Podcast
Questions and Answers
Which organism is associated with producing an enterotoxin and causing food poisoning?
Which organism is associated with producing an enterotoxin and causing food poisoning?
Which microscopy technique is optimal for observing spirochetes?
Which microscopy technique is optimal for observing spirochetes?
What characteristic differentiates Borrelia from other spirochetes?
What characteristic differentiates Borrelia from other spirochetes?
In terms of motility, which genus is known for being non-motile?
In terms of motility, which genus is known for being non-motile?
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What is the typical morphology of Bacillus species when viewed under a microscope?
What is the typical morphology of Bacillus species when viewed under a microscope?
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What color indicates a positive result for the Tween 80 hydrolysis test?
What color indicates a positive result for the Tween 80 hydrolysis test?
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Which of the following organisms can reduce nitrate to either nitrite or nitrogen gas?
Which of the following organisms can reduce nitrate to either nitrite or nitrogen gas?
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What is the expected result when zinc is added to a nitrate broth that has no color change?
What is the expected result when zinc is added to a nitrate broth that has no color change?
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What is the result of tellurite reduction in mycobacterial testing?
What is the result of tellurite reduction in mycobacterial testing?
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Which mycobacterial species is inhibited by P-nitroacetylamino-β-hydroxypropiophenone (NAP)?
Which mycobacterial species is inhibited by P-nitroacetylamino-β-hydroxypropiophenone (NAP)?
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Which temperature is not typically used for isolating mycobacterial growth in culture media?
Which temperature is not typically used for isolating mycobacterial growth in culture media?
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How is the Mycobacterium tuberculosis complex primarily spread?
How is the Mycobacterium tuberculosis complex primarily spread?
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What color change is observed when nicotinic acid reacts with cyanogen bromide in testing?
What color change is observed when nicotinic acid reacts with cyanogen bromide in testing?
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What is the minimum volume of sputum required for collection to assist in diagnosing Hansen's disease?
What is the minimum volume of sputum required for collection to assist in diagnosing Hansen's disease?
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Which method is employed to collect bronchial washings if sputum cannot be produced?
Which method is employed to collect bronchial washings if sputum cannot be produced?
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Which species is primarily associated with actinomycosis?
Which species is primarily associated with actinomycosis?
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In the MB/BacT system, what indicates the generation of CO2?
In the MB/BacT system, what indicates the generation of CO2?
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What is the first test performed to confirm the presence of mycobacteria in AFB identification?
What is the first test performed to confirm the presence of mycobacteria in AFB identification?
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What characteristic is associated with the Gram stain of Actinomyces species?
What characteristic is associated with the Gram stain of Actinomyces species?
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Which of the following is a notable feature of A. israelii cultures?
Which of the following is a notable feature of A. israelii cultures?
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What type of growth parameters would classify an organism that forms visible colonies within 7 days?
What type of growth parameters would classify an organism that forms visible colonies within 7 days?
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Which of the following species is characterized as being a common isolate in anaerobic cultures?
Which of the following species is characterized as being a common isolate in anaerobic cultures?
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Which of the following tests would NOT be considered a biochemical test for identifying mycobacteria?
Which of the following tests would NOT be considered a biochemical test for identifying mycobacteria?
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In which situation is aluminum foil wrap used during the incubation of isolates?
In which situation is aluminum foil wrap used during the incubation of isolates?
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What kind of morphology is noted in Bifidobacterium species?
What kind of morphology is noted in Bifidobacterium species?
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What indicates a positive result in the heat-stable catalase test?
What indicates a positive result in the heat-stable catalase test?
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What is a distinguishing characteristic of Bacteroides fragilis group?
What is a distinguishing characteristic of Bacteroides fragilis group?
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Which of the following describes Propionibacterium acnes?
Which of the following describes Propionibacterium acnes?
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Which anaerobic microorganism is known for producing brown to black colonies on Bacteroides Bile-Esculin agar?
Which anaerobic microorganism is known for producing brown to black colonies on Bacteroides Bile-Esculin agar?
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Which of the following best describes how Mycobacterium bovis is typically transmitted?
Which of the following best describes how Mycobacterium bovis is typically transmitted?
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What does the presence of necrotic centers with a soft, cheesy appearance in lung granulomas indicate?
What does the presence of necrotic centers with a soft, cheesy appearance in lung granulomas indicate?
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What percentage of individuals infected with MTB are at risk for future reactivation of tuberculosis?
What percentage of individuals infected with MTB are at risk for future reactivation of tuberculosis?
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In which body sites can extrapulmonary TB potentially cause disease?
In which body sites can extrapulmonary TB potentially cause disease?
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What does a positive result on the PPD tuberculin skin test indicate?
What does a positive result on the PPD tuberculin skin test indicate?
