Medical Microbiology Quiz
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Questions and Answers

What is the primary characteristic of mycobacteria regarding their physical properties?

  • They are non-spore forming. (correct)
  • They are highly motile.
  • They can be easily gram stained.
  • They require anaerobic conditions.

    • Where is endemic syphilis primarily found?

  • Hot, arid areas of the Middle East (correct)
  • Central and South America
  • Humid tropical areas
  • Cold, mountainous regions

    • Which species is associated with Yaws, a disease affecting skin and bones?

  • T.pertenue (correct)
  • T.carateum
  • M.tuberculosis
  • M.leprae

    • What is the main reason mycobacteria do not gram stain well?

    <p>They have high lipid content in their cell wall.</p> Signup and view all the answers

    What biosafety level is required when working with cultured mycobacterial organisms?

    <p>BSL3</p> Signup and view all the answers

    What is a significant consequence of untreated bacterial vaginosis?

    <p>Pelvic inflammatory disease (PID)</p> Signup and view all the answers

    Which test is NOT recommended for diagnosing bacterial vaginosis?

    <p>Culture</p> Signup and view all the answers

    What type of discharge is characteristic of bacterial vaginosis?

    <p>Foul-smelling with an amine odor</p> Signup and view all the answers

    Which organism is known to cause pulmonary infections in immunocompromised patients?

    <p>Nocardia spp.</p> Signup and view all the answers

    How do Nocardia spp. appear under a gram stain?

    <p>Beaded, branching filaments</p> Signup and view all the answers

    Which of the following claims about Bacillus is FALSE?

    <p>It increases survival by being metabolically active.</p> Signup and view all the answers

    What stain is used to identify Nocardia as weakly acid fast?

    <p>Kinyoun stain</p> Signup and view all the answers

    Which condition is associated with Tropheryma whipplei infection?

    <p>Whipple disease</p> Signup and view all the answers

    What condition is associated with Clostridium perfringens in infected tissues?

    <p>Myonecrosis</p> Signup and view all the answers

    Which symptom is NOT associated with food poisoning caused by Clostridium perfringens?

    <p>Fever</p> Signup and view all the answers

    What characteristic is used in the laboratory to help identify Clostridium difficile?

    <p>Yellow ground glass colonies on CCFA</p> Signup and view all the answers

    What type of toxin is primarily produced by all toxigenic strains of Clostridium difficile?

    <p>Enterotoxin</p> Signup and view all the answers

    What is the main cause of infant botulism?

    <p>Ingestion of honey</p> Signup and view all the answers

    What is the preferred method to diagnose Clostridium botulinum in a patient?

    <p>Isolation of organism or toxin detection</p> Signup and view all the answers

    What is a notable characteristic of Clostridium tetani?

    <p>Enters through wound and produces neurotoxin</p> Signup and view all the answers

    Which condition is NOT typically caused by Clostridium septicum?

    <p>Asymptomatic carrier state</p> Signup and view all the answers

    Which type of anaerobic bacteria requires anaerobic conditions for growth and is killed in the presence of oxygen?

    <p>Obligate anaerobes</p> Signup and view all the answers

    What nutritional components do anaerobes require for growth?

    <p>Vitamin K, hemin, and yeast extract</p> Signup and view all the answers

    Which of the following is NOT an appropriate specimen collection method for anaerobic bacteria?

    <p>Hooving sputum sample</p> Signup and view all the answers

    What is a characteristic clue to diagnose anaerobic infections?

    <p>Foul-smelling, gaseous discharge</p> Signup and view all the answers

    What type of anaerobic bacteria can survive in oxygen but prefers anaerobic environments?

    <p>Facultative anaerobes</p> Signup and view all the answers

    Which culture media is suitable for growing obligate anaerobes?

    <p>Anaerobic Broth</p> Signup and view all the answers

    Which organisms are typically involved in an endogenous anaerobic infection?

    <p>Normal flora of the host</p> Signup and view all the answers

    What characteristic does PRAS media provide for anaerobic transport?

    <p>Prevents exposure to oxygen</p> Signup and view all the answers

    Which of the following conditions is optimal for the growth of anaerobic bacteria?

    <p>Anaerobic conditions with reducing agents</p> Signup and view all the answers

    What is the role of superoxide dismutase in strict aerobes?

    <p>To detoxify superoxide anions</p> Signup and view all the answers

    Which species is the most common anaerobe associated with actinomycosis?

    <p>Actinomyces israelii</p> Signup and view all the answers

    What is a characteristic feature of Actinomyces spp. when viewed under a Gram stain?

    <p>Beaded appearance</p> Signup and view all the answers

    Which of the following bacteria is NOT typically associated with actinomycosis?

    <p>Nocardia spp.</p> Signup and view all the answers

    What type of morphology does Bifidobacterium exhibit?

    <p>Branched or bifurcated</p> Signup and view all the answers

    Which feature distinguishes Bacteroides fragilis from other bacteria?

