Microbiology: Anaerobic Bacteria Quiz
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Questions and Answers

What type of agar is best suited for supporting facultative and obligate anaerobes?

  • Nutrient agar
  • Cycloserine-cefoxitin-fructose agar
  • Kanamycin-vancomycin laked blood agar
  • Brucella blood agar (correct)

    • Which two antibiotics are used in Cycloserine-cefoxitin-fructose agar?

  • Vancomycin and kanamycin
  • Cycloserine and cefoxitin (correct)
  • Tetracycline and penicillin
  • Amoxicillin and nitrofurantoin

    • What gas mixture is typically used in anaerobic incubation?

  • Hydrogen and argon
  • Oxygen and carbon monoxide
  • Nitrogen, hydrogen, and carbon dioxide (correct)
  • Nitrogen and oxygen

    • What is the purpose of the palladium pellets in anaerobic jars?

    <p>To catalyze the removal of oxygen</p> Signup and view all the answers

    Which of the following statements is true regarding anaerobic bags used in culture?

    <p>They contain an oxygen removal system.</p> Signup and view all the answers

    What is the primary identification test for anaerobes?

    <p>Gram-stain</p> Signup and view all the answers

    At what temperature should anaerobic incubation typically occur?

    <p>35 to 37°C</p> Signup and view all the answers

    What is the color indication of methylene blue in an anaerobic jar when it is oxidized?

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

    At what temperature does L. monocytogenes exhibit tumbling motility?

    <p>25°C</p> Signup and view all the answers

    What type of growth pattern does L. monocytogenes display on semisolid agar at 25°C?

    <p>Umbrella-like growth pattern</p> Signup and view all the answers

    Which test distinguishes L. monocytogenes from other Listeria species?

    <p>CAMP test</p> Signup and view all the answers

    Which organism is a major reservoir for Erysipelothrix rhusiopathiae?

    <p>Domestic swine</p> Signup and view all the answers

    What is the catalase reaction for Lactobacillus species?

    <p>Catalase negative</p> Signup and view all the answers

    Which of the following bacteria is linked to bacterial vaginosis?

    <p>Gardnerella vaginalis</p> Signup and view all the answers

    Which statement accurately describes the motility of Erysipelothrix rhusiopathiae?

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

    What type of hemolysis is exhibited by Arcanobacterium haemolyticum?

    <p>Beta hemolysis</p> Signup and view all the answers

    What organism causes Lyme disease?

    <p>Borrelia burgdorferi</p> Signup and view all the answers

    Which stage of Lyme disease is characterized by flu-like symptoms and a target lesion?

    <p>Stage 1</p> Signup and view all the answers

    What is a common laboratory test for relapsing fever caused by Borrelia recurrentis?

    <p>Giemsa or Wright’s stain</p> Signup and view all the answers

    What is the main reason antigenic variation is significant in the context of relapsing fever?

    <p>It allows the bacterium to evade the immune system.</p> Signup and view all the answers

    Which type of tick is primarily responsible for the transmission of Lyme disease?

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

    Which laboratory method is considered labor-intensive for culturing Borrelia?

    <p>Kelly medium</p> Signup and view all the answers

    What are the complications associated with late-stage Lyme disease?

    <p>Chronic arthritis and neurologic defects</p> Signup and view all the answers

    Why is serology less practical in diagnosing relapsing fever?

    <p>Antigenic variation of the bacteria</p> Signup and view all the answers

    What is the minimum volume of sputum required for collection?

    <p>5-10 ml</p> Signup and view all the answers

    Which method is performed if sputum cannot be produced?

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

    How does the MB/BacT system detect CO2 generation?

    <p>Through a colorimetric sensor</p> Signup and view all the answers

    What type of hemolysis is typically observed with C. diphtheriae on Sheep Blood Agar (SBA)?

    <p>Small zone of beta hemolysis</p> Signup and view all the answers

    Which of the following tests is included in the biochemical testing for identifying AFB?

    <p>Catalase test</p> Signup and view all the answers

    Which medium enhances the development of metachromatic granules for C. diphtheriae?

    <p>Loeffler medium</p> Signup and view all the answers

    What characterizes slow growers in the context of AFB identification?

    <p>Visible colonies within 14 days</p> Signup and view all the answers

    What additional preparation is needed for photoreactivity tests?

    <p>Wrapping the tube in aluminum foil</p> Signup and view all the answers

    What characteristic does Tinsdale agar exhibit when C. diphtheriae is cultured on it?

    <p>Brown or black colonies</p> Signup and view all the answers

    What is the purpose of the Elek test?

    <p>To detect toxin production</p> Signup and view all the answers

    Which enzyme do most mycobacteria possess regarding the niacin test?

    <p>Niacin ribonucleotide synthase</p> Signup and view all the answers

    What indicates a positive result in the heat-stable catalase test?

    <p>Formation of bubbles</p> Signup and view all the answers

    Which Corynebacterium species is most commonly associated with infections related to catheters or prosthetic devices?

    <p>C. jeikeium</p> Signup and view all the answers

    What is the fatality rate of Listeriosis in neonates?