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What type of tuberculosis is characterized by small tubercles scattered throughout the body?
What type of tuberculosis is characterized by small tubercles scattered throughout the body?
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Which characteristic is NOT associated with Mycobacterium tuberculosis in lab identification?
Which characteristic is NOT associated with Mycobacterium tuberculosis in lab identification?
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Which of the following symptoms is commonly associated with MTB infection?
Which of the following symptoms is commonly associated with MTB infection?
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What is the virulence factor of C. diphtheriae that contributes to its pathogenicity?
What is the virulence factor of C. diphtheriae that contributes to its pathogenicity?
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Which of the following describes the appearance of Corynebacterium under a Gram stain?
Which of the following describes the appearance of Corynebacterium under a Gram stain?
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How is a C. diphtheriae infection primarily treated?
How is a C. diphtheriae infection primarily treated?
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What is the primary characteristic that differentiates Veillonella from other anaerobic gram-negative cocci (GNC)?
What is the primary characteristic that differentiates Veillonella from other anaerobic gram-negative cocci (GNC)?
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What role does the Diphtheria toxin play in the respiratory disease caused by C. diphtheriae?
What role does the Diphtheria toxin play in the respiratory disease caused by C. diphtheriae?
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Which of the following is NOT a characteristic of non-spore-forming Gram-positive rods?
Which of the following is NOT a characteristic of non-spore-forming Gram-positive rods?
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What test would likely show darker staining areas within C. diphtheriae cells?
What test would likely show darker staining areas within C. diphtheriae cells?
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C. diphtheriae can cause which of the following diseases?
C. diphtheriae can cause which of the following diseases?
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Study Notes
Anaerobic Bacteria
- Anaerobic bacteria do not require oxygen to grow
- Different types have varying tolerances to oxygen
- Obligate anaerobes require anaerobic conditions to grow, oxygen is toxic and kills them
Aerotolerant Anaerobes
- Can grow in atmospheres with oxygen, but grow best without oxygen
- These bacteria are not harmed by oxygen
Facultative Anaerobes
- Do not require oxygen, but use it if available
- They can survive with or without oxygen
Oxygen Reduction
-
Superoxide anion and hydrogen peroxide are toxic to obligate anaerobes
-
Strict aerobes and facultative anaerobes possess enzymes: superoxide dismutase/catalase to neutralize these toxic byproducts
Oxidation-Reduction (Redox) Potential
- Anaerobic bacteria need a low redox potential
- In high redox environments, oxygen is present, so these bacteria have a hard time surviving
- Normal human tissue and aerobic media have a high redox potential
Normal Habitat
- Soil, water, and animals
- Human Normal Flora (NF) in oral cavity, upper respiratory tract (URT), intestinal tract, genitourinary tract, and skin
- Facultative organisms use up oxygen in protected areas
- They reduce redox potential and inactivate harmful oxygen molecules
Diseases
- Exogenous Infections: Organisms, spores, or toxins enter the body through ingestion or trauma (e.g., tetanus, botulism)
- Endogenous Infections: Infections involving host's NF near mucosal surfaces (e.g., bacteremia, abscesses, gas gangrene, pneumonia)
Clues to Anaerobic Diseases
- Trauma (deep or puncture wounds)
- Animal or human bites
- Foul-smelling, gaseous discharge
- Necrotic tissue/vascular stasis
- Presence of sulfur granules (actinomycosis)
- Black or fluorescent red color
- Previous therapy with ineffective aminoglycosides
- Failure to grow the organism when stained with a gram stain
Specimen Collection
- Appropriate Specimens: Aspiration with a needle and syringe (e.g., blood, sterile body fluids, CSF, bone marrow, cavity fluid, urine, abscesses, ulcers, draining wounds, biopsy material) -Inappropriate Specimens: Sites containing resident flora (oral, GI, GU) e.g., superficial skin sites, voided or catheterized urine, expectorated sputum, throat, nasopharyngeal swabs, bronchial washings, vaginal, cervical, or urethral swabs, stool, or rectal swabs
Transport and Processing
- Inoculate media and immediately place in an anaerobic environment
- Limit exposure to room air
- Do not allow to dry out
- Do not refrigerate
- Minimize time at room temperature
- Use an anaerobic transport system (PRAS media): prereduced, anaerobically sterilized transport medium with: Agar transport medium (modified Cary-Blair or Amies) & Rezasurin (oxygen indicator) & Reducing substances
- An anaerobic chamber, or anaerobic bags can be used
Culture
- Specimens are cultured aerobically on BAP, CHOC and MAC plates first to determine if this organism is an aerotolerant anaerobe.
- Anaerobes require Vitamin K, hemin, and yeast extract to grow.
Nonselective Media
- CDC or Anaerobic Blood Agar Plates (anaBAP) support obligate and facultative anaerobes
- Anaerobic Broth, Thioglycollate, cut (cooked) meat support obligate and facultative anaerobes; anaerobes grow toward bottom (facultative grow throughout)
Anaerobic-BAP / Thioglycollate Broth
- Visual aids describing the growth pattern of different types of anaerobic organisms in respective media
Selective Media
- Phenylethyl alcohol blood agar (anaPEA) supports GN and GP obligate anaerobes; inhibits enteric GNRs
- Colistin nalidixic acid blood agar (anaCNA) supports GN and GP obligate anaerobes; inhibits enteric GNRs
- Bacteroides Bile-Esculin (BBE) agar: Selective / differential for esculin hydrolysis/ bile tolerant organisms including B. fragilis
- Kanamycin-vancomycin laked blood (KVLB) agar: selects for Bacteroides and Prevotella; kanamycin inhibits most facultative GNRs; vancomycin inhibits most GPRs; laked blood encourages Prevotella to produce brown-black pigments
- Cycloserine-cefoxitin-fructose agar (CCFA): selective/differential for Clostridium difficile; cycloserine & cefoxitin are antibiotics; fructose is a CHO source; neutral red (red in acid; yellow in alkaline); C. difficile causes alkaline (yellow) reactions when metabolizing proteins
Other Selective Media
- List of additional selective media for various anaerobic and facultative organisms. The different medias are tailored to different requirements concerning what compounds should inhibit or promote growth.
Anaerobic Incubation
- Maintaining a controlled anaerobic environment is critical for growing anaerobic organisms
- Usual incubation conditions include: temperatures of 35 to 37°C for 48 hours; 80-90% nitrogen gas; 5-10% hydrogen gas; and 5-10% carbon dioxide
Anaerobic Systems
- These systems include: jars, bags, and chambers to create and maintain anaerobic conditions
Anaerobic Jars
- Jar technique, or gas pak jar: An anaerobic jar utilizes a catalyst (palladium pellets) generating H₂ and CO₂ when water is added. To remove oxygen, a sealed chamber is incubated at 35°C. Indicators (Methylene blue or resazurin) change to white when reduced(anaerobic).
Anaerobic Bags
- Commercially available systems holding 1-3 plates, that contain an oxygen removal system and indicator. The gas-impermeable plastic bag is the primary component of the system.
Anaerobic Chamber
- An optimized anaerobic incubation system that provides an oxygen-free environment for inoculation, incubation, and inspection of media for growth. The chamber contains a palladium catalyst, desiccant, and an indicator and an anaerobic gas mixture
Culture Examination
- Cultures are examined in the specified anaerobic chamber at specified times.
- Maintaining anaerobic conditions from incubation to examination is required to avoid contaminations.
Anaerobic Identification Tests
- Presumptive identification using gram staining and colony morphology
- Rapid tests such as catalase, motility, urease, indole, and esculin hydrolysis assays.
Anaerobes Gram Stain RXN
- Flowchart for classifying anaerobic organisms based on their gram stain characteristics
- Includes spore-forming vs. non-spore-forming and gram positive/negative categories
Rapid Identification Tests
- Aerotolerance, fluorescence, disk tests, lecithinase, naglar test, lipase, reverse CAMP assays
Aerotolerance
- Determining if an isolate is a strict anaerobe, aerotolerant anaerobe, or facultative anaerobe
- Incubating specimens aerobically & anaerobically in parallel
- (i.e., anaBAP anaerobically; CHOC aerobically in CO2). The plates are examined in 48 hours
Fluorescence
- Organisms that fluoresce under UV light (366 nm)
- Demonstrating the use of UV light in analyzing organisms
Antibiotic Disk Test
- Confirming gram stain through special potency disks (kanamycin, colistin, vancomycin)
- Tests to distinguish organisms based on reaction/resistance to various antibiotics
- (e.g., Van S, Col R = Clostridia, Porphyromonas), (Van R = GN, not Porphyromonas)
Disk Tests
- Sodium polyanethol sulfonate (SPS) disk : Differentiating gpc based on susceptibility to sodium polyanethol sulfonate
- Nitrate disk: Nitrate is reduced in differing ways in the presence of reagents
- Bile disk: Determining whether an organism will grow in elevated bile concentrations (i.e., bile tolerant anaGNR = Bacteroides fragilis group)
Lecithinase
- Determining if the isolate produced a lecithinase
- Positive lecithinase reaction: Opaque, white zone surrounding the bacterial colonies on the egg yolk agar plate
- Negative lecithinase reaction: No change in the egg yolk agar surrounding bacterial colonies
Nagler Test
- Detecting the specific alpha toxin produced by C. perfringens through the use of antiserum
- A specific type of lecithinase produced by C. perfringens
Lipase
- Identifying bacteria capable of hydrolyzing triglycerides into glycerol and fatty acids
- Identifying positive/negative reaction based on whether an oil/water sheen is visible and the general morphology of each colony of bacteria
Reverse CAMP
- Alpha toxin of C. perfringens synergistically works with beta-hemolytic group of S. agalactiae causes hemolysis
- A positive test results in an arrowhead-shaped zone of hemolysis surrounding a perpendicular streak of C. perfringens
Definitive Identification Tests
- Biochemical tests
- Commercial systems
- Gas-liquid chromatography
- Fatty acid analysis
- 16S rRNA gene sequencing
Biochemical and Commercial Systems
- PRAS or non-PRAS biochemical media
- Commercial biochemical and pre-existing bacterial enzyme minisystems
Gas-Liquid Chromatography
- Analysis of cellular fatty acids & metabolic end products (volatile acids) to produce a characteristic pattern for identification purposes
Gene Sequencing
- 16S rRNA sequencing
- Extracting and amplifying ribosomal DNA (rRNA)
- Sequenced through PCR analysis to identify organisms
Clostridium Species
- This group of bacteria include catalase-negative, motile (except C. perfringens), anaerobic gram-positive rods (GPR), and some gram-variable or gram-negative
- Gram positive bacteria that are susceptible to vancomycin
- Produce endospores (i.e., they appear as unstained refractile structures in gram stain and are oval to round, terminal to subterminal)
- Aerotolerant, cause exogenous infections via ingestion or wounds (e.g., tetanus, gas gangrene, botulism, food poisoning)
- Produce potent toxins
Genera Differences
- Chart comparing Clostridium, Bacillus, and Lactobacillus in terms of optimal growth conditions, sporulation conditions, and catalase test results
Clostridium perfringens
- Most common Clostridium species; found in undercooked meat, soil, water
- Gram-positive, boxcar-shaped rods producing a double zone of beta hemolysis
- Positive reverse CAMP, lecithinase, and Nagler reactions
- Spore production is seldom observed or not common
C. perfringens Clinical Significance
- Isolated from tissue infections and bacteremia
- Produces gas in infected tissue (Myonecrosis; Gas gangrene)
- Deep penetrating wounds or surgical procedures
- Diabetic foot ulcers
C. perfringens Clinical Significance (Food poisoning/gravy)
- Food poisoning comes from meat, meat products, or gravy
- Ingested spores that germinate in intestines & cause enterotoxin
- Symptoms such as nausea, vomiting, diarrhea, and abdominal pain
Clostridium difficile
- May be a normal flora member of the stool
- Antibiotic-associated diarrhea and pseudomembranous colitis
- C. difficile increases in number when antibiotic use disrupts the natural intestinal ecosystem.
- 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 be tested for toxin production
C. difficile Toxin Tests
- Cytotoxin test: Evaluating the toxic effects of stool on cultured human cells
- Kit testing: Determines if toxin A, toxin B, or glutamate dehydrogenase (not a virulence factor) is present in the stool; using an enzyme immunoassay
- Molecular testing: Still under development
Clostridium botulinum
- Botulism toxin ingestion from home-canned vegetables, home-cured meats
- Infant botulism associated with honey
- Neuormuscular toxins cause paralysis or death
- Foodborne botulism, wound botulism, infant botulism (lacking NF)
C. botulinum Diagnosis
- Diagnosed clinically based on symptoms
- Isolating the organism or detecting toxin requires a reference laboratory
Clostridium tetani
- Found in soil and intestinal tracts of animals
- Enters the body through a wound site
- Produces the potent neurotoxin (tetanospasmin) causing severe muscle spasm in unimmunized individuals
- Can cause tetanus (lockjaw).
C. tetani Diagnosis
- Usually diagnosed clinically
Terminal Spores
- Tennis-racquet-shaped
C. septicum
- Aerotolerant
- Swarming colonies
- Subterminal spores
- Can cause myonecrosis and bacteremia
- Associated with leukemia, lymphoma, and large bowel carcinoma
Non-Spore-Forming Anaerobic GPR
- Actinomyces spp., Bifidobacterium spp., Propionibacterium spp., Eggerthella and Eubacterium spp.
- All can cause actinomycosis
Actinomyces
- Includes aerobic and anaerobic bacteria.
- Nocardia is an aerobic actinomycete
- Causes mycetomas and nocardiosis
- A. israelii is the most common anaerobe, causes actinomycosis
- Inhabit human and animal mucosal surfaces
- Anaerobic actinomycetes are not weakly acid fast like Nocardia spp.
Actinomyces Gram Stain
- Aero- and anaerobic Actinomyces spp.
- GPR irregularly stained (beaded appearance)
- Coccoid to filamentous
- Filaments branch
A. israelii Actinomycosis
- Can cause actinomycosis
- Forms granulomatous infection usually in the jaw
- Develops fistulae & drains pus with sulfur granules
A. israelii Cultures
- Associated with "lumpy jaw"
- Pus and sulfur granules observed in anaBAP
- Growth is slow, taking 7-9days
- Colony morphology resembles molar teeth
- Anaerobic, branching GPR
Bifidobacterium
- Actinomyces-like morphology
- Gram stain is branched or bifurcated GPR
- Normal flora member of the intestines and oral cavity
- Rarely cause disease, but can cause actinomycosis
Propionibacterium
- Anaerobic diphtheroid like GPR
- Resembles corynebacteria
- Normal flora member of skin
- Most common anaerobe isolated in the lab as a contaminant in blood cultures & specimens collected from the skin penetration
- P. acnes linked to acne
- Catalase and indole positive
- May cause subacute bacterial endocarditis (SBE) & bacteremia
Eggerthella and Eubacterium spp.
- Eggerthella previously Eubacterium
- Anaerobic diphtheroid like GPR
- No branching
Anaerobic GPC
- Peptostreptococcus: GPC in chains
- P. anaerobius is susceptible to sodium polyanethol sulfonate (SPS) disk. Other anaerobic cocci are resistant
- Peptococcus: GPC in clusters
Anaerobic GNR
- Bacteroides, Prevotella, Prophyromonas, Fusoacterium, Veillonella (Normal flora member of the mucous membrane)
Bacteroides fragilis Group
- Anaerobic GNR
- Bacteroides fragilis is the most common
- Causes intra-abdominal infections, bacteremia, & soft tissue infections
- B. thetaiotaomicron is the 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 is stimulated by bile
- Brown to black colonies indicate esculin hydrolysis
Bacteroides ureolyticus Group
- Anaerobic GNR
- Bile sensitive and bile-tolerant, non-pigmented organisms
- Some GNRs pit the agar
- Growth in formate and fumarate are observed
- Resistant to vancomycin
- Sensitive to kanamycin and colistin
Prevotella
- Anaerobic GNR
- Bile susceptible
- Resistant to kanamycin & vancomycin
- Growth on KVLB, but not BBE
- Some produce protoporphyrin pigmenting colonies
- Colonies fluoresce a “brick red” under UV light
Fusobacterium
- Susceptible to kanamycin and colistin
- Vancomycin-resistant
- F. nucleatum: Thin fusiform rods; bread-crumb, speckled colonies
- F. necrophorum: Lipase positive
Mobiluncus
- Associated with bacterial vaginosis, pelvic inflammatory disease, & abdominal infections
- Curved bacilli
- Gram variable
- Motile, catalase, and indole-negative
- Inhibited by vancomycin
Selective Anaerobic GNR ID
- Flowchart to aid in identifying gram-negative anaerobic rods
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
- Acranobacterium
- Lactobacillus
- Gardnerella
Corynebacterium
- Normal skin and mucous membrane flora
- Called diphtheroids or coryneforms, are club-shaped
- Gram positive rods (GPR), are pleomorphic (variable in size), non-spore forming
- Gram stain: looks like "Chinese letters" (V, L, Y formation) or palisades (line up side by side)
- Facultative anaerobic
- Small gamma colonies on SBA
- Catalase-positive
- Nonmotile
- Frequent contaminants
C. diphtheriae
- Significant pathogen
- Virulence factor is diphtheria toxin
- In strains infected with bacteriophage carrying tox gene
- Blocks protein synthesis, destroys host cells
- Absorbed in heart and nervous system; lethal at 130 ng/kg of body weight (150 lb person = 0.3 ounces)
- Causes cutaneous (non-healing ulcer) and respiratory (diphtheria) diseases
Diphtheria
- URT infection (tonsils, pharynx)
- Bacteria multiply, release toxin
- Tissue necrosis
- Exudate (inflammation → pseudomembrane)
- Necrotic epithelial cells, WBCs, fibrin, bacteria involved in suffocation
- Treatment with antitoxin
- Prevention 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 colony)
- Elek Test: Immunodiffusion test for toxin production
Corynebacterium spp.
- Normal skin flora
- May contaminate cultures
- C. jeikeium: devices/prosthetic valve endocarditis
- C. urealyticum: Urinary pathogen
Other Non-spore-forming GPR
- Rothia dentocariosa: oral NF, endocarditis, and wound infections
- Filamentous or coryneform groups
- Undesignated CDC coryneforms groups: opportunistic or nosocomial infections
Listeria monocytogenes
- Vaginal and intestinal NF in humans
- Clinical infections via contaminated food
- Virulence Factors: Hemolysis (listeriolysin O), others
Listeria monocytogenes
- Listeriosis (sepsis)
- Neonates (50% mortality)
- Pregnant women (spontaneous abortion/stillbirth)
- Immunocompromised individuals
- CNS predilection
L. monocytogenes Cultures
- CSF, blood, amniotic fluid
- Facultative anaerobe
- Growth 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
- Hemolysis may be hidden under colonies
- Catalase positive
- Bile-esculin positive
- Motile at 25°C
L. monocytogenes Motility
- Tumbling motility at 25°C but not 35°C
- Results in umbrella-like growth patterns
- Positive CAMP test differentiates L. monocytogenes from other Listeria spp.
- Block vs. arrowhead pattern
Differentiation of Listeria
- Table comparing L. monocytogenes, Corynebacterium spp., S. agalactiae, and Enterococcus spp. based on catalase, esculin hydrolysis, motility, β-hemolysis, and 6.5% NaCl results
Erysipelothrix rhusiopathiae
- Reservoir: domestic swine
- Occupational risk to butchers, vets, and fishermen
- Causes erysiploid (red skin lesion)
- Can disseminate into bacteremia/endocarditis in humans
Erysipelothrix rhusiopathiae
- GPR
- Catalase negative
- Alpha or gamma hemolysis
- Nonmotile
- H₂S positive in TSI
Culture
- Skin biopsies, blood
- Routine media (BAP, CHOC, CNA, PEA)
- Small colonies visible after 48 hours
Arcanobacterium haemolyticum
- Formerly Corynebacterium
- Significant: A. haemolyticus, A. pyogenes, A. bernardiae cause pharyngitis
- Facultative anaerobe
- Beta hemolysis
- Catalase negative
- Reverse CAMP positive
- Inhibits S. aureus hemolysis
Lactobacillus
- Normal vaginal flora
- Produce lactic acid
- Reduce vaginal pH
- Inhibits growth of other organisms
- Rarely causes disease (e.g., L. acidophilus probiotic), may assist in inhibiting other species
Lactobacillus
- Non-spore forming GPR
- Medium to long rods
- Aerotolerant anaerobes
- Catalase-negative
- Nonmotile
Gardnerella vaginalis
- Pleomorphic coccobacilli
- Cell wall with GPR characteristics, stains gram-variable
- Non-motile
- Facultative anaerobe
- NF in 50-70% women
- Associated with bacterial vaginosis
Bacterial Vaginosis (BV)
- Change in NF (lack of lactobacillus)
- Increase in pH, allowing G. vaginalis growth
- Foul-smelling discharge
- Untreated leads to PID and UTIs
Clue Cells
- Squamous cells with bacteria clustered at the edges
Cultures
- Appropriate for extravaginal sites
- Grows on CHOC, BAP, but not MAC
- Small pleomorphic gram-variable coccobacilli
- Catalase and oxidase negative
- Hippurate hydrolysis positive
Non-Spore-Forming Branching GPR (Aerobic Actinomycetes)
- Nocardia spp.
- Tropheryma whipplei
- Streptomyces
- Actinomadura
- Gordonia
- Tsukamurella
- Rhodococcus equi
Nocardia spp.
- Found in soil
- Usually infects immunocompromised individuals
- May cause pulmonary or cutaneous diseases
Nocardia spp.
- Pulmonary: N. asteroides complex, pneumonia → abscess → necrosis
- Cutaneous: N. brasiliensis, abscess → invasive, draining pus with sulfur granules
- Aerobic GPR morphologically similar to fungi
- Form beaded, branching filaments on gram stain
- Weakly acid-fast
Nocardia spp.
- Growth is slow, with colonies described as waxy, chalky, or crumbly
- Can be identified using acid-fast staining
Nocardia ID
- Filamentous, branching organism
- Weakly acid-fast
- Acid-fast stains (Ziehl-Neelsen or Kinyoun)
- Primary stain is carbolfuchsin
- Rinsed, decolorized with acid
- Counterstained with methylene blue
- Reddish purple filaments (partially acid fast) are positive
Other Actinomycetes
- Tropheryma whipplei: Whipple Disease, intracellular pathogen; diarrhea, weight loss, malabsorption
- 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, resistant to biocides; thick outer wall
- Found in nature, metabolically diverse (lab contaminants)
- B. anthracis, B. cereus: Important pathogens
Bacillus
- Large aerobic GPR
- Spore-formers
- Spores appear as holes in gram stain
- Catalase positive (B. cereus is beta-hemolytic and motile; B. anthracis isn't)
Bacillus
- Catalase positive
- Aerobic spore formation distinguishes from Clostridia
- Growth on SBA and PEA
- Does not grow on enteric agars; is a GPR
B. anthracis
- Causes anthrax (usually in herbivores)
- Bioterrorism agent; virulence factors include glutamic acid capsule, protective antigen, edema factor, and lethal factor
B. anthracis
- Clinical significance includes cutaneous, pulmonary, and gastrointestinal forms
- Cutaneous: pimple → vesicles → erythematous ring → necrotic lesion (black eschar)
- Pulmonary (woolsorter's disease): flu-like symptoms progressing to respiratory distress, coma, death
- Ingestion: pain, nausea, vomiting
- Gram stain: very long chains ("bamboo shoots"), square-ended
- Colonies: non-motile, non-hemolytic, large, gray, flat, irregular edges, medusa head
B. anthracis Identification
- Aerobic/anaerobic
- Spore-forming GPR
- Nonhemolytic on SBA
- Nonmotile
- Catalase-positive
- Requires a biological safety hood
B. cereus
- Beta hemolytic and motile; distinguishes it from B. anthracis
- Produces enterotoxin
- Causes food poisoning
- Self-limiting
Identification of GPR
- Flowchart summarizing the identification of gram-positive rods based on spore formation, catalase reaction, motility, bile-esculin reaction, and other specific characteristics
Spirochetes
- Borrelia, Leptospira, Treponema: Long, slender spiral-shaped, motile, not seen in gram stain, require silver stains
Borrelia
- Several species of spirochetes
- Transmitted by arthropods (ticks and lice)
- 3 to 10 spirals per organism
- Most cause relapsing fever
- B. burgdorferi causes Lyme disease
Borrelia recurrentis
- Causes relapsing fever (repeated febrile episodes of spirochetemia)
- Cyclic relapses caused by antigenic variation
- Episode ends when immune system responds
Relapsing Fever Lab Tests
- Microscopic examination of blood during febrile periods (Giemsa or Wright's stain)
- Culture in Kelly medium (rare)
- Serology (inadequate due to antigenic variation)
Borrelia burgdorferi
- Lyme borreliosis (Lyme disease)
- Originated in the Northeast, spreading
- Transmitted by Ixodes ticks (deer or mouse ticks)
Three Stages of Lyme Disease
- Stage 1: Erythema chronicum migrans (ECM), lymphoadenopathy, flu-like symptoms
- Stage 2: Dissemination, fever, bone and joint pain, splenomegaly, malaise
- Stage 3: Late or persistent infection; months to years; chronic arthritis, neurological defects, cerebrovascular lesions
Lyme Disease Lab Tests
- Serologic tests (most common & fastest): antibody detection tests (immunoassays)
- Direct microscopic examination of skin or blood (usually negative & labor intensive)
- Cultures are labor intensive & use Kelly medium
Leptospira
- L. biflexa: Nonpathogenic
- L. interrogans: Pathogenic
- Transmitted to humans via contaminated water with animal urine
- Occupational hazard to vets, farmers, sewer workers
Leptospira interrogans
- Spirals with hooked ends
- Tightly coiled
Leptospirosis
- Zoonotic infection
- Spirochetes infect kidneys & enter urine → contaminated water
- Leptospires enter through breaks in skin or intact mucosa
- Symptoms range from asymptomatic to severe
Leptospirosis
- Clinical manifestation range from asymptomatic to severe
- Incubation period: 3-30 days
- Abrupt onset of influenza like symptoms & nonspecific symptoms
- Severe systemic diseases. Weil's disease (CSF positive); renal failure; hepatic failure; icteric leptospirosis; intravascular disease
Leptospirosis Lab Tests
- Blood & CSF (1st week); urine (later)
- Darkfield microscopy or DFA
- Cultures (Fletcher's semisolid media, 30°C dark, 6 weeks)
- Darkfield microscopy in media
- Serology (ELISA)
Treponema
- 4 to 14 spirals/organism
- 4 pathogenic organisms: T. pallidum subsp. pallidum, T. pallidum subsp. pertenue, T. pallidum subsp. endemicum, T. pallidum subsp. carateum
Treponema pallidum subspecies pallidum
- Venereal syphilis
- Great imitator
- Variety of clinical presentations
- Transmitted sexually or via non-genital lesions (mouth or skin)
- Can cross placenta (congenital syphilis)
- Three stages (primary, secondary, tertiary)
Primary Syphilis
- Few days to months after organism acquisition
- Painless firm chancre (lesion) at inoculation site
- Highly infectious due to numerous spirochetes present
Secondary Syphilis
- 2-12 weeks after primary lesion
- Organisms disseminate throughout the body
- Fever, lymphoadenopathy, headache
- Secondary infectious lesions on skin and mucous membranes
- Widespread skin rashes (may include palms and soles)
Tertiary or Late Syphilis
- 1/3 of untreated cases develop tertiary syphilis years after initial infection
- Not infectious
- Many body sites affected (gummas, 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 infection; infected fetus often dies from congenital infection
- Affects many body systems, with skin and mucous membrane lesions, anemia, hepatosplenomegaly, meningitis, & bone lesions
Late Onset Congenital Syphilis
- Mother has chronic, untreated syphilis
- Symptoms in children > 2 years old: blindness, deafness, mental retardation, bone/tooth deformities
- Prevention by screening pregnant women and necessary treatment
Serological Laboratory Diagnosis
- Nontreponemal tests (screening): VDRL, RPR
- Treponemal tests (confirmation): EIA, TPPA, FTA-ABS
Nontreponemal Tests
- Detects antibodies (reagin/reaginic antibodies) against treponemal lipids (e.g., cardiolipin-lecithin)
- Sensitive but not specific; can have false positives from Lyme, viral infections, autoimmune diseases, and pregnancy
- Monitor therapy & detect reinfection
VDRL and RPR
- Visual aids describing the different results of the VDRL and RPR tests
Treponemal Tests
- Detects treponemal specific antibodies
- Remain positive after treatment
- Enzyme immunoassay (EIA)
- Treponema pallidum particle agglutination assay (TPPA)
- Previously FTA-ABS (fluorescent treponemal antibody absorption)
TPPA
- Positive control wells, negative control wells
Direct Microscopic Exam
- Darkfield microscopy (fluid from lesions); patience and experience
- Look for corkscrew motility
- Oral lesions are not ideal due to prevalence of nonpathogenic treponema
Other Pathogenic Treponemes
- Spread via direct contact or utensils, with contaminated drinking/eating utensils
- May exhibit primary, secondary, or tertiary disease; Tertiary syphilis and congenital syphilis are rare
- T. endemicum (Endemic syphilis in Middle East/arid areas), T. pertenue (Yaws in humid/tropical areas), T. carateum (Pinta in Central and South America)
Mycobacteria
- Aerobic; require increased CO₂
- Non-spore forming
- Non-motile
- Cultures held for 6-weeks before considered negative
- Slm gram-variable rods
- High lipid content (mycolic acid) in cell wall, so they do not gram stain well
Mycobacteria Gram Stain
- Gram stain poorly due to cell wall lipids
- Crystal violet and safranin do not properly penetrate
- Resulting stains: absence of organisms, beaded gram-positive rods, ghost cells
Mycobacteria Safety
- High incidence of positive tuberculin skin tests
- Labs should be separate, nonrecirculating ventilation, negative air pressure, and work within a biosafety cabinet
Mycobacteria Safety
- Airborne hazard
- BSL-2 for specimen processing (gloves and gowns); BSL-3 for cultured organisms
- Specimens double sealed during centrifugation
Mycobacteria Species
- Major pathogens: M. tuberculosis complex, Nontuberculous mycobacteria (NTM/MOTT), M. leprae (Hansen's disease/leprosy)
Specimen Collection
- Sputum, bronchial washings, gastric contents, urine, stool, and tissue samples
- First morning sputum on 3 consecutive mornings
- 5-10 ml sputum minimum; refrigerate overnight
- If sputum can't be obtained, proceed to bronchoscopy or biopsy
- Gastric aspirates and washings; young patients (cannot produce sputum); young children (swallow sputum); neutralize with sodium bicarbonate; mycobacteria damaged by stomach acid
- Stool specimens: AIDS patients get M. avium screen; Neutralize gastric aspirates/urine with sodium bicarbonate (refrigerate overnight)
Inappropriate Specimens
- Insufficient material for identification
- Contamination from other organisms
- Interference from prolonged exposure (e.g., urine) to specimens interfering with growth of mycobacteria
Digestion and Decontamination
- Specimens from sterile sites (CSF): concentrate for inoculation
- Specimens contain NF and mucous membranes: Digestion with N-acetyl-L-cysteine (NALC), decontamination with NaOH, concentration
Specimen Processing
- Digestion: frees mycobacteria from protein clumps using NALC (mucolytic agent)
- Decontamination: removes NF
- May use NALC, timed and controlled to remove contaminants with minimal effects on mycobacteria
- Concentration: increases the number of organisms per mL using centrifugation
Staining for AFB Smear
- Ziehl-Neelsen (hot stain, heat): stains acid-fast bacilli (AFB)
- Kinyoun (cold stain, detergent/phenol): stains acid-fast bacilli (AFB)
- Primary stain: carbolfuchsin; rinse and decolorize with acid; counterstain with methylene blue
- Reddish filaments = AFB (acid-fast positive); blue filaments are negative
Staining for AFB Smear
- Heat/phenol forces special dye into mycobacterial cells
- High lipid content resists decolorization = acid fastness
- Scan at least 300 oil immersion fields before reporting a specimen as negative
- Any number of AFB bacteria on the slide is significant
Acid-Fast Stain Carbolfuchsin
- Microscopic image of acid-fