et al., 2007).
The microbiomes of marine mammals (phylum Chordata) (Figure 3G) have recently been investigated and offer a comparative study system to terrestrial mammals (reviewed within Nelson et al., 2015). Marine mammals are often viewed as sentinel species of the ocean, because they appear to rapidly respond to ocean conditions, disturbances, and pathogens similarly to humans (Bossart, 2011). Several studies have examined the skin (Figure 3H), gut and respiratory microbiomes of diverse marine mammal species, and describe species-specific relationships (Johnson et al., 2009; Apprill et al., 2014; Bik et al., 2016). Connections between the community composition of the microbiome and animal health (Apprill et al., 2014) and diet (Nelson et al., 2013; Sanders et al., 2015) have been made, and more detailed studies are needed to understand these specific connections. While there are very limited resources available for studying host–microbiome interactions in marine mammals, there are some animals in captivity as well as well-studied populations that will heighten investigations of host–microbiome symbiosis and dysbiosis in these sentinel species.
Challenges and Emerging Solutions to Studying Animal–Microbiome Interactions
A number of the systems highlighted above are currently examining animal–microbiome interactions, but these are generally most developed in systems such as O. algarvensis that offer lower complexity microbiomes, or within the single host–symbiont relationship between E. scolopes and V. fisheri. As such, a major challenge to the field is exploring host–microbiome interactions within the context of a diverse microbiome, and especially if the microbiome includes members such a uncharacterized protists, fungi, and viruses, which have generally not been described in most marine animal systems. Therefore, a through description of the microbiome is a first necessity, but this still presents many challenges on a variety of levels. For example, amplifying or shotgun sequencing microbial DNA with the presence of abundant host cells often requires optimization or high sequencing output (e.g., Rocha et al., 2014; Weber et al., 2017). Taxonomic databases generally contain few microbial sequences from many of these animals, and therefore simple tasks such as assigning taxonomy can be challenging. Developing animal-specific databases (Simister et al., 2012b), which include the next-generation supplied sequences generally not available in curated taxonomic databases, could help alleviate this problem. There are also a number of new tools for metagenomics-based analysis, including advancements in binning genomes from complex samples (Kang et al., 2015; Graham et al., 2017) as well as new visualization methods for comparing genomes (Eren et al., 2015; Wagner et al., 2017). A challenging issue that has received less attention is how to gain information from unknown genes and gene families, which can make up over half of the environmental microbial genomes. Algorithms utilizing gene function predictions do provide some assistance with this problem (Mi et al., 2015), and these tools may improve as more environmental microbial genomes are available. Lastly, computational tools are emerging to facilitate identifying associations between host genetic variation and microbiome composition (Lynch et al., 2016).
Once some of these hurdles are overcome and a comprehensive view of the microbiome is available, researchers can then explore the nature of the host–microbiome relationship. Visualization using a variety of different microscopy-based techniques is a powerful tool to recognize the physical relationship between a host and the microbiome, as well as the organization of cells within the microbiome. Electron microscopy provides the most detailed information about this organization, but this is less useful for complex microbiomes because taxonomically distinct microbial cells with similar appearances cannot be distinguished. Fluorescent in situ hybridization (FISH), and especially using a multi-taxonomic, simultaneous probing technique such as Combinatorial Labeling and Spectral Imaging FISH (CLASI-FISH) (Valm et al., 2011) can provide significant insight into host–microbe and microbe–microbe interactions. FISH techniques do require optimization for some animal systems, such as corals that possess autofluorescent host tissues (Wada et al., 2016). Visualization techniques can also be paired with isotope probing, to provide opportunities to trace the transfer of specific molecules between the host and microbiome, as well as within the microbiome using Nano-SIMS and Nano-SIP approaches (Musat et al., 2016). There have also been many recent instrumental and database advances in the field of metabolomics (Beisken et al., 2015), and this approach is beginning to be applied to examine host–microbiome interactions (Gomez et al., 2015; Sogin et al., 2016). An understanding of specific microbial metabolites will help facilitate targeted investigations of how these products affect the host nutritional and immune systems.
Lastly, experimental manipulation is a challenge to the study of host–microbial interactions in the ocean. Studying the animals in their natural environment is the most ideal approach because it ensures that the surrounding seawater microbial community is maintained. However, natural experiments are only as helpful as the natural variability in the host-microbe system, and generally only afford the opportunity to study events such as seasonality, animal growth or other life history events. Artificial systems such as aquaria or mesocosms offer opportunities to manipulate environmental conditions or expose the animal to antibiotics or other molecules that are difficult to dose in the wild. However, not all animals are ideal for these systems (e.g., large whales, hydrothermal vent worms), and it can be challenging to reproduce some environmental conditions. Advances in aquaria design that offer consistency in environmental conditions and the ability to manipulate complex environmental interactions, such as the Australian Institute of Marine Science’s National Sea Simulator, provide opportunities to conduct more realistic experiments. As the need to understand how host–microbiome interactions will alter with the forecasted changes in ocean temperature and pH, facilities such as this will become critical to animal–microbiome research in the ocean.
While studies of marine animal–microbiome interactions are certainly plagued by a number of challenges, the future is also very bright for this emerging field. Many of the new bioinformatics and methodological advancements now available to marine biologists stem from the biomedical field, and thus marine animal microbiome research, as well as other environmental-based fields, are profiting from the elevation in microbiome research funding and attention. There could also be growing interest in using marine animals as models for examining resilience, promoted by the fact that alterations in the ocean conditions are often outpacing those in terrestrial environments. Given the phylogenetic breath of animals in the ocean, coupled with the many diverse ocean environments, there is certainly a wealth of research opportunities available to study host–microbiome interactions in the ocean.
Author Contributions
The author confirms being the sole contributor of this work and approved it for publication.
Funding
Funding was provided by the WHOI’s Andrew W. Mellon Foundation Endowed Fund for Innovative Research.
Conflict of Interest Statement
The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Acknowledgments
Many thanks to Margaret McFall-Ngai and Evan D’Alessandro for use of images and to Laura Weber for early comments on this review.
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Studying Ocean Microbiomes Data
Query \(1\)
Prevalence of certain marine topics appearing in combination with the term holobiont in the title, abstract, and/or keywords of scientific publications from 1990 to February 2021 (N = 1269). Bars indicate the percentage of publications that contain terms related to a certain marine topic (cnidarians, corals, sponges, etc.). It is important to note that the different topics may overlap in several publications. For example, many publications treat of the topics of coral and sponge holobionts together. Data retrieved from the curated citation and abstract database Scopus on the date 10/03/2021. (Stevenne et al., 2021)
Check your Understanding
• Which environmental factor is the biggest driver of change for the community composition of free-living oceanic microbes?
• What factors affect the normal symbiotic state between marine host and their microbiome?
• Describe one well-studied marine animal-microbial symbiosis and its importance.
• What are some challenges to studying marine host-associated microbiomes?
References
1. Apprill, A. (2017). Marine Animal Microbiomes: Toward Understanding Host–Microbiome Interactions in a Changing Ocean. Frontiers in Marine Science, 4. https://www.frontiersin.org/article/10.3389/fmars.2017.00222
2. Doney, S. C., Ruckelshaus, M., Emmett Duffy, J., Barry, J. P., Chan, F., English, C. A., Galindo, H. M., Grebmeier, J. M., Hollowed, A. B., Knowlton, N., Polovina, J., Rabalais, N. N., Sydeman, W. J., & Talley, L. D. (2011). Climate Change Impacts on Marine Ecosystems. Annual Review of Marine Science, 4
3. Moran, M. A. (2015). The global ocean microbiome. Science, 350(6266), aac8455. https://doi.org/10.1126/science.aac8455
4. Stévenne, C., Micha, M., Plumier, J.-C., & Roberty, S. (2021). Corals and Sponges Under the Light of the Holobiont Concept: How Microbiomes Underpin Our Understanding of Marine Ecosystems. Frontiers in Marine Science, 8. https://doi.org/10.3389/fmars.2021.698853
5. Sunagawa, S., Pedro, C. L., Samuel, C., Roat, K. J., Karine, L., Guillem, S., Bardya, D., Georg, Z., R, M. D., Adriana, A., M, C.-C. F., I, C. P., Corinne, C., Francesco, d’Ovidio, Stefan, E., Isabel, F., M, G. J., Lionel, G., Falk, H., … Didier, V. (2015). Structure and function of the global ocean microbiome. Science, 348(6237), 1261359. https://doi.org/10.1126/science.1261359 | textbooks/bio/Microbiology/Microbiomes_-_Health_and_the_Environment_(Parks)/04%3A_Environmental_Microbiomes/4.02%3A_The_Ocean_Microbiome_and_Marine_Life.txt |
Soil Microbiomes
The terrestrial landscape on Earth harbors countless microbes that have major responsibilities in shaping their surrounding environment. One gram of soil can contain up to 10 billion microorganisms and consist of thousands of different species. Each ecosystem has unique soil properties that cultivate a diverse array of microbial communities, which are primarily composed of bacteria, however archaea, protists, fungi, viruses, and other microscopic organisms can be found in varying abundances too. The composition and health of the surrounding environment and its macro inhabitants are dependent on their soil borne microbial partners, and a disturbance in the balance of the soil microbiome can have far-reaching effects (Omotayo and Babalola, 2021).
Environmental and agricultural sustainability is important in a number of regards, especially maintaining biodiversity, plant and animal health, ecosystem homeostasis, and even human health. Anthropogenic effects on the climate and environment have become abundantly apparent, and it is imperative to adjust mindsets to become more environmentally conscious, not only to preserve aspects of nature, but to accommodate an increasing human population. The soil is foundational to agricultural practices, and therefore food production, and so a better understanding of various soil microbiomes could serve to benefit future applications and practices (Tosi et al., 2020). One possibility, is to become less reliant on synthetic nitrogen fertilizers for soil, and utilize symbiotic nitrogen-fixing bacteria as sources for plant nitrogen.
Microbe to Microbiome: A Paradigm Shift in the Application of Microorganisms for Sustainable Agriculture
Article by Ray et al., 2020 licensed under the terms of the Creative Commons Attribution License (CC BY).
Light, water and healthy soil are three essential natural resources required for agricultural productivity. Industrialization of agriculture has resulted in intensification of cropping practices using enormous amounts of chemical pesticides and fertilizers that damage these natural resources. Therefore, there is a need to embrace agriculture practices that do not depend on greater use of fertilizers and water to meet the growing demand of global food requirements. Plants and soil harbor millions of microorganisms, which collectively form a microbial community known as the microbiome. An effective microbiome can offer benefits to its host, including plant growth promotion, nutrient use efficiency, and control of pests and phytopathogens. Therefore, there is an immediate need to bring functional potential of plant-associated microbiome and its innovation into crop production. In addition to that, new scientific methodologies that can track the nutrient flux through the plant, its resident microbiome and surrounding soil, will offer new opportunities for the design of more efficient microbial consortia design. It is now increasingly acknowledged that the diversity of a microbial inoculum is as important as its plant growth promoting ability. Not surprisingly, outcomes from such plant and soil microbiome studies have resulted in a paradigm shift away from single, specific soil microbes to a more holistic microbiome approach for enhancing crop productivity and the restoration of soil health. Herein, we have reviewed this paradigm shift and discussed various aspects of benign microbiome-based approaches for sustainable agriculture.
Introduction
The health of soil plays an essential role in the ability of plants to produce food, fuel, and fiber for a growing world population. To keep pace, the total area of cultivated land worldwide has increased over 500% in the last five decades with a 700% increase in fertilizer use and a several-fold increase in pesticide use (Banerjee et al., 2019). In addition to being the world’s largest agricultural producers and exporters, the EU, Brazil, United States, and China also are some of the world’s largest pesticide users – each using 827 million, 831 million, 1.2 billion, and 3.9 billion pounds of pesticides, respectively, in 2016 (Donley, 2019). However, these numbers are not sustainable from either a supply-chain or environmental perspective. Thus, because natural resources are limited and their overuse pollutes the environment, the continued use of fertilizers and water to meet the demand of future global food requirements is not sustainable. Of relevance here is that agricultural intensification with high resource use and low crop diversity can negatively affect soil- and plant-associated microbiota (the so-called “phytobiome”) with subsequent impacts on critical ecosystem services (Matson et al., 1997).
There is growing evidence that aboveground plant diversity supports belowground microbial biodiversity, primarily through root exudation and rhizo-deposition (Bais et al., 2006; Eisenhauer et al., 2017; Morella et al., 2020). These more simple carbohydrates released into the soil primarily feed bacteria (Gunina and Kuzyakov, 2015) and are the most abundant near the root surface and diffuse along a gradient as distance from the root increases (Gao et al., 2011). The microbial composition is more abundant and complex in the rhizosphere, the narrow zone surrounding plant roots, with up to 109 cells per gram in typical rhizospheric soil, comprising up to 106 taxa (Lakshmanan et al., 2017). The more complex carbohydrates (e.g., lignin, cellulose) are largely degraded by decomposer fungi that break down these recalcitrant compounds into forms that can be used by other microbes. This conversion is largely decoupled from conventional agricultural practices, wherein the organic matter content is often lost to the system (Craven and Ray, 2019), and the carbon flux is at least partially unregulated in this regard. Again, defining nutrient fluxes with techniques like Stable Isotope Labeling (SIP) holds great potential to define and construct resilient, functioning and beneficial microbiomes that can contribute to future holistic agriculture. Thus, applying an efficient and diverse soil microbiome backed by these new technologies can facilitate and promote sustainable agriculture and can effectively contribute to meet the triple requirements of economic, social and environmental sustainability (Ray and Craven, 2016).
Historically, microorganisms that promote plant growth and nutrient acquisition have been used largely as single strains in agriculture to offset such fertilizer inputs as nitrogen and phosphorous. However, studies of natural populations suggest that groups of microbes with distinct function niches play pivotal roles in adhering and desorbing inorganic nutrients to physical surfaces, as well as breaking down organic residues and incorporating them into the soil (Lakshmanan et al., 2014; Finkel et al., 2017; Kumar and Dubey, 2020). Conceptually, such observations support the idea of the microbiome as a second genome or an extended genome of the plant (Vandenkoornhuyse et al., 2015). It is now evident that improving plant performance in a sustainable manner is beyond the binary interaction between a specific microbe or a consortium of beneficial microbes and a targeted host plant. This is a much more complex set of interactions than previously thought that requires modeling for improving predictable outcomes. In this review, we will highlight the current state of the art for the incorporation of specific plant growth-promoting microorganisms and discuss the principles and management practices for next-generation, microbiome-based approaches for sustainable agriculture.
Application of Beneficial Microbes in Sustainable Agriculture: Past, Present and Future
Since the early 1800s, the United States Department of Agriculture has recommended the use of certain rhizobacteria to improve nitrogen fertility in leguminous crops (Schneider, 1892). Since that time, a great deal of research has been conducted on this relationship between legumes and these bacteria, now termed rhizobia, that inhabit unique structures, the nodules, that form on the roots. Rhizobia infecting these nodules are now capable of “biological nitrogen fixation,” whereby di-nitrogen is fixed into forms that can be used by the plant. Symbiotically, the bacteria trade these nitrogenous compounds to the host plant in exchange for photosynthetically derived carbon. Despite these limited applications, much remains to be learned regarding both the functional and taxonomic diversity of these symbiotic bacteria and their host plants, the role they play in the global nitrogen cycle, and ultimately, how they can best be harnessed for improving plant productivity. This is particularly true for marginal lands that are not suited for row crop production but will need to be incorporated into global food and forage production approaches moving forward. Further, such degraded lands must but regenerated with the goal of restoring soil health and productivity. Any successful endeavor in this regard must include a characterization of the soil microbiome, both taxonomically and functionally. Attempts currently are underway to fix nitrogen in such non-legumes as wheat, corn and other staple crops that produce the bulk of human food by engineering symbiotic relationships using synthetic biology approaches (Rogers and Oldroyd, 2014; Ryu et al., 2020). Such approaches would significantly impact global food supplies, and may function adequately to reduce the arable land required to meet productivity goals.
Plant growth-promoting microbes not only play critical and diverse roles in growth promotion per se, but also in improving various aspects of plant resilience against a wide array of biotic and abiotic stresses (Arnold et al., 2003; Sun et al., 2010; Agler et al., 2016; Azad and Kaminskyj, 2016; Singh, 2016; Oleńska et al., 2020; Rai et al., 2020). In this context, researchers globally have worked over the last several decades on plant growth-promoting microorganisms, such as root-associated mycorrhizal fungi, across a broad range of crops and encompassing a wide range of agro-climatic conditions. For perspective, Brundrett and Tedersoo (2018) recently reviewed 135 years of mycorrhizal research and reported that merely 8% of the vascular plants are non-mycorrhizal, suggesting that plant families associating with mycorrhizae have been very successful over the evolution of the plant kingdom.
Traditionally, agricultural application of beneficial microorganisms involves a few types of well-characterized microbes, such as mycorrhizal fungi or rhizobia bacteria, for which the mechanisms underlying the plant growth promotion effects are well understood. Further, most of these studies focused solely on the ability of the applied microorganisms to facilitate such specific plant growth-promoting traits as phosphate solubilization, nitrogen fixation, ACC deaminase production (Sarkar et al., 2018), siderophore production, biofilm formation, plant hormone production, biotic, and abiotic stress tolerance or resistance, among others (Weyens et al., 2009; Bhattacharyya and Jha, 2012; Singh et al., 2019). While these beneficial microorganisms can impart considerable benefits to plant growth and fitness, they are typically documented in simple, one-on-one studies, often conducted in sterile soils in greenhouse conditions. As a consequence, the effects found in such simplified conditions often fail to translate to more complex field situations (Chutia et al., 2007; Nicot et al., 2011; Parnell et al., 2016). Soil in field plots have more complex microbial environments that are presumably adapted to the local eco-environment.
In recent years, next-generation sequencing has revolutionized our understanding of microbial community composition and function, and together with improved culturing methodologies has greatly facilitated the use of biologicals in the field (Schweitzer et al., 2008; Panke-Buisse et al., 2014; Mueller and Sachs, 2015). Specifically, metagenomics-based approaches have uncovered vast, previously unrecognized populations of microbes that may have new or enhanced properties that could be used for agriculture, bioremediation, and human health. For example, comparative analyses of rhizosphere metagenomes from resistant and susceptible tomato plants enabled the identification and assembly of a flavobacterial genome that was far more abundant in the resistant plant rhizosphere microbiome than in that of the susceptible plants. Such findings certainly reveal a role for native microbiota in protecting plants from phytopathogens, and pave a way forward for the development of probiotics to ameliorate plant diseases akin to human health (Kwak et al., 2018). In another study, a 16S rRNA gene amplicon sequencing analysis of maize root microbiome led to the identification of bacteria that promote growth under low temperature conditions (Beirinckx et al., 2020). Additionally, principles of consortium design that rely on cross-talk, cross-feeding and/or substrate channeling between different microorganisms offer new opportunities for “intelligent” consortia design (Calvo et al., 2014; Vorholt et al., 2017; Paredes et al., 2018). We propose that the manipulation of the plant microbiome holds tremendous potential for agricultural improvement (Table 1). Through recent years of research, it is elucidated how microbes worked in nature before, and how decades of chemical fertilizer use have silenced their ability to improve plant fitness and soil health. Therefore, designing a microbial consortium that carefully weighs and evaluates the relationship between inoculants and the resident microbiome would substantially improve the plant growth-promoting potential and resilience of agricultural biologicals to boost plant growth. In this review, we will discuss the key considerations that would improve the likelihood of microbial products to improve crop yield, decrease disease severity and/or ameliorate abiotic stress response. Further, it is likely that such considerations would reduce the inconsistency between the performances of beneficial microbes from controlled greenhouse conditions and more natural environments.
Microbes for Plant Growth Promotion: A Reductionist Approach
Sustainable agriculture primarily focuses on reducing the dependency of plants on chemical fertilizers and improving their ability to grow on marginal soil types. For such purposes, individual microorganisms for plant growth-promotion have largely focused on those that facilitate growth and development by enhancing acquisition of nutrient resources from the environment, including fixed nitrogen, iron and phosphate, or modulating growth by altering plant hormone levels (Figure 1) (Hayat et al., 2010). Another approach aimed at reducing yield losses to disease relies on microbes that decrease or prevent the deleterious effects of plant pathogens by several different mechanisms (Glick, 2012), i.e., by acting as a biocontrol agent. Microbe-based plant growth-promoting products, more popularly marketed as biofertilizer, has been commercially available in many countries since the 1950s (Timmusk et al., 2017). Application of such plant growth-promoting microbes in agricultural context and more specifically as inoculants has been nicely reviewed by Souza et al. (2015). However, under certain cases, the results obtained in the laboratory could not be reproduced in the field primarily due to the presence of many crop species and crop varieties, variable environmental conditions between fields, (Timmusk et al., 2017; Saad et al., 2020), occasionally due to the low quality of the inocula, and their inability to compete with the indigenous population. In that context, it is important to consider the fact that there is always greater likelihood of success by introducing mixed cultures of compatible microorganisms, rather than single, pure cultures. This is simply because each strain in the multi-strain consortium can compete effectively with the indigenous rhizosphere population and enhance plant growth with its partners. For example, sequential inoculation of nitrogen fixing bacterium Azotobacter vinelandii, followed by plant growth-promoting root-endophytic fungus Serendipita indica demonstrated better growth in rice (Dabral et al., 2020). Dual inoculation of S. indica and Mycolicibacterium strains boosted the beneficial effects in tomato (del Barrio-Duque et al., 2019) and that of arbuscular mycorrhizal fungus with plant growth-promoting bacteria Bacillus subtilis demonstrated better growth in wheat (Yadav et al., 2020) as compared to the singly inoculated plants. There also are numerous other reports that showed two strains used in a consortium promoted plant growth in a more effective manner (Nadeem et al., 2013; Fatnassi et al., 2015; Priyadharsini and Muthukumar, 2016). Nevertheless, to unlock the full potential of soil microbes for such nutrient cycling as nitrogen or phosphorus and providing plant protection against biotic and abiotic stress microbiomes, it is necessary to develop strategies to comprehend the functional capabilities of soil microbial communities. Irrespective of the approach, persistence is the first and foremost principle underlying the design of a successful microbial consortium for conferring plant growth promotion. This is not surprising, as the survival and activity of microbes in any soil system face a monumental task of competing with the myriad of microbes naturally adapted to that same soil. Thus, in addition to establishment of a compatible interaction with the host, a successful microbial inoculant has to subsequently compete and persist in the context of indigenous microbes as well as local abiotic conditions (Finkel et al., 2017). It has been reported that bacterial inoculations can persist in soil up to 7 weeks, but whether this inoculum also can provide plant growth benefits is not clear (Schreiter et al., 2014). While persistence or resilience of any microbial inoculum is more dependent on biotic components of a specific soil type, their persistence can be improved by inoculating crops with consortia rather than single strains (Verbruggen et al., 2012; Nemergut et al., 2013). Thus, it can arguably be stated that the diversity of a microbial inoculum, in addition to its plant growth-promoting traits, is critical for enhancing productivity and longevity (Cordero and Polz, 2014).
To improve the likelihood of success for such a management strategy, a priori knowledge of indigenous microbial populations competing with the introduced plant growth-promoting agent(s) is critical. While a reductionist approach can define the currency of individual plant-microbe interactions, the concepts of microbial community survival and functioning require, a more holistic, microbiome-based approach empowered by next-generation sequencing technology to study plant-microbe interactions at the community level (Figure 2). Indeed, this will enable researchers to design more robust, synthetic microbial consortia capable of reliably enhancing agricultural productivity.
Microbiomes for Plant Growth Promotion: The Holistic Approach
Soil is a vastly heterogeneous growth medium, providing a wide spectrum of ecological niches for microorganisms that enable diverse strains to coexist and form complex microbial communities. When the earliest plants extended their roots into primordial soils, they encountered a habitat already teeming with bacterial and fungal life (Bulgarelli et al., 2013; Kemen, 2014). Since that early time, plants have interacted with rhizosphere microbes, evolving strategies to forge beneficial alliances with some while keeping others at bay. Such early associations certainly had consequences on plant growth and development. Therefore, a more holistic approach is needed to understand better these microbes and the roles they play in the overall health of plant and soil (Figure 1). Again, recent advances in next-generation sequencing technology and the decreasing costs associated with that technology now allow us to evaluate how microbial populations fluctuate in both space and time or to identify core microbiomes that appear conserved among host genotypes or species (Sergaki et al., 2018). Thus, although culture-independent methods have contributed tremendously to our understanding of plant-associated fungal and bacterial community structures, the study of microbiome functions remains challenging because of the inherent noise of plant-associated microbial communities. It is now well known that there are core sets of microbes that, depending on the host, are recognized as keystone taxa that consistently associate with healthy plants (Banerjee et al., 2018). Consequently, researchers working with specific plant-microbe interactions have increasingly acknowledged the mitigating impact these larger microbial communities have on individual plant-microbe outcomes for plant growth promotion or fitness. Now, plant-associated fungal and bacterial stains from various plant species are being isolated, which will provide in the near future an inestimable resource for assembling taxonomically defined microbial communities with increasing complexity. Therefore, it is now imperative to take advantage of this knowledge to design consortia of microbes to maintain a sustainable rhizosphere community, with key functional properties that include plant protection, nutrient acquisition, and alleviating biotic and abiotic stress responses. From that perspective, synthetic community (SynCom) approaches can provide functional and mechanistic insights into how plants regulate their microbiomes (Figure 1). Not surprisingly, recent culture-independent analyses thus have paved the way for developing SynComs more often (Bodenhausen et al., 2014; Armanhi et al., 2018; Carlström et al., 2019).