    <p>50%</p> Signup and view all the answers

    What type of specimen is typically used to culture Listeria monocytogenes?

    <p>CSF, blood, amniotic fluid</p> Signup and view all the answers

    Which of the following characteristics differentiates Listeria monocytogenes from Streptococci?

    <p>Catalase positive</p> Signup and view all the answers

    What indicates a positive result for nitrate reduction in mycobacteria?

    <p>Red color after adding reagents</p> Signup and view all the answers

    Which of the following is true regarding tellurite reduction?

    <p>Black color indicates positive reduction.</p> Signup and view all the answers

    What is the result of Tween 80 hydrolysis when it is positive?

    <p>The media turns red.</p> Signup and view all the answers

    Which organism is most commonly associated with the Mycobacterium tuberculosis complex?

    <p>Mycobacterium tuberculosis</p> Signup and view all the answers

    What is the primary route of transmission for Mycobacterium tuberculosis?

    <p>Airborne respiratory droplets</p> Signup and view all the answers

    Which component inhibits the growth of the Mycobacterium tuberculosis complex?

    <p>P-nitroacetylamino-β-hydroxypropiophenone</p> Signup and view all the answers

    Which of the following is not a member of the Mycobacterium tuberculosis complex?

    <p>Mycobacterium kansasii</p> Signup and view all the answers

    At which temperature is the mycobacterial growth NOT typically assessed?

    <p>50°C</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 (strict) anaerobes: require anaerobic conditions for growth; oxygen is toxic and kills them.
    • Aerotolerant anaerobes: can grow in an atmosphere with oxygen, but grow best in anaerobic environments.
    • Facultative anaerobes: do not require oxygen but will use it if available.

    Oxygen Reduction

    • Superoxide anion and hydrogen peroxide are toxic to anaerobic bacteria.
    • Strict aerobes and facultative anaerobes have superoxide dismutase and/or catalase enzymes to detoxify these molecules.

    Oxidation-Reduction (Redox) Potential

    • Anaerobic bacteria thrive in low redox potential environments.
    • Normal human tissue and aerobic culture media have high redox potentials.
    • Reducing agents (thioglycollate, cysteine, dithiothreitol) are added to anaerobic media to maintain the low redox environment.

    Normal Habitat

    • Anaerobic bacteria are found in soil, water, and animals.
    • They can also be found in the human body's natural flora.
    • Organisms in the human natural flora use up oxygen in protected areas and reduce the redox potential to eliminate harmful oxygen molecules.

    Diseases

    • Exogenous infections: these occur when outside organisms, spores, or toxins enter the body through ingestion or trauma
      • Examples include tetanus and botulism.
    • Endogenous infections: these occur when the body's natural flora near mucosal surfaces cause infections in the body.
      • Examples include bacteremia, abscesses, gas gangrene, and pneumonia.

    Clues to Anaerobic Diseases

    • Trauma (deep or puncture wounds)
    • Animal or human bite
    • Foul-smelling, gaseous discharge
    • Necrotic tissue, vascular stasis
    • Contains sulfur granules (actinomycosis)
    • Black or fluorescent red color
    • Previous therapy with aminoglycosides (aminoglycosides are ineffective against anaerobes)
    • Failure to grow organism seen on a gram stain

    Specimen Collection

    • Appropriate specimens: aspirate with a needle and syringe

      • Blood, CSF, bone marrow, cavity fluid
      • Urine when collected as a suprapubic aspirate
      • Abscesses, ulcers, and draining wounds
      • Biopsy material

    • Inappropriate specimens: site containing resident flora (oral, GI, GU)

      • Superficial skin sites
      • Voided or catheterized urine
      • Expectorated sputum, throat, or nasopharyngeal swabs, and bronchial washings
      • Vaginal, cervical, and urethral swabs
      • Stool or rectal swabs

    Transport and Processing

    • Inoculate media and place immediately in anaerobic environment.
    • Limit exposure to room air.
    • Do not allow to dry out.
    • Do not refrigerate.
    • Minimize time at room temperature.
    • PRAS media: prereduced, anaerobically sterilized transport medium
      • Agar transport medium (modified Cary-Blair or Amies)
      • Rezasurin (oxygen indicator)
      • Reducing substances (to protect against oxygen, and inhibit NF)
      • A sterile container.

    Culture

    • Specimens for anaerobic culture should be cultured aerobically on BAP, CHOC, MAC and on anaerobic media to correlate aerotolerance.
    • Anaerobes require Vitamin K, hemin, and yeast extract.

    Nonselective Media

    • CDC or Anaerobic Blood Agar Plates (anaBAP)
      • Support the growth of obligate and facultative anaerobes.
    • Anaerobic Broth, Thioglycollate or chopped (cooked) meat
      • Support all obligate and facultative anaerobes
      • Anaerobes grow toward bottom (facultative grow throughout).

    Selective Media

    • Phenylethyl alcohol blood agar (anaPEA): contains phenylethyl alcohol; supports GN and GP obligate anaerobes, supports GP facultative anaerobic, inhibits enteric GNR
    • Colistin nalidixic acid blood agar (anaCNA): supports GN and GP obligate anaerobes, supports GP facultative anaerobic, inhibits enteric GNR
    • Bacteroides Bile-Esculin (BBE) agar: selective (gentamicin and bile), differential (esculin hydrolysis), supports growth of bile-tolerant organisms like B. fragilis.
    • Brucella blood agar: supports facultative and obligate anaerobes, best for GN
    • Kanamycin-vancomycin laked blood (KVLB) agar: selects for Bacteroides and Prevotella; kanamycin inhibits most facultative GNR; vancomycin inhibits most GP; laked blood encourages Prevotella to produce brown-black pigments
    • Cycloserine-cefoxitin-fructose agar (CCFA): selective and differential for Clostridium difficile; cycloserine and cefoxitin are antibiotics; fructose is a source of CHO; neutral red is the pH indicator (red in acid, yellow in alkaline); C. difficile metabolize proteins, making the medium alkaline (yellow).

    Anaerobic Incubation

    • 35 to 37°C for at least 48 hours.
    • Nitrogen gas (80–90%) removes oxygen.
    • Hydrogen gas (5–10%) removes oxygen.
    • Carbon dioxide (5–10%). Some anaerobes are capnophilic (require CO2).

    Anaerobic Systems

    • Anaerobic jars
    • Anaerobic bags
    • Anaerobic chambers

    Anaerobic Jars - (Gas Pak Jars)

    • Jar technique
    • Classic principle of anaerobic culture
    • Catalyst - palladium pellets
    • Envelope generates H2 and CO2 when water is added.
    • Sealed, incubated at 35°C.
    • Methylene blue or resazurin indicator is blue when oxidized, white when reduced (anaerobic).

    Anaerobic Bags

    • Commercially available
    • Hold 1-3 plates
    • Contains oxygen removal system
    • Indicator
    • Gas-impermeable plastic bag

    Anaerobic Chamber

    • An optimal anaerobic incubation system
    • Provides an O2-free environment for inoculation, incubation, and examination of media.
    • Contains a palladium catalyst, desiccant, indicator, and anaerobic gas mixture.

    Culture Examination

    • Examine samples in the chamber at any time during the 48-hour incubation period.
    • Keep sealed bags & jars sealed for 48 hours.
    • Minimize any time samples are out of anaerobic atmosphere.

    Anaerobic Identification Tests

    • Presumptive ID
      • Gram stain is the key identification test.
      • Use colony morphology.
      • Rapid tests

    Anaerobes Gram Stain RXN

    • Tree diagram showing results of anaerobic bacteria by gram stain, and grouping of gram positive rods, gram positive cocci and gram negative rods.

    Rapid Identification Tests

    • Catalase test
    • Motility test
    • Urease test
    • Indole
    • Esculin hydrolysis
    • Aerotolerance
    • Fluorescence
    • Disk tests
    • Lecithinase
    • Naglar test
    • Lipase
    • Reverse CAMP

    Aerotolerance

    • Determines if isolate is a strict anaerobe or a facultative anaerobe
    • Incubate the isolate in both aerobic and anaerobic environments; anaBAP anaerobically and CHOC aerobically in CO2.
    • Examine plates at 48 hours.

    Fluoresence

    • Fluorescent under ultraviolet light (366 nm).
    • Red, orange, pink, chartreuse

    Antibiotic Disk Test

    • Confirm gram stain
    • Special potency disks - Kanamycin, Vancomycin, colistin
    • Most GN are resistant to vancomycin
    • Most GP are sensitive to vancomycin, and resistant to colistin
    • Van S, Col R = Clostridia, Porphyromonas (Kan R)
    • Van R = GN or not Porphyromonas

    Disk Tests

    • Sodium polyanethol sulfonate (SPS) disk
      • Identify ana GPC, Peptostreptococcus are sensitive
    • Nitrate disk: Reduction of nitrate
    • Bile disk: Ability to grow in 20% bile
      • Bile-tolerant anaGNR = Bacteroides fragilis group

    Lecithinase

    • Detects lecithinase and lipase activity
    • Clostridium spp. ID
    • Cleaves lecithin in EYA
    • Releases insoluble fat, making an opaque zone
    • Positive reaction: white opaque zone in agar surrounding growth
    • Negative reaction: no change in agar surrounding growth

    Nagler Test

    • Detects alpha toxin of C. perfringens on EYA.
    • Alpha toxin is a specific form of lecithinase produced by C. perfringens
    • Antiserum to alpha toxin neutralizes the activity of the alpha toxin type of lecithinase by C. perfringens (but not that produced by other clostridia).

    Lipase

    • Hydrolyzes triglycerides in EYA producing glycerol + free fatty acids
    • Positive reaction: oil-on-water surface or multicolored sheen in colony
    • Negative reaction: no sheen observed, normal colony morphology

    Reverse CAMP

    • Alpha toxin of C. perfringens works synergistically with beta hemolytic group of S. agalactiae.
    • Test organism on anaBAP
    • GBS perpendicular to test organism streak (close but not touching)
    • Arrowhead-shaped zone = positive.

    Definitive Identification Tests

    • Biochemical tests
    • Commercial systems
    • Gas-liquid chromatography
    • Cellular fatty acid analysis
    • 16S rRNA gene sequencing

    Biochemical and Commercial Systems

    • PRAS or non-PRAS biochemical test media
    • Commercial biochemical and preexisting bacterial enzymes minisystems.

    Gas Liquid Chromatography

    • Analysis of cellular fatty acids or metabolic end products (volatile acids)
    • Produce characteristic patterns

    Gene Sequencing

    • 16S rRNA gene sequencing
    • Ribosomal DNA extracted, amplified via PCR, sequenced

    Clostridium Species

    • Catalase negative
    • Motile (except C. perfringens)
    • Anaerobic GPR
    • Some gram variable or GN
    • Susceptible to vancomycin
    • Spore forming
      • Appear as unstained refractile structures in a gram stain
      • Oval to round, terminal to subterminal

    Clostridium Species

    • Some species are aerotolerant.
    • Clostridia cause exogenous infections
      • Gain access to body via ingestion and wounds.
      • Examples include tetanus, gas gangrene, botulism, and food poisoning.
    • Produce potent toxins.

    Genera Differences

    • Compares Clostridium, Bacillus, and Lactobacillus based on optimal growth conditions, sporulation conditions, and catalase reaction
    • Each bacteria genus has different characteristics

    Clostridium perfringens

    • Most common Clostridium spp.
    • Found in undercooked meat, soil, water.
    • GP boxcar-shaped rods.
    • Double zone of beta hemolysis on anaBAP
    • Positive reverse CAMP, lecithinase, Nagler
    • Spore seldom observed.

    C. perfringens Clinical Significance

    • Isolated from tissue infections and bacteremia.
    • Produce gas in infected tissue.
      • Myonecrosis (gas gangrene)
        • Deep penetrating wound or surgery
        • Diabetic foot ulcers
    • Food poisoning from meat or meat products (gravy)
      • Ingested spores germinate in intestines
      • Produce enterotoxin causing nausea, vomiting, diarrhea, and abdominal pain

    Clostridium difficile

    • May be NF of stool; antibiotic-associated diarrhea and pseudomembranous colitis.
    • Antibiotics upset intestinal ecosystem by killing indigenous NF.
    • Resistant C. difficile increase in number
    • All 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 GPR
    • Chartreuse fluorescence.
    • Must test for toxin production

    C. difficile Toxin Tests

    • Cytotoxin test (detects toxin B).
    • Kit testing: determines if toxin A, toxin B or glutamate dehydrogenase is present in stool sample.
    • Enzyme immunoassay
    • Molecular testing (under development)

    Clostridium botulinum

    • Botulism
      • Ingestion of botulinum toxin.
        • Home canned veggies, home cured meat
      • Infant botulism associated with honey.
    • Neuromuscular toxins cause paralysis or death.
      • Foodborne botulism (ingested)
      • Wound botulism (inoculated)
      • Intestinal infant botulism (infants lacking NF)

    C. botulinum Diagnosis

    • Diagnosed clinically (patient history and symptoms).
    • Isolate organism or detect toxin (reference lab).

    Clostridium tetani

    • Soil and intestinal tract of animals
    • Enters body through wound site.
    • Produces potent neurotoxin (tetanospasmin).
    • Severe muscle spasm in unimmunized individuals (lockjaw).

    C. tetani Diagnosis

    • Diagnosed clinically. (Clinical symptoms)

    Terminal Spores (Tennis Racquet Shaped)

    • Appearance under a microscope

    C. septicum

    • Aerotolerant.
    • Swarming colonies.
    • Sub-terminal spores
    • Myonecrosis and bacteremia
    • Associated with leukemia, lymphoma, large bowel carcinoma.

    Other Species

    • Cause bacteremia, intra-abdominal infections, wound infections, myonecrosis

    Non-Spore-Forming Anaerobic GPR

    • Actinomyces spp.
      • Causes actinomycosis
    • Bifidobacterium spp.
    • Propionibacterium spp.
    • Eggerthella and Eubacterium spp. (All can cause actinomycosis)

    Actinomyces

    • Actinomyces spp. include aerobic and anaerobic bacteria.
    • Nocardia is an aerobic Actinomyces.
    • Causes mycetomas and nocardiosis.
      • A. israelii is the most common anaerobe that causes actinomycosis.
    • Inhabit human and animal mucosal surfaces.
    • Anaerobic actinomyces is not weakly acid fast like Nocardia spp.

    Actinomyces Gram Stain

    • Aerobic and anaerobic Actinomyces spp.
    • G.P.R. irregularly stained (beaded appearance), coccoid to filamentous, filaments branch.

    A. israelii Actinomycosis

    • Actinomycosis
    • Primarily A. israelii.
    • Usually in the jaw
    • Chronic granulomatous infection
    • Develop fistulae that drain pus with sulfur granules (colonies of bacteria).

    A. israelii Cultures

    • Associated with "lumpy jaw".
    • Pus and sulfur granules grow on anaBAP.
    • Grow slowly (7–9 days).
    • Molar-tooth colonies
    • Anaerobic branching GPR

    Bifidobacterium

    • Actinomyces-like morphology.
    • Gram stain branched or bifurcated GPR.
    • NF intestines and oral cavity.
    • Rarely cause diseases.
    • Can be a cause of actinomycosis.

    Propionibacterium

    • Anaerobic diphtheroid-like GPR, resembles corynebacteria.
    • NF of skin.
    • Most common anaerobe isolated in the lab as a contaminant of blood cultures.
    • P. acnes is linked to acne.
    • Catalase and indole positive.
    • May cause endocarditis and bacteremia.

    Eggerthella and Eubacterium spp.

    • Previously called Eubacterium.
    • Anaerobic diphtheroid-like GPR.
    • No branching

    Anaerobic GPC

    • Peptostreptococcus: GPC in chains.
      • P. anaerobius: susceptible to sodium polyanethol sulfonate (SPS) disk. All other anaerobic cocci are resistant.
    • Peptococcus: GPC in clusters.

    Anaerobic GNR

    • NF mucous membrane (includes species below)
      • Bacteroides
      • Prevotella
      • Porphyromonas
      • Fusobacterium
      • Veillonella

    Bacteroides fragilis Group

    • Anaerobic GNR
    • NF of GI tract.
      • B. fragilis: most common.
    • Intra-abdominal infections, bacteremia, soft tissue infections.
    • B. thetaiotaomicron: second most common.

    Bacteroides fragilis Group

    • Bile resistant.
    • Resistant to kanamycin, vancomycin, and colistin disks.
    • Growth on KVLB agar.
    • Growth on Bacteroides Bile-Esculin (BBE) agar (growth stimulated by bile).
    • Growth=bile tolerance
    • Brown to black colonies = esculin hydrolysis

    Bacteroides ureolyticus Group

    • Anaerobic GNR.
    • Bile sensitive and bile tolerant nonpigmented organisms.
    • Some organisms pit the agar.
    • Growth in formate and fumarate.
    • Resistant to vancomycin.
    • Sensitive to kanamycin and colistin.

    Prevotella

    • Anaerobic GNR.
    • Bile susceptible.
    • Resistant to kanamycin and vancomycin.
    • Growth on KVLB but not BBE
    • Some produce protoporphyrin.
    • Dark pigmented colonies.
    • Colonies fluoresce a "brick red" under UV light.

    Fusobacterium

    • Susceptible to kanamycin and colistin.
    • Vancomycin resistant
    • Fluoresce chartreuse
      • F. nucleatum: thin fusiform rods, bread-crumb, speckled colonies
      • F. necrophorum: lipase positive

    Mobiluncus

    • Associated with BV, PID, abdominal infections.
    • Curved bacilli.
    • Gram variable.
    • Motile, catalase, and indole negative.
    • Inhibited by vancomycin.

    Selective Anaerobic GNR ID

    • Tree diagram of identification of anaerobic GNR based on characteristics

    Veillonella

    • Only commonly encountered anaerobic GNC.
    • Sensitive to kanamycin and colistin.
    • Resistant to vancomycin.
    • Smallest gram-negative cocci

    Gram Positive Rods

    • Non-Spore-Forming GPR
    • Non-Spore-Forming Branching GPR
    • Spore-Forming Non-Branching GPR

    Non-Spore-Forming GPR

    • Corynebacterium and Coryneforms, Listeria, Erysipelothrix, Acrranobacterium, Lactobacillus, Gardnerella

    Corynebacterium

    • C. diphtheriae,C. jeikeium, C. urealyticum, C. pseudodiphtheriticum, C. striatum, C. xerosis.
    • Normal skin and mucous membrane flora.
    • Called diphtheroids or coryneforms, "club shaped"
    • Gram stain looks like "Chinese letters" (V, L, Y formation) or palisades (line up side by side).
    • Facultative anaerobes.
    • Small gamma colonies on SBA.
    • Catalase positive.
    • Nonmotile.
    • Frequent contaminants.

    C. diphtheriae

    • Significant pathogen.
    • Virulence factor is diphtheria toxin.
      • Tox gene in strains infected with bacteriophage carrying the tox gene.
      • Blocks protein synthesis and destroys host cells.
      • Can absorb in heart, nervous system damage.
      • Lethal at 130 ng/kg of body weight
    • Two forms
      • Cutaneous: non-healing ulcer
      • Respiratory: diphtheria
    • URT infection (tonsils, pharynx); tissue necrosis.
    • Exudate → pseudomembrane (necrosis of epithelial cells, WBCs, fibrin, bacteria).
    • Suffocation
    • Treated with antitoxin.
    • Prevented by immunization (DIP/TET).

    C. diphtheriae Lab Diagnosis

    • Pleomorphic GPR
    • Darker staining area of cell (metachromatic granules)
    • Methylene blue stain.
    • Pockets of inorganic phosphates or nutrient reserves

    C. diphtheriae Media

    • SBA – small zone of beta hemolysis.

    • Serum or blood containing media (enhances development of metachromatic granules)

    • Pai agar (egg-based agar)

    • Tinsdale agar (cystine-tellurite blood agar)

      • Selective (potassium tellurite inhibits non-coryneform bacteria)
      • Differential (tellurite reduction produces brown or black colonies; cystinase activity = halo around colonies).

    • WARNING: Staphylococcus can also produce brown colonies.

    Elek Test

    • Immunodiffusion test for toxin production
    • Strip of filter paper saturated with antitoxin embedded in agar medium.
    • Isolate, positive, and negative control are streaked on agar parallel to each other and perpendicular to filter paper.
    • Incubate (1–2 days).
    • Line of precipitate forms in agar when toxin interacts with antitoxin (arc of identity).

    Corynebacterium spp.

    • Normal skin flora, most isolates are considered contaminants.
    • C. jeikeium: catheters or prosthetic devices, diphtheroid prosthetic valve endocarditis
    • C. urealyticum: Urinary pathogen

    Other Non-spore-forming GPR

    • Rothia dentocariosa: oral NF; endocarditis and wound infections
      • Filamentous or coryneform.
    • Undesignated CDC Coryneform Groups: Opportunistic or nosocomial infections

    Listeria monocytogenes

    • Vaginal and intestinal NF in humans
    • Clinical infections due to ingestion of contaminated food.
    • Virulence factors:
      • Listerolysin O (hemolysis)
      • Others

    Listeria monocytogenes

    • Listeriosis (sepsis)
    • Neonates: fatality rate ~50%
    • Pregnant women
    • Can cause spontaneous abortion and stillbirth
    • Immunocompromised
    • Predilection for CNS

    L. monocytogenes Cultures

    • Specimens (CSF, blood, amniotic fluid)
    • Facultative anaerobe.
    • Grow on routine media (BAP, CHOC, prefers increased CO2;
    • Colonies resemble S. agalactiae.
    • Cold enrichment (growth at 4°C)

    L. monocytogenes ID

    • GPR, non-spore forming
    • Small beta colonies on BAP; sometimes hemolysis is hidden under colonies.
    • Catalase positive (differentiates it from streptococci)
    • Bile-esculin positive
    • Motile at 25°C.

    L. monocytogenes Motility

    • Tumbling motility at 25°C, not at 35°C
    • One tube incubated at 35°C, other at RT.
    • Tumbling motility on broth wet mount in broth incubated at 25°C.
    • Umbrella pattern at 25°C, not at 35°C
    • Semisolid Agar Motility: Umbrella-like growth pattern
    • Positive CAMP test distinguishes L. monocytogenes from other Listeria spp.
    • Block vs. arrowhead

    L. monocytogenes CAMP

    • Image of a positive test.

    Differentiation of Listeria

    • Table comparing L. monocytogenes, Corynebacterium spp., S. agalactiae, and Enterococcus spp. based on catalase reaction, esculin hydrolysis, motility, β-hemolysis, and 6.5% NaCl tolerance.

    Erysipelothrix rhusiopathiae

    • Domestic swine major reservoir.
    • Occupational hazard to butchers, vets, and fishermen.
    • Causes erysiploid (red skin lesion) in animals and humans.
    • Can disseminate into bacteremia and endocarditis.

    Erysipelothrix rhusiopathiae

    • GPR
    • Catalase-negative
    • Alpha or gamma hemolysis
    • Nonmotile
    • H2S positive in TSI

    Cultures

    • Specimens (skin biopsies, blood)
    • Grow on routine media (BAP, CHOC, CNA, PEA)
    • Very small colonies (48 hours).

    Arcanobacterium haemolyticum

    • Formerly Corynebacterium.
    • Significant: A. haemolyticum, A. pyogenes, and A. bernardiae
    • Causes pharyngitis.
    • Facultative anaerobe
    • Beta hemolysis
    • Catalase negative.
    • Reverse CAMP positive; inhibits S. aureus hemolysis

    Reverse CAMP

    • Image of a positive test.

    Lactobacillus

    • Normal vaginal flora.
    • Produce lactic acid.
      • Reduces vaginal pH, inhibits the growth of other organisms.
    • Rarely cause disease.
    • L. acidophilus: probiotic.
      • Tiny alpha colonies on BAP.

    Lactobacillus

    • Non-spore-forming GPR
    • Medium to long rods
    • Aerotolerant anaerobes
    • Catalase negative
    • Nonmotile

    Gardnerella vaginalis

    • Pleomorphic coccobacilli.
    • Cell wall with GP characteristics, stains gram variable.
    • Nonmotile.
    • Facultative anaerobe.
    • NF in 50–70% of women.
    • Associated with bacterial vaginosis

    Bacterial Vaginosis (BV)

    • Change in NF (lactobacillus), increase in pH allows growth of G. vaginalis.
    • Foul-smelling discharge – amine odor.
    • Untreated can lead to PID, UTIs.
    • Culture not recommended.
    • Perform wet prep or gram stain - "clue cells".

    Clue Cells

    • Image of clue cells.

    Cultures

    • Appropriate for extravaginal sites.
    • Grow on CHOC, BAP, not MAC.
    • Small pleomorphic gram variable coccobacilli.
    • Catalase and oxidase negative.
    • Hippurate hydrolysis positive (46).

    Non-Spore-Forming Branching GPR (Aerobic Actinomycetes)

    • Nocardia spp.
    • Tropheryma whipplei
    • Streptomyces
    • Actinomadura
    • Gordonia
    • Tsukamurella
    • Rhodococcus equi

    Nocardia spp.

    • Found in soil
    • Usually infect immunocompromised individuals.
    • Pulmonary: example N. asteroides complex; pneumonia → abscess → may disseminate → necrosis
    • Cutaneous: (mycetomas), example N. brasiliensis; abscess → invasive, draining pus with sulfur granules.

    Nocardia spp.

    • Aerobic GPR morphologically similar to fungus.
    • Form beaded, branching filaments on gram stain.
    • Weakly acid fast. (Microscopic image of Nocardia spp.)

    Nocardia spp.

    • Grow slowly (1 week or more) on nonselective media.
    • Waxy, chalky, crumbly colonies. (Colony morphology image of Nocardia spp.)

    Nocardia ID

    • Filamentous, branching organism.
    • Weakly acid fast.
    • Acid-fast stains (Ziehl Neelsen or Kinyoun) using heat or detergent respectively; primary stain with carbolfuchsin (minutes); rinse and decolorize with acid; rinse and counterstain with methylene blue; reddish purple filaments (acid fast) are positive, blue is negative. Nocardia is positive with Modified acid fast stain

    Other Actinomycetes

    • Tropheryma whipplei: Whipple Disease. Intracellular pathogen, diarrhea, weight loss, malabsorption. Identify via PCR or 16S rRNA.
    • Streptomyces, Actinomadura, Gordonia, Tsukamurella, Rhodococcus equi

    Spore-Forming Non-Branching GPR

    • Bacillus: spore-forming, non-branching bacilli; spores protective, metabolically inactive; increase survival in nature; resist biocides; thick outer wall.

    Bacillus

    • Found in nature
    • Metabolically diverse
    • B. anthracis and B. cereus: lab contaminants, important pathogens.

    Bacillus

    • Large aerobic GPR, spore-formers.
      • Spores not stained by gram stain. Appear as holes.
      • Beta hemolytic (except for B. anthracis).
      • Catalase positive.

    Bacillus

    • Catalase positive and aerobic spore formation distinguishes them from Clostridium .
    • Growth on SBA and PEA.
    • Does not grow on enteric agars, is GPR.

    B. anthracis

    • Causes anthrax (usually in herbivores)
    • Bioterrorism agent.
    • Virulence factors
      • Endospores.
      • Protective antigen
      • Edema factor
      • Lethal factor

    B. anthracis Clinical Significance

    • Three forms (cutaneous, pulmonary, or gastrointestinal).
    • Cutaneous anthrax: pimple, vesicles, erythematous ring, necrotic lesion (black eschar) (1–2 weeks to heal).
    • Inhalation anthrax: pulmonary, flu-like phase followed by respiratory distress, coma, death
    • Gl anthrax: ingestion, pain, nausea, vomiting. (Clinical significance images)

    B. anthracis Gram Stain

    • Very long chains, “bamboo shoots”
    • Square ended

    B. anthracis

    • Nonmotile and nonhemolytic.
    • Large, gray, flat colony with irregular edges
    • Medusa-head colonies

    B. anthracis Identification

    • Aerobic/anaerobic

    • Spore-forming GPR.

    • Nonhemolytic on SBA.

    • Nonmotile.

    • Catalase positive.

    • Biological safety hood needed; send to reference lab if not rule out immediately (no clinical signs).

    B. cereus

    • B. cereus is beta-hemolytic and motile; distinguishes it from B. anthracis; produces enterotoxin, causes food poisoning. (Colony morphology images)

    Identification of GPR

    • Tree diagram that classifies Gram-positive Rods based on spores, catalase, motility, and esculin hydrolysis reactions.

    Spirochetes

    • Borrelia, Leptospira, Treponema - long, slender, spiral-shaped microbes that are motile (cannot be seen in gram stain, use silver stains in wet preparations).

    Borrelia

    • Contains several species (e.g., Borrelia burgdorferi).
    • Transmitted by arthropods (e.g., ticks, lice).
    • 3-10 spirals/organism
    • Causes relapsing fever; B. burgdorferi causes Lyme disease. (Image of Borrelia under dark-field microscopy)

    Borrelia recurrentis

    • Causes relapsing fever—repeated febrile episodes of spirochetemia; fever, headache, muscle pain.

      • Cyclic relapses caused by antigenic variation; evades the immune system

      • Episodes end when the immune system responds to new antigen makeup

      • Epidemic relapsing fever (louse-borne)

    • Endemic relapsing fever (tick-borne) (Relapsing Fever Lab image)

    Relapsing Fever Lab Tests

    • Microscopic examination of blood during febrile periods via Giemsa or Wright's stain (preferred method)
    • Can be cultured in Kelly medium (rare)

    Borrelia burgdorferi

    • Lyme borreliosis.
    • Originally from northeast, spreading.
    • Transmitted by Ixodes ticks: deer or mouse ticks. (Image of Borrelia risk map)

    3 Stages of Lyme Disease

    • Stage 1: Erythema chronicum migrans (target lesion), bull's-eye lesion; lymphadenopathy and flu-like symptoms
    • Stage 2: Dissemination of fever, bone and joint pain, splenomegaly, and malaise.
    • Stage 3: Late or persistent infection, months to years later; chronic arthritis, neurologic defects, meningioencephalitis or cardiac problems. (Images of 3 stages of Lyme disease.)

    Lyme Disease Lab Tests

    • Serologic tests (most common and fastest; antibody detection tests with western blot confirmation)
    • Direct microscopic exam (skin or blood)
    • Cultures (labor intensive, Kelly medium) which are usually negative

    Leptospira

    • L. biflexa (nonpathogenic)
    • L. interrogans: animal pathogen; passed to humans via water contaminated with animal urine (e.g., an occupational hazard for vets, farmers, sewer workers).

    Leptospira interrogans

    • Spirals with hooked ends.
    • Tightly coiled

    (Scanning electron micrograph of Leptospira interrogans)

    Leptospirosis

    • Zoonotic infection (dogs, rats, cattle)
    • Spirochete that infects animal kidneys → urine.
    • Leptospires enter humans via breaks in skin or intact mucosa. (Diagram of mode of infection)

    Leptospirosis

    • Clinical manifestations: asymptomatic to severe
    • Incubation period: 3-30 days
    • Abrupt onset of nonspecific influenza-like symptoms
    • Severe systemic disease
    • Weil's disease: CSF positive.
    • Renal and hepatic failure
    • Icteric leptospirosis.
    • Intravascular disease

    Leptospirosis Lab Tests

    • Blood & CSF (first week), urine (after)
    • Direct examination (darkfield microscopy, DFA)
    • Cultures (Fletcher's semi-solid media, incubate in the dark at 30°C for 6 weeks).
    • Darkfield microscopy from media.
    • Serology (ELISA assay) (Image of Leptospira ELISA kit)

    Treponema

    • 4 to 14 spirals/organism
    • Four types of pathogenic organisms
      • Treponema pallidum subsp. pallidum
      • Treponema pallidum subspecies pertenue
      • Treponema pallidum subsp. endemicum
      • Treponema pallidum subsp. carateum

    Treponema pallidum subspecies pallidum

    • Venereal syphilis - Great imitator before serological tests
    • Variety of clinical presentations
    • Transmitted sexually or via non-genital lesions
    • Can cross the placenta (congenital syphilis)
    • Three stages: primary, secondary, and tertiary

    Primary Syphilis

    • Few days to months after organism acquired
    • Firm chancre forms at inoculation site
    • Lesion contains many spirochetes and is highly infectious.
    • Painless

    Secondary Syphilis

    • 2 to 12 weeks after primary lesion
    • Organisms disseminate throughout body; fever, lymphoadenopathy, headache
    • Secondary infectious lesions of skin and mucous membrane
    • Widespread unusual skin rashes (may include palms and soles).

    Tertiary or Late Syphilis

    • 1/3 of untreated individuals develop tertiary syphilis years after initial infection.
    • Not infectious.
    • Many body sites affected (gummas in skin, liver, bones).
    • CNS: neurosyphilis
      • Deafness, blindness, partial paralysis, shuffling gait, mental disturbances
    • Cardiovascular lesions: syphilitic aortitis

    Early Onset Congenital Syphilis

    • Mother has early syphilis; treponemes cross placenta.
    • Severe, infected fetus often dies.
    • Affects many body systems, including skin and mucous membrane lesions, anemia, hepatosplenomegaly, meningitis, bone lesions.

    Late Onset Congenital Syphilis

    • Mother has chronic, untreated syphilis
    • Symptoms present in individuals >2 years old
      • Blindness, deafness, mental retardation, bone/tooth deformities.
    • Prevented by screening pregnant women and necessary treatment.

    Serological Laboratory Diagnosis

    • Nontreponemal tests (screening)
      • VDRL (Venereal Disease Research Laboratory)
      • RPR (Rapid Plasma Reagin)
    • Treponemal tests (confirmation)
      • EIA (Enzyme Immunoassay)
      • TPPA (Treponema pallidum particle agglutination assay)
      • FTA-ABS (Fluorescent treponemal antibody absorption)

    Nontreponemal Tests

    • Detects antibodies formed against lipids as the result of infection with Treponema.
    • Antigen is cardiolipin-lecithin.
    • Sensitive but not specific; false positive can occur in Lyme disease, certain viral infections, autoimmune disease, or pregnancy
    • Screening, monitor therapy (titers), detect reinfection

    VDRL and RPR

    • Images of VDRL and RPR tests

    Treponemal Tests

    • Detects treponemal-specific antibodies
    • Confirmation
    • Remain positive after treatment.
    • Enzyme immunoassay
    • Treponema pallidum particle agglutination assay (TPPA) Previous FTA-ABS

    TPPA

    • Image of a TPPA test result

    Direct Microscopic Exam

    • Darkfield microscopy (requires patience and experience)
    • Fluid from lesions look for corkscrew motility
    • Oral lesions not appropriate; nonpathogenic treponemes may be present

    Other Pathogenic Treponemes

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