    <p>Bile resistance</p> Signup and view all the answers

    What is a common clinical manifestation of actinomycosis caused by Actinomyces israelii?

    <p>Fistulae draining pus</p> Signup and view all the answers

    Which of the following statements about anaerobic Peptostreptococcus is true?

    <p>It appears in chains when viewed under a microscope.</p> Signup and view all the answers

    Which characteristic aids in the growth of Bacteroides fragilis on specific media?

    <p>Stimulation by bile</p> Signup and view all the answers

    What is the stage of syphilis characterized by a firm chancre at the inoculation site?

    <p>Primary Syphilis</p> Signup and view all the answers

    Which of the following is NOT a symptom of secondary syphilis?

    <p>Gummas</p> Signup and view all the answers

    At which stage of syphilis is the infection considered not infectious?

    <p>Tertiary Syphilis</p> Signup and view all the answers

    What is the primary cause of early onset congenital syphilis?

    <p>Treponemes cross the placenta</p> Signup and view all the answers

    Which test is primarily used for screening in syphilis diagnosis?

    <p>VDRL</p> Signup and view all the answers

    Which of the following is a characteristic of nontreponemal tests?

    <p>Sensitive but not specific</p> Signup and view all the answers

    What is a potential outcome for a fetus if the mother has early syphilis?

    <p>Severe infection leading to death</p> Signup and view all the answers

    Which of the following is true regarding treponemal tests?

    <p>Remain positive after treatment.</p> Signup and view all the answers

    What complication may arise from untreated tertiary syphilis?

    <p>Gummas in various body sites</p> Signup and view all the answers

    Which symptom is associated with late onset congenital syphilis?

    <p>Bone deformities</p> Signup and view all the answers

    Study Notes

    Anaerobic Bacteria

    • Anaerobic bacteria do not need oxygen to grow.
    • They vary in their ability to tolerate oxygen.
    • Obligate anaerobes require anaerobic conditions for growth.
    • Oxygen is toxic to obligate anaerobes, killing them.
    • Aerotolerant anaerobes can grow in atmospheres with oxygen, but grow best anaerobically.
    • Facultative anaerobes do not require oxygen but can use it if available.
    • Superoxide anion and hydrogen peroxide are toxic byproducts of oxygen metabolism.
    • Strict aerobes and facultative anaerobes have enzymes like superoxide dismutase and/or catalase to neutralize these toxic compounds.
    • Anaerobic bacteria require a low redox potential, while normal human tissue and aerobic culture media have a high redox potential.
    • Reducing agents (thioglycollate, cysteine, dithiothreitol) are included in anaerobic media to maintain the low redox potential.
    • Anaerobic bacteria are found in soil, water, and animals, as well as the human oral cavity, upper respiratory tract, intestinal tract, genitourinary tract, and skin.
    • Facultative bacteria use up oxygen in protected areas and reduce redox potential, inactivating harmful oxygen molecules.
    • Diseases caused by anaerobic infections can be exogenous or endogenous.
    • Exogenous infections are those where the organisms enter the body from outside sources. Infections at the host's normal flora (NF) near the mucosal surfaces are endogenous infections.
    • Clues to anaerobic diseases include trauma, animal/human bites, foul-smelling gaseous discharges, necrotic tissue, vascular stasis, and black or fluorescent red coloring.

    Specimen Collection

    • Best specimens for anaerobic cultures are aspirated with a needle and syringe.

    • Examples of appropriate specimens include blood, CSF, bone marrow, and cavity fluid.

    • Other appropriate specimens include urine (suprapubic aspirate), abscesses, ulcers, draining wounds, and biopsy materials.

    • Examples of inappropriate specimens include those from areas with resident flora, such as the oral cavity, gastrointestinal tract, and genitourinary tract.

    • Further examples include superficial skin sites, and voided or catheterized urine. This is because these specimens can have resident flora present that will make analysis difficult.

    Transport and Processing

    • Inoculate media in anaerobic environment immediately.
    • Limit exposure to room air and prevent drying.
    • Do not refrigerate specimens.
    • Minimize time at room temperature.
    • PRAS media, a prereduced, anaerobically sterilized transport medium is used.
    • Transport media includes modified Cary-Blair or Amies agar.
    • Rezasurin is an oxygen indicator and reducing substances prevent oxygen exposure.

    Culture

    • Specimens for anaerobic cultures should first be aerobically cultured on plates like BAP, CHOC, or MAC to assess their aerotolerance.
    • Anaerobes need vitamin K, hemin, and yeast extract.

    Nonselective Media

    • CDC (or anaBAP) Blood Agar Plates support obligate and facultative anaerobes.
    • Anaerobic Broth, Thioglycollate, or Chopped (Cooked) Meat support the growth of obligate and facultative anaerobes. Anaerobes tend to grow toward the bottom, while facultative anaerobes grow generally throughout the media.

    Anaerobic Incubation

    • Incubation times are typically 35-37°C for 48 hours.
    • Environments include nitrogen gas (80-90%), hydrogen gas (5-10%), and carbon dioxide (5-10%).
    • Hydrogen and carbon dioxide remove oxygen, and some anaerobes thrive in these conditions (capnophilic).

    Anaerobic Systems

    • Anaerobic jars, bags, and chambers are used.

    Anaerobic Jars

    • The technique uses a jar and palladium pellets inside an envelope.
    • H2 and CO2 are generated by adding water and heating
    • methylene blue or resazurin as indicators turn white when reduced (anaerobic or oxygen-free environment).

    Anaerobic Bags

    • Commercially available, they hold 1-3 plates and contain an oxygen removal system and indicator.
    • A gas-impermeable plastic bag holds the media.
    • The system creates an oxygen-free environment for the media.

    Anaerobic Chamber

    • Providing optimal anaerobic growth conditions, the system inoculates, incubates, and examines media.
    • The system includes a palladium catalyst, desiccant, and indicator for gas mixture.

    Culture Examination

    • Examination in chamber can happen at any time within 48 hours.
    • The sample's oxygen exposure after 48 hours is minimized.

    Anaerobic Identification Tests

    • Gram-staining, colony morphology, and rapid tests are used for presumptive identification.

    Anaerobes Gram Stain RXN

    • Gram staining is the key identification test for anaerobes.

    Rapid Identification Tests

    • Tests can include catalase, urease, indole, motility, esculin hydrolysis, lecitinase, lipases, and reverse CAMP.
    • Aerotolerance, fluorescence, and disk tests are also possible rapid identification tests.

    Aerotolerance

    • This test determines if an organism is strict, aerotolerant, or facultative.
    • The isolate is incubated in both aerobic and anaerobic environments (e.g., anaBAP and CHOC agar with CO2)
    • Organisms are examined for growth at 48 hours.
    • The growth patterns (anaerobic vs aerobic) determine aerotolerance type.
    • Differentiating characteristics include growth only on anaBAP (strict anaerobic), growth on anaBAP > CHOC (aerotolerant), and growth on both anaBAP and CHOC (facultative anaerobic).

    Fluorescence

    • Fluorescent organisms will fluoresce a color (Red, orange, pink, chartreuse) under UV light.

    Antibiotic Disk Test

    • Helpful for confirming gram stain results and may test for susceptibility to various classes of antibiotics including kanamycin, vancomycin, and colistin.
    • Results of the antibiotic susceptibility test can help with categorization.

    Disk Tests

    • Sodium polyanethol sulfonate (SPS) disk identify ana GPC and Peptostreptococcus which are sensitive to this disk.
    • Nitrate disk measures nitrate reduction.
    • Bile disk measures growth in 20% bile to test for bile tolerance. Some anaGNR (e.g. Bacteroides fragilis group) are bile tolerant.

    Lecithinase

    • Lecithinase activity is identified using egg-yolk agar.
    • Positive reaction creates opaque zones in the agar around the colony.
    • A negative reaction shows no change, resulting in a normal colony morphology.

    Nagler Test

    • Used to detect alpha toxin production by C. perfringens.
    • Alpha toxin is a type of lecithinase.
    • An antiserum neutralizes the alpha toxin from C. perfringens, and not alpha toxins produced by other clostridia.

    Lipase

    • Lipase hydrolyzes triglycerides on egg yolk agar (EYA), producing glycerol and free fatty acids.
    • A positive reaction will show an oil-on-water surface or multicolored sheen. Negative reactions will not show sheen or coloring changes on the medium.

    Reverse CAMP

    • Alpha toxin of C. perfringens, in conjunction with beta hemolysis of Group B streptococcus (GBS) will show as an arrowhead-shaped hemolytic zone.
    • The test organism is inoculated onto anaBAP and GBS is streaked perpendicularly to the test organism, but not touching.

    Definitive Identification Tests

    • Testing includes biochemical tests, commercial systems, gas-liquid chromatography, cellular fatty acid analysis, and 16S rRNA sequencing.

    Biochemical and Commercial Systems

    • The tests utilize PRAS or non-PRAS biochemical test media, and commercial biochemical/pre-existing bacterial enzymes mini-systems.

    Gas Liquid Chromatography

    • Analysis of cellular fatty acids or metabolic end products (volatile acids) using gas-liquid chromatography produces characteristic patterns.

    Gene Sequencing

    • 16S rRNA gene sequencing is used on amplified ribosomal DNA to determine the type of organism.

    Clostridium Species

    • Catalase is negative. Organisms are typically motile, except C. perfringens.
    • They may be gram variable or gram negative.
    • Most (but not all) are susceptible to vancomycin.
    • Most form spores (refractive structures) that are oval, round or terminal/subterminal.
    • Some species are aerotolerant, causing exogenous infections (e.g., tetanus, gas gangrene, botulism, and food poisoning), gaining access through wounds or ingestion.

    Genera Differences

    • A comparison of optimal conditions, sporulation conditions, and catalase production for various Bacillus, Clostridium, and Lactobacillus genera.

    Clostridium perfringens

    • The most common Clostridium sp.
    • Commonly found in food such as undercooked meat, soil, and water; and in wound infections from injury or surgery.
    • Gram positive rod forms a box-car shape.
    • Produces a double beta-hemolytic zone on anaBAP.
    • Demonstrates positive reactions for reverse CAMP, lecithinase, and Nagler test.
    • Rarely seen in spore form.

    Myonecrosis

    • Myonecrosis (gas gangrene) is a common clinical manifestation of C. perfringens infections commonly arising from deep or penetrating wounds or surgical procedures in addition to diabetic foot ulcers.

    C. perfringens Clinical Significance

    • C. perfringens can cause food poisoning, primarily associated with contaminated meat, gravy, and other foods high in protein.

    • Spores are ingested, and germinate in the intestines producing enterotoxin, resulting in nausea, vomiting, diarrhea, and abdominal pain.

    Clostridium difficile

    • May be found in the feces and can cause antibiotic-associated diarrhea and pseudomembranous colitis.
    • A resistant C. difficile strain increase in number if antibiotics upset the normal flora of the gut.
    • Toxigenic strains produce enterotoxin (toxin A) and cytotoxin (toxin B).

    C. difficile Characteristics

    • Yellow ground-glass colonies on cycloserine-cefoxitin-fructose agar (CCFA).
    • Smells like horse manure.
    • Spore forming gram positive rod.
    • Fluorescent with a chartreuse color.

    C. difficile Toxin Tests

    • Cytotoxin tests are used to assess the effects of stool on human cell cultures to examine presence of toxins A or B.
    • Additional tests like kit testing and molecular testing can also determine toxin types.

    Clostridium botulinum

    • C. botulinum causes botulism, a potentially fatal condition.
    • Common foodborne and wound sources are home canned foods, cured meats, and contaminated wounds.
    • Toxins cause neuromuscular paralysis.
    • Infant botulism associated with eating honey can occur if the infant's gut microbiome is not fully developed and lacks the necessary competition for beneficial bacteria.

    C. botulinum Diagnosis

    • Clinically diagnosed based on patient history and symptoms.
    • Isolating the organism or detecting the toxin in reference labs also aids in diagnosis.

    Clostridium tetani

    • C. tetani is an anaerobic, spore-forming rod that leads to tetanus, also known as lockjaw, with severe muscle spasms and affecting many body parts.
    • The organism enters the body through wound sites.
    • Tetanospasmin (potent neurotoxin) is produced by the organism.

    C. septicum

    • C. septicum is an important aerotolerant species.
    • Forms swarming colonies.
    • Forms sub-terminal spores.
    • Important in cases of Myonecrosis and bacteremia.
    • Associated with leukemia, lymphoma, and large bowel carcinoma.

    Other Species (Non-Spore-Forming Anaerobic GPR)

    • Actinomyces, Bifidobacterium, Propionibacterium, Eggerthella and Eubacterium can cause actinomycosis.

    Actinomyces spp.

    • Includes aerobic and anaerobic bacteria.
    • Nocardia is an aerobic Actinomyces species.
    • A. israelii is a common anaerobic Actinomyces causing actinomycosis.
    • Actinomyces inhabit human and animal mucosa and are not weakly acid fast.
    • Gram positive rods are irregularly stained (beaded appearance) in specimens. The appearance is coccoid (spherical) to filamentous (thread-like) or form filaments that branch.

    A. israelii Actinomycosis

    • A. israelii causes actinomycosis, a chronic granulomatous infection usually affecting the jaw.
    • Can manifest as "lumpy jaw."

    A. israelii Cultures

    • Pus and sulfur granules are frequently associated with A. israelii infections.
    • Cultures grow slowly (7-9 days).
    • Molar-tooth colonies are seen.
    • Organisms are anaerobic, and gram-positive, branching rods.

    Bifidobacterium

    • Bifidobacterium inhabits intestines and the oral cavity.
    • Its morphology resembles Actinomyces, appearing as gram staining, branched, or bifurcated GPR.
    • It rarely causes disease.

    Propionibacterium

    • Propionibacterium is an anaerobic, diphtheroid-like GPR.
    • It resembles corynebacteria.
    • Commonly found on the skin, as a contaminant in blood cultures or specimens.
    • It is associated with acne (e.g., P. acnes).
    • Catalase and indole are positive.

    Eggerthella and Eubacterium spp.

    • Eggerthella (previously Eubacterium) are anaerobic, diphtheroid-like GPR.
    • They do not branch.

    Anaerobic GPC

    • Peptostreptococcus are GPC in chains.
    • P. anaerobius is susceptible to sodium polyanethol sulfonate (SPS).
    • Other anaerobic cocci are resistant.
    • Peptococcus are GPC in clusters.

    Anaerobic GNR

    • Bacteroides, Prevotella, Porphyromonas, Fusobacterium, and Veillonella are found in the NF mucous membranes of the human body.

    Bacteroides fragilis Group

    • Bacteroides fragilis is an anaerobic GNR in the NF of the GI tract.
    • Common cause of intra-abdominal infections, bacteremia, and soft tissue infections.
    • B. thetaiotaomicron is common and second in occurrence, after B. fragilis.

    Bacteroides ureolyticus Group

    • Anaerobic GNR.
    • Bile sensitive and bile tolerant, non-pigmented organisms.
    • Some organisms pit the agar.
    • Show growth in formate and fumerate.
    • Resistant to vancomycin but sensitive to kanamycin and colistin.

    Prevotella

    • Anaerobic GNR
    • Bile susceptible
    • Resistant to kanamycin and vancomycin.
    • Grows on KVLB agar but not BBE agar
    • Some produce protoporphyrin. Colonies are dark pigmented, fluorescing a bright brick red under UV light. Colonies are often a bright reddish hue.

    Porphyromonas

    • Anaerobic GNR
    • Require hemin and vitamin K .
    • Resistant to kanamycin and colistin.
    • Sensitive to bile and vancomycin.
    • Does not grow well on KVLB agar.
    • Colonies produce pigmented colonies and fluoresce brick red.

    Fusobacterium

    • Susceptible to kanamycin and colistin.
    • Vancomycin resistant
    • F. nucleatum is a thin fusiform (spindle-shaped) rod.
    • Forms bread-crumb, speckled colonies.
    • F. necrophorum is lipase-positive.

    Mobiluncus

    • Associated with bacterial vaginosis (BV), pelvic inflammatory disease (PID), and abdominal infections.
    • Curved bacilli.
    • Gram variable.
    • Motile, catalase-negative, indole-negative.
    • Inhibited by vancomycin.

    Selective Anaerobic GNR ID

    • Uses sensitivities to various antibiotics and other factors to categorize the organisms.

    Veillonella

    • Only commonly encountered non-spore-forming anaerobic GNC.
    • Sensitive to kanamycin and colistin, resistant to vancomycin.
    • Smallest gram-negative cocci.

    Gram Positive Rods

    • Further categorization into those with and without spores, and those that branch.

    Non-Spore-Forming GPR

    • Corynebacterium and Coryneforms, Listeria, Erysipelothrix, Acanobacterium, Lactobacillus, and Gardnerella.

    Corynebacterium

    • Facultative anaerobe.
    • Forms small gamma-shaped colonies on SBA.
    • Can form "Chinese letter" formations.
    • Catalase-positive.
    • Nonmotile.
    • Frequently a contaminant.
    • C. diphtheriae, a significant pathogen, produces diphtheria toxin, causing exotoxin, destroying host cells leading potentially to heart and nervous system damage in cutaneous or respiratory infections. (Typically treated with antitoxin. Prevented by immunization.)
    • C. diphtheria, can be diagnosed based on pleomorphic GPR, darker staining area of cell (metachromatic granules), and methylene blue stain, also looking for pockets of inorganic phosphates or nutrient reserves.
    • Using selective media such as SBA, serum or blood containing media (Loeffler or Pai medium), and selective media such as Tinsdale (cystine-tellurite blood agar) may aid in diagnosis.

    Listeria monocytogenes

    • Vaginal and intestinal NF in humans.
    • Clinical infections due to ingestion of contaminated food.
    • Virulence factors include hemolysin (listeriolysin O).
    • Usually causes listeriosis (sepsis), especially in neonates ( ~50% fatality rate), pregnant women, and immunocompromised individuals.
    • Organisms are GPR, non-spore forming, small beta colonies on BAP, catalase positive, bile-esculin positive, motile at 25°C. Motility can be identified by use of tumbling or umbrella patterns in a tube motility test.
    • Positive CAMP test can differentiate Listeria monocytogenes from other Listeria spp

    Erysipelothrix rhusiopathiae

    • Domestic swine are a major reservoir.
    • Erysipelothrix rhusiopathiae causes occupational risk among butchers, vets, and fishermen (in addition to other animals and humans).
    • Erysipelothrix rhusiopathiae is a GPR, catalase-negative bacterium with alpha or gamma hemolysis.
    • Organisms are usually nonmotile.

    Arcanobacterium haemolyticum

    • Previously known as a Corynebacterium
    • Significant pathogen, causes pharyngitis.
    • Facultative anaerobe, beta hemolytic.
    • Catalase negative
    • Reverse CAMP test positive to distinguish from other organisms.

    Lactobacillus

    • Normal vaginal flora, produces lactic acid.
    • Reduces vaginal pH, inhibits the growth of other organisms.
    • Rarely causes disease.
    • L. acidophilus is a probiotic.

    Gardnerella vaginalis

    • Pleomorphic coccobacilli with variable Gram stains, not motile and are facultative anaerobes.
    • Common cause of bacterial vaginosis (BV) in 50-70% of women.

    Bacterial Vaginosis (BV)

    • Characterized by a change in the composition of the normal vaginal flora.
    • Increase in pH that allows for growth of Gardnerella vaginalis.
    • Foul-smelling discharge (amine odor).
    • Clue cells (squamous vaginal epithelial cells with bacteria clustered along their edges and surfaces) are often seen in specimens.
    • Culture is not recommended.

    Non-Spore-Forming Branching GPR (Aerobic Actinomycetes)

    • Important groups which include Nocardia, Tropheryma whipplei, Streptomyces, Actinomadura, Gordoni, Tsukamurella, and Rhodococcus equi.

    Nocardia spp.

    • Found in soil and frequently infect immunocompromised individuals.
    • Pulmonary and cutaneous forms are common.
    • Pulmonary infections can disseminate, leading to abscess formation.
    • Cutaneous form (mycetoma) involves invasive abscesses containing sulfur granules.

    Tropheryma whipplei

    • This intracellular pathogen causes Whipple's disease, and its diagnosis can be confirmed via PCR or 16S rRNA. The disease is uniformly fatal if not treated, manifesting as diarrhea, weight loss, and malabsorption.

    Spore-Forming Non-Branching GPR

    • The important group is Bacillus.
    • Spore-forming, non-branching bacilli are protective and metabolically inactive.
    • Spores enhance organism survival in nature.
    • Spores are resistant to biocides and have a thick outer wall.

    Bacillus

    • Primarily found in the environment. It is metabolically diverse.
    • B. anthracis, a significant pathogen, causes anthrax, which occurs in herbivores and can be transmitted to humans via contaminated animal products, wounds, inhalation, or ingestion.
    • B. cereus can cause food poisoning via consumption of foods high in protein.

    M. tuberculosis Complex

    • Pathogenic species of the M. tuberculosis complex (MTC) include M. tuberculosis (most common), M. bovis, and others such as M. africanum, M. canettii and M. microti. M. tuberculosis infects lungs, and other tissues such as lymph nodes, kidneys, and bones or other tissue sites and has a person-to-person transmission.
    • An important finding is M. bovis, which infects humans from unpasteurized milk products.
    • M. tuberculosis complex is characterized via colony morphology (rough, buff-colored colonies in serpentine cords) and is a slow-growing organism. These organisms are tested in respiratory specimens to determine if M. tuberculosis complex is present.
    • M. tuberculosis complex has a high risk of reactivation if the immune system is compromised.

    Extrapulmonary TB, Miliary TB

    • Extrapulmonary TB can infect meningeal, bone (Pott's disease), kidney, and genital tracts.
    • Miliary TB is a disseminated form of TB with widespread small tubercles throughout the body.

    PPD Tuberculin Skin Test

    • The Mantoux test is used for screening and diagnosing TB infection.
    • Positive PPD results imply the presence of an immunological response to M. tuberculosis. It is a delayed hypersensitivity reaction, with a red hard area at the injection site in 48–72 hours, indicating previous or current infection with M. tuberculosis. Negative PPD results suggest no infection with or no response to the test organism.

    MTB Symptoms

    • Common symptoms associated with MTB infection include coughing, hemoptysis, weight loss, and low-grade fevers. The disease can affect multiple sites within the body.

    M. tuberculosis ID

    • Slow grower; rough, buff-colored colonies with serpentine cords (due to cord factor).
    • Positive results for niacin and nitrites, and negative at 68°C catalase, further suggest that the organism is susceptible to NAP and T2H testing.

    Acid-Fast Stain

    • Key to the staining process is maintaining appropriate temperatures, and reagents to stain mycobacterium (acid-fast bacilli or AFB).

    AFB Culture Media

    • AFB culture methods utilize liquid or solid growth media which commonly include egg-based, and agar-based media.
    • Other media includes Tween 80. and instrumentation broth

    Solid Egg-Based Media

    • Lowenstein-Jensen (LJ) media is a common example of solid egg-based media.
    • Its composition includes eggs and inhibits the growth of Gram-positive bacteria, slowing down the growth of AFB.

    Solid Agar-Based Media

    • Middlebrook 7H10, 7H11, and other modified media are used; these media are non-selective and contain nutrients, vitamins, and albumin to prevent inhibition or neutralize toxins.

    Culture Examination

    • Agar-based specimens are examined twice weekly in the first four weeks, and weekly thereafter.
    • Egg-based specimens are examined weekly; colonies on egg-based specimens are examined microscopically (4x - 10x), using a hand-lens.

    Liquid Media

    • Tween 80 media aids in dispersing AFB colonies, improving visualization of growth, assisting with sub-culturing procedures.

    Instrumentation - Liquid Media

    • Automated systems are frequently used to culture and detect AFB growth in liquid medium; these systems include the Bactec 460TB and 9000MB instruments.
    • Bactec MB/BacT systems detect CO2 production.
    • ESP Myco systems detect pressure changes as gases are produced.

    Approach to ID AFB

    • A presumptive diagnosis of AFB is made through culture and acid-fast staining.
    • Once AFB is confirmed, additional, more complex procedures including biochemical testing and cell wall lipid analysis are used for precise identification; such tests may use commercial systems or require lab procedures.
    • Growth parameters, biochemical tests, and lipid profiles further aid in AFB species classification.

    Growth Parameters

    • Growth rate, including rapid and slow growers, differentiates non-tuberculous mycobacteria.

    Photoreactivity

    • Some non-tuberculous mycobacteria produce carotenoid pigments, dependent on exposure to light.

    Identification Testing

    • AFB species are identified via a variety of tests, such as biochemical tests (catalase, niacin, nitrate, and Tween 80 hydrolysis) and tellurite reduction.
    • Additional miscellaneous tests like NAP and growth on MAC are used to identify characteristics.

    Catalase

    • Heat-stable catalase is verified using a 68°C water bath and hydrogen peroxide/tween 80.
    • Bubbles = positive result, no bubbles = negative result.

    Niacin

    • Most mycobacteria convert niacin to niacin ribonucleotide. This process is absent in M. tuberculosis.
    • Accumulation of niacin in the media leads to a yellow coloration in the test specimens.

    Nitrate Reduction

    • Nitrate is reduced to nitrite/nitrogen gas in media by adding reagents including sulfanilamide and N-naphthylenediamine dihydrochloride.
    • A lack of color change indicates a negative result. Red color indicates a positive result.
    • Added zinc detects if nitrate reduced to nitrogen.

    Tween 80 Hydrolysis

    • Some non-pathogenic mycobacteria hydrolyze Tween 80 to turn the media a red color.
    • Amber/no color = negative.

    Tellurite Reduction

    • Tellurite is reduced to tellurium.
    • Black color in media = positive result.

    Growth Inhibition

    • Tests like NAP and T2H inhibit growth of microorganisms.

    Growth on Special MAC

    • Rapid growers such as M. fortuitum-chelonae complex grow on MAC without crystal violet.

    Growth Temperatures

    • Growth temperatures of 24, 30, 37 and 42 °C vary based on organism type.

    Classification of Mycobacteria

    • The M. tuberculosis complex (MTC) includes M. tuberculosis (most common), M. bovis and others such as M. africanum, M. canettii and M. microti.
    • Atypicals or Nontuberculous mycobacteria (NTM) or Mycobacterium other than tuberculosis (MOTT) include species such as those seen in the M. avium-intracellulare complex and M. ulcerans.
    • Uncultivable mycobacteria such as M. leprae is important due to the disease leprosy.

    M. tuberculosis Complex

    • M. tuberculosis is important pathogen. Rapid growers, and slow growers of M. tuberculosis affect lung and other tissues in the body.
    • Important information on transmission is also identified.

    M. tuberculosis Complex

    • Spread of M. tuberculosis is from respiratory droplets. The infection is person-to-person transmissions. M. tuberculosis complex is non-pigmented on growth and considered slow growers.

    M. tuberculosis Complex

    • M. bovis is the atypical form of M. tuberculosis. It is transmitted by consuming unpasteurized milk. Less common due to pasteurization measures of milk products, M. bovis can be found in cattle and can be transferred to humans.
    • Colony morphology includes the rough and buff appearance, cording and cauliflower shapes.
    • Requires long-term MDR treatment with multiple antibiotic regimens.
    • Skin tests like PPD are performed in US and Canada and globally to assist with diagnosis.

    Primary TB

    • Primary TB infection, in many but not all cases, is an important step in the formation granuloma lesion in the lungs.
    • Exposure to respiratory droplets, where many patients will form lung granuloma that is described caseous, with tubercles present and may require long-term treatment. Risk of disease if immune system is suppressed.

    Tubercles

    • Tubercles are the key anatomical areas of infection. They represent a type of granulomatous lesion characterized by the masses of inflammatory cells that clump around the M. tuberculosis organisms. These tubercles can be seen via radiographic or other imaging techniques.

    Primary TB

    • Risk of TB reactivation is high if the immune system is suppressed.
    • 15–20% of the affected individuals that are infected with M. tuberculosis will develop the disease.

    Extrapulmonary TB

    • Extrapulmonary TB involves infections in tissues and organs other than the lungs.
    • Common extrapulmonary TB infections include kidney, bone, and genital tract.

    Miliary TB

    • Disseminated TB infection involves widespread lesions throughout the body, particularly the lungs.
    • The appearance resembles millet seeds.

    PPD Tuberculin Skin Test

    • The PPD detects the presence of M. tuberculosis via an immunoassay, that elicits a delayed hypersensitivity reaction (an inflammatory response seen 48–72 hours post initial injection.)
    • In the positive case, a hard red area at injection site indicating an exposure or infection with M. tuberculosis (may not be active infection).
    • A negative PPD implies no response to the test and no known infection or presence of M. tuberculosis in the patient

    MTB Symptoms

    • Common symptoms of MTB infection include coughing, hemoptysis, weight loss and low-grade fevers.

    M. tuberculosis ID

    • Rapid identification requires confirming an AFB. Non-tuberculous mycobacteria (NTM) can be detected by slow growth, colony morphology (rough, buff, or serpentine cords), niacin test and nitrate reduction testing, in addition to temperature susceptibility.

    Colonies on LJ

    • M. tuberculosis and other MTC pathogens are visibly visualized on growth media. The growth may be slow and form rough, buff, or beaded colonies in serpentine cords on LJ and other media.

    Acid Fast Stain - Cord Factor

    • Staining organisms via heat or phenol-based stains will produce an appropriate image to allow for the detection of the presence of these organisms and associated cord factors.

    M. bovis ID

    • M. bovis colonies resemble M. tuberculosis.
    • M. bovis is negative for nitrate, niacin, and 68°C catalase; however, it is susceptible to NAP and T2H.

    Runyon NTM Classification

    • Nontuberculous mycobacteria (NTM) are further categorized into photochromogens, scotochromogens, nonphotochromogens, and rapid growers.

    I Slow Growing Photochromogens

    • M. kansasii: Pulmonary disease, infection of other body tissues like joints, bone, and lymph nodes; positive for nitrate and Tween 80.
    • M. marinum: Swimming-pool granuloma; acquired via contaminated water, grows well at 30°C and poorly at 37°C, and is positive for Tween 80.

    II Slow Growing Scotochromogens

    • M. scrofulaceum: Mycobacterial cervical (neck) lymphadenitis; negative for nitrate and Tween 80.
    • M. gordonae: Tap water bacillus; negative for nitrate and Tween 80.

    III Slow Growing Nonphotochromogens

    • M. avium complex (MAC): Common NMT that is an increasing prevalence of infection in patients with AIDS affecting the lungs, and disseminated throughout the body. The AFB is positive for 68 °C catalase and requires additional methodologies for identification such as GLC, HPLC, and nucleic acid probe testing.

    III Slow Growing Nonphotochromogens

    • M. ulcerans: Skin ulcer form of infection. Optimum temperature is 32°C.

    IV Rapid-Growers

    • This group includes M. fortuitum, M. chelonae, and M. smegmatis.
    • Environmental organisms, causing infections like wound infections, abscesses, osteomyelitis, and pulmonary infections; grow well on special MAC, and negative for nitrate; M.chelonae is an important rapid grower demonstrating nitrate positive.

    Uncultivable

    • M. leprae: Causes Hansen's disease (leprosy), and affects peripheral nerves and skin and mucous membranes.
    • Grows best at a temperature of 30°C. Two forms of leprosy exist-- tuberculoid leprosy, affecting skin; peripheral nerves and is treatable; and leprmatous leprosy which is more progressive, not treatable and can be fatal.

    Uncultivable Mycobacteria

    • Rare but treatable in the US is detected and confirmed via clinical and microscopic techniques.

    Other Pathogenic Species

    • M. genavense, M. simiae, M. xenopi, and M. szulgai are additional pathogenic species.

    Newer Identification Tests

    • Newer testing techniques for Mycobacteria, including chromotography (GLC or HPLC), are based on the unique characteristics of mycolic acids found in the organism's cell walls.
    • Molecular ID of AFB requires techniques such as hybridization and nucleic acid amplification by nucleic acid probe.

    Antimicrobial Susceptibility Tests

    • M. tuberculosis is tested for susceptibility to various antimicrobial drugs such as isoniazid, rifampin, ethambutol, and streptomycin; these are helpful in determination of treatment regimens.

    Susceptibility Testing Methods

    • Several methods including absolute concentration, resistance ratio, and proportional methods are used to detect susceptibility to antimicrobial agents. The results can assist medical professionals to administer the most effective and appropriate treatment regimen.

    Antimicrobial Susceptibility Tests

    • MDR variants are prevented using two to three drugs in conjunction (such as INH and Rifampin) for a minimum of 9 months; treatment of other non-tuberculosis mycobacteria are also considered, depending on the infecting organism and severity.
    • Prior exposure to any antibiotic may also contribute to the drug resistance profile of a pathogenic organism. Antibiotic-susceptibility testing is not often performed on NTM due to the nature of these organisms and the need for precise methodology.

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