Mycorrhizal fungi, at least the arbuscular type, were early symbiotic partners of most land plant species, improving nutritional conditions through soil exploration and pathogen resistance of host plants (Klironomos et al., 2000). In reward for the essential physiological services, they receive ca. 20% of net photosynthetic products from plants (HoÈgberg et al., 2001). Other mycorrhizal systems may have different nutritional benefits and costs, as has been proposed for the serendipitous system (Craven and Ray, 2019). Additionally, third-party partners can modulate the outcome of the tripartite interaction, such as the case of mycorrhizal helper bacteria (Frey-Klett et al., 2007), fungal endobacteria (Bonfante and Desirò, 2017; Bonfante et al., 2019) like Candidatus Moeniiplasma glomeromycotorum within the spores and hyphae of Glomeromycotina (Naito et al., 2017), Rhizobium radiobacter within Serendipita indica (Guo et al., 2017), and N2-fixing endobacteria Pseudomonas stutzeri inside basidiomycetes yeast endophyte Rhodotorula mucilaginosa (Paul et al., 2020). Hence, it is imperative to consider the composition and functioning of these microbe–microbe interactions to understand plant–microbiome associations in a holistic manner.
Principles and Management of Rhizosphere Microbiomes for Sustainable Agriculture
Competence and Resilience of the Rhizosphere Microbiome: Impact of Introduced Microbes on Native Microbiomes
In 1904, the German agronomist and plant physiologist Lorenz Hiltner coined the term rhizosphere (Hartmann et al., 2008) to describe the area around a plant root inhabited by a unique population of microorganisms. Since then, numerous studies have been undertaken to decipher the interplay between plants and rhizosphere microorganisms, encompassing a wide variety of plant growth-promoting bacteria, fungi, insects, protozoans, viruses, etc. (Marschner, 2012; McNear, 2013). The majority of these studies have traditionally followed a simple principle for maximizing successful host infection by pre-inoculation onto the targeted crop of choice to provide a competitive advantage for a desired microbe. Conceptually, this increases the relative abundance of a given beneficial microbe in the rhizosphere, at least temporarily, to achieve the desired benefit. Such studies typically take place in a controlled, artificial condition, such as a defined growth medium in a greenhouse, where competition from a native rhizosphere community is relatively low or non-existent. As mentioned above, this approach occasionally has failed once field application is attempted or the benefits are dramatically reduced in amplitude and/or endurance.
As an example, Lekberg and Helgason (2018) conducted a literature survey of research papers published on mycorrhizal functioning spanning a 30-year period (1987–2017). The most striking finding of this survey was that less than 5% of the work scientifically manipulated mycorrhizal abundance in the field. While we are not arguing the merit of greenhouse-based studies where the number of variables can be controlled and accounted for, yield gains in field conditions will continue to be modest with such an approach. Rhizosphere competence must be evaluated in a field situation if the true power of this approach is to be realized.
Over the last few decades, mycorrhiza-based bio-fertilizers containing one or several species of fungi were developed in forestry and agriculture (Jeffries and Rhodes, 1987; Baraza et al., 2016; Igiehon and Babalola, 2017). These inoculants are generally effective in plant growth promotion under controlled lab and greenhouse conditions. However, few targeted efforts have been made to measure interactions between the introduced microbe(s) and the native mycorrhizal community, let alone the more complex rhizosphere microbiome (Svenningsen et al., 2018; Turrini et al., 2018). To optimize outcomes from these interactions, targeted research must be undertaken to understand how such mycorrhiza-based biofertilizer integrate themselves within the context of the native microbiome.
Integration of Rhizosphere Microbiomes in Plant-Microbe-Nutrient Relationships
The soil microbial community often assists plants by weathering minerals from rock surfaces and degrading recalcitrant soil organic matter whereby soil microbes break down soluble and insoluble organic matter and convert it into inorganic, plant-available forms. Soil organic matter turnover is thus considered a net positive, as it liberates the nutrients locked up in organic matter. For this reason, conventional farming has always relied heavily on soil tillage, along with such other intensive agricultural practices as usage of inorganic fertilizers, herbicides and pesticides. However, it is already clear that such practices have negative consequences on the functional diversity of soil microbiomes. Long-term chemical fertilization has been shown to dramatically decrease the soil pH, which leads to a decrease in bacterial diversity and other changes in microbial community structure (Sun et al., 2015). This was well documented in the work of Kumar et al. (2017), who showed that long-term application of high doses of inorganic nitrogenous fertilizers severely reduces relative abundance, diversity and structure of diazotrophs, which play a key role in converting atmospheric N2 to plant-available ammonium.
As mentioned above, soil bacterial communities play a pivotal role in soil organic matter decomposition. In particular, soil carbon and nitrogen are critical factors for bacteria that rely on soil organic C and N decomposition to obtain energy (Chen et al., 2014; Wild et al., 2014; Tian et al., 2018). Further, different types of soil C selectively manipulate soil microbial community composition, resulting in changes in such belowground ecosystem functions as decomposition and nutrient transfer and creating feedbacks that may affect overall plant growth and productivity (Orwin et al., 2006). For example, bacteria belonging to the genera Chloroflexi, Nitrospirae, and Planctomycetes preferentially feed on recalcitrant organic C, whereas Proteobacteria and Bacteroidetes prefer labile organic C present in the soil (Nie et al., 2018). For this reason, amending the soil with such organic fertilizers as compost or manure contributes to higher microbial diversity and biomass compared to mineral-fertilized soils, which in turn positively impacts soil health (Schmid et al., 2018; Banerjee et al., 2019). Unfortunately, only a few agroecosystem experiments exist that compare organic and conventional management strategies over an extended period for evaluation of impact on soil health and restoration (Raupp et al., 2006; Khatoon et al., 2020). Hartmann et al. (2015) took a metagenomics approach to assess microbial diversity of soil in response to more than 20 years of continuous organic and conventional farming. Not surprisingly, they found that organic farming increased richness, decreased evenness, and shifted the structure of the soil microbiota when compared with conventionally managed soils under mineral fertilization (Hartman et al., 2018; Li et al., 2020b). There also are reports of significant alterations in the microbial community composition of both summer maize and winter wheat in response to increased nitrogen fertilization dose (Wang et al., 2018; Li et al., 2020a). Clearly, a better understanding of the interactions between the soil microbiome and conventional agricultural practices is crucial for the development of sustainable management of soil fertility and crop production.
Managing the Rhizosphere Microbiome to Induce Disease Suppression in Soil
Disease suppressive soils were originally defined by Baker and Cook (1974) as “soils in which the pathogen does not establish or persist, establishes but causes little or no damage, or establishes and causes disease for a while but thereafter the disease is less important, although the pathogen may persist in the soil.” Disease suppressive soils are the best examples of microbiome-mediated protection of plants against root infections by soil-borne pathogens. Such disease-suppressive soils have been described for various soil-borne pathogens, including fungi, bacteria, oomycetes, and nematodes (Mazzola, 2007; Kwak et al., 2018). To date, several microbial genera have been proposed as key players in disease suppressiveness of soils, but the complexity of the microbiome, as well as the underlying mechanisms and microbial traits, remain elusive for most disease suppressive soils (Toyota and Shirai, 2018).
Recently, Carrión et al. (2019) showed that upon pathogen invasion, members of the Chitinophagaceae and Flavobacteriaceae became enriched within the plant endosphere. They proposed that this bacterial population shift led to the induction of enzymatic activities associated with fungal cell-wall degradation, as well as secondary metabolite biosynthesis, all aimed at accelerating and augmenting the plant defense response(s). Although the disease suppressive abilities of certain soils can be at least partially attributed to their physico-chemical properties, the capacity of a soil to suppress disease progression is more often attributed to agri-management practices and crop rotation (Weller et al., 2002). In classic studies by Gerlagh (1968) and Shipton et al. (1973), the authors have shown soil to become disease suppressive after mono-culturing wheat over time. More recently, a comparative metatranscriptome analysis of wheat rhizosphere microbiome grown in fields suppressive and non-suppressive to the plant pathogen R. solani AG8 clearly revealed distinct dominant taxa in these two soil types. Additionally, suppressive samples showed greater expression of polyketide cyclase, terpenoid biosynthesis, and cold shock proteins (Hayden et al., 2018). While development of probiotics for the human gut microbiome has already been an established field of research, the use of probiotics that comprises naturally occurring bacterial antagonists and competitors that suppress pathogens has recently emerged as a promising strategy for disease suppression in soil. A study on application of probiotic consortia that comprised predefined Pseudomonas species reported suppression of the bacterial plant pathogen Ralstonia solanacearum in the tomato rhizosphere microbiome (Hu et al., 2016). In another study, amendment of Metarhizium, an insect-pathogenic fungus that is commonly employed as biological control agents against crop pests, in the rhizosphere of common bean (Phaseolus vulgaris) significantly increased the relative abundance of plant growth promoting such taxa as Bradyrhizobium, Flavobacterium, Chaetomium, and Trichoderma while suppressing the root rot disease symptoms Fusarium solani (Barelli et al., 2020). Soil suppressive properties are mostly derived from the biological functions of soils. Therefore, elucidation of microbial functions in suppressive soils by a next-generation sequencing approach will facilitate the development of effective, consistent and durable disease management tools.
Impact of Agriculture Management Practices on the Soil Microbiome
One important context for plant-microbe interactions is soil structure, as it can vary greatly depending on land-use history, plant species composition and successional stage (Erktan et al., 2016). Besides playing pivotal roles in soil organic matter decomposition, carbon cycling, nutrient mobilization, etc., saprotrophic fungi also are involved in creating soil structure through the secretion of extracellular compounds and physical binding of soil via hyphal networks (Bergmann et al., 2016). Interestingly, studies on the impact of tillage on the soil fungal communities have shown mixed results. Reports in no-till systems have varied from increased ratios of fungal to bacterial biomass (Acosta-Martínez et al., 2010) to decreased ratios (Mbuthia et al., 2015), as well as no change at all (Mathew et al., 2012). More recent studies have shown that soil fungal communities are negatively impacted by tillage, as they typically would be responsible for degrading crop residue left on the surface with no-till (Yin et al., 2017). More specifically, soil bacterial communities were primarily found to be structured by tillage, whereas soil fungal communities responded mainly to management type with additional effects by tillage (Hartman et al., 2018). Additionally, it is acknowledged that organically managed systems increased taxonomic and phylogenetic richness, diversity and heterogeneity of the soil microbiota when compared with conventional farming systems (Lupatini et al., 2017). In a simple definition, organic farming system consists of low-input agro-ecosystem farms in which plant productivity and ecosystem functionality are based on the natural availability of plant nutrients (Lammerts van Bueren et al., 2002). A study aimed at comparing the soil microbiome in conventional and organic farming systems in central Europe revealed no major differences among the main phyla of bacteria between the two farming styles (Armalytë et al., 2019), whereas another study that investigated the effects of 12 years of organic farming on soil microbiomes in northern China reported shifting of the community composition of dominant phyla and significant alterations of functional groups associated with ammonia oxidation, denitrification and phosphorus recycling when compared to conventional farming systems (Ding et al., 2019).
In addition to tillage, crop rotation also plays a pivotal role in increasing belowground microbial diversity compared to intensive mono-cropping practices. Although the United States Department of Agriculture has advocated [via the Conservation Reserve Program (CRP)] crop rotation to improve eroded land as early as 1985 (Allen and Vandever, 2005), its benefit on soil health has only been recognized recently. Several studies reported increases in such soil quality parameters as organic matter content, microbial biomass and respiration under crop rotation management when compared with a mono-cropping system (Campbell et al., 1991; Luce et al., 2013). A meta-analysis of 122 studies that examined crop rotation revealed similar findings, namely that adding one or more crops in rotation to a monoculture substantially increased the soil microbial biomass along with increases in total soil C and N, respectively (McDaniel et al., 2014). In another study, soil microbial communities of corn and switchgrass in mono-cropping systems when compared with mixed prairie grasses demonstrated that bacterial and fungal biomass, especially arbuscular mycorrhizal fungi, were higher in plots with mixed prairie grasses (Jesus et al., 2016). A 16S amplicon-based metagenomic analysis of an almost 20-year-old field trial in Bernburg, Germany revealed a significant effect of tillage practice and the preceding crop on prokaryotic community structures (Babin et al., 2019)
Cover crops are typically unharvested crops planted between cash crops that augment C provisioning to the soil system not only via unharvested residues, but also as root exudates that can support many rhizosphere microbes during the active growing season of the cover crop. Other benefits attributed to cover cropping include improved N fertility by incorporating legumes as a cover crop, reduced soil compaction via deep-rooted plants, and reduced erosion by keeping a plant and its root system in the field year round (Fernandez et al., 2016). Of various crop rotation management practices, those that include cover crops sustain soil quality and productivity by enhancing soil C, N and microbial biomass (Kim et al., 2020), making them a cornerstone for sustainable agroecosystems. Nonetheless, very few studies have assessed the relationship between cover crop stands and their associated belowground microbial communities. Early research in unfertilized grasslands demonstrated that fungal communities respond positively to plant-derived C inputs, suggesting that inclusion of cover crops in a rotation may promote fungal community development (Denef et al., 2009). More recently, a field study tested this hypothesis by specifically examining the impact on soil microbial communities of eight fall-sown cover crop species grown singly and in multispecies mixtures following a spring oats (Avena sativa L.) cropping season and found that certain cover crops selectively favored particular microbial functional groups. Arbuscular mycorrhizal fungi were more abundant beneath oat and cereal rye (Secale cereale L.) cover crops, while non-AM fungi were positively associated with hairy vetch (Vicia villosa L.) (Finney et al., 2017). Beyond positively affecting soil C and increasing the diversity of such beneficial fungi as arbuscular mycorrhiza, clover as a cover crop is often reported to suppress the relative abundance of pathogenic fungi (Benitez et al., 2016). Contrarily, in a 2-year field study, cover crops reportedly increased overall phylogenetic diversity of fungi but did not change the relative abundance of saprophytes, symbionts or pathogens, implying that cover cropping does not always appear to contribute to functional changes in the fungal community (Schmidt et al., 2019).
Reassessment of Plant Responsiveness to Symbiosis
It is now increasingly evident that plants employ fine-tuned mechanisms to shape the structure and function of their microbiome, with different genotypes of the same plant species growing in the same soil yet associating with distinct microbial communities (Berendsen et al., 2012). This is demonstrated in the findings of Bazghaleh et al. (2015), who clearly demonstrated the importance of intraspecific host variation in the association of chickpea cultivars with AM and non-AM fungi. Therefore, specific traits of a plant that modulate its microbiome should be considered as a trait for plant breeding (Wallenstein, 2017).
Despite the obvious importance of beneficial microorganisms for plant growth and fitness, and the impact of plant genotype on shaping their microbiome composition, plant germplasm is typically screened in the absence of microbes, and the selection of best breeding lines made solely based on the interaction between plant genotype and performance under various abiotic factors. We propose that an a priori examination of the interaction between a plant genotype(s) and the symbiotic microbes upon which it likely depends is an important factor in the selection of plant breeding lines. It seems very likely that a subset of rejected germplasm could outperform others, but only when coupled with a beneficial microbe or microbiome (Figure 2). Arguably, current breeding and selection efforts most likely result in decoupling of the soil microbiome from plant fitness. As a result, modern varieties may have lost their ability to support diverse microbiomes and thus, fail to gain the most from these interactions (Wallenstein, 2017).
It is now acknowledged that transitioning from a highly intensive mono-cropping system to a more diversified cropping system consisting of multiple host genotypes leads to increased bacterial and fungal diversity (Calderon et al., 2016). Hence, future plant breeding efforts should incorporate plant characteristics that are related to microbiome diversity. For example, efforts focusing on manipulating plant root exudates likely play a critical role in selective recruitment of the rhizosphere microbiome (Bakker et al., 2012). In support of this notion, it has been shown that plants can select which microbial populations receive the lion’s share of root exudates, demonstrating a capacity by the host to refine its microbial composition. Hence, an unbiased screening of plant genotypes for responsiveness in the presence of a beneficial microbe or microbiome can set forth a new and potentially transformative paradigm in selecting microbes for plant growth promotion (Figure 2).
Significance of Mycorrhizas: A Critical Component of Healthy Soil Rhizospheres
Mycorrhizae are mutualistic associations between soil fungi and plant roots that gradually evolved to be reciprocally beneficial to both partners (Brundrett, 2002). The benefits are generally assumed to involve an exchange of photosynthetically derived carbon from the host plant in exchange for soil nutrients provided by the foraging mycorrhiza. While likely true of arum-type arbuscular types of mycorrhizae, there are other types that can derive carbon from organic matter in the soil, or even “steal” it from one host plant to supply to another (Allen and Allen, 1991). A recent study has reported that in contrast to Arum maculatum, in which carbon is entirely derived from photo-assimilation, the green leaves of Paris quadrifolia contain a striking 50% carbon of fungal origin. Such partial mycoheterotrophy could thus potentially be widespread among the roughly 100,000 plant species that are known to develop a Paris-type AM, with far-reaching implications for our understanding of C trading in plant-microbe communities (Giesemann et al., 2019). Exactly what the mycorrhiza gains from this interaction is still under debate, but benefits may involve a safe haven from the open, more competitive soil space and a second, more reliable carbon source (Sapp, 2004).
Mycorrhizae not only shape plant communities, but they also affect the functional diversity of their cohabitants in the rhizospheric microbiome. The mycelium of mycorrhizal fungi transports plant-derived carbon into the soil in the form of sugars, amino acids and polyols to help sustain the microbiome (Tarkka et al., 2018). More recent studies focusing on soil microbial ecology revealed that mycorrhizal fungi mediate many diverse interactions within the soil “mycorrhizosphere,” including pathogens and mutualists that fix atmospheric nitrogen, take up phosphorus, produce vitamins, and/or protect against antagonists (Buée et al., 2009; Tedersoo et al., 2020). The “ectomycorrhizosphere,” which forms a very specific interface between soil and many trees, hosts a large and diverse community of microorganisms that likely play roles in mineral weathering and solubilization processes (Uroz et al., 2007). This carbon-rich mycorrhizosphere also supports large communities of root-associated microorganisms that further accelerate weathering of minerals by excreting organic acids, phenolic compounds, protons, and siderophores (Drever and Vance, 1994; Illmer et al., 1995).
Similarly, the extraradical hyphae of arbuscular mycorrhiza provide a direct pathway for the translocation of photosynthetically derived carbon to the soil, leading to the development of nutrient-rich niches for other soil microorganisms, particularly bacteria. A quantitative real-time PCR method detected significantly higher 16S rDNA abundance in both the bulk and the rhizosphere soils of zucchini (Cucurbita pepo L.) inoculated with Acaulospora laevis and Glomus mosseae (Qin et al., 2014). Additionally, arbuscular mycorrhizae have been reported to increase the relative abundance of Firmicutes, Streptomycetes, Comamonadaceae, and Oxalobacteraceae inhabiting the mycorrhizosphere (Offre et al., 2007; Nuccio et al., 2013). While there is clear evidence that microbial communities in the rhizosphere function cohesively with their mycorrhizal partner in nutrient mobilization from soil minerals, nitrogen cycling and protection of plants against root pathogens, such bidirectional synergy is not always universal. There are reports that indicate suppressive effects of bacterial communities on mycorrhizal functioning and vice versa. While one study reported (Svenningsen et al., 2018) that soil with a higher abundance of Acidobacteria suppresses the normal functioning of extra-radical mycelium in arbuscular mycorrhizae, another study found that Glomus intraradices and Glomus mosseae suppressed most of the associated soil microbial community (Welc et al., 2010).
A Novel Type of Endophytic Symbiont: The Serendipitaceae
A diverse group of fungi in the Basidiomycota, the Serendipitaceae (formerly Sebacinales Group B) (Oberwinkler et al., 2014) encompasses endophytes and lineages that repeatedly evolved ericoid, orchid and ectomycorrhizal types. Accordingly, in many natural ecosystems these fungi form mycorrhizal symbioses with an astounding variety of host plants – every mycorrhizal type, in fact, except for arbuscular. Previous research performed in our lab with a strain of this group, Serendipita vermifera, demonstrated plant growth-promoting properties in a variety of plants (Ghimire and Craven, 2011; Ray et al., 2015; Ray and Craven, 2016; Ray et al., 2020). Unfortunately, the agronomic utility of these fungi is hampered by the paucity of strains available, the large majority isolated from Australian orchids. We have begun to address this constraint by isolating the first North American strain of Serendipita, named Serendipita vermifera subsp. bescii NFPB0129, from the roots of a switchgrass plant in Ardmore, Oklahoma (Craven and Ray, 2017; Ray et al., 2018).
As mentioned above, soil organic matter has a tremendous influence on the biological, chemical, and physical properties of soils, making it a vital component of healthy agricultural systems. Whether a natural soil or an agricultural one, the release of the nutrients locked within SOM requires decomposers, primarily insects, fungi, and bacteria, to secrete organic acids and enzymes that can loosen and break down the cellulose and the recalcitrant lignin into nutritive forms that can be used by other microbes and plants. Unlike arbuscular mycorrhizae, which exchange inorganic, mineralized nutrients mined from the soil for carbon derived from host photosynthesis, members of the Serendipitaceae studied thus far have a complete arsenal of carbohydrate-active enzymes (CAZymes), representing approximately 4% of the entire gene set and rivaling the more well-studied saprophytic white and brown wood rotters, and much more than other symbiotic fungi. Additionally, genome analysis of S. bescii and S. vermifera suggests that Serendipitaceae fungi have the metabolic capacity to assimilate N from organic forms of N-containing compounds (Ray et al., 2019). We hypothesize that this carbohydrate-degrading enzyme complement endows these Serendipitaceae fungi with saprotrophic abilities (Craven and Ray, 2019). Unlike free-living decomposers that maintain a solitary lifestyle, seeking only dead or dying plant tissues as their source of subsistence, Serendipitaceae fungi seem to maintain a largely symbiotic lifestyle with the roots of living host plants. It currently is unclear whether there is expression of CAZymes while strains of Serendipita are in symbiosis with host plants, and if so, whether there is spatial or temporal separation from more mutualistic traits. Still, the capacity of some strains to form mycorrhizal relationships with orchids, where the seeds require carbon from the fungus for germination and often well into the plant’s lifespan, suggests that these Serendipitaceae symbionts may be less of a carbon cost to their host plant. Presumably, this saved carbon could potentially be used for other symbiotic relationships or developmental processes. In any case, these intriguing fungi and their seemingly unlimited host range provide a novel symbiosis that could be used in a broad variety of cropping systems.
Conclusion
Soil-dwelling microorganisms are critical components of soil health, itself a determinant of plant productivity and stress tolerance. Deploying microbes to improve agriculture productivity is an extremely attractive approach that is non-transgenic and can be viewed collectively as the extended plant genome. Because these same microbes can contribute to restoring soil health and productivity, they have a bright future in low-input, sustainable agriculture that extends beyond more classically defined plant-microbe symbioses.
Author Contributions
PR and KC conceived and planned the overall idea of the review manuscript. PR, VL, JL, and KC wrote the manuscript. All authors contributed to the article and approved the submitted version.
Funding
This work was supported by the Center for Bioenergy Innovation (CBI) project. The Center for Bioenergy Innovation (CBI) was a United States Department of Energy (DOE) Bioenergy Research Center supported by the Office of Biological and Environmental Research (OBER) in the DOE Office of Science.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Acknowledgments
The authors thank Josh Meo for graphic design.
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Check your Understanding
• What roles does the soil microbiome have in the movement towards sustainable agriculture?
• What is a rhizosphere?
• What type of soil fungi are associated with promoting plant health as well as the soil microbiome? How do they do so?
• What are the challenges with studying plant growth promoting microorganisms?
• Explain the difference between a reductionist vs. holistic approach for improving plant growth with microorganisms.
• What types of agricultural practices can alter the soil microbiome?
• How could the use of ‘probiotics’ suppress disease-causing soil microorganisms?
References
1. Gopal, M., & Gupta, A. (2016). Microbiome Selection Could Spur Next-Generation Plant Breeding Strategies. Frontiers in microbiology, 7, 1971. https://doi.org/10.3389/fmicb.2016.01971
2. Omotayo, O. P., & Babalola, O. O. (2021). Resident rhizosphere microbiome’s ecological dynamics and conservation: Towards achieving the envisioned Sustainable Development Goals, a review. International Soil and Water Conservation Research, 9(1), 127–142. https://doi.org/10.1016/j.iswcr.2020.08.002
3. Ray, P., Lakshmanan, V., Labbé, J. L., & Craven, K. D. (2020). Microbe to Microbiome: A Paradigm Shift in the Application of Microorganisms for Sustainable Agriculture. Frontiers in Microbiology, 11. https://www.frontiersin.org/article/10.3389/fmicb.2020.622926
4. Tosi, M., Mitter, E. K., Gaiero, J., & Dunfield, K. (2020). It takes three to tango: the importance of microbes, host plant, and soil management to elucidate manipulation strategies for the plant microbiome. Canadian Journal of Microbiology, 66(7), 413–433. https://doi.org/10.1139/cjm-2020-0085 | textbooks/bio/Microbiology/Microbiomes_-_Health_and_the_Environment_(Parks)/04%3A_Environmental_Microbiomes/4.03%3A_Soil_Microbiomes.txt |
Plant Microbiomes
Plant health is influenced by a variety of environmental factors, and though the soil content (including the microbial community present) is a major element, the distinct plant microbiome serves an important role. Similar to organ systems in the human body (e.g. skin, gut, etc.), the components of various plants (e.g. internal tissues, leaves, roots, etc.) can have unique microbial communities that contribute to its respective and overall vitality. A better understanding of the corresponding roles of plant microbiomes can be combined in a synergistic effort with other environmental microbiomes to maintain and promote sustainable agricultural practices, ecosystem biodiversity, and research model studies.
Plant microbiome–an account of the factors that shape community composition and diversity
Article by Dastogeer et al., 2020 licensed under the terms of the Creative Commons Attribution License (CC BY).
Abstract
Plants live in association with diverse microorganisms, collectively called the microbiome. These microbes live either inside (endosphere) or outside (episphere) of plant tissues. Microbes play important roles in the ecology and physiology of plants. Significant progress has been made in revealing structure and dynamics of plant microbiome in the last few years. Various factors related to host, microbes as well as environment influence the community composition and diversity of plant microbiome. This review aimed to provide a general account of the factors (host, microbe and environment) that drive the microbial community composition in plant. First, we gave an overview of the aboveground and belowground plant microbiomes. Next, we discussed which host factors are involved in variation in plants followed by importance of microbe-microbe interactions and the elements of environment that influence composition and community structuring of plant microbiomes.
Introduction
A diverse kind of microorganisms associated with a higher organism (human, animal, plants etc.) is together defined as its microbiome. All higher organisms examined to date, including plants, insects, fish, rats, apes, and humans, harbor microbiomes [1,2]. Research on the human microbiome has progressed very quickly. Recently, researchers have also paid much attention to elucidating the composition and functions of plant and soil microbiomes. It is now believed that plants are not separate entities, but rather they live in association with a large variety of microbes. These microbes live either inside (endosphere) or outside (episphere) of plant tissues. Among these microorganisms, bacteria and fungi are predominant. About a few thousand bacterial and fungal taxa have been reported from plant tissues [3,4]. They play important roles such as increased nutrient availability, uptake by plants and increased plant stress tolerance. Thus, plant fitness (growth and survival) is the result of physical and physiological functions of the plant per se as well as the associated microbiome, which together are known as a plant holobiont [5].
The study of the association of plants with microorganisms precedes that of the animal and human microbiomes, notably the roles of microbes in nitrogen and phosphorus uptake. The most notable examples are plant root-arbuscular mycorrhizal (AM) and legume-rhizobial symbioses, both of which greatly influence the ability of roots to uptake various nutrients from the soil. Some of these microbes cannot survive in the absence of the plant host (the ‘obligate symbionts’ including viruses, some bacteria and fungi), which provides space, oxygen, proteins, and carbohydrates to the microorganisms. The association of AM fungi with plants has been known since 1842, and over 80 % of land plants are found associated with them [6]. It is thought that AM fungi helped in the domestication of plants [7]. Traditionally, culturable microbes have been used for plant-microbe interaction studies with the enormous unculturable microbes remain uninvestigated and consequently, our knowledge of the roles of these unculturable microbes remains largely unknown.
Unraveling the types and outcomes of plant-microbe interactions has received considerable interest among ecologists, evolutionary biologists, plant biologists, and agronomists [4,8,9]. Recent developments in meta-omics and the establishment of large collections of microorganisms have dramatically increased our knowledge of the plant microbiome composition and diversity. The sequencing of marker genes of entire microbial communities, referred to as metagenomics, sheds light on the phylogenetic diversity of the microbiomes of plants. It also adds to the knowledge of the major biotic and abiotic factors responsible for shaping plant microbiome community assemblages [8].
However, our understanding on the roles of microbiomes, with respect to their impact on plant ecology and physiology, is still far from complete, and we are at the beginning of knowing their functions [10]. The outcome of this improved knowledge will have significant bearings on a variety of experiments and applications, such as development of biofertilizer and biopesticides for sustainable agricultural production with less reliance on agrochemicals, while augmenting yield and nutritional value [11].
In this review, we will present an account of recent studies and prospects of studying the plant microbiome. Firstly, we will present an overview of aboveground and belowground plant microbiomes. Next, we will discuss the factors driving the composition and community structuring of plant microbiomes. We will not address plant pathogenic microbes, although inclusion of this fraction of microbiome would make sense from a broader and more holistic viewpoint.
Rhizosphere microbiome
The rhizosphere comprises the 1–10 mm zone of soil immediately surrounding the roots that is under the influence of the plant through its deposition of root exudates, mucilage and dead plant cells [12]. A diverse array of organisms specialize in living in the rhizosphere, including bacteria, fungi, oomycetes, nematodes, algae, protozoa, viruses, and archaea [13]. The most frequently studied beneficial rhizosphere organisms are mycorrhizae, rhizobium bacteria, plant growth promoting rhizobacteria (PGPR), and biocontrol microbes. Gans, Wolinsky [14] projected that one gram of soil could harbor more than a million distinct bacterial genomes. İnceoğlu, Al-Soud [15] reported 55,121 OTUs (operational taxonomic unites) from the potato rhizosphere. Among the prokaryotes in the rhizosphere, the most frequent bacteria are within the Acidobacteria, Proteobacteria, Planctomycetes, Actinobacteria, Bacteroidetes, and Firmicutes [3,16]. In some studies, no significant differences were reported in the microbial community composition between the bulk soil (soil not attached to the plant root) and rhizosphere soil [17,18]. Certain bacterial groups (e. g. Actinobacteria, Xanthomonadaceae) are less abundant in the rhizosphere than in nearby bulk soil [3].
Mycorrhizal fungi are abundant members of the rhizosphere community, and have been found in over 200,000 plant species, and are estimated to associate with over 80 % of all plants [19]. These mycorrhizae–root associations play profound roles in land ecosystems by regulating nutrient and carbon cycles. Mycorrhizae are integral to plant health because they provide up to 80 % of N and P requirements. In return, the fungi obtain carbohydrates and lipids from host plants [20]. Recent studies of arbuscular mycorrhizal fungi using sequencing technologies show greater between-species and within-species diversity than previously known [21].
Phyllosphere microbiome
The aerial surface of a plant (stem, leaf, flower, fruit) is called the phyllosphere and is considered comparatively nutrient poor when compared to the rhizosphere and endosphere. The environment in the phyllosphere is more dynamic than the rhizosphere and endosphere environments. Microbial colonizers are subjected to diurnal and seasonal fluctuations of heat, moisture, and radiation. In addition, these environmental elements affect plant physiology (such as photosynthesis, respiration, water uptake etc.) and indirectly influence microbiome composition. Rain and wind also cause temporal variation to the phyllosphere microbiome [22]. Overall, there remains high species richness in phyllosphere communities. Fungal communities are highly variable in the phyllosphere of temperate regions and are more diverse than in tropical regions [23]. There can be up to 107 microbes per cm2 present on leaf surfaces of plants, and thus the bacterial population of the phyllosphere on a global scale is estimated to be 1026 cells [24]. The population size of the fungal phyllosphere is likely to be smaller [25]. Phyllosphere microbes from different plants appear to be somewhat similar at high levels of taxa, but at the lower levels taxa there remain significant differences. This indicates that microorganisms may need finely tuned metabolic adjustment to survive in phyllosphere environment [24]. Proteobacteria seems to be the dominant colonizers, with Bacteroidetes and Actinobacteria also predominant in phyllospheres [26]. Although there are similarities between the rhizosphere and soil microbial communities, very low similarity has been reported between phyllosphere communities and those in open air [27].
Endosphere microbiome
Some microorganisms, such as endophytes, penetrate and occupy the plant internal tissues, forming the endospheric microbiome (Fig. 1). The AM and other endophytic fungi are the dominant colonizers of the endosphere [28]. Bacteria, and to some degree Archaea, are important members of endosphere communities. Some of these endophytic microbes interact with their host and provide obvious benefits to plants [[29], [30], [31]]. Unlike the rhizosphere and the rhizoplane, the endospheres harbor highly specific microbial communities. The root endophytic community can be very distinct from that of the adjacent soil community. In general, diversity of the endophytic community is lower than the diversity of the microbial community outside the plant [18]. The identity and diversity of the endophytic microbiome of above-and below-ground tissues may also differ within the plant [28].
Drivers of plant microbiome composition
Plant microbiome structure is influenced by complex interactions between hosts, microbes, and associated environmental factors such as climate, soil, cultivation practices etc. (Fig. 2). Below, we provide an assessment of current knowledge of these factors, providing insight to plant-microbe interactions in a broader-sense.
Host factors that influence plant microbiome community composition
Plant species
The identity of the host plant has a significant influence on the identity of its microbiome. Different plant species growing adjacent to one another can harbor distinct microbiomes. A comparative survey of root microbiomes in maize, sorghum, and wheat showed different community composition among these plants [32]. Samad et al. [33] investigated the microbiome compositions of roots and rhizospheres using 16S rRNA gene from grapevines and some weed species growing in the same field, and their findings suggested that these species hosted significantly different microbiomes in the roots and rhizosphere, with the more pronounced difference in the root communities. Plants that are distantly-related phylogenetically show greater variation in associated microbiome compositions, suggesting a role of plant phylogeny in structuring root microbiomes [32]. Plant species also influences the identity and diversity of endophytic communities. Manter et al. [34] reported differences in endophytic community composition in potato and Eucalyptus plants. The most abundant bacterial root endophytes in potato were rare or absent in Eucalyptus and vice-versa [34] suggesting that the host plant selects its endophytic microbes. Analysis of endophytic fungi of three native Australian Nicotiana species revealed that they are host-specific but not plant organ- or host location-specific [28]. In addition to the rhizospheric and endophytic microbiomes, phyllosphere community composition also depends on plant identity [24]. Kembel et al. [35] showed that the leaf microbiome community is highly correlated with plant evolutionary relatedness similar to the endospheric microbiome.
The effects of host plant species in recruiting microbes from the surrounding environment indicate that plants have evolved traits that govern root microbiome assemblages [36]. For example, endosphere, rhizosphere community composition are correlated with host taxonomy [36]. Xiao et al. [37] found that the rhizosphere and root microbiomes are mostly influenced by soil type, and the nodule and root endophytes are influenced by plant species. Differential microbiome assembly in different plant species is attributed to variation in plant resource consumption [36]. Plant traits such as leaf permeability, wettability and topography and physicochemical properties, cuticle chemistry, root exudates, antibiotic production, and inherent plant immunity to invasion by microbes may also to play a role.
Plant genotypes
Evidence suggests a difference in microbe community composition between genotypes of a particular species [17,26,38]. Genetics of the host is one of the factors that shape the plant-microbiome structure. For example, OTUs in three different potato varieties were cultivar-specific [36]. Similarly, cultivar-dependent effects have been reported for the bacterial communities in young potato rhizospheres [15]. Peiffer et al. [39] demonstrated that OTU richness and β-diversity are influenced by plant genotypes in maize. Bulgarelli et al. [38] reported that genotype contributed to about 6% of the variation of the microbiome composition in the rhizosphere region. A larger influence of host genotype on community composition has been reported [40]. Genotype-dependent microbiome community structuring has been reported for sweet potato, wheat, pea, and oat [9,41]. Bacteria such as Acinetobacter, Chryseobacterium, Pseudomonas, Sphingobium, and Stenotrophomonas were more abundant in low-starch cultivars than those having high-starch contents [41]. The rhizosphere communities of different genetic clones of wild-type and transgenic lines have been reported to be distinct in Populus [42]. Within-species genetic variability can influence microbiome composition in leaf tissues [43]. Wagner et al. [44] conducted an extensive field experiment to unravel drivers of community composition of bacteria associated with leaves and roots of Boechera stricta. Their findings suggested that the host genotype influences leaf community, but the root microbiome was variable at different collection sites.
Agler et al. [45] proposed that host genotype influences on keystone microbes, which then pass these effects onto the total microbiome by changing microbe–microbe interactions and altering plant fitness [3]. The host specificity of plant microbiome could also be attributed to the nutrient preferences of plants [46]. However, whether the observed host influences are heritable, how strong the effect is, and whether these associations will be actionable for plant breeding is yet to be established.
Plant organ
Different plant tissues host distinct microbiome communities. Edwards et al. [47] demonstrated that each surface and internal tissue of plants may harbor distinct microbial communities and that the role of tissue-type was greater than host type and the microbiome of the soil. This may be because the adaptation strategies of various tissues may affect the microbes in colonizing them for community composition. For instance, surface tissues are exposed to constant fluctuations of weather and have relatively poor nutritional status compared to the root or internal tissues. Therefore, microbes colonizing the leaf surface need to be adapted in these conditions [25]. Other studies found very little or no effect of plant organ in community composition of fungi and bacteria [28].
Study with agaves determined that prokaryotes are largely influenced by the plant compartment, whereas the rhizosphere, the phyllosphere, and endosphere communities are clearly different from each other and from adjacent soils [48]. Many studies have reported that fungal communities show a different pattern, where the biogeography of host were the major influencing factor [48]. This may be explained by the dispersal limitation in fungi [49], because fungi are eukaryotes like plants and animals, but bacteria are different in this respect.
Plant age and developmental stage
In the cases of interactions between plants and pathogens, ontogenic resistance (age-related resistance) is widely reported and correlated with plant developmental stage [50]. Symbiosis research has also indicated that plant age and developmental stage are important factors affecting microbial communities [51]. Analysis of the bacterial rhizosphere community of Arabidopsis revealed that the seedling stage selects distinct microbiomes at developmental time points. Plants produce mixtures of compounds and specific phytochemicals in the root exudates. Some of these chemicals are indeed distinct at plant developmental stages and appear to shape microbiome community assemblages [52]. The effect of the plant age on microbiome using DGGE fingerprint analysis revealed that it significantly influences bacterial community composition of all groups investigated for all three sweet potato cultivars [41]. The effect of plant age on the composition of bacterial microbiome in the rhizosphere has been demonstrated in potato, maize and soybean [15,53,54]. Age-related microbiome differentiation may be associated with root growth, physiology, root architecture, root morphology, root exudate, and its composition [51,52]. However, further research is needed on the identity and effect of root exudates at different plant developmental stages to determine how plants communicate in the rhizosphere. This knowledge might offer a basis for augmenting agricultural crops by the application of rhizosphere microbes.
Plant canopy type
Plant canopy type also influences microbiome community composition. For example, bacterial communities in sugar maple leaf samples are correlated with canopy composition [55]. The microbial migration through rain runoff may be an important factor for variation in microbial colonization in different canopy types. Canopy structure influences the composition of endophytic community but not the rhizospheric community, indicating less effect of rain runoff, and there may be other mechanisms such as soil factors or some unknown factors involved in this variation [55].
Plant immunity and signaling
Plant health status may influence microbiome composition. Plants employ two layers of defense against pathogens: pattern-triggered immunity (PTI), which is triggered by conserved molecular structures such as microbe/pathogen-associated molecular patterns (MAMPs/PAMPs), and damage-associated molecular patterns, which are recognized by plasma membrane-localized pattern recognition receptors [56]. It is unknown whether plants recognize non-pathogenic microbes in the similar way as they recognize pathogens and modulate their response. When plants are challenged with herbivory or pathogens they release hormones and exogenous volatiles that alter the composition of root exudates (for review, see [57]), and these in turn modify the microbiome community. Aphid infestation and pathogenic microbial infection increaseed populations of the non-pathogenic rhizobacterium Bacillus subtilis in the sweet pepper rhizosphere [58]. In Arabidopsis thaliana plants infected by Pseudomonas syringae, the expression of root malate transporter is altered, indicating a change in secretion of malic acid that increased the number of the beneficial rhizobacterium Bacillus subtilis [59,60]. Cucumber roots infected by Fusarium oxysporum f. sp. cucumerinum exhibited augmented secretion of fumaric and citric acid, which led to the formation of biofilms (aggregates of living bacteria in a slimy extracellular polymeric substances) of Bacillus amyloliquefaciens. Most investigations, however, have focused on one-to-one interactions (plant-microbe), although in reality, plants are subjected to attack by numerous microbial pathogens and insect pests. Therefore, it would be interesting to see how multiple herbivory and/or pathogens modify the community composition of plant microbiomes. Recent studies have reported a change in rhizosphere microbiome community composition as influenced by specific compounds such as sugars, sugar alcohols, or mixtures of various chemical compounds in root exudates [52]. Plants use various strategies in response to pathogenic infection and insect attack. One of them is activation of defense responses in roots, which may influence microbiome composition in the rhizosphere and roots [61]. When aphids feed on foliage both SAR (Systemic Acquired Resistance) and ISR (Induced Systemic Resistance) signaling are activated throughout the plants, which elicits sweet pepper plants to attract B. subtilis in the rhizosphere [58]. Again, increased JA signaling in plant either by injury or exogenously in Medicago truncatula caused higher colonization of beneficial mycorrhizae [62]. Different root and phyllosphere endophyte microbial communities have been reported when altered SA signaling was induced [63]. Plants lacking in jasmonate-mediated defense have shown more diverse epiphytic colonization [64]. It is evident from these studies that the role of plant defense systems on the microbial composition are inconstant, and that SAR is an important factor in regulating some bacterial community composition. Chemical signals released by plants for example, flavonoids, activate varied responses in plant rhizosphere microbiomes [65]. Branching in mycorrhizal hyphae is affected by strigolactones, which enhance and promote seed germination by parasitic plants [66].
Plant derived compounds
A diverse array of antimicrobial compounds are produced in plants [67]. Some of them, such as different alkaloids, phenolics, and terpenoids, are common in plants. Some are specific to particular groups [68], for instance, Brassicales produce glucosinolates. It was found that transgenic Arabidopsis producing an exogenous glucosinolate had different microbiomes in the rhizosphere and root tissues [69]. Voges et al. 2018 reported a significant role of plant derived coumarins in structuring the rhizosphere community. They suggested that iron-mobilizing coumarins are involved in redox reactions that can mobilize ferric iron and generate reactive oxygen species (ROS) with detrimental effects on microbial proliferation and thus selectively inhibit certain microbial growth while allowing proliferation other more beneficial partners. Triterpenoid saponins, which are known as avenacins, are found in oat (Avena strigosa), and have antifungal properties [70]. Oat plants lacking avenacin production attracted different culturable fungi in roots [71] and were more vulnerable to pathogenic infections than wild-type oat [72]. Interestingly, however, a recent comprehensive analysis of the rhizosphere community of these two genotypes reported little difference between the fungal communities. The effect of avenacins on the Amoebozoa and Alveolata was profound but has not been reported for bacterial communities [73]. This revealed that a small difference in plant genotype might exert multifaceted and unpredicted effects on the plant microbiome composition and diversity. These plants derived compound may affect microbiome assembly in different ways. For example, root exudates may be specific for host plant and can modulate rhizosphere community as well as selects specific root microbiome and thus contributing host specific plant microbiome. Also, antimicrobial compound may selectively enhance microbial growth by restricting certain microbes which is a kind of ‘balance’ in mutualism.
Microbial factors in shaping plant microbiome structure
Microorganisms play important roles in shaping microbial community structures in plants. However, our knowledge on how microorganisms influence microbiome structuring is limited.
Microbial manipulation of hosts
Microorganisms can affect host plants, for example host root exudations, which in turn affect the permeability of roots and root metabolism. Some microbes in soil where the plant if growing can also absorb certain compounds in root exudates and excrete other compounds. Soil microbes can produce compounds that affect plant signaling and metabolism, which lead to production of microbe-derived compounds in plants. Some microbes produce antibiotics (e.g., penicillin and polymyxin) which increase the exudation of organic materials, altered cell permeability, and increased leakage [74] and results in a variable microbiome assembly.
Microbe-microbe interaction
The extent to which microbe–microbe interactions can play roles in the microbiome composition is not well understood. The outcome of microbe-microbe interactions could be explained as cooperation, parasitism, and competition. In cooperation, at least one species benefits, while others are not harmed. When both species benefit, the term mutualism is used, whereas, when one partner benefits while the other is not affected, the term commensalism is used. In contrast, parasitism and competition are harmful for at least one species [75].
We know from recent studies that microbial communities harbor highly connected taxa called keystone taxa [76]. These taxa independently or in a group show a substantial effect on microbiome composition and functions regardless of their spatial and temporal dynamics. They play a unique and vital role on microbiomes, and their absence could cause a significant alterations in microbiome composition and functioning [76]. They use various strategies to impact on host microbiome. For instance, they might cause changes in intermediate or effector groups which in turn regulate microbiome community composition and functioning [77]. Production of a secondary metabolite (2,4-diacetyl phloroglucinol) was reported for some strains of Pseudomonas fluorescens that suppress Gaeumannomyces graminis var. tritici, responsible for wheat take-all [78]. Similarly, many fungi (e.g. Trichoderma and bacteria (e.g. Bacillus) produce various secondary metabolites that suppress microbial growth [79,80]. The keystone taxa may produce bacteriocins to shift microbiota structure selectively. Again, by synergism, keystone taxa may alter the abundance of their partners, and influence community structure and performance. For example, certain species of Burkholderia are symbiotic with arbuscular mycorrhizae and may change abundance and community composition of AM fungi, thereby influencing plant community richness, diversity, and production [19]. Agler et al. [45] studied the roles of microorganisms (bacteria, fungi and oomycetes) in the community composition of phyllosphere microbiomes of Arabidopsis thaliana using a systems biology approach. They described an interkingdom interactions network with a profound influence on community structure. They identified a few taxa, termed “microbial hubs’, which are highly interlinked and have a significant impact on communities. They used two “hub” microbes (Albugo, an oomycete pathogen and Dioszegia, a basidiomycete yeast) in detail. Albugo strongly affected epiphytic and endophytic bacterial colonization. Many symbiotic microbes (including pathogens) produce effector proteins to suppress, activate, or alter host defense mechanisms [81], and some can entirely reprogram the host metabolism [82]. These host adjustments can lead to alterations of microbiome composition because some microorganisms and not others can benefit from altered conditions. Actually, the niches of some microorganisms is dependent on others. For instance, primary colonizers can aid subsequent colonizers against hazardous abiotic factors [83] or can enhance the competitive ability of following colonizers by producing secondary metabolic compounds [84]. There can be direct microbe-microbe interactions, such as the hyper-parasitism (parasite of parasite) of primary colonizers [85] and opportunists that utilize host’s compromised plant defenses to colonize them [86]. Such phenomena point out why some colonizers can affect the development of microbes on the host even if they may be distantly related [84] and highlight a crucial functions of such interactions in shaping microbiome composition and structure.
One of the major strategies by which PGPR augments plant growth is by its influence on rhizosphere microbes. For example, Pseudomonas sp. DSMZ 13134 alters the composition of dominant bacteria in barley roots [87]. In some cases, however, the effects of PGPRs on resident microbiome may not be prominent. For example, no substantial changes in rhizosphere community were noticed after the application of Bacillus amyloliquefaciens FZB42 [88]. Supporting results have also been reported in soybean with the application of B. amylolique faciens BNM122 [89]. The recent investigation on the effect of B. amyloli quefaciens on the lettuce microbiome using 454-amplicon sequencing revealed no or only transient and minor effects in the rhizosphere zone [90]. Interestingly, a decrease in the bacterial number was reported. In the field only 55 % of the inoculated bacterial DNA could be traced after a month [91]. The effects of Bacillus subtilis strain PTS-394 on the rhizosphere microbiome has been examined by metagenomic profiling. Similar to the results above for B. amyloliquefaciens FZB42 [90,91], only a minor effect on the composition was reported. However, up until now, the impact of Bacillus PGPR on other plant microbiota, such as fungi, has not been investigated, and investigation on this could reveal a more general effect of inoculated bacteria on resident microbiota and thus on host physiology and ecology.
Environmental factors as drivers of plant microbiome assembly
Environmental elements, such as soils, climatic conditions, geography, farming activities, and plant domestication, could result in the differences of plant associated microbial community composition [92]. A change in an environmental component results in plant phenotypic changes (e.g., [93], which consequently also change the assemblage of distinct microbiomes harboring plant compartments [94]. Plants grown under controlled conditions provide specific environments for microorganisms. For instance, when lettuce was grown under a glasshouse, a distinct bacterial signature was found from those grown in open fields [95]. Whitaker, Reynolds et al. [96] reported the community composition of endophytic fungi local environment (i.e., site), but not by host ecotype, pointing that environmental factors are major drivers of the endophytic mycobiome of switchgrass.
Soil
Plants recruit root microbes mainly from the soils where they grow. Various soil factors viz. soil types, soil pH, and the C/N ratio, as well as available P and K are frequently reported to be the determinants of root microbial community composition by affecting plant growth and immunity [8,9]. Innumerable studies using high throughput sequencing have proved that soil type is a major factor for root microbiome structure, which is evident from the differential initial microbial inocula present in different soil types [18,47]. Dombrowski et al. [97] collected the arctic-alpine Arabis alpine samples from the native location as well as from those grown under controlled conditions and investigated the root microbiome by 16S rRNA amplicon sequencing analysis. They reported that soil type and length of time the plants remained in the soil are the most important drivers, causing variation of up to 15 % of root microbiota. In addition, in the same soil, the root microbiome of perennial A. alpina was similar to A. thaliana and Cardamine hirsuta, the annual relatives of A. alpina. The root microbiome communities are strongly influenced by the composition of the soil microbiome close to roots. A strong correlation between the soil and root bacterial communities in A. thaliana has been reported by various authors [3,18]. Similarly, the structure of fungal communities is influenced more predominately by soil type than by host plant [98]. The type of soil influenced the rhizosphere bacterial microbiota composition in lettuce [99], oak [16], Arabidopsis [3], and maize [100].
Several studies have shown that environmental variability, such as soil pH, C: N ratio, soil carbon, water content and biogeography may influence the microbiota composition [39]. Lauber et al. [101] described that the impact of soil pH on total community composition was obvious even at a very high taxonomic level. Analyses revealed that pH has substantial correlation with the structure of these microbiome phyla in all soil types studied. The effects of soil pH on soil bacterial community composition has also been reported in other studies using various methods [102]. Zarraonaindia et al. [103] reported that the composition of soil and root microbiome of grapevine significantly influenced by soil pH and C:N ratio but leaf- and grape-associated microbiota were mostly influenced by soil carbon. Hartman, Richardson et al. [104] reported from their study that pH was the most important factor in predicting the alteration of soil bacterial communities, and they detected changes in phylum-level abundances across the pH levels. However, contrasting results have been reported by Fierer et al. [105], where they noted soil carbon was more important than soil pH for Bacteroidetes, Betaproteobacteria, and Acidobacteria. This difference of the findings was possibly linked to sample sizes and number of soil types investigated as well as to differences in methodologies. The exact mechanism(s) of the role of soil pH on microbial community composition and diversity is unknown. Two general explanations have been given [101]. Firstly, soil pH may indirectly alter bacterial community structure with its influences on soil characteristics. Secondly, soil pH may directly impose a physiological limit on soil bacteria, changing outcomes of competition or reducing survival of taxa intolerable to condition. In addition, we hypothesize that soil pH may alter plant microbiome by its influence on plant growth and physiology, which also may influence soil microbiome composition. Other soil properties, for example soil temperature and contaminants in the soil, also influence microbiome composition. Heat disturbance of soil results in a shift in rhizobacterial microbiome composition [106]. Heat disturbances due to natural wildfires can cause a decrease in microbial activity and significant alterations in microbial communities [107]. Increasing petroleum hydrocarbon contamination levels results in the alteration of willow microbiome structure. These alterations were less extreme in the rhizosphere and plant tissues, but they were prominent in the bulk soil. These could be because plants provide more controlled conditions and shield microbes against an enhanced contamination gradient [108].
Cultivation practices
Land use and cultivation practices are the most important causes of declines in biodiversity, leading to undesirable consequences for the environment [109]. Changes in the vegetation influence the diversity and structure of soil microbiomes. Agricultural activities do not necessarily have negative consequences in soil bacterial community diversity and structure, but they may have positive or neutral feedback (effect not perceived) [110]. For example, the intensity of land use (LUI) has been reported to influence the pattern of bacterial communities. Estendorfer et al. [111] found that under low LUI, there remains a strong interaction between plants and adjacent soil. In contrast, no influence of LUI on microbial diversity has been found in the rhizosphere, which indicates that plant species have much more influence on the rhizosphere community than soil properties do [112]. However, Suleiman et al. [113] reported that plants may have robust core microbiome compositions that are less prone to alteration due to variation of land use, soil type or edaphic factors. It was found that microbiome of in relatively untouched deciduous forest and long-term mowed grassland soils were comparable, although there were significant differences in soil properties and vegetation [114], Continuous cultivation causes changes in soil properties, which in turn might affect the soil microbiome communities. As per the reports of Allison and Martiny [115], there are three potential impacts caused by land disturbance such as the microbial structure might be affected, it might be changed but quickly return to the original composition (resilient), or might remain unchanged.
Climatic variables
Climate is an important driver of plant and soil microbiome composition. The role of climate in influencing plant microbiome community composition has to date been largely unheeded or found to be of little importance [116]. A recent study, however, across various ecosystems in Britain showed that rainfall and temperature gradients are two major climatic factors in shaping the bacterial community composition in plants [117]. Researchers de Vries et al. [118] reported that precipitation is an important driver of soil microbial community composition. The biomass of fungi and bacteria has been reported to increase with increasing mean annual precipitation (MAP), and the effect was more prominent for fungi, resulting in comparatively higher fungal abundance (increased F/B ratio) under higher precipitation levels. The above trends could be linked to higher soil organic matter contents in higher rainfall areas. In particular, in uplands where harsh climatic conditions prevail, a higher organic matter build-up is noticeable, which leads to fungi-dominated microbiome communities [119].
Conclusions and future perspective
The factors that influence microbiome assemblages and dynamics in plant and soil are now better understood, and research in this aspect is increasing. However, our knowledge on the underlying mechanism(s) of microbiome assemblages and how they influence the host plants is still lacking. Connecting the microbiome composition and diversity to their function is a great challenge for future research. For instance, to what extent could we use and manipulate the plant microbiome to boost sustainable agricultural production and environmental protection? We now know that that host genetic factor has a significant influence on microbiome diversity and structure, indicating that breeding and trait selection provide opportunities to select for desired microbiomes [45]. To have a more profound and broader knowledge on plant microbiome, there is a need to integrate novel molecular approaches (e.g., meta-omics), ecological models (e.g., food web theory, assembly of communities, or coexistence theory), and recent bioinformatics and statistical advances with a view to correlating community assemblages with ecological functions.
In the future, more emphasis should be placed on identifying the underlying mechanisms that drive microbiome community composition and assembly. We need to know the contributions of (1) the microbe-microbe interactions, (2) soil and other environmental variable and (3) various host traits in shaping community structure. Future research will direct toward solving some pertinent questions. For example, how stable are the drivers of microbiome community? To what extent do agricultural practices affect the microbiome of plant species? How predictable are these divers? With high throughput technologies, such as next-generation sequencing and metagenomics, we can begin to study endophyte microbiomes across hosts, environmental conditions, and at different time points and focus on the mechanisms of the plant-endophyte association.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We thank Stephen J Wylie, PhD, Murdoch University, Australia for kindly checking the manuscript in part.
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K.M.G. Dastogeer, et al. Current Plant Biology 23 (2020) 100161 Google Scholar
Check your Understanding
• What are the various types of plant-associated microbiomes and how does each affect plant health?
• How are the compositions of plant microbiomes influenced by the environment?
• What aspects of the host plant can shape its microbiome?
• In what ways can the plant microbiome be governed by microbe-microbe interactions?
References
1. Dastogeer, K. M. G., Tumpa, F. H., Sultana, A., Akter, M. A., & Chakraborty, A. (2020). Plant microbiome–an account of the factors that shape community composition and diversity. Current Plant Biology, 23, 100161. https://doi.org/10.1016/j.cpb.2020.100161 | textbooks/bio/Microbiology/Microbiomes_-_Health_and_the_Environment_(Parks)/04%3A_Environmental_Microbiomes/4.04%3A_Plant_Microbiomes.txt |
Pollution and Bioremediation
Achieving sustainable life, for humans, animals, and the environment, requires a plan of action to mitigate anthropogenically-induced damage and develop future practices to maintain planetary homeostasis. One of the biggest threats to ourselves and the environment is the buildup of human waste and pollution (e.g. plastics, oil, synthetic products, etc.) that take a tremendously long time to naturally breakdown. With the human population predicted to continually climb, continuing the same destructive practices will only result in more waste generation in a fraction of the time that it takes for decomposition. One alternative means to solve the pollution problem is to use specialized microbiomes for bioremediation. That is, a specially designed microbial consortia could clean up (i.e. breakdown and assimilate) various toxic molecules much more quickly. However, this will take time to understand how and which type of microbes could perform these tasks and if there are any associated (negative) effects.
Alternative Strategies for Microbial Remediation of Pollutants via Synthetic Biology
Article by Jaiswal and Shukla, 2020 licensed under the terms of the Creative Commons Attribution License (CC BY).
Continuous contamination of the environment with xenobiotics and related recalcitrant compounds has emerged as a serious pollution threat. Bioremediation is the key to eliminating persistent contaminants from the environment. Traditional bioremediation processes show limitations, therefore it is necessary to discover new bioremediation technologies for better results. In this review we provide an outlook of alternative strategies for bioremediation via synthetic biology, including exploring the prerequisites for analysis of research data for developing synthetic biological models of microbial bioremediation. Moreover, cell coordination in synthetic microbial community, cell signaling, and quorum sensing as engineered for enhanced bioremediation strategies are described, along with promising gene editing tools for obtaining the host with target gene sequences responsible for the degradation of recalcitrant compounds. The synthetic genetic circuit and two-component regulatory system (TCRS)-based microbial biosensors for detection and bioremediation are also briefly explained. These developments are expected to increase the efficiency of bioremediation strategies for best results.
Introduction
The remediation processes aided by microorganisms present at the various contaminated scenarios constitute bioremediation (Basu et al., 2018; Kumar et al., 2019). Microbial remediation uses multiple metabolic pathways responsible for enzyme production (Sharma B. et al., 2018; Dangi et al., 2019). These enzymes mainly take part in the degradation pathways of xenobiotics (Junghare et al., 2019). There are different customary methods for bioremediation, primarily based on the site of bioremediation, in and ex situ (Tomei and Daugulis, 2013). In situ is applied to the site to minimize soil disturbance. This method is mostly adopted due to less expenditure from avoiding excavation and transport of contaminated soil (Khan et al., 2004). According to Khan et al., 2004 less disruption in in situ bioremediation causes less dust dispersion and hence better degradation (Joshi et al., 2016) of contaminant. Bioaugmentation, bioventing, biosparging, and engineered in situ bioremediation are main in situ bioremediation methods (Azubuike et al., 2016). Ex situ bioremediation methods are solid phase system (composting, landfarming, and biopiling) and slurry phase system (bioreactors) (Kumar et al., 2011). Transportation of soil to accelerate microbial degradation are done by solid and slurry phase systems, whereby treatments of domestic, industrial, and organic waste are done by ex situ bioremediation (Juwarkar et al., 2010). These traditional bioremediation methods take time and consume much cost expenditure, giving less result output. Traditional bioremediation (Duarte et al., 2017) processes showed the above limitations of extra time taking, less removal or dissimilation of pollutants, (Bharagava et al., 2019) disturbance to nature delicacy such as more land coverage for a long time, and a foul smell in the environment (Dangi et al., 2019; Kumar, 2019). Therefore, researchers are eager to discover new bioremediation technologies for best results. Dvoøák et al. (2017) described bioremediation via synthetic biology for boosting bioremediation strategies. This approach can catch the catabolic (Jacquiod et al., 2014) and metabolic complexities for reviewing the potential of the microbial population synthetically. The preliminary information for developing synthetic microbial models for bioremediation can be obtained by mining genes from the databases (Fajardo et al., 2019). The computer logics involvement can determine the microbial cell interactions with recalcitrant compounds (Kim et al., 2015). These strategies can together grasp the natural metabolic potential of microorganisms to transform into novel biological entities of interest (Dangi et al., 2019). Furthermore, the regulation of metabolic pathways (Alves et al., 2018) in a controlled manner can also be achieved for bioremediation processes (Rochfort, 2005). This transition via synthetic biology application (Figure 1) for remediation purposes would improve the bioremediation processes via the involvement of potent (Zhu et al., 2017) dissimilating particular contaminants (Trigo et al., 2008). Synthetic biological systems mediate cellular modulations for efficient functioning and working of existing processes. They permit the modification of cellular processes viz. metabolic pathway acting for a particular chemical compound. The advancement of synthetic biology for bioremediation of various contaminants is attaining the focus of scientists and researchers. For instance, a sustainable synthetic microbial community’s establishment for bioremediation is being investigated. Microbial interactions and quorum sensing within communities are vastly studied for application in the area of bioremediation with synthetic biology applications. Achievement of the synthetic genetic circuit of Pseudomonas putida proved to be the golden gadget for degradation studies. Besides this, genome editing by CRISPR-Cas, TALEN, and ZFNs adds knowledge for reviewing the progression in bioremediation studies. Synthetic microbial biosensors and metabolic engineering of cellular processes for utilization and detection of contaminant residues will remediate the environment from persistent recalcitrant pollutants. This review is focused on the above mentioned strategies and their elements (Figure 2) applicable for bioremediation purposes and research.
Metabolic Reconstruction for Designing Synthetic Models
A computational platform is utilized for the reconstruction of cellular metabolism (Agapakis et al., 2012) via metabolic pathway analysis (MPA) (Banerjee et al., 2016). MPA mathematically represents the reactions of metabolism. This method is based on stoichiometric balance reactions so as to propose steady-state metabolic flux during cellular growth. The stoichiometry matrix imposes constraints of flux, making the consumption and production of the compound at a steady state (Bordbar et al., 2014). The maximum and minimum flux of a reaction can also be determined by providing the topper and least bound. This helps to define the extent of permissible flux supply (Richelle et al., 2016; Rawls et al., 2019). The next step is defining the objective according to the biological problem to be studied. This objective mathematically represents the reactions responsible for the phenotype appearance. The mathematical reactions and phenotype are combined with linear equations and solved by computational algorithms such as COBRA Toolbox11 and Matlab toolbox (Orth et al., 2010; Bordel, 2014). FBA is fundamentally simple, having immense applications in studying gaps, physiology, and genomes via systems biology approach (Kim et al., 2015; Hellweger et al., 2016). These gaps are missing metabolic reactions, making the genome partially known. FBA uses computational algorithms that can predict missing reactions viz. OptKnock and OptCom, which can knock out the genes responsible for producing the desired compound (Biggs et al., 2015). These approaches are beneficial for constructing microbial communities for bioremediation of particular contaminants (Khandelwal et al., 2013). However, MPA is the most challenging method when metabolic information is incomplete, making it difficult to obtain a real model (Covert et al., 2001). But this method shows cellular functions in the dynamic community, and thus is very useful for the prediction and exchange of metabolic flux in communities of microorganisms (Khandelwal et al., 2013). Recently, a metabolic model has been constructed by using two Geobacter species with parameterized electron transfer and metabolic exchange to characterize syntrophic growth dynamics. Such a system may have useful applications in the field of bioremediation and degradation of particular contaminants (Butler et al., 2010). A computational platform is also needed for better prediction of engineered genetic pathways for community dynamics. A graph-based tool Metabolic Tinker was developed by McClymont and Soyer to identify thermodynamically feasible biochemical routes for compounds deterioration (Johns et al., 2016). This may be applied to identify the routes for degradation of recalcitrant compounds by microbial consortia. These computational tools are utilized along with omics (Kim et al., 2014; El Amrani et al., 2015) and biological data for desired output (Berger et al., 2013) and toxicity prediction (i.e., META-CASETOX System) (Peijnenburg and Damborský, 2012). These are also applied for functional gene identification and their profile analysis, PCR analysis and drug discovery, etc (Dangi et al., 2019). Computer-aided drug discovery and development (CADDD) is used effectively with chemical and biological aspects, i.e., chemical structures accounting the biological role and its activity via ligand-based drug design, structure-based drug design, quantitative structure-property relationships, and quantitative structure-activity (Kapetanovic, 2008). Furthermore, Table 1 depicts similar methodologies applicable to bioremediation studies. De Jong (2002) analyzed the multicellular feedback control strategy in a bacterial consortium (Bruneel et al., 2011) to define the robustness conceivable under desired conditions.
They utilized an ordinary differential equations (ODE)-based model and agent-based simulation on a consortium (Hawley et al., 2014; Atashgahi et al., 2018) of interacting species population for increasing the efficacy of the proposed feedback control strategy. The application of bioinformatics (Arora and Bae, 2014) resources is a prerequisite dimension for obtaining the data to begin the microbial bioremediation studies of recalcitrant compounds (Gong et al., 2012; Ofaim et al., 2019). This involves the information related to the degradation of xenobiotics by microbes and their pathways for dissimilation (Dao et al., 2019; Salam and Ishaq, 2019; Thelusmond et al., 2019; Wei et al., 2019). The data related to end products and intermediate metabolites released throughout degradation pathways can also be retrieved (Dvoøák et al., 2017). An extended information source linked to degradation is MetaRouter, allowing data (Singh and Gothalwal, 2018) for life sciences laboratories to explore degradation possibilities of recalcitrant compounds (Mohanta et al., 2015). The information on oxygenic degradation of xenobiotics can be retrieved from OxDBase, a biodegradative oxygenase database (Chakraborty et al., 2014; Shah et al., 2018). Oxygenase is a class of enzyme which transfers the oxygen molecule for oxidizing the chemical compound. They play a role in the degradation of organic compounds by aromatic ring cleavage (Jadeja et al., 2014). OxDBase is very particular in providing knowledge of oxygenases-catalyzed reactions, and is a powerful tool applicable to bioremediation studies (Singh, 2018). Bioconversion and biodegradation of persistent and toxic xenobiotics (Desai et al., 2010; Bao et al., 2017) compounds catalyzed by oxygenases decrease the compound sustainability and toxicity in the environment (Kües, 2015; Kondo, 2017). Therefore, OxDBase is very helpful in acknowledging the degradation processes involved in bioremediation (Shah et al., 2012). The transcriptional characterization of genes responsible for the biodegradation and biodissimilation of a particular compound has great significance in proposing molecular methodologies. This can be done by the Bionemo (Biodegradation Network Molecular Biology) database (Libis et al., 2016a). Bionemo contains the entries for sequences of genes coding for biodegradation (Carbajosa and Cases, 2010). It also links the gene transcription and its regulation (Libis et al., 2016b). The data retrieved from Bionemo can be used for designing cloning experiments and primers (Arora and Shi, 2010). Garg et al. (2014) used eMolecules and the EAWAG-BBD PPS database for the prediction of pathways involved in the biodegradation of 1-naphthyl-N-methyl carbamate. These above findings empower the researchers to analyze and establish what prerequisites must be fulfilled for developing synthetic bioremediation models.
Designing the Synthetic Microbial Communities
Recent advancements in the field of synthetic biology for environmental issues have shown a great impact. The use of GMOs in environmental biotechnology for remediation (Malla et al., 2018) of toxic compounds, xenobiotics, and pesticidal compounds are being done. To design a synthetic community, it is important to understand natural microbial communities (Schloss and Handelsman, 2008). In a natural community, it is difficult to find out which species are actually taking part in bioremediation (Großkopf and Soyer, 2014). Thus, a synthetic microbial community is a promising method for constructing an artificial microbial community with function-specific species for bioremediation purposes. These communities may act as a model system for the study of functional, ecological, and structural characteristics in a controlled manner. Großkopf and Soyer (2014), defined synthetic community by the culturing of two microbial species under well-defined conditions on the basis of interaction and function (Bruggeman and Westerhoff, 2007). These factors determine the dynamics and structure of the community. It is based upon the identification of processes and patterns engaged in by bacterial species. These microbial interaction patterns are metabolism-driven and responsible for community interaction (Wintermute and Silver, 2010). Social-based microbial interactions (i.e., mutualism, cooperation, and competition, etc.) and the total outcome of these interactions between two microbial populations can be +/+ and −/+ or +/−, respectively (Foster and Bell, 2012). It is said that community structure and function majorly depends on cooperation. The effect of cooperation on community dynamics is determined by engineered cooperation resulting in the synthetic community. Engineered cooperation between two microbial strains (Singh et al., 2016) can be done by manipulation of environmental conditions, i.e., knocking the genes out and in Zuroff et al. (2013). Beyond this, other interaction patterns have been analyzed with engineered microbial species in the synthetic community. Such an application of engineered interaction is highly recognizable in bioremediation strategies (Sharma, 2012). Synthetic biology provides greater potential for the sustainable existence of microorganisms (Dellagnezze et al., 2014) acting together in a large population. Thus, synthetic microbial communities are proved as a key strategy for the bioremediation of contaminants, i.e., pesticides, petroleum (Kachienga et al., 2018), oil spill, acid drainage (Serrano and Leiva, 2017), etc. For building the synthetic microbial communities, the engineered interspecies and intraspecies interactions can make cellular functions robust and enhance the capabilities of microbial consortia in various contaminated scenarios. Quorum sensing is a bacterial signaling mechanism, which is a density-dependent phenomenon via cell-cell communication and population level behavior. The signaling is done by the release and reception of chemical compounds by microbial candidates in a population. This leads to multicellular behavior (Obst, 2007), offering engineerable tasks to design function specific synthetic communities. These synthetic models can also be exploited to obtain a rational design that can lose the function when subjected to competition with other species in the natural environment. With the evolution of genomic constituents and gene transfer, the possibility of the gradual extinction of genetic circuits is present. Thus, strategies are required to maintain the robustness of the synthetic community, achieved via the synthetic models by the development of synergistic and cooperative properties that reduce instability and loss of function (Johns et al., 2016). A recent study by Coyte et al. (2015) suggests that competition among species is significant in determining the stability of communities, acting as a limiting factor in the stability of the synthetic community. Thus, these dynamics must be accelerated in order to design particular function specific synthetic communities for bioremediation purposes (Coyte et al., 2015).
Genetic and Metabolic Engineering
Enríquez (2016) said that genome editing is an umbrella term that refers to “scientific technological advances that enable rational genetic engineering at a local (gene) or global (genome) level to facilitate precise insertion, removal, or substitution of fragments of Deoxyribonucleic acid (“DNA”) molecules, comprising one or more nucleotides into the cell(s) of an organism’s genome.” Transcription-activators like effector nucleases (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR-Cas), and zinc finger nucleases (ZFNs) are major gene editing tools used (Table 2). The most efficient and simple technique of gene editing has been described as CRISPR-Cas (Kanchiswamy et al., 2016). These tools can boost the process of bioremediation. TALEN has a DNA-binding modular which is sequence-specific for the host genome (Utturkar et al., 2013). TALEN binding to DNA creates a double stranded break (DSB) in the target sequence and leaves sticky ends for stability. Similarly, ZFNs is also a DNA-binding domain composed of 30 amino acids. It introduces DSB at the target site of the host genome by the Fok1 cleavage domain. A new sense of using hybrid nucleases containing TALENs and ZFNs nucleases came to act for solving the molecular complications. The CRISPR-Cas system, on the other hand, has unique action properties of high sequence specificity and multiplex gene editing. This unique property is derived from bacteria Streptococcus pyogenes as immunity against the virus. The CRISPR-Cas system consists of crisper derived RNA (crRNA) and trans acting antisense RNA (trcRNA) joined by guide RNA (gRNA). gRNA directs the Cas9 enzyme to introduce DSB in the target DNA sequence by recognizing it. These gene editing tools create the knock-in and knock-out and are under processing for implementation in bioremediation studies (Kumar V. et al., 2018). Recent reports indicate though that the CRISPR-Cas system is mostly adopted and performed by researchers in model organisms i.e., Pseudomonas (Karimi et al., 2015; Nogales et al., 2020) or Escherichia coli (Chen et al., 2018; Marshall et al., 2018; Pontrelli et al., 2018). Nowadays, the new insights toward CRISPR tool kits and designing gRNA for expression of function-specific genes related to remediation in non-model organisms (i.e., Rhodococcus ruber TH, Comamonas testosteroni and Achromobacter sp. HZ01) are also suggested in the field of bioremediation (Mougiakos et al., 2016; Jusiak et al., 2016; Hong et al., 2017; Stein et al., 2018; Tang et al., 2018; Liang et al., 2020). For gene editing and metabolic engineering, the contaminant-inhabited bacteria are the most suitable candidates because they are used to survive and harbor in stress conditions of various toxic, recalcitrant and non-degradable xenobiotics, as discussed above. Moreover, understanding metabolic pathways seems to be important in studying the microbial bioremediation (Plewniak et al., 2018), i.e., bioremediation of toxic pollutants by the haloalkane dehalogenases production pathway and decontamination of pyrethroid from the soil via the biodegradation pathway of fenpropathrin studied in Bacillus sp. DG-02 (Chen et al., 2014). Metabolic engineering leads to modification of the existing pathway for the betterment of the bioremediation process (Michel et al., 2007). This approach majorly covers the study of microbial enzymes, i.e., oxidases, esterases, monooxygenases, oxidoreductases, phenoloxidases involved at various degradation steps (Figure 3A; Mónica and Jaime, 2019; Mujawar et al., 2019). Enzyme-based bioremediation has many advantages because it is an eco-friendly process. The genetic approach increases the perspective of getting recombinant enzymes. There are research reports of extracellular enzymes having a role in enzymatic bioremediation. For instance, arsenic bioremediation (Andres and Bertin, 2016; Choe and Sheppard, 2016; Akhter et al., 2017; Biswas et al., 2019) (bioaccumulation and biotransformation) is achieved via arsenite oxidase coded by aioA gene of Klebsiella pneumonia (Mujawar et al., 2019); enzymes released by white rot fungi degrade PAHs (polycyclic aromatic hydrocarbon) (Zhao and Poh, 2008; Košnár et al., 2019), dyes, TNT (2,4,6- Trinitrotoluene) and PCBs (polychlorinated biphenyls) (Gupte et al., 2016; Kutateladze et al., 2018; Sadańoski et al., 2018). Esterase D enzyme acts on β-endosulfan (organochlorine pesticide), producing simpler compounds (Mehr et al., 2017; Chandra et al., 2019). LiPs encoding hemoproteins from Phanerochaete chrysosporium degrade PAHs. However, incomplete or partial degradation of contaminants lead to simpler non-toxic degradable compounds which can be consumed by microbes (Kumavath and Satyanarayana, 2014) as intermediates in biological pathways or substrate, i.e., LiP (lignin peroxidase) dissimilate benzopyrene to three compounds of quinine, namely 1,6- quinone, 6,12- quinine and 3,6- quinine (Gupta and Pathak, 2020). Furthermore, MnP (Manganese peroxidase) oxidizes organic compounds in the presence of Mn(II) (Xu et al., 2018; Singh et al., 2019). Laccase, MFO (mixed function oxidases), glutathione S transferase, cytochrome P450 also acts in biodegradation of recalcitrant compounds (Singh, 2019; Boudh et al., 2019). Catechol 1,2-dioxygenase (intracellular enzyme) from Pseudomonas NP-6 dissimilate catechol to muconate compounds (Guzik et al., 2011). Also, enzyme immobilization (Cavalca et al., 2013; Sharma B. et al., 2018) increases the half-life, stability, and enzyme activity at a notable level. The enzymatic bioremediation is an elementary, expeditious, and environmental friendly method for microbial removal and degradation of persistent xenobiotics compounds (Sharma B. et al., 2018). Isolation and characterization of microorganisms with enzymatic capabilities have been done with the limitation of less productivity (Rayu et al., 2012). Organophosphates (OP) and organochlorines (OC), major constituents of insecticides, accumulate in the agricultural soil (Panelli et al., 2017) and reach the water bodies via agricultural run-off. Effective bioremediation of γ-hexachlorocyclohexane (OC) and methyl parathion (OP) has been reported by genetically engineered microorganisms (Gong et al., 2016). Moreover, bioremediation of organophosphates and pyrethroids has been experimented with using genetically modified P. putida KT2440 (Zuo et al., 2015). With the advent of metabolic engineering, the catabolism and degradation of various persistent compounds has been reported. The degradation pathways of methyl parathion and γ-hexachlorocyclohexane in Sphingobium japonicum and Pseudomonas sp. WBC-3 witnessed the bioremediation strategy (Liu et al., 2005; Miyazaki et al., 2006). Furthermore, 1, 2, 3-trichloropropane, a persistent constituent of fumigant, is dissimilated into the environment (Techtmann and Hazen, 2016) via heterologous catabolism by the assembly of three enzymes from two different microorganisms in E. coli (Dvorak et al., 2014). A metabolic pathway (Bertin et al., 2011) degrading organophosphorus and paraoxon is engineered by inserting the organophosphorus hydrolase gene (opd) and pnp operon encoding enzymes that convert p-nitrophenol into β-ketoadipate in P. putida (de la Pena Mattozzi et al., 2006). A study showed pobA and chcpca gene clusters of Rhodococcus opacus R7 take part in the bioremediation of naphthenic acid; more specifically, expression aliA1 gene codes for fatty acid CoA ligase for degrading long chains of linear as well as alicyclic naphthenic acid (Zampolli et al., 2020). To minimize the accumulation, the above-mentioned strategy is attained using microbes for partial or complete mineralization of persistent compounds (Miyazaki et al., 2006).
Synthetic Genetic Circuit and Microbial Biosensor
The synthetic genetic circuit requires chassis for implantation. The P. putida is a HVB (Host Vector Biosafety) strain recognized as safe by the Recombinant DNA Advisory Committee. It is also referred to as GRAS (Generally Recognized as Safe) to release in the environment. It is ideal for the next generation of synthetic biology chassis panel because it can withstand high intolerant changing conditions including temperature, pH, solvents, toxins, osmotic, and oxidative stress. Also, P. putida has versatile metabolism and low nutrient requirements (Pabo and Nekludova, 2000). These qualities make this organism the best bacterial model for environmental bioremediation applications (Tanveer et al., 2018). Recently, the P. putida synthetic genetic circuit has been established for the designing of promoter genes and expression of the gene responsible for the degradation of persistent compounds (Adams, 2016). An extension of synthetic biology is the integration of genome with reporter system, and synthetic promoters of P. putida may be developed for synthetic bioremediation pathways. Elmore et al. use serine integrases for synthetic genetic circuit development. Microbial cells have the advantage of a cellular system, which controls cell growth and response to external factors like temperature, light, pH, and oxygen (Tropel and Van Der Meer, 2004). The external environment of microbes inhabiting the contaminated site will respond to concentrations of various persistent compounds present (Ray et al., 2018; Antonacci and Scognamiglio, 2019). Whole cell biosensors for checking the presence, detection and biodegradation potential of xenobiotics compounds (pharmaceutical residues, pesticides, paraffin, PAHs and PCBs, etc.) present (Adhikari, 2019) in environmental samples are attaining attention (Wynn et al., 2017; Heng et al., 2018; Patel et al., 2019). The reporter proteins acting microbe makes a color signal at the detection of particular contaminants via transducer (Zhang and Liu, 2016). A biosensor aiming for detection and bioremediation purposes must have enhanced contact between microbe and contaminant (Dhar et al., 2019). This helps the bacterium to adjust their cellular pathways in response to external environmental conditions and encodes the genes for utilizing the recalcitrant compounds as substrate (Bilal and Iqbal, 2019; Skinder et al., 2020). Synthetic biology strategies are feasible for removing a particular toxic compound because the genetic circuits can be developed against the exogenous environmental toxin (Checa et al., 2012; Tay et al., 2017). The synthetic genetic circuits are assembled via a two-component regulatory system (TCRS) in bacteria (Futagami et al., 2014; Uluşeker et al., 2017). This system acts upon environmental change, and thereby, cells respond to these changes. A prokaryotic TCRS has histidine kinase (HK) and response regulator (RR). The HK is a sensor domain with an extracellular loop present as an integral membrane protein. HK also has a transmitter domain in the last cytoplasmic transmembrane, which is a highly conserved domain. Histidine phosphotransfer (DHp) and catalytic ATP-binding domain (CA) acts for molecular recognition of RR and ATP hydrolysis. The transmitter domain transmits the signal from periplasm to cytoplasm via PAS (Periodic circadian proteins, Aryl hydrocarbon nuclear translocator proteins, and single minded proteins), HAMP (HKs, Adenyltatecyclases, Methyltransferases, and Phosphodiesterases) and GAF (cGMP-specific phosphodiesterases, adenylyl cyclases, and formate hydrogenases) (Casino et al., 2010). Thus, HK senses the external environmental change and adds phosphate to conserved histidine. The HK also regulates RR by phosphorylating the aspartate residues. This promotes the promoter (Figure 3B) binding to activate the gene expression or vice-versa (Ravikumar et al., 2017). Therefore, TCRS-based synthetic biology application for biosensor development for cell-mediated detection and bioremediation can prove to be a new advancement.
Ecological Safety and Risk Assessment
The scientists and researchers are performing the experimental setup to study the bioremediation (Yergeau et al., 2012) potential against various pollutants like oil spill, plastics, synthetic dyes, organic hydrocarbons (Yadav et al., 2015), pesticides (Jaiswal et al., 2019a), heavy metals (Hemmat-Jou et al., 2018; Lebrazi and Fikri-Benbrahim, 2018), and other xenobiotics, etc (Rucká et al., 2017; Paniagua-Michel and Fathepure, 2018; Wu et al., 2020). Considering that bioremediation is performed in an open environment rather than in a closed fermentation tank, the ecological safety of bioremediation performing bacteria must be considered. Economic safety is justified by the metabolic aptness (Gillan et al., 2015) of microorganisms as compared to other traditional physical and chemical bioremediation methods. Besides, regulation for using genetically and metabolically modified bacteria is released to evaluate the possible risks (Khudur et al., 2019). The risk assessment is mainly done by regulatory agencies, i.e., Organization for Economic Cooperation and Development (OECD) at the application level for environmental safety (Russo et al., 2019; Alam and Murad, 2020; Pastor-Jáuregui et al., 2020). The possible risks are gene contamination in the native member of microbial consortium, leading to mislaying of the natural trait (Mills et al., 2019; Pineda et al., 2019; Rycroft et al., 2019). The competitiveness between natural and genetically modified species can give rise to selection pressure on non-target microflora (Kumar N. M. et al., 2018; Mohapatra et al., 2019). Moreover, environment and ecosystem risk assessments infer unpredictable and adverse effects, as discussed above (Cervelli et al., 2016; van Dorst et al., 2020). Particularly, the ecological risk assessment behind addition of GEMs (Genetically Engineered Microorganisms) (Benjamin et al., 2019; Ahankoub et al., 2020) to the native environment rather than a laboratory (Fernandez et al., 2019) is done mainly because of unnecessary delivery of antibiotic resistance marker along with recombinant genome of interest (Davison, 2002; Nora et al., 2019), and unintentional uptake or transfer of induced genes to other microorganisms (French et al., 2019; Janssen and Stucki, 2020). This phenomenon is definitely disturbing the microbial native genome entity (Gangar et al., 2019; Petsas and Vagi, 2019). Another aspect to consider is the change in microbial metabolism (Okino-Delgado et al., 2019), which may release uncertain toxic compounds into the environment, indirectly affecting (negatively) microbial candidates in this context (Myhr and Traavik, 2002). Under the TSCA (Toxic Substances Control Act) (Gardner and Gunsch, 2020), the Office of Pollution Prevention and Toxics (OPPT) programs (Pietro-Souza et al., 2020) of the United States Environmental Protection Agency (Leong and Chang, 2020; Saxena et al., 2020) monitors the environmental and health risks and releases premanufacture legal notice for the accreditation of field research outlines and prototypes (McPartland and McKernan, 2017; Khan et al., 2020). A magnificent example is given by University of Tennessee. In 1995, they gave application and suggested the risk evaluation of microbial bioremediation agents (mainly Pseudomonas fluorescens HK44) on the environment and health (Sayler et al., 1999; Khan et al., 2016; Sharma J. K. et al., 2018; El Zanfaly, 2019). Remarkably, the literature survey points toward a biowar weapon for humanity (Gómez-Tatay and Hernández-Andreu, 2019; Wang and Zhang, 2019), stating that gene editing tools left in bad hands could mislead ethical and moral duties (Khan, 2019; Thakur et al., 2019; Sharma et al., 2020).
Conclusion and Future Perspectives
The microbial bioremediation process for removal and detoxification of contaminants from the environment has now emerged as the best option. Synthetic biology is addressing the decontamination and remediation strategies for xenobiotics and related compounds from the environment. It has been observed that the requisite for understanding existing metabolic pathways is a must for developing synthetic models of bioremediation. Moreover, genomics reconstruction methods (Luo et al., 2014; Marco and Abram, 2019) and technologies made a solid platform for bioremediation studies. Satisfactory progress has been witnessed in the field of bioremediation among various contaminants with the role of specific genes and enzymes applicable via synthetic biology methodologies. Therefore, it is concluded that microbial synthetic biology remediation strategies not only increase the efficiency of microbial bioremediation processes for a particular contaminant, but also provide the best methodologies for researchers.
Author Contributions
SJ contributed to this article under the guidance of PS. SJ acknowledges the support and guidance of PS in pursuing a doctorate under his guidance.
Funding
The author SJ acknowledges Maharshi Dayanand University, Rohtak, India, for providing University Research Scholarship (URS). PS acknowledges the infrastructural support from Department of Science and Technology, New Delhi, Government of India, through FIST grant (Grant No. 1196 SR/FST/LS-I/2017/4).
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Check your Understanding
• Explain bioremediation and the roles that microorganisms can have in this process.
• What is synthetic biology and how can it be applied for bioremediation?
• What are some important considerations and challenges for designing a synthetic microbiome?
• Compare the major techniques for genetic and metabolic engineering.
• How could microbial biosensors contribute to bioremediation efforts?
• What aspects of ecological safety and risk assessment must be addressed for application of synthetic microbiomes for bioremediation in open environments? Why?
References
1. Jaiswal, S., & Shukla, P. (2020). Alternative Strategies for Microbial Remediation of Pollutants via Synthetic Biology. Frontiers in Microbiology, 11. https://www.frontiersin.org/article/10.3389/fmicb.2020.00808 | textbooks/bio/Microbiology/Microbiomes_-_Health_and_the_Environment_(Parks)/04%3A_Environmental_Microbiomes/4.05%3A_Pollution_and_Bioremediation.txt |
Forensic Microbiomes
Characterizing human and environmental microbiomes is not only important to maintain host and ecological health, but can have other far-reaching applications such as forensic science. Determination of unexplained situations and phenomenon can be quite challenging, and with advances in microbiomics, there is a possibility to generate sufficient data and evidence to provide viable answers and solutions.
Forensic Applications of Microbiomics: A Review
Article by Robinson et al., 2021 licensed under the terms of the Creative Commons Attribution License (CC BY).
The rise of microbiomics and metagenomics has been driven by advances in genomic sequencing technology, improved microbial sampling methods, and fast-evolving approaches in bioinformatics. Humans are a host to diverse microbial communities in and on their bodies, which continuously interact with and alter the surrounding environments. Since information relating to these interactions can be extracted by analyzing human and environmental microbial profiles, they have the potential to be relevant to forensics. In this review, we analyzed over 100 papers describing forensic microbiome applications with emphasis on geolocation, personal identification, trace evidence, manner and cause of death, and inference of the postmortem interval (PMI). We found that although the field is in its infancy, utilizing microbiome and metagenome signatures has the potential to enhance the forensic toolkit. However, many of the studies suffer from limited sample sizes and model accuracies, and unrealistic environmental settings, leaving the full potential of microbiomics to forensics unexplored. It is unlikely that the information that can currently be elucidated from microbiomics can be used by law enforcement. Nonetheless, the research to overcome these challenges is ongoing, and it is foreseeable that microbiome-based evidence could contribute to forensic investigations in the future.
Introduction
For over 100 years, microbiology has played a relatively diminutive role in forensic science (MacCallum and Hastings, 1899). In the early 1990s, the sequencing of amplified viral DNA was used to support a case alleging the transmission of Human Immunodeficiency Virus from a dentist to several patients in Florida, United States (Smith and Waterman, 1992). The emergence of PCR-mediated genotyping of bacteria was considered to be a valuable forthcoming tool in forensics—e.g., van Belkum (1994) suggested that forensic science would soon be a major area for the application of PCR-mediated genotyping due to the rapidity of technological advances at the time (van Belkum, 1994). In the mid-1990s, fungal and pollen spore analyses were also developed, allowing investigators to differentiate between soil types, which in turn allowed linking substrate items to particular sites (Bruce and Dettmann, 1996; Bryant and Mildenhall, 1998). However, it was not until the early 2000s and the rise of bioterrorism that microbial forensics—the “scientific discipline dedicated to analyzing evidence from a bioterrorism act, biocrime, or inadvertent microorganism/toxin release for attribution purposes”—emerged in response to the new threat (Budowle et al., 2003; Carter et al., 2017).
Many forensic applications have been limited to individual taxa analyses, and microbial forensics has, historically, been constrained by a lack of available and cost-effective sequencing technologies (Berglund et al., 2011; Kuiper, 2016). This approach has changed dramatically in the last decade as advances in genomic sequencing technology, and new methods for processing complex community datasets (and often low biomass samples) have led to the advent of a new field of microbiomics. The science and study of the microbiome (Statnikov et al., 2013; Capasso et al., 2019) combined with metagenomics (all genomes from a sample) have enhanced the development of the microbial forensic toolkit (Clarke et al., 2017; Hampton-Marcell et al., 2017).
As of March 2019, the conviction rate for homicides in England and Wales (United Kingdom) was only 79% (Office for National Statistics, 2019), slightly higher than in the US (∼70%) (Bureau for Justice Statistics., 2019). Across the globe, there is also a high prevalence of wrongful convictions and often insufficient evidence to convict a perpetrator of a crime (Sangero and Halpert, 2007; Ingemann-Hansen et al., 2008; LaPorte, 2017; Walsh et al., 2017). According to the Innocence Project, a national litigation and public policy organization dedicated to exonerating wrongfully convicted individuals, to date, 375 people in the United States have been exonerated by DNA testing, including 21 who served time on death row (Innocent Project, 2020). There is thereby a strong interest from the public, lawmakers, and the law enforcement system to augment and expand the forensic toolkit, including molecular methods. Microorganisms are abundant in and on the human body (microbial cells can outnumber or equal the total number of human somatic cells) (Noel et al., 2014; Sender et al., 2016; Vázquez-Baeza et al., 2018), in surrounding environments, and on objects associated with a crime (Desmond et al., 2018; Oliveira and Amorim, 2018). A growing body of evidence suggests that forensically relevant microbial profiles could be used as evidence or, at the very least, complement traditional investigative methods (Metcalf et al., 2017; Schmedes et al., 2017; Richardson et al., 2019; Phan et al., 2020). This use of microbial profiles as evidence is done using computational tools that are being developed alongside new approaches in bioinformatics, processing tools, and refined protocols. However, since the field is still in its infancy (Goudarzi et al., 2016; Komaroff, 2018) and historically underfunded (Morgan and Levin, 2019), there is much uncertainty as to the true potential of microbiomic tools in forensics.
In this review, we provide an overview of past, current, and future potential applications of microbiomics in forensics. Specifically, we will discuss the six most comprehensively researched themes (Figure 1): including geolocation (e.g., substrate analysis and different spatial dimensions and the power of machine learning), personal identification, biological sex determination, trace evidence, manner and cause of death (e.g., death by drowning), PMI, and other applications (e.g., localization through animal microbiomes).
Major Themes in the Forensic Microbiology Literature
Geolocation
In the past few years, intensive work has been carried out to characterize environmental microbiome, particularly in urban environments and transit systems. These studies have demonstrated that unique community profiles may exist in certain areas of a city (Afshinnekoo et al., 2015; Rosenfeld et al., 2016), as well as “molecular echoes” of environmental events, and even a forensic capacity for geospatial microbiomic data (MetaSUB International Consortium, 2016; Danko et al., 2019a). In the following, we focus on two leading aspects of geolocalization.
Substrate Analysis
The potential of analyzing microbial profiles from the soil is increasingly being recognized in forensic microbiological research. Both the rhizosphere and bulk soil microbiomes exhibit a high level of heterogeneity between different sites. As such, with methodological refinement, soil microbiome samples could provide valuable biogeographic data to localize the origin of the soil sample. Another potential application is the acquisition of information to help determine the provenance of an item(s) associated with a crime.
Habtom et al. (2019) demonstrated distance-decay relationships between microbial samples from the soil at a local scale (25–1,000 m) (n = 5 sites, n = 2–4 soil types, and five replications). The results showed that the greater the distance between the samples, the more they differed, suggesting that both soil type and geographic location are important factors in determining microbial community composition. Indeed, patch discrimination using the soil microbiome has previously been demonstrated (Macdonald et al., 2011), and Jesmok et al. (2016) correctly classified 95.4% of soil bacterial profiles to their location of origin using various methods including abundance charts, non-metric multidimensional scaling, analysis of similarity, and k-nearest neighbor. However, this was a feasibility study with a modest sample size (n = 19). Further studies with larger sample sizes and replications are needed to explore the full potential of this approach.
Evaluating the microbial communities in an assemblage of soil samples (e.g., soils from a crime scene or alibi site and other intermediary sites) could be useful in forensics. Samples originating from a mixture of different soil substrates have been correctly differentiated by using a combination of Ribosomal Intergenic Spacer Analysis and 16S rRNA gene sequencing (Demanèche et al., 2017). Recent evidence suggests that 18S rRNA gene sequencing can also provide greater discriminatory power over traditional Mid-Infrared spectroscopy at fine scales for eukaryotic species (Young et al., 2015). Furthermore, Sanachai et al. (2016) demonstrated that the site origin of soil, obtained from the sole of a shoe, could be elucidated by comparing the similarity of soil bacterial 16S rDNA profiles acquired by the denaturing gradient gel electrophoresis technique.
Despite the potential in this field, further limitations need to be identified and addressed. For example, Pasternak et al. (2019) identified several potentially limiting factors to consider when interpreting the results of microbiomic analyses. For instance, soil samples are incredibly complex and highly heterogeneous even at short spatial scales, which presents a major issue to using these in a forensic context, and microbiomes exhibit a high level of physical, chemical, and biological diversity in both space and time. Pasternak et al. (2013) showed that actinobacterial fingerprints significantly differed between two seasons (summer and winter) at the same sites—implying that temporally-associated issues could arise. Keet et al. (2019) have also pointed out that soil microbiome composition can change as a result of abiotic soil conditions and plant community patterns—which poses a considerable challenge to the accuracy of results.
Metagenomic analysis of gravesoil and the soil around human and non-human animal cadavers has been undertaken for forensic purposes. Carter et al. (2015) investigated microbial community succession in soils associated with swine cadavers across two seasons (summer and winter). They demonstrated that postmortem microbial communities changed in specific and reproducible ways, but decomposition effects on soil microbial communities differed significantly between seasons. The authors suggest that the ecological succession of microbial communities will be useful for forensic investigations, but future research should aim to gain a greater understanding of seasonality on decomposition. The sample size for this study was not explicitly stated, but according to the ordination plots, it appears to be modest (n ≤ 10 per treatment). Therefore, results should be interpreted with caution.
Adserias-Garriga et al. (2017) investigated daily thanatomicrobiome changes in the soil as an approach to estimate PMI. They collected soil samples from around human cadavers (n = 1 male and n = 2 females) and demonstrated successional changes on a daily basis. Rapid growth of Firmicutes was observed from the bloat stage to advance decay (<5% relative abundance at day 1 to 75% relative abundance day 12), and the authors proposed a Firmicutes growth curve to estimate PMI. However, the authors state that the growth curve results may only apply under Tennessee summer conditions and that confirmatory research is needed using a larger number of cadavers and under different environmental conditions. The results do, however, corroborate those of Finley et al. (2016), who evaluated microbial communities associated with gravesoil human cadavers (n = 18). The researchers allowed the cadavers to decompose over a range of decomposition time periods (3–303 days) and showed increases in the relative abundance of Firmicutes in surface bodies over the decomposition period (from ∼10% at 0–3 months to ∼40% at 7–9 months).
Singh et al. (2018) investigated the spatial (0, 1, and 5 m) dynamics of human cadaver decomposition on soil bacterial community structure. They collected soil samples from each spatial buffer (n = 14 for the 0 m, n = 17 for both 1 and 5 m) and observed evidence of a predictable response to cadaver decomposition that varied over space. Bacterial community composition (beta diversity) at 0 m was significantly different from the 1 and 5 m communities, whereas there were no significant differences between the 1 and 5 m communities. The researchers also found that bacterial alpha diversity was significantly lower in the 0 m samples, suggesting that the additional nutrient input from the cadavers may reduce bacterial alpha diversity. This study provides additional spatial-compositional insights to complement the growing body of knowledge in this area of forensic microbiome applications.
Soil microbiome analysis has the potential to be used in forensics; however, additional research is required to validate the sensitivity and reproducibility of results (Young et al., 2017). Overall, to gain a greater understanding of both spatial and temporal dynamics associated with the microbiome and to develop techniques to mitigate similar pitfalls, further microbiome surveys are essential.
Different Spatial Dimensions and the Power of Machine Learning
The growing interest in sampling and predicting environmental microbiome profiles at different spatial scales and orientations (e.g., between households, cities, states, and altitudes) to provide information on the location and provenance of people and objects resulted in the development of a multitude of approaches. Chase et al. (2016) identified the three cities where nine offices were located with 85% accuracy based on analyzing office microbiome samples using sampling plates, and although this study suffers from a small sample size (n = 3 per office), it demonstrates potential with further refinement. Lax et al. (2014) analyzed samples from household occupants (n = 1625 from 18 participants in 10 houses) and their built environments. The authors matched feet microbiome samples to the house with 82.9% accuracy—a relatively low degree of accuracy from an evidentiary perspective but demonstrating the potential of such methods for fine-scale biolocalization. Walker and Datta (2019) analyzed whole-genome sequenced microbiota sampled from 12 cities in seven different countries as part of the 2018 CAMDA MetaSUB Forensic Challenge. The CAMDA dataset (n = 30) included three mystery samples. The authors applied machine learning techniques to identify the geographical provenance of the microbiome samples. Up to 90% of the samples were correctly classified, demonstrating the potential of machine learning applications to biogeography, although further evidence is necessary to employ these applications in an evidentiary context. In a related study, Ryan (2019) applied a random forest classifier built on a dataset of 311 city microbiome samples. Their method correctly classified 83.3% of the mystery samples. Grantham et al. (2019) presented a different algorithm for predicting the geolocation of fungal samples from dust (n = 1300) in the United States using deep neural network classifiers. Applied to a global dataset of samples from 28 countries, the authors state that their algorithms make “good point predictions” with >50% of the geolocation errors under 100 km for US-wide analysis and nearly 90% classification accuracy of a sample’s country of origin for the global analysis. This particular field, combining microbiomics and machine learning, is in its infancy, and future studies would benefit from larger sample sizes and improved classification accuracy before such approaches can be used with confidence in a forensic context.
Another important spatial factor to consider is that microbiome compositions do not only differ in horizontal space. Skin microbiomes have also been shown to differ between humans living in high and low altitudes. For example, Zeng et al. (2017) collected skin microbiome samples from humans (n = 99) and pigs (n = 82) in Tibet. They found enrichments of several bacterial taxa (e.g., Arthrobacter sp., Paenibacillus sp., and Carnobacterium sp.) in samples collected from higher altitudes. Alpha diversity was also significantly lower in skin samples collected from individuals living at higher altitudes. This suggests a potential future route to determine geolocation based on altitudinal parameters via the analysis of skin microbiome samples in the future—although here too, methodological refinement will be essential. Furthermore, understanding how skin microbiomes may fluctuate throughout the life course will also be an essential factor to consider.
Overall, all the models prioritize classification over prediction abilities. To enable real-time prediction of geographical coordinates from sampling data, increasing the sample sizes geographically and temporally, and developing more rigorous methods is essential.
Personal Identification
A growing body of evidence suggests that human individuals may be uniquely identified based on stable autochthonous (i.e., native to a given environment) microbial profiles. This could have a substantial impact on forensic science—for example, in situations where the investigator cannot retrieve sufficient amounts of human DNA (i.e., from human somatic and germ cells). Yet it is unknown whether the variation in microbial communities between people is sufficient to identify individuals within large populations uniquely or stable enough to place them over time.
To answer some of these questions, Franzosa et al. (2015) tested different body site-specific microbial profiles and attempted to match them with 25–105 microbiome profiles during the person’s first and second visits to the sampling site. The authors reported that these profiles were useful in distinguishing individuals at the initial sampling time point and that 30% of the individuals were still uniquely identified several months later. In this study, gut microbiome samples were used to pinpoint 80% of individuals (n = 120) up to a year later. These results are encouraging—particularly in shorter timescales—however, they still suffer from relatively high variability. As such, greater improvements, e.g., in methods and sampling effort, will be needed before such approaches can be useful in a forensic setting.
High resolution melting analysis that targeted the 16S rRNA gene from oral swab samples have also been used to demonstrate its potential in distinguishing between individuals (Wang et al., 2019), albeit with a very small sample size in this study (n = 5). Schmedes et al. (2018) demonstrated accurate identification of individuals (n = 12) based on skin swab samples from different body sites (n = 14). They achieved 97% accuracy by sampling shirts and 96% accuracy using palm samples based on 1-nearest neighbor classification on nucleotide diversity of the bacterial genome. In another recent study, the researchers utilized a similar approach to identify individuals (n = 51). They analyzed microbiome samples collected from three different body sites—the manubrium (i.e., the upper-most segment of the sternum), the palmar surface of the hand, and the ball of the foot (Woerner et al., 2019). The researchers achieved 100% classification accuracy when conditioned on a maximum nearest neighbor distance for diversity, suggesting strong potential should these results be replicable in studies with much larger sample sizes.
Watanabe et al. (2018) suggested that minor taxa are one of the key factors for distinguishing between individuals. Their study analyzed microbiome samples (n = 66) from individuals (n = 11) over 2 years and achieved 85% accuracy in distinguishing individuals. They also used the same analytical methods to classify publicly available skin microbiome samples from individuals (n = 89) with a 78% identification accuracy. However, this level of accuracy is unlikely to be sufficient for forensic applications. The authors suggested that although personal identification is possible, the estimation of the accuracy decreases for larger cohorts due to increments of similar microbiome patterns. Overall, the use of microbiomics as a forensic tool to determine personal identification shows potential and technological viability and might be useful in situations where the investigator is unable to retrieve sufficient amounts of human DNA. Nonetheless, the findings fall short of the burden of proof. Improvements in the model’s sensitivity and specificity are required, and a methodology to address potential contamination issues. Furthermore, a better understanding of the microbial dynamics across time and space is essential for the findings to have a forensics value.
Biological Sex Determination
Recent evidence supports another contribution of microbiomics toward personal identification –the discrimination of biological sex, which could be useful where sufficient quantities of human DNA are unable to be retrieved. For example, airborne bacteria communities have previously been characterized in indoor environments (Chan et al., 2009). Luongo et al. (2017) investigated airborne bacterial and fungal diversity (i.e., constituents of the “aerobiome”) from different University dormitory rooms (n = 91). They used machine learning techniques and were able to predict the biological sex of room occupants with 79% accuracy based on relative abundances of the microbiota. Curiously, rooms occupied by males exhibited higher relative abundances of the microbiota. The authors suggested that it could be because males may shed more biological particles or use fewer cosmetic barriers such as skin lotions.
Biological sex-related differences in the human thanatomicrobiome—the microbial communities colonizing organs following death (thanatos, Greek for death) (Zhou and Bian, 2018)—have also been demonstrated by Bell et al. (2018). The authors compared amplicon signatures (using the 16S rRNA gene V1-2 and V4 regions) in the corpse heart tissue of 10 individuals and discovered key differences between males (n = 6) and females (n = 4). For example, Streptococcus sp. was found exclusively in male heart tissues, whereas females had a significantly higher prevalence of Pseudomonas sp. With refinement, such an approach could help to determine the biological sex of a corpse and the provenance of body parts.
In a study by Tridico et al. (2014), the authors “readily distinguished” male (n = 3) and female (n = 4) subjects based on the analysis of their pubic hair microbiomes. They identified Lactobacillus spp. that were unique to female participants. They also suggested that pubic hair is relatively insulated from the environment and colonized with niche-specific microbiota, which could be useful in forensic investigations. Unfortunately, the modest sample size of this study limits the conclusions that can be drawn from it. Nonetheless, the findings were supported by another small study by Williams and Gibson (2017), who identified individuals (n = 9) and their biological sex from pubic hair microbiota with an error ratio of 0.027 ± 0.058 and 0.017 ± 0.052, respectively. However, the sample sizes for all these studies are modest, and as such, further validation studies with larger sample sizes are needed before reliable conclusions can be drawn.
Interestingly, Phan et al. (2020) analyzed skin microbiome samples from both genders (n = 45) and found that the absence of the bacterial genus Alloiococcus could be useful in predicting female biological sex. The study showed a correlation between certain bacterial species and personal characteristics (e.g., biological sex). They specifically explored the presence/absence of microbiota from fingermarks left behind on surfaces and achieved a relatively modest 67% sex prediction accuracy using leave-one-out cross-validation analysis. Improvements in sample sizes and machine learning accuracy are necessary to explore the potential of this approach further. Additional research into whether certain bacteria (and other microorganisms) are distinct to females or simply related to external factors (such as cosmetic products on hands) would also be necessary.
Trace Evidence
There is an increasing interest in studying forensically relevant microbial profiles left behind on objects and surfaces. For instance, several studies showed that there is often a high level of bacterial presence on personal objects such as mobile phones (Koroglu et al., 2015; Kõljalg et al., 2017; Koscova et al., 2018; Kurli et al., 2018). Furthermore, human-associated items such as shoes and mobile phones have been shown to support distinct microbiomes (Lax et al., 2015; Coil et al., 2019).
Meadow et al. (2014) investigated the potential utility of mobile phones as “personal microbiome sensors.” They selected 17 individuals and collected three samples (the cell phone’s touch surface, their index finger, and their thumb). They demonstrated that bacterial communities sampled from mobile phones were more similar to their owners than other people. They found that about 22% of the taxa on participants’ fingers were also found on their phones, whereas only 17% were shared with other people’s phones. An individual’s index finger shared approximately 5% more OTUs with their mobile phone than with everyone else’s mobile phone in the study. Furthermore, 82% of the OTUs were shared between a person’s index finger and their phone. Although promising, here again, the sample size and accuracy of results need to be increased in future studies.
Kodama et al. (2019) found that postmortem skin microbiomes could be associated with personal objects with a high degree of accuracy. Several of the objects in the study were associated with 100% accuracy (i.e., medical devices, eyeglasses, bottles, and steering wheels), whereas objects like computer devices, remote controls, and cell phones were associated with over 67% accuracy, suggesting that with refinement, skin microbiome samples could be reliably linked to objects at the scene. Furthermore, studies have found that the postmortem skin microbiomes were stable and similar to antemortem skin microbiomes for up to 60 h postmortem (Kodama et al., 2019).
Salzmann et al. (2019) investigated the microbial profiles of different bodily fluids (n = 22). They identified source-specific microbial signatures from various bodily fluids. For example, the phyla Proteobacteria was associated with skin and semen sources, whereas Firmicutes showed a higher prevalence in saliva and vaginal secretions. Dobay et al. (2019) suggest that even when body fluid is exposed to indoor conditions for 30 days, samples continue to harbor body-site-specific microbial signatures. Hanssen et al. (2017) also demonstrated promising results, albeit with a small sample size (n = 6), for the microbially-mediated classification of body fluids. They performed pattern recognition by fitting a linear discriminant analysis model using Principal Component scores and were able to classify saliva samples in 94% of the cases.
Neckovic et al. (2020) recently investigated the potential transfer of skin microbiomes between individuals and substrates (i.e., allochthonous microbiota). They found that skin microbiota has been reliably transferred through direct contact, that is, between individuals shaking hands. Microbiota also transmits through indirect contact, as demonstrated by individuals rubbing a substrate and then swapping substrates with another person. The authors suggested that such analysis could be useful to corroborate sexual assault cases or other contact-related crimes. They also suggested that further research should consider the relative surface area of contact, pressure, friction, and the duration of the contact.
Manner and Cause of Death
The ‘manner of death’ is a determination made by an expert following an investigation (e.g., a coroner, the police, or a medical examiner). Five manners of death are generally considered: natural, accidental, suicide, homicide, and undetermined (Advenier et al., 2016). Lutz et al. (2019) recently collected microbiome samples from 265 corpses from Finland, Italy, and the United States. The inspected cadavers differed in the manners of death: accidental death (n = 88), natural death (n = 106), homicide (n = 23), and suicide (n = 45). Their results suggested that Lactobacillus, Enterobacteriaceae, Sediminibacterium, and Rhizobiales were associated with different manners of death. With further research, these associations could be developed into predictive markers that help to determine the manner of death. However, as noted by the authors, Sediminibacterium and Rhizobiales bacteria may also represent environmental contamination, which needs to be controlled, and further validation through controlled experiments is needed to improve the reliability of their approach to determine the manner of death.
The potential of this microbiomics approach to determining the manner of death was corroborated in a recent study by Zhang et al. (2019) who, by obtaining samples during routine death investigations at the Wayne County Medical Examiner’s Office (Detroit, Michigan, United States), found different biomarkers associated with the manner of death. In this study, Xanthomonadaceae was more prevalent in cases related to hospital deaths, whereas Actinomyces sp. tended to be more prevalent in suicide cases. Increasing the numbers of samples generally increased the accuracy of the models. The authors cautioned that the prediction accuracy depends on the machine learning methods used and the number of anatomical sites analyzed. The authors suggest this study provides baseline information, and it could be possible to use machine learning to develop reference databases that allow microbially-mediated manner of death predictions in the future.
Kaszubinski et al. (2020) modeled beta-dispersion to test for manner and cause of death association using a microbiome data set of n = 188 postmortem cases (five body sites per case). The researchers demonstrated that beta-dispersion and demographic data could distinguish among manner and cause of death. In particular, they found that cardiovascular disease and drug-related deaths were correctly classified in 79% of cases. They found that binary logistic regression models were most effective at improving model success. This was an improvement over multinomial logistic regression models, which confirmed the manner and cause of death assessment only 62% of the time. The results of this study show promise for using postmortem microbiomes to indicate the manner of death. However, as the researchers’ highlight, sample sizes need to be greater. Moreover, the development of large databases will likely be required to train models with high success rates prior to being used in practical forensic contexts.
In terms of cause of death (i.e., the disease or injury that produces physiological disruption in the body leading to death), researchers such as Christoffersen (2015) have investigated the importance of microbiological testing. Studying autopsy results (n = 42), the author reported that the cause of death could be determined in 42% of the cases via microbiological analysis. The study highlighted factors indicative of a microbiologically related cause of death, such as a raised CRP measurements. Raised CRPs have also been implicated in SIDS as a cause of death (Rambaud et al., 1999; Szydlowski et al., 2013) and even for astronauts returning from space (Garrett-Bakelman et al., 2019). Deadly bacterial infections, such as infection or sepsis, may also occur following neonatal circumcision (Elhaik, 2016, 2019).
A specific forensic microbiome application for determining the cause of death is the diagnosis of ‘death by drowning,’ which is one of the leading causes of unnatural deaths worldwide (Domínguez et al., 2018; Cenderadewi et al., 2019). Analyzing the presence of diatoms (single-celled algae) has been the ‘gold standard’ for well over a decade; however, its reliability has been questioned (Kakizaki et al., 2009; Huys et al., 2012). Several studies have provided support for death by drowning diagnoses by designing real-time PCR assays with primers to detect bacterial species associated with aquatic environments, such as Aeromonas spp. (Aoyagi et al., 2009; Uchiyama et al., 2012; Rutty et al., 2015; Voloshynovych et al., 2019). These studies provided support for this cause of death diagnosis based on relatively high detection rates of microbiota, for example, 84% (n = 32), 75% (n = 20), and 84% (n = 43)—although to strengthen the cause of death diagnoses, the accuracy levels, and sample sizes could again be much improved. It has also been suggested that bioluminescent bacteria may be biomarkers for death by drowning in seawater. For example, Kakizaki et al. (2009) developed a simple assay targeting the 16S rRNA gene to identify bioluminescent colonies such as Vibrio fischeri and Vibrio harveyi. More recently, Lee et al. (2017) analyzed microbiome composition and pulmonary surfactant protein (SP-A) expression to develop a marker for diagnosis of death by drowning. They analyzed microbiota and histological appearance of both drowned and postmortem groups of experimental rats, comparing freshwater vs. marine water treatments. The authors found that 5513 and 5480 OTUs were unique to marine and freshwater, respectively. They also found that expression levels of SP-A were higher in the lungs of drowning victims compared to postmortem submersion. These findings could have important forensic value (e.g., determining both the type of environment and the timing of death) and demonstrate good potential for future applications. Marella et al. (2019) point out that other studies have focused on the presence of fecal bacteria, coliforms, and streptococcal bacteria to help determine the cause of death by drowning. These bacteria are sampled from the femoral artery and vein and the right and left ventricles. Fecal bacteria are considered to be always present in subjects who drowned compared with those with other cause of death diagnoses (Lucci et al., 2008; Marella et al., 2019). For example, Lucci and Cirnelli (2007) found fecal streptococcal presence in 100% of the freshwater drowning cases they studied (n = 22) and coliforms present in 90.91%. In this study, the control subjects (n = 30) uniformly showed an absence of fecal bacteria. In a later study, Lucci et al. (2008) assessed if the presence of these bacteria in the drowning medium could be detected in victims submerged after death. The researchers collected samples from drowned victims (n = 5 freshwater and n = 5 in seawater) and victims who were submerged after death (n = 3). Coliforms and streptococci were detected in all drowned victims but not in those submerged after death. These findings suggest that fecal coliforms and streptococci could be used as markers of drowning. However, the minuscule sample sizes must be interpreted with caution and increased considerably in future studies.
Postmortem Interval
The Thanatomicrobiome
Determining the PMI (the time elapsed since a person has died) is often an essential part of a criminal investigation. To improve PMI prediction accuracy, researchers have begun examining the thanatomicrobiome (Javan et al., 2016; Burcham et al., 2019). Postmortem, these communities overwhelm the immune system allowing for subsequent colonization (Javan et al., 2019). Preliminary studies suggest that these microbial communities may undergo important successional changes in organs that could aid in determining the PMI (Adserias-Garriga et al., 2017).
Early studies on model animals suggest that this is feasible. For over a 48-day period of decomposition, Metcalf et al. (2013) aimed to uncover a “microbial clock” to provide an estimate of PMI by sequencing the 16S rRNA gene for bacterial and archaeal communities and the 18S rRNA gene for microeukaryotes. Their model provided reliable PMI estimates (±3 days) (n = 223). However, the study was conducted in controlled conditions using experimental mouse models—thereby necessitating a degree of caution when extrapolating the data to ‘real-life’ situations. Another study investigated the decomposition of pig cadavers. Their model predicted the PMI within 2–3 h of the time of death with 94.4% accuracy (Pechal et al., 2014), demonstrating promise with further methodological refinement. Pechal et al. (2018) carried out a large-scale study of body microbiome samples (n = 188) that found postmortem microbiomes were stable, reflecting antemortem microbiomes 24–48 h after death. The researchers also found that specific bacterial taxa were important in predicting health status. For example, Haemophilus and Fusobacterium were twice as abundant in healthy individuals, whereas Rothia was 0.09 times more abundant in heart disease cases. With further development, this could be used to indicate the state of human health during clinical investigations into a range of deaths, from chronic and natural to sudden and violent (Pechal et al., 2018). It is important to note that, although appropriate at the time, the bioinformatics approach used to process OTUs and to make functional predictions (e.g., QIIME 1.8 and PICRUSt 1) is now considered to be outdated. Furthermore, Amplicon Sequence Variants (ASV) may provide a richer taxonomic picture (Callahan et al., 2017).
Studying human subjects, Johnson et al. (2016) sampled the skin microbiome of decomposing human cadavers and developed an algorithm to estimate PMI. The authors achieved low error rates for skins samples and a PMI estimation accuracy of ±2 days (n = 144 from 21 cadavers), a substantial improvement compared to prior efforts (e.g., via entomological analysis). Belk et al. (2018) used 16S rRNA amplicon sequencing and found that creating models with the class or phylum taxonomic levels provided the most accurate predictions of PMI. This finding corroborated the study by Johnson et al. (2016) and illustrated its potential usefulness for forensics.
Another study using 454 pyrosequencing to determine abundances and diversity of the postmortem microbiome in several key organs such as the brain, heart, liver, and spleen found varying PMIs ranging from 29.5 to 240 h (Can et al., 2014). This study revealed that the most abundant taxa in postmortem microbial communities were the anaerobic, spore-forming Firmicute bacteria, Clostridium sp. Javan et al. (2017) confirmed that Clostridium sp. dominated at long PMIs, adding evidence to support the use of microbiomics in PMI determination in the future.
Localization Through Animal Microbiomes
Several studies have shown that animals from different taxonomic groups and environments possess unique microbial profiles. For example, Tibetan chickens Gallus gallus, Chinese Rhesus macaques Macaca mulatta, and plateau sheep Ovis spp. have unique gut microbiomes (Zhou et al., 2016; Huang et al., 2017; Zhao et al., 2018) shaped by genetic, geographical, and altitudinal factors. It has been demonstrated that the skin microbiomes of Estrildid finches, amphibians, bats, cetaceans, and dogs Canis lupus familiaris are unique (McKenzie et al., 2012; Avena et al., 2016; Erwin et al., 2017; Torres et al., 2017; Engel et al., 2018; Russo et al., 2018). Interestingly, Song et al. (2013) found that humans share microbial communities with their dogs.
With further investigations and methodological refinement, such capabilities point to the potential feasibility of linking a person with a site based on shared microorganisms with animals. Although further studies are needed, there is potential for forensic pathways to associate trace microbial profiles obtained from other species (unique to the given species) to a given environment and/or occupation (e.g., animal industries) or to pet ownership. For example, non-human animal-specific microbiota could potentially be detected on the body or clothing of a suspect or victim, which may be useful in the absence of sufficient animal DNA (i.e., from somatic and germ cells) evidence. This profile could then conceivably be traced to the point of contact with an animal or animal-based environments such as equine stables, pet shops, or zoos, thus complementing other traditional forensic evidence. However, this approach is mostly theoretical at the moment, and future research will be needed to test its feasibility.
Discussion
As of today, microbiome-based forensics are almost absent from criminal investigations and courts. To explain why this is so, we may divide the different possible applications of microbial forensics into two groups: first, reconstruction issues such as the cause of death and PMI, which ask “what happened?” and help elucidate the circumstances of the crime; and second, comparison issues such as geolocation and personal identification, which ask “how similar are these two DNA profiles?” and may (dis)connect a suspect from an object or a place (e.g., murder weapon or crime scene). Reconstruction applications for forensic use are easier to develop since competing propositions are usually well-defined and limited in number; if sufficient research is invested in ascertaining the microbial characteristics associated with each combination of possible environmental, spatial and temporal conditions, then reconstruction becomes straight-forward. For example, if a cadaver is found buried at a depth of 1 m in a desert in summertime, and the temporal succession of the gut microbial community for these environmental conditions has previously been established, then PMI can be inferred with a high degree of accuracy and certainty. Thus, reconstruction microbiomics can readily pass the Daubert standard set by the US supreme court (Daubert v. Merrell Dow Pharmaceuticals Inc, 1993) to be recognized as admissible evidence bearing sufficient scientific foundation, including general acceptance in the scientific community, known and acceptable rate of error, and so on.
Comparison microbiomic tools, on the other hand, may provide greater benefit to the criminal investigation but are harder to develop to a level that would satisfy the Daubert standard. The most beneficial way to employ such tools would be in a “one-to-many” configuration, similar to forensic human DNA analysis: a DNA profile from trace evidence is compared to all the profiles (from known persons and locations) in a database, and if it exists in the database, its frequency in the relevant population (e.g., of soils) is calculated to enumerate the probability of encountering this profile by chance (i.e., originating from a location or person unrelated to the crime). At this time, however, there are hardly any relevant forensic databases of microbiomes that can be compared to trace evidence. In their absence, the only way to proceed in a forensic context is in a “one-to-one” configuration. For each criminal case, questioned samples (e.g., from a suspect’s shoe) are compared to context samples from the crime scene, alibi area, and other relevant sites. This approach provides less benefit to the investigation because it can only give conclusions of exclusion, that two samples do not share the same origin, or relative conclusions, such as “sample A is more similar to sample B than it is to sample C.” Inclusionary conclusions such as “sample A is very similar to sample B; the probability of a different sample, from another place, person or time, being this similar to sample B at random is 1 in X Million” is impossible without either an extensive database (which is costly to build and maintain) or thorough theoretical knowledge (which we do not have yet) regarding the factors that shape microbiomes. However, even for “one-to-one” analyses, we can provide a statistical evaluation of the evidence (Habtom et al., 2019) based on the Likelihood Ratio framework as recommended by the European Network of Forensic Science Institutes (Willis et al., 2015).
There are three more major hurdles to forensic tools of comparative microbiomics. The first one involves samples of mixed origins, containing substrates or DNA from different locations or persons. DNA analysis of mixtures is difficult even with simple human DNA profiles, and certainly more so with microbiomic DNA profiles which are far more complex. It is often impossible to tell with certainty which, or even how many, disparate microbiomes are present in the mixture, let alone accurately infer the DNA profile of each one. The famed 2016 report of the US President’s Council of Advisors on Science and Technology [President’s Council of Advisors on Science and Technology (US), 2016] found that the prevalent subjective analysis of complex human DNA mixtures by forensic experts is not a reliable methodology. Consequently, several computer programs were developed to interpret complex human DNA mixtures in an objective manner, and these are slowly being validated and accepted for routine forensic use. Software for objective analysis of microbiomic DNA mixtures may be built on this basis, but these are still years in the future. The second major hurdle is temporal variation. Contrary to human DNA, which can remain unchanged for years, microbial communities (both on the body and deposited as trace evidence) can fluctuate over time, often in correlation with changes in environmental parameters like moisture and pH (e.g., Pasternak et al., 2013). In cases where two samples for comparison are obtained at different times when markedly different environmental conditions prevail, mitigating the temporal changes in community structure is needed before analysis can ensue. So far, only a few studies have addressed this topic experimentally, mainly by applying various carbon sources to force the different microbial communities to “converge” (Pasternak et al., 2019), but so far with limited success. The third, and perhaps most challenging hurdle, is the problem of DNA transfer. In the past decade, human DNA evidence gained widespread credibility and acceptance in the courts so that the identification of a DNA profile from trace evidence as originating from a specific person is rarely disputed nowadays. Instead, it is becoming more and more common for the defense to challenge the method of deposition of the DNA, suggesting that it reached the crime scene by a legitimate activity (before or after the crime occurred) or by DNA transfer (e.g., when the innocent suspect shook the hand of the real perpetrator). This hurdle can sometimes be overcome by using the likelihood-ratio approach with activity-level propositions (Mayuoni-Kirshenbaum et al., 2020); however, similar to the former hurdles, this one is also very much still an open question, and it will take more time, effort, and research before microbiomics is ready to be employed and accepted within the legal system.
Conclusion
Over the last decade, advances in genomic sequencing and bioinformatics have given rise to microbiomics, which fructified in a growing compendium of tools seeking to explore the panoply of microorganisms present in our bodies and environment. The evidence examined in this review indicates that microbiomics could be a forensically relevant and promising discipline with a multitude of applications—from determining substrate provenance and acquiring trace evidence to identifying individuals and estimating PMI. These advances may allow various microbiomic data, like those obtained from thanatomicrobiome analysis, to be used by forensic scientists to address questions related to criminal investigations, or at least be used alongside other forensic methods.
Throughout their life-course, humans and their microbiomes undergo complex interactions and co-adaptation processes involving nutrient intake and resulting in the production of decomposition products such as metabolites. Following a person’s death, these interactions change dramatically, and the microbiome composition and dynamics fluctuate accordingly. Understanding these colonizations and fluctuations represent major conceptual, methodological, and computational challenges—as do antemortem microbial dynamics. Related microbiome-based research in a forensics context and greater exploration of fungal and viral communities may also lead to an important enhancement in the forensic toolkit in the future.
Many challenges remain to overcome, such as contamination issues, modest study sample sizes, model over-specification and misspecification, prediction accuracies of machine learning techniques, understanding complex spatial and temporal variations in environmental microbiome dynamics, as well as risks and ethical concerns (Shamarina et al., 2017). Notably, even human DNA-based evidence, which is far better understood, is not error-proof, as indicated by the Phantom of Heilbronn case (Daniel and van Oorschot, 2011). Moreover, the vast majority of published work used 16S or targeted sequencing approaches, which have known limitation for taxonomic resolution and could likely benefit from metagenomics methods (Ogilvie and Jones, 2015; McIntyre et al., 2017) and/or methods that utilize longer reads (Danko et al., 2019b; Foox et al., 2020). Also, more field-based testing and deployment of these sequencing methods could benefit from rigorous, titrated standards for ensuring accuracy (McIntyre et al., 2019). Given this, many of the applications reported in the literature should be considered proof of concepts rather than full-fledged forensic applications. Nonetheless, as Ogilvie and Jones (2015, p. 1) have summarized: “it is clear that we remain in a period of discovery and revelation, as new methods and technologies begin to provide [a] deeper understanding of the inherent ecological characteristics of this [microbial] ecosystem.”
Author Contributions
EE initiated the study. JR carried out the review. EE, JR, ZP, and CM wrote the manuscript. All authors approved the manuscript.
Funding
Payment for open access publication was made by Lund University. JR was undertaking a Ph.D. through the White Rose Doctoral Training Partnership (WRDTP), funded by the Economic and Social Research Council (ESRC). Grant code: ES/J500215/1.
Conflict of Interest
EE consults the DNA Diagnostics Center. CM is a co-founder of Biotia, Inc.
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Acknowledgments
We would like to thank the Starr Cancer Consortium (I13-0052), the Vallee Foundation, the WorldQuant Foundation, The Pershing Square Sohn Cancer Research Alliance, and the National Institutes of Health (R01AI151059), the NSF (1840275), and the Alfred P. Sloan Foundation (G-2015-13964).
Abbreviations
PCR, polymerase chain reaction; MetaSUB, metagenomics and metadesign of subways and urban biomes; PRISMA, preferred reporting items for systematic reviews and meta-analyses; CAMDA, critical assessment of massive data analysis; PMI, postmortem interval; OUT, operational taxonomic unit; CRP, C-reactive protein; SIDS, sudden infant death syndrome.
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Check your Understanding
• What types of microbiomes can be analyzed in regards to geolocation for forensic science?
• How could microbiome analytical techniques be improved for personal identification?
• How are various microbiomes different between biological sexes?
• What are some common objects that can have microbiomes applicable for trace evidence?
• What is the thanatomicrobiome and how can it be used to determine postmortem interval?
• What are the major difficulties with comparative microbiomes concerning forensic applications?
References
1. Robinson, J. M., Pasternak, Z., Mason, C. E., & Elhaik, E. (2021). Forensic Applications of Microbiomics: A Review. Frontiers in Microbiology, 11. https://www.frontiersin.org/article/10.3389/fmicb.2020.608101 | textbooks/bio/Microbiology/Microbiomes_-_Health_and_the_Environment_(Parks)/05%3A_Other_Microbiome_Applications/5.01%3A_Forensic_Microbiomes.txt |
The following ‘Journal Club’ articles can be used as supplementary information for various microbiome topics and included in group discussions:
7.01: Case Study 1 - Human Health
Case study #1 – The Farmer’s Flu
Disclaimer: This is a fictitious scenario created for the purposes of microbiome, health, disease, and environmental education. Any names, characters, places and incidents either are products of the author’s imagination or are used fictitiously. Any resemblance to actual events or locales or persons, living or dead, is entirely coincidental.
Part I – Background and Problem
During one week at the end of January of 2019, a high number of flu-like cases have appeared in the small city of Bogart, Iowa which has a population of approximately 50,000 people. 157 individuals were hospitalized over a two-week period, and large proportion included children under the age of 5 years old. Many common symptoms were initially observed in the majority of the patients, however, other less common symptoms manifested in some ill patients after about a week and earlier in those who were immunocompromised and with co-morbidities.
Common symptoms included:
• fever
• headache
• muscle pain or body aches
• shortness of breath
• vomiting
• diarrhea
• cough
• congestion or runny nose
• fatigue
Less common symptoms included:
• stiff neck
• lethargy
• chest pain
• swelling of the throat
• severe joint pain
• green, yellow, or bloody mucus production
• facial redness and swelling
Initial epidemiological data showed that diseased individuals all were at or were in contact with someone who visited the farmer’s market the previous weekend. The market was established more than 20 years ago, and each weekend merchants open their stalls selling everything from pottery, jewelry, and linens to produce, homemade jams, and even livestock. The market is considered to be the staple of town commerce and entertainment, giving Bogart its cozy home-town feel, and even many consumers and merchants come in from the smaller surrounding towns to benefit from the commerce.
Questions
1. What specific pathogens could be responsible for the observed symptoms and why would there be differences in the observed effects in different individuals?
2. Which microbiomes may be implicated in this disease and why?
3. Do you think this situation is a major public concern? Why or why not?
Case study #1 – The Farmer’s Flu
Disclaimer: This is a fictitious scenario created for the purposes of microbiome, health, disease, and environmental education. Any names, characters, places and incidents either are products of the author’s imagination or are used fictitiously. Any resemblance to actual events or locales or persons, living or dead, is entirely coincidental.
Part II – Approach, Implementation, Reasoning
Medical personnel took nasopharyngeal (NP) swabs of sick patients for testing. For children and older adults and those who were averse to NP swabs, nasal and throat swabs or aspirate specimens were collected (Flu specimen collection – CDC). Physicians prescribed various antiviral medication; either two doses per day of oral oseltamivir, four oral doses per day of umifenovir, or inhaled zanamivir for 5 days, and for patients in the hospital, one dose of intravenous peramivir or oral baloxavir for one day. They also prescribed prophylactic antimicrobials, including quinolones (moxifloxacin), cephalosporins (ceftriaxone and cefepime), or macrolides (azithromycin), or a glycopeptide (vancomycin). Other over the counter drugs were suggested to treat symptoms like fever and headache. Public health officials advised the community to get the flu vaccine as well.
Over the next few weeks, the number of cases increased and symptoms began to worsen for many. The initial assumption is a seasonal flu outbreak. Interestingly, more cases began to pop up in surrounding rural towns, and many patients were transported to the larger hospitals in the city to be put on ventilators. As mortality rates also began to rise, the CDC declared this situation a flu epidemic.
Questions
1. What types of laboratory tests and analytical techniques do you think were being conducted? What problems could have arisen with the analysis of the patient’s samples?
2. Why do you think both antibacterial and antiviral medication was prescribed? How would you explain the difference between treatments of a viral and a bacterial infection to a patient?
3. What reasons or factors could cause a high mortality rate in those infected with the influenza virus?
Case study #1 – The Farmer’s Flu
Disclaimer: This is a fictitious scenario created for the purposes of microbiome, health, disease, and environmental education. Any names, characters, places and incidents either are products of the author’s imagination or are used fictitiously. Any resemblance to actual events or locales or persons, living or dead, is entirely coincidental.
Part III – Discussion
After several weeks of patient study and a rising number of mortality and cases, infections were confirmed to be from a mutated variant of influenza A (H1N1; i.e. swine flu). Medical officials believe the high mortality rate and other severe cases may be due to a secondary bacterial infection caused by a pathogen that is resistant to antimicrobials.
Further epidemiological analysis shows that the initial original cases were specifically in individuals who visited the livestock section of the farmer’s market for an extended period of time.
***Apologies, the video and audio are a bit glitchy.***
Last line of defense antibiotics, polymixin (colistin) and carbapenems, were prescribed for patients not responding to the initial antibiotics. These patients were also put under quarantine in ICU wards as their symptoms began to worsen, which additionally included abdominal pain, bloody stool, and severe diarrhea that persisted in patients even after being discharged from the hospital.
Questions
1. How do you think antibiotic resistance of the secondary bacterial pathogen came about?
2. How could chemotherapy affect various microbiomes and potentially contribute to pathogenesis and other disease symptoms?
3. How would you inform the public about this situation, and what measures would you suggest to prevent transmission or recurrence?
Case study #1 – The Farmer’s Flu
Disclaimer: This is a fictitious scenario created for the purposes of microbiome, health, disease, and environmental education. Any names, characters, places and incidents either are products of the author’s imagination or are used fictitiously. Any resemblance to actual events or locales or persons, living or dead, is entirely coincidental.
Part IV – Resolution
Research scientists, medical professionals, and epidemiologists finally pieced the ‘Farmer’s Flu’ epidemic puzzle together using next generation sequencing technology and keeping detailed records of observational data.
Two farmers from a nearby rural town contracted the novel swine flu variant. They then brought the pigs to market in Bogart, where the flu spread. However, they not only spread the flu variant, but also a highly contagious antibiotic-resistant strain of Haemophilus influenza type B (Hib). One of the farmers was sick the week prior and therefore immunocompromised which allowed the development of this secondary infection. It is likely that this strain of H. influenza acquired antibiotic resistance via horizontal gene transfer from Haemophilus parasuis, which is commonly found in pigs.
As this respiratory disease was treated with multiple ineffective prophylactic antimicrobial drugs, patients’ resident microbiomes became depleted and allowed for another infection by opportunistic pathogens. In the case of those who were hospitalized, many developed another secondary infection by Clostridioides difficile, resulting in gastrointestinal distress.
After a few months, with the use of both old and new treatment options, outreach to the public with information about the diseases, and proper community compliance, the epidemic came to an end. Bogart resumed as a quiet cozy town, and still holds its locally famed farmer’s market.
Questions
1. How is the gut microbiome linked with the oral and lung microbiomes? Explain how both of the secondary infections by H. influenza and C. difficile in this case could be related by their respective microbiomes.
2. What other diseases, conditions, or treatments have similar multi-microbiome effects?
3. What are some examples of novel microbiome diagnostic and treatments that could work to restore the affected microbiomes? | textbooks/bio/Microbiology/Microbiomes_-_Health_and_the_Environment_(Parks)/06%3A_Journal_Club/6.01%3A_Journal_Club_Articles.txt |
Case study #2 – The Land is Sick
Disclaimer: This is a fictitious scenario created for the purposes of microbiome, health, disease, and environmental education. Any names, characters, places and incidents either are products of the author’s imagination or are used fictitiously. Any resemblance to actual events or locales or persons, living or dead, is entirely coincidental.
Part I – Background and Problem
Coffee production in South America has drastically decreased in the 2021-23 growing seasons. Specifically, the major farms in Columbia and Brazil which collectively contribute over half of each countries coffee (both specialty and commercial) production, have had severe crop failures. As a result, the price of a cup of coffee has noticeably increased, putting financial strain on suppliers and businesses.
Interestingly, the few years prior to this decline, coffee production was at an all-time high, with more coffee in the market than demanded. This dropped the price of coffee, and in many cases original farmers were not fairly compensated.
The International Coffee Organization (ICO) has tasked a team of researchers to identify potential problems to South American coffee plant failure and provide quick and efficient resolution.
Questions
1. As a scientific researcher, what are some factors you would take into consideration when addressing this problem?
2. What type of environmental microbiomes could be implicated in coffee plant crop failure and why?
3. Should other coffee farms in countries besides those in South America be worried about similar failure in their crops? Why or why not?
Case study #2 – The Land is Sick
Disclaimer: This is a fictitious scenario created for the purposes of microbiome, health, disease, and environmental education. Any names, characters, places and incidents either are products of the author’s imagination or are used fictitiously. Any resemblance to actual events or locales or persons, living or dead, is entirely coincidental.
Part II – Approach, Implementation, Reasoning
Upon initial inspection, the unhealthy coffee plants exhibited yellowing of leaves, appearance of red-brown lesions, abnormal shape, damaged coffee berries, and little new growth or production. Researchers decided to take samples of healthy and unhealthy leaves and berries, as well as soil and root samples.
Video 1 – #2 Producer Crossover 2019
Over the next year, other plantations in Columbia and Central American begin to experience similar declines in coffee plant health. Farmers were questioned about any changes in practices, and while there have been some adjustments do to the fluctuating coffee prices, traditional farming techniques have remained the same for the most part in smaller plantations. Larger plantations with intensive farming made more changes due to trader suggestions on improving yields including application of different fertilizers and hiring less experienced workers.
Questions
1. What type of analytical techniques do you think were or should be performed on the samples taken?
2. How could the observed symptoms of the unhealthy plants be connected to an associated microbiome?
3. How could human intervention exacerbate or ameliorate this situation?
Case study #2 – The Land is Sick
Disclaimer: This is a fictitious scenario created for the purposes of microbiome, health, disease, and environmental education. Any names, characters, places and incidents either are products of the author’s imagination or are used fictitiously. Any resemblance to actual events or locales or persons, living or dead, is entirely coincidental.
Part III – Discussion
Analysis of the negatively affected plantations’ soil showed a decline in plant growth promoting bacteria and mycorrhizae and an increase in bacterial and fungal pathogens like Pseudomonas syringae and Cercospora coffeicola.
Over the next few years (2022-24) coffee plants still struggle to grow in Central and South America. Other parts of the world, such as Vietnam and other Asian countries, are seeing a decline in crop health also, further contributing to global coffee market troubles. Many of these farms switched from shade-grown to intense sun-grown coffee to try to meet needs for demand. Larger corporations and roasters have purchased many of the smaller family farms to try to recoup losses and implement ‘new and improved’ growing strategies for increased production. This included switching to primarily sun-grown coffee, and application of large amounts of synthetic nitrogen fertilizer.
Video 2 – How coffee destroys the environment
Questions
1. What factors could cause these changes in the soil microbiomes?
2. Why do you think coffee plantations across the globe are beginning to fail as well?
3. How does natural biodiversity benefit ecosystem health on both a micro and macro scale?
Case study #2 – The Land is Sick
Disclaimer: This is a fictitious scenario created for the purposes of microbiome, health, disease, and environmental education. Any names, characters, places and incidents either are products of the author’s imagination or are used fictitiously. Any resemblance to actual events or locales or persons, living or dead, is entirely coincidental.
Part IV – Resolution
In 2030, most varieties of coffee are extinct, and only small amounts of coffee are produced on shade grown farms further from the equator. It appears that coffee plantations were severely impacted by increasing global temperatures, which altered ecosystem dynamics and promoted pathogen and pest invasion. Fungi, bacteria, and arthropods devastated already struggling coffee farms and with a major switch to sun-grown coffee, soil microbiomes were depleted and disease spread rapidly through monoculture crops. This switchover was prompted by an energy drink corporation, KAPOW!, which wanted to corner the caffeine market and boost production, though their expertise in growing coffee was lacking and the excessive use of synthetic fertilizers on already sun baked land which requires much more watering further doomed plantations. At least they now produce a cheap “coffee” flavored energy drink. It tastes terrible.
Questions
1. In what ways are environmental microbiomes impacted by changing environmental factors and how can this promote diseases within an ecosystem?
2. How could various environmental microbiomes be utilized to improve sustainable practices and technology?
3. How are environmental and human microbiomes interconnected?
7.03: Case Study 3 - Synthesis (Create Your Own)
Develop your own case study related to microbiomes!
Group members (4-5) will develop and write an original case study involving a microbiome and health or environmental condition. Topics must be approved by the instructor beforehand. The case study should consist of 3 parts: (i) background and problem/issue, (ii) approach to solve, implementation, and reasoning, (iii) discussion of progression of the case study. The final part, (iv) resolution and conclusion will be completed by another group and presented over. The case study should have an interesting title, include references, images, figures, etc. from relevant resources (be sure to use in-text citations and include references at the end), and 3 critical thinking questions concluding each part (9 total). | textbooks/bio/Microbiology/Microbiomes_-_Health_and_the_Environment_(Parks)/07%3A_Case_Studies/7.02%3A_Case_Study_2_-_Environment.txt |
The Integrative Human Microbiome Project is an open access article published in Nature under the Creative Commons: By Attribution 4.0 License (http://creativecommons.org/licenses/by/4.0/).
The Integrative Human Microbiome Project
The Integrative HMP (iHMP) Research Network Consortium
Abstract
The NIH Human Microbiome Project (HMP) has been carried out over ten years and two phases to provide resources, methods, and discoveries that link interactions between humans and their microbiomes to health-related outcomes. The recently completed second phase, the Integrative Human Microbiome Project, comprised studies of dynamic changes in the microbiome and host under three conditions: pregnancy and preterm birth; inflammatory bowel diseases; and stressors that affect individuals with prediabetes. The associated research begins to elucidate mechanisms of host–microbiome interactions under these conditions, provides unique data resources (at the HMP Data Coordination Center), and represents a paradigm for future multi-omic studies of the human microbiome.
Main
Although the ’omics era has accelerated all aspects of biological research, its effects have been particularly apparent in studies of microbial communities and the human microbiome. In the 18 years since the publication of the first human genome, studies of the microbiome have grown from culture-based surveys of the oral cavity and gut to molecular profiles of microbial biochemistry in all ecological niches of the human body1,2,3. Epidemiology and model systems have been used to identify associations between changes in the microbiome and conditions ranging from autism4 to cancer5,6,7, and microbial and immunological mechanisms have been identified that affect, for example, the efficacy of drugs used to treat cardiac conditions8 or survival during graft-versus-host disease9.
Contemporary studies of the human microbiome have also been a source of basic biological and translational surprises, exposing a compelling range of novel findings and open questions. Every human being appears to carry their own, largely individual, suite of microbial strains10,11, which are acquired early in life12,13,14, differ between environments and populations15,16, and can persist for years17 or undergo relatively rapid transitions18. Microbial diversity manifests differently in different ecological niches of the body; for example, greater diversity is generally expected in the gut, but can be associated with dysbiotic states and risk of adverse events in the female reproductive tract. The microbiome can be perturbed by conditions such as inflammatory bowel disease and diabetes, but a variety of microbiome-linked health states, and the underpinnings of these links, remain unexplored. How dynamic is the microbiome during processes such as pregnancy or viral infection? Which changes in the microbiome represent causes rather than effects of changes in health? Which molecular elements of a personalized microbiome might be responsible for health outcomes, and how do they integrate with and maintain physiological processes such as the immune system and metabolism? And what ecological elements dictate the success of a microbiota transplant, and why are they successful in treating some individuals and conditions, but not others?
The National Institutes of Health Human Microbiome Project was one of the first large-scale initiatives to address a subset of these questions19 (Fig. 1). Launched in 200720, the first phase of the program sought to determine whether there were common elements to ‘healthy’ microbiomes, in the absence of overt disease. Studies of both a baseline adult population21,22,23 and ‘demonstration’ populations with specific disease states established typical ranges (for some populations) of microbial membership and enzymatic repertoires across the body, combinations of metabolic functions that were either prevalent or strain-specific, and some of the host factors (such as race or ethnicity) that determine this variation. Studies of targeted populations identified ecological states of niches such as the vagina24,25, skin26,27,28, and gut29,30,31,32,33, among many others (https://www.hmpdacc.org/health/projectdemos.php). This first phase of the HMP (HMP1) thus yielded a wealth of community resources: nucleotide sequences of microorganisms and communities from a large number of isolates, individuals, and populations (http://hmpdacc.org)34,35,36,37; protocols to support reproducible body-wide microbiome sampling and data generation38,39,40; and computational methods for microbiome analysis and epidemiology41,42,43,44,45,46,47.
One of the main findings of the HMP1 was that the taxonomic composition of the microbiome alone was often not a good correlate with host phenotype—this tended to be better predicted by prevalent microbial molecular function or personalized strain-specific makeup21. This finding served as the foundation for the development of the second phase of the HMP, the Integrative HMP (iHMP or HMP2)48, which was designed to explore host–microbiome interplay, including immunity, metabolism, and dynamic molecular activity, to gain a more holistic view of host–microbe interactions over time. This multi-omic program sought to expand the resource base available to the microbiome research community, to begin to address the relationship between host and microbiome mechanistically, and to address the questions introduced above. Disease-targeted projects within the HMP2 were therefore encouraged to use multiple complementary approaches in order to assess the mechanisms of human and microbial activity longitudinally and to provide protocols, data, and biospecimens for future work. These projects included three studies that followed the dynamics of human health and disease during conditions with known microbiome interactions, thus addressing important health outcomes directly while also serving as models of ‘typical’ microbiome-associated conditions of broad interest to the research community. These comprised pregnancy and preterm birth (PTB); inflammatory bowel diseases (IBD); and stressors that affect individuals with prediabetes. These studies, which have now reached the first stage of completion49,50,51, together provide a wealth of information and insights about not only microbial dynamics, but also associated human host responses and microbial inter-relationships. A collection of more than 20 manuscripts to date describe some of these results at https://www.nature.com/collections/fiabfcjbfj, and together they provide a rich multi-omic data resource to be mined by future work (http://www.ihmpdcc.org).
The vaginal microbiome, pregnancy and preterm birth
Preterm birth can have devastating consequences for newborn babies, including death and long-term disability. In the United States, approximately 10% of births are premature52, and the incidence is even greater in lower-resource countries. Environmental factors, including the microbiome of the female reproductive tract, are important contributors to prematurity. Notably, these factors have a greater effect in women of African ancestry, who also bear the highest burden of PTB53. Infant mortality has been reduced in recent decades, but the incidence of PTB has not decreased54, and progress in predicting individual risk of PTB has stalled. During pregnancy, the maternal immune system maintains a delicate balance of pro- and anti-inflammatory effectors55, and contributors to PTB include breakdown in maternal–fetal tolerance, vascular disorders, stress, cervical insufficiency, premature rupture of the fetal membranes, and intra-amniotic infection56. Microbial ascension into the uterus is thought to precipitate PTB by disrupting the maternal immune balance, leading to spontaneous preterm labour, and/or by the release of microbial products (for example, collagenases, proteases or toxins) that compromise the integrity of fetal membranes and lead to premature rupture of the membranes57.
The Multi-Omic Microbiome Study: Pregnancy Initiative (MOMS-PI) research group, as part of HMP2, characterized the microbiomes of pregnant women to gauge their effects on risk of PTB (Fig. 2). The project followed 1,527 women longitudinally through pregnancy and involved the collection of 206,437 specimens, including maternal vaginal, buccal, rectal, skin and nares swabs, blood, urine, and birth products, as well as infant cord and cord blood, meconium and first stool, buccal, skin and rectal swabs. Subsets of these specimens underwent 16S rRNA gene taxonomic analysis, metagenomic and metatranscriptomic sequencing, cytokine profiling, lipidomics analysis, and bacterial genome analysis. The MOMS-PI team analysed 12,039 samples from 597 pregnancies to investigate the dynamics of the microbiome and its interactions with the host during pregnancy leading to PTB50.
These multi-omic investigations identified temporal changes in the vaginal microbiome associated with full-term pregnancies. Women who often began pregnancy with a vaginal microbiome of greater ecological complexity generally converged towards a more homogeneous Lactobacillus-dominated microbiome by the second trimester58. Interestingly, this trend was most pronounced in women of African ancestry with lower socioeconomic profiles. Although the overall MOMS-PI cohort was demographically diverse, most women who experienced spontaneous PTB at less than 37 weeks of gestation were of African ancestry. The MOMS-PI team (http://vmc.vcu.edu/momspi) also identified signatures of higher risk for PTB in women who experienced spontaneous preterm birth at less than 37 weeks of gestation50. Women who went on to experience spontaneous PTB were less likely to exhibit a vaginal microbiota dominated by Lactobacillus crispatus, as previously reported in other populations59,60,61,62, and were more likely to exhibit an increased proportional abundance of several taxa including Sneathia amnii, Prevotella-related clades, a Lachnospiraceae taxon known as BVAB1, and a Saccharibacteria bacterium known as TM7-H1. Notably, these taxa were also associated with low levels of vitamin D63, suggesting that the vaginal microbiome might mediate a link between PTB risk and vitamin D deficiency64. The signatures of PTB were also reflected in metagenomic and metatranscriptomic measurements, and vaginal pro-inflammatory cytokines (including IL-1β, IL-6, MIP-1β and eotaxin-1) were positively correlated with PTB-associated taxa. Conveniently for future possible interventions, the vaginal microbiomes of mothers who experienced PTB were most distinct from those of control mothers early in pregnancy, and a preliminary model to predict risk of PTB was most sensitive and specific using vaginal microbiome profiles from samples collected before 24 weeks of gestation.
The MOMS-PI research group identified intriguing associations between the vaginal microbiota, host response, and pregnancy outcomes that are consistent with the involvement of microorganisms ascending from the vagina in at least some cases of spontaneous PTB. As an essential next step, the contribution of racial and demographic background to the vaginal microbiome in pregnancy with relation to pregnancy outcomes must be fully explored through harmonized, large-scale studies50. It is clear that PTB has a complex aetiology56. The relative contributions of fetal and maternal genetics and epigenetics, particularly as related to genetic variation of the innate immune system, should be explored. Harmonized large-scale studies would permit the development of population-specific risk assessment algorithms using vaginal microbiome profiles, features from genetic and prenatal (fetal) genetic screens, biomarkers such as cytokines and metabolites, and key clinical features from classic markers of risk including maternal age, body mass index, pregnancy history (including history of PTB), cervical length, and measures of stress and other environmental exposures. With the addition of new data from the microbiome, other environmental factors, and multi-omic inputs, new algorithms promise to improve our ability to predict risk of PTB early in pregnancy, to facilitate clinical trials by identifying high-risk patients, and ultimately to stratify patient populations into treatment groups.
The gut microbiome and inflammatory bowel disease
Studies of the gut microbiome in gastrointestinal disease have a particularly long and detailed history, especially in complex chronic conditions such as the inflammatory bowel diseases (IBD). IBD, including Crohn’s disease and ulcerative colitis, affects millions of individuals worldwide, with increasing incidence over the past 50 years or more coinciding with multiple factors such as westernization, urbanization, shifts in dietary patterns, antimicrobial exposure, and many more that could influence host–microbiome homeostasis65. The microbiome has long been implicated in IBD, potentially as a causative or risk factor66,67, as an explanation for heterogeneity in treatment response (that is, some individuals respond well to relatively benign aminosalicylates or corticosteroids whereas others still experience severe inflammation even after surgical intervention)68, or as a novel point of therapeutic intervention (for example, by transplantation of faecal microbiota69,70). Although meta-omic techniques have been used to identify functionally consistent microbial responses that help to explain the gut microbiome’s role as part of a pro-inflammatory feedback loop in the gut during disease71, and a few strains of microorganisms have been shown to be IBD-specific72, no comprehensive model of specific microbial, molecular, and immune interactions yet exists to explain the disease’s onset and dynamic progression.
Therefore, to better characterize mechanisms of host–microbiome dysregulation during disease, the Inflammatory Bowel Disease Multi’omics Database (IBDMDB) project followed 132 individuals from five clinical centres over the course of one year each as part of HMP2 (Fig. 3). Integrated longitudinal molecular profiles of microbial and host activity were generated by analysing 1,785 stool samples (self-collected and sent by mail every two weeks), 651 intestinal biopsies (collected colonoscopically at baseline), and 529 quarterly blood samples. To the extent possible, multiple molecular profiles were generated from the same sets of samples, including stool metagenomes, metatranscriptomes73, metaproteomes, viromes, metabolomes74,75, host exomes, epigenomes, transcriptomes, and serological profiles, among others, allowing concurrent changes to be observed in multiple types of host and microbial molecular and clinical activity over time. Protocols and results from the study, further information about its infrastructure, and both raw and processed76,77 data products are available through the IBDMDB data portal (http://ibdmdb.org), from the HMP2 Data Coordination Center (DCC; http://ihmpdcc.org), and in the accompanying manuscript49.
This unique study design allowed the IBDMDB to identify a variety of differences in the microbiome and host immune response over time during the course of the disease. Indeed, these dynamic changes were of much greater magnitude than were cross-sectional differences among clinical phenotypes, which have been emphasized by previous studies67,71,78,79. This was due in part to the prospective nature of the cohort, which recruited patients with Crohn’s disease or ulcerative colitis during both active and quiescent periods of disease, showing that microbial compositions in patients with IBD often revert to more control-like, ‘baseline’ configurations when the disease is not active. By identifying the gut microbial configurations that were most different from baseline—regardless of specific disease state—the study defined a dysbiosis score that called out highly divergent microbial compositions, which share many features common to an overall inflammatory response (for example, tolerance to oxidative stress). This dysbiosis was not unique to the microbial response to inflammation, however, and was associated with other host and biochemical alterations, pointing to new potential directions for management of systemic dysregulation in IBD. These included large shifts in acylcarnitine pools and bile acids, increased serum antibody levels, and alterations in transcription for several microbial species. Concurrent transcriptomics and 16S amplicon mucosal community profiling from biopsies also identified potential host factors that might be able to shape the microbial community, in particular several chemokines, highlighting these as being involved in a potentially dysregulated interaction during periods of disease activity49.
The study’s longitudinal multi-omic profiles further allowed researchers to characterize the stability and dynamics of host–microbiome interactions during disease, in particular highlighting ways in which community state and immune responses are distinctly less stable in participants with IBD than in control, healthy individuals. In numerous cases, the microbiome of a participant with IBD changed completely over the course of only weeks (measured as maximal Bray–Curtis dissimilarity to earlier samples from the same subject), whereas such shifts were rare in individuals without IBD. The main microbial contributors to these large-scale shifts from one time point to the next largely mirrored the differences observed in dysbiosis, and the shifts frequently marked the entrance into or exit from periods of dysbiosis. Finally, the study’s long-term, complementary molecular measurements enabled the construction of a network of more than 2,900 significant host and microbial cellular and molecular interactors during IBD, ranging from specific microbial taxa to human transcripts and small molecule metabolites. This network of mechanistic associations identified several key components that are central to the alterations seen in IBD, highlighting octanoyl carnitine, several lipids and short-chain fatty acids, the taxa Faecalibacterium, Subdoligranulum, Roseburia, Alistipes, and Escherichia, some at both the metagenomic and metatranscriptomic levels, and host regulators of interleukins49. Networks of mechanistic associations such as this may provide the key to disentangling the complex system of interactions that results in chronic inflammation in IBD and in other systemic microbiome-linked immune diseases.
Multi-omics profiling in prediabetes
Type 2 diabetes mellitus (T2D) affects more than 10% of the adult US population, and another 30% show early signs of the disease (referred to as prediabetes)80; 70% of the latter will develop diabetes in their lifetime. T2D is characterized by complex host–microbiome interactions81,82, but little is known about systemic alterations during prediabetes, their effects on biological processes, or the critical transition to full-blown T2D. Prediabetes and T2D are often associated with insulin resistance, and thus studies of individuals with prediabetes or insulin resistance offer unique opportunities to investigate the earliest stages of diabetes. It is essential to create a global and simultaneous profile of both host and microbial molecules in individuals with prediabetes over time, in order to fully understand the molecular pathways that are affected in people with prediabetes and/or insulin resistance and how these conditions affect both biological responses to environmental challenges (for example, viral infections83,84) and the onset of T2D.
To better understand T2D at its earliest stages, as part of iHMP, the Integrated Personal ’Omics Project (IPOP)85 followed 106 healthy and prediabetic individuals during quarterly periods of health, respiratory viral infection (RVI) and other perturbations over about four years51 (Fig. 4). In one such perturbation, a subset of 23 individuals underwent a directed weight gain followed by weight loss86. In total, 1,092 collections across all participants were profiled. For each visit, blood was assayed for host molecular ’omics profiling and two types of samples, nasal swabs and faeces, were collected for microbial profiling. Each participant’s exome was sequenced once; otherwise, for each visit, 13,379 transcripts were profiled from peripheral blood mononuclear cells, 722 metabolites and 302 proteins from plasma, and 62 cytokines and growth factors from serum. In addition, thousands of gut and nasal microbial taxa and computationally predicted genes were profiled using 16S rRNA amplicons. All visits were also intensively characterized by 51 clinical laboratory tests. In addition, because of the focus on T2D, a number of glucose dysregulation tests were performed, including measurements of fasting glucose and haemoglobin A1C levels, oral glucose tolerance tests, and tests of insulin resistance.
Baseline measurements were generally stable within individuals, even for long periods of time, with only some analytes changing significantly over time51. However, many analytes, such as clinical laboratory measurements, cytokine profiles, and gut microbial taxa (mostly those of low abundance) were highly variable between individuals. Participants who were ultimately insulin-resistant had distinguishable molecular and microbial patterns at baseline from those who were ultimately insulin-sensitive, and an analyte test was devised as part of the study in order to differentiate them. Notably, individuals undergoing RVI or changes in weight showed thousands of specific molecular and microbial changes during these perturbations, and insulin-resistant and insulin-sensitive individuals responded very differently to perturbations. For example, during RVI, insulin-resistant participants showed substantially decreased and delayed inflammatory responses (for example, the acute phase response and IL-1 signalling) and altered gut microbial changes when compared with insulin-sensitive participants (for example, in Lachnospiraceae and Rikenellaceae but not bacilli). Accordingly, there were fewer changes in nasal microbiota in insulin-resistant participants, and both the richness and the diversity of nasal microorganisms decreased during RVI in insulin-sensitive but not insulin-resistant participants. Furthermore, global co-association analyses among the thousands of profiled molecules revealed specific associations in insulin-resistant individuals that differ from those seen in insulin-sensitive participants and vice versa, indicating different patterns of host–microbiome interactions in the two groups51.
Another important goal of the study was to assess how host–microbiome multi-omics and related emerging technologies can be used to better manage patients’ health. We found that taking millions of measurements per individual over time enabled the early detection of potential disease states51,87. These included early detection of T2D, which developed differently among participants and was better detectable with varied assays; for example, some individuals first exhibited measurements in the diabetic range on tests of fasting glucose, whereas others did so on tests of haemoglobin A1c, oral glucose tolerance tests, or even continuous glucose monitoring. These results, together with detailed characterization of glucose dysregulation over time, illustrate the heterogeneity of T2D development. Overall, the data led to microbially linked, clinically actionable health discoveries in a number of diseases in addition to T2D, including metabolic disease, cardiovascular disease, haematological or oncological conditions, and other areas; these signs were often present before symptom onset, demonstrating the power of using big data, including the microbiome, to better manage human health.
Resources from the HMP2
Together, the HMP1 and HMP2 phases have produced a total of 42 terabytes of multi-omic data, which are archived and curated by the DCC at at http://ihmpdcc.org and in public and/or controlled-access repositories such as the Sequence Read Archive (SRA; https://www.ncbi.nlm.nih.gov/sra), the Database of Genotypes and Phenotypes (dbGaP; https://www.ncbi.nlm.nih.gov/gap/), Metabolomics Workbench (https://www.metabolomicsworkbench.org/), and others (Fig. 5). All data on the DCC is available for unrestricted use, with a subset of project metadata also being shared when permitted by institutional review boards (IRBs), and other restricted data (for example, human genome sequences and protected metadata) available through controlled access at dbGaP (projects PRJNA398089, PRJNA430481, PRJNA430482, PRJNA326441, phs001719, phs000256, phs001626, phs001523, and others). The formal data models and associated entity relationship schemas produced by all phases of the HMP are freely available at https://github.com/ihmpdcc/osdf-schemas. The DCC website allows users to find, query, search, visualize, and download data from thousands of samples with associated metadata. Once a user has identified a set of files, conditions, subjects, or phenotypes of interest, he or she can add this set to a shopping cart for further operations. Files can then be directly downloaded for use at the user’s local site or in the cloud. The HMP DCC efforts are thus by design consistent with the NIH’s stated goals to make all data generated from NIH funding findable, accessible, interoperable, and reusable88. The success of these efforts is evidenced by a consistently high rate of user access to the web resources, with 9,000–12,000 user sessions each month, and a greater throughput anticipated after the publication of these resources here.
Complementary host–microbiome interactions
Although each of the three HMP2 studies revealed new biology within their respective areas of health and disease, a surprising range of host–microbiome immune and ecological features were common among them. The combination of shotgun metagenomics, untargeted metabolomics, and immunoprofiling was particularly effective, as in all projects this subset of molecular measurements tended to efficiently capture interpretable host and microbial properties that are linked to disease. Conversely, genetic variants were generally difficult to link to the microbiome in such small populations, which were necessary in order to deeply profile multi-omics over time, and we anticipate host sequencing to be more useful when integrated into larger cross-sectional surveys. Another notable property was that, as in most microbiome studies, changes that occurred within individuals, populations, or phenotypes were often much smaller than baseline variation between individuals. This is particularly true at microbiome-relevant time scales, for which repeated measures as rapid as days to weeks were necessary to capture the most specific host–microbiome interactions. Health-associated microbiome interactions can thus manifest in extremely diverse ways among individuals, making a combination of large-scale population surveys with within-subject longitudinal profiles essential for understanding the mechanisms of microbiome-linked disease.
As a result, other aspects of host–microbiome interactions were highly localized and subject-specific within each of the three studies. In all three conditions, microbial changes and associated host responses were strongest when captured at the time the changes occurred, and often within the tissue of origin. It is thus clear from these and other studies that host–microbiome interactions have both localized and systemic effects. Strong local perturbations initiated from either the host or microbial side can induce subsequent spatiotemporal responses that can continue over time and/or in other tissues, presumably with signals carried spatially by circulating small molecules and/or temporally by gene regulation or microbial growth, and involving regulatory circuits with both host and inter-microbial components. Continued coordinated efforts to measure the diverse host and microbial properties involved in each condition will thus be important for developing targeted and, when necessary, personalized therapies for microbiome-associated conditions, as well as for uncovering general principles that govern host–microbiome interactions. Other dynamic interactions that were not measured in all studies, such as an individual’s first microbial exposures near birth and subsequent immune development, may also represent key contributors to baseline microbiome personalization and help to explain disease-linked dynamics based on events that took place years or even decades earlier.
Next steps in microbiome multi-omics
The collective results of the NIH HMP projects, alongside many other studies, show that the microbiome is an integral component of human biology, with a major role in health and well-being. Inter-individual variability and highly diverse host–microbiome responses over time have driven the development of new methods for population microbiome studies using multiple, complementary longitudinal measurements, as well as highlighting the need to follow such studies up with mechanistic models in order to validate causative associations. The successful close of the HMP program itself has left an enduring legacy of multiple scientific generations of trained human microbiome investigators; provided the resulting community with a wealth of data, analytical, and biospecimen resources; and positioned the NIH and other funding agencies to continue work in a broad range of microbiome-linked conditions89. Funding for microbiome science, human and otherwise, is now being coordinated among NIH Centers and Institutes (https://www.niaid.nih.gov/research/trans-nih-microbiome-working-group); other US government agencies including the National Science Foundation, Environmental Protection Agency, Department of Energy, National Institute of Standards and Technology, Department of Agriculture, National Oceanographic and Atmospheric Administration, National Aeronautics and Space Administration, and Department of Defense (https://commonfund.nih.gov/hmp/programhighlights); philanthropic organizations including the Bill and Melinda Gates Foundation, the March of Dimes, the Burroughs Wellcome Fund, the Sloan Foundation, the Keck Foundation, the Juvenile Diabetes Research Foundation, the Crohn’s and Colitis Foundation, and others; and industry and public–private partnerships. Moreover, as complex global projects are launched to tackle aspects of personalized medicine, it is now obvious that it is informative to include components focused on the effect of the human microbiome.
As with any large study, the HMP2 has raised more new questions than it has answered. The aetiologies of baseline inter-individual differences in the microbiome, and of its dynamic changes over time, were not apparent even from the wide range of measurement types incorporated into these three studies and populations. Many immune and biochemical responses appear to be associated with specific strains that are unique to one or a few individual hosts, but it is not clear whether such strains are sufficient or necessary for their associated disease phenotypes. A few mechanisms were identified by which signals in the gut can be transmitted to systemic conditions such as diabetes, but not the specific small molecules or immune cell subsets by which they are likely to be transmitted—particularly in other health conditions that have not yet been studied in such detail. Finally, each HMP2 study was necessarily carried out within a geographically and genetically constrained population, and global differences in early life events, infectious disease exposure, or diet may change how microbiome dynamics contribute to human disease. Human-associated microbiology now clearly extends beyond infectious and gastrointestinal diseases to areas barely imaginable a few decades ago, including metabolism, neoplasia, maternal and child health, and central nervous system function. As the NIH HMP comes to an end, it is clear that its results have revealed a multitude of new avenues of research and technologies for future investigation, and we look forward to new discoveries based on resources from the program and exciting findings yet to come.
Acknowledgements
We thank the NIH Common Fund (particularly M. E. Perry), the Trans-NIH Microbiome Working Group (TMWG) and the HMP Science Advisors (iHMP advisors: J. Davies, F. Ouellette, E. Chang, and the late S. Falkow) for their support throughout the HMP program, and additional project principal investigators K. Jefferson and R. Xavier. We acknowledge funding from NIH grants UH2/UH3AI083263 and U54HD080784 (G.A.B., J.F.S., K. Jefferson) supported with funds from the Common Fund, the National Center for Complementary and Integrative Health, and the Office of Research on Women’s Health, grant U54DK102557 (C.H., R. Xavier), including funds from the Common Fund, the National Institute of Diabetes and Digestive and Kidney Diseases, the National Center for Complementary and Integrative Health, and the Office of Dietary Supplements and grant U54DK102556 (M.P.S., G.M.W.), with funds from the Common Fund and the National Institute of Diabetes and Digestive and Kidney Diseases.
Reviewer information
Nature thanks Frederic Bushman and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Author information
A list of participants and their affiliations appears at the end of the paper.
These authors contributed equally: Lita M. Proctor, Heather H. Creasy, Jennifer M. Fettweis, Jason Lloyd-Price, Anup Mahurkar, Wenyu Zhou.
Contributions
All authors contributed manuscript text and created or edited figures. Individual HMP2 projects discussed were implemented and managed by J.M.F., G.A.B. and J.F.S. (PTB); J.L.-P. and C.H. (IBD); W.Z., M.P.S. and G.M.W. (T2D); H.H.C., A.M. and O.W. (DCC).
Corresponding authors
Correspondence to Gregory A. Buck, Michael P. Snyder, Jerome F. Strauss III, George M. Weinstock, Owen White or Curtis Huttenhower.
Ethics declarations
Competing interests
M.S. is a cofounder of Personalis, Qbio, Sensomics, January, Filtricine and Akna and advisor for Genapsys. The other authors declare no competing interests.
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8.02: BMC Microbiome Open Collections
The following is a link to BMC Microbiome Open Collections where literature pertaining to various microbiomes can be accessed:
https://microbiomejournal.biomedcentral.com/articles/collections | textbooks/bio/Microbiology/Microbiomes_-_Health_and_the_Environment_(Parks)/08%3A_Additional_Resources/8.01%3A_The_Integrative_Human_Microbiome_Project.txt |
Learning Objectives
At the end of this section, students should be able to meet the following objectives:
1. Define “financial accounting.”
2. Understand the connection between financial accounting and the communication of information.
3. Explain the importance of learning to understand financial accounting.
4. List decisions that an individual might make about an organization.
5. Differentiate between financial accounting and managerial accounting.
6. Provide reasons for individuals to be interested in the financial accounting information supplied by their employers.
Question: This textbook professes to be an introduction to financial accounting. A logical place to begin such an exploration is to ask the obvious question: What is financial accounting?
Answer: In simplest terms, financial accounting is the communication of information about a business or other type of organization (such as a charity or government) so that individuals can assess its financial health and prospects. Probably no single word is more relevant to financial accounting than “information.” Whether it is gathering financial information about a specific organization, putting that information into a structure designed to enhance communication, or working to understand the information being conveyed, financial accounting is intertwined with information.
In today’s world, information is king. Financial accounting provides the rules and structure for the conveyance of financial information about businesses (and other organizations). At any point in time, some businesses are poised to prosper while others teeter on the verge of failure. Many people are seriously interested in evaluating the degree of success achieved by a particular organization as well as its prospects for the future. They seek information. Financial accounting provides data that these individuals need and want.
organization → reports information based on the principles of financial accounting → individual assesses financial health
Question: Every semester, most college students are enrolled in several courses as well as participate in numerous outside activities. All of these compete for the hours in each person’s day. Why should a student invest valuable time to learn the principles of financial accounting?Why should anyone be concerned with the information communicated about an organization?More concisely, what makes financial accounting important?
Answer: Many possible benefits can be gained from acquiring a strong knowledge of financial accounting and the means by which information is communicated about an organization. In this book, justification for the serious study that is required to master the subject matter is simple and straightforward: obtaining a working knowledge of financial accounting and its underlying principles enables a person to understand the information conveyed about an organization so that better decisions can be made.
Around the world, millions of individuals make critical judgments each day about the businesses and other organizations they encounter. Developing the ability to analyze financial information and then using that knowledge to arrive at sound decisions can be critically important. Whether an organization is as gigantic as Wal-Mart or as tiny as a local convenience store, a person could have many, varied reasons for making an assessment. As just a single example, a recent college graduate looking at full-time employment opportunities might want to determine the probability that Company A will have a brighter economic future than Company B. Although such decisions can never be correct 100 percent of the time, knowledge of financial accounting and the information being communicated greatly increases the likelihood of success. As Kofi Annan, former secretary-general of the United Nations, has said, “Knowledge is power. Information is liberating1.”
Thus, the ultimate purpose of this book is to provide students with a rich understanding of the rules and nuances of financial accounting so they can evaluate available information and then make good choices about those organizations. In the world of business, most successful individuals have developed this talent and are able to use it to achieve their investing and career objectives.
Question: Knowledge of financial accounting assists individuals in making informed decisions about businesses and other organizations. What kinds of evaluations are typically made? For example, assume that a former student—one who recently graduated from college—has been assigned the task of analyzing financial data provided by Company C. What real-life decisions could a person be facing where an understanding of financial accounting is beneficial?
Answer: The number of possible judgments that an individual might need to make about a business or other organization is close to unlimited. However, many decisions deal with current financial health and the prospects for future success. In making assessments of available data, a working knowledge of financial accounting is invaluable. The more in-depth the understanding is of those principles, the more likely the person will be able to use the available information to arrive at the best possible choice. Common examples include the following:
• The college graduate might be employed by a bank to work in its corporate lending department. Company C is a local business that has applied to the bank for a large loan. The graduate has been asked by bank management to prepare an assessment of Company C to determine if it is likely to be financially healthy in the future so that it will be able to repay the money when due. A correct decision to lend the money eventually earns the bank profit because Company C (the debtor) will be required to pay an extra amount (known as interest) on the money borrowed. Conversely, an incorrect analysis of the information could lead to a substantial loss if the loan is granted and Company C is unable to fulfill its obligation. Bank officials must weigh the potential for profit against the risk of loss. That is a daily challenge in virtually all businesses. The former student’s career with the bank might depend on the ability to analyze financial accounting data and then make appropriate choices about the actions to be taken. Should a loan be made to this company?
• The college graduate might hold a job as a credit analyst for a manufacturing company that sells its products to retail stores. Company C is a relatively new retailer that wants to buy goods (inventory) for its stores on credit from this manufacturer. The former student must judge whether it is wise to permit Company C to buy goods now but wait until later to remit the money. If payments are received on a timely basis, the manufacturer will have found a new outlet for its merchandise. Profits will likely increase. Unfortunately, another possibility also exists. Company C could make expensive purchases but then be unable to make payment, creating significant losses for the manufacturer. Should credit be extended to this company?
• The college graduate might be employed by an investment firm that provides financial advice to its clients. The firm is presently considering whether to recommend acquisition of the ownership shares of Company C as a good investment strategy. The former student has been assigned to gather and evaluate relevant financial information as a basis for this decision. If Company C is poised to become stronger and more profitable, its ownership shares will likely rise in value over time, earning money for the firm’s clients. Conversely, if the prospects for Company C appear to be less bright, the value of these shares might be expected to drop (possibly precipitously) so that the investment firm should avoid suggesting the purchase of an ownership interest in Company C. Should shares of this company be recommended for acquisition?
Success in life—especially in business—frequently results from making appropriate decisions. Many economic choices, such as those described above, depend on the ability to understand and make use of the financial information that is produced and presented about an organization in accordance with the rules and principles underlying financial accounting.
Exercise
Link to multiple-choice question for practice purposes: http://www.quia.com/quiz/2092614.html
Question: A great number of possible decisions could be addressed in connection with an organization. Is an understanding of financial accounting relevant to all business decisions?What about the following?
• Should a business buy a building to serve as its new headquarters or rent a facility instead?
• What price should a data processing company charge customers for its services?
• Should advertisements to alert the public about a new product be carried on the Internet or on television?
Answer: Organizational decisions such as these are extremely important for success. However, these examples are not made about the reporting organization. Rather, they are made within the organization in connection with some element of its operations.
The general term “accounting” refers to the communication of financial information for decision-making purposes. Accounting is then further subdivided into (a) financial accounting and (b) managerial accountingThe communication of financial information within an organization so internal decisions can be made in an appropriate manner2.. Financial accounting is the subject explored in this textbook. It focuses on conveying relevant data (primarily to external parties) so that decisions can be made about an organization (such as Motorola or Starbucks) as a whole. Thus, questions such as the following all fall within the discussion of financial accounting:
• Do we loan money to Company C?
• Do we sell on credit to Company C?
• Do we recommend that our clients buy the ownership shares of Company C?
They relate to evaluating the financial health and prospects of Company C as a whole.
Managerial accounting is the subject of other books and other courses. This second branch of accounting refers to the communication of information within an organization so that internal decisions (such as whether to buy or rent a building) can be made in an appropriate manner. Individuals studying an organization as a whole have different goals than do internal parties making operational decisions. Thus, many unique characteristics have developed in connection with each of these two branches of accounting. Financial accounting and managerial accounting have evolved independently over the decades to address the specific needs of the users being served and the decisions being made. This textbook is designed to explain those attributes that are fundamental to attaining a usable understanding of financial accounting.
It is not that one of these areas of accounting is better, more useful, or more important than the other. Financial accounting and managerial accounting have simply been created to achieve different objectives. They both do their jobs well; they just do not have the same jobs.
Exercise
Link to multiple-choice question for practice purposes: http://www.quia.com/quiz/2092571.html
Question: Financial accounting refers to the conveyance of information about an organization as a whole and is most frequently directed to assisting outside decision makers. Is there any reason for a person who is employed by a company to care about the financial accounting data reported about that organization?Why should an employee in the marketing or personnel department of Company C be interested in the financial information that it distributes?
Answer: As indicated, financial accounting is designed to portray the overall financial condition and prospects of an organization. Every employee should be quite interested in assessing that information to judge future employment prospects. A company that is doing well will possibly award larger pay raises or perhaps significant end-of-year cash bonuses. A financially healthy organization can afford to hire new employees, buy additional equipment, or pursue major new initiatives. Conversely, when a company is struggling and prospects are dim, employees might anticipate layoffs, pay cuts, or reductions in resources.
Thus, although financial accounting information is often directed to outside decision makers, employees should be vitally interested in the financial health of their own organization. No one wants to be clueless as to whether their employer is headed for prosperity or bankruptcy. In reality, employees are often the most avid readers of the financial accounting information distributed by their employers because the results can have such an immediate and direct impact on their jobs and, hence, their lives.
Key Takeaway
Financial accounting encompasses the rules and procedures to convey financial information about an organization. Individuals who attain a proper level of knowledge of financial accounting can utilize this information to make decisions based on the organization’s perceived financial health and outlook. Such decisions might include assessing employment potential, lending money, granting credit, and buying or selling ownership shares. However, financial accounting does not address issues that are purely of an internal nature, such as whether an organization should buy or lease equipment or the level of pay raises. Information to guide such internal decisions is generated according to managerial accounting rules and procedures that are introduced in other books and courses. Despite not being directed toward the inner workings of an organization, employees are interested in financial accounting because it helps them assess the future financial prospects of their employer.
2Tax accounting serves as another distinct branch of accounting. It is less focused on decision making and more on providing the information needed to comply with all government rules and regulations. Even in tax accounting, though, decision making is important as companies seek to take all possible legal actions to minimize tax payments. | textbooks/biz/Accounting/Accounting_in_the_Finance_World/01%3A_Why_Is_Financial_Accounting_Important/1.01%3A_Making_Good_Financial_Decisions_about_an_Organization.txt |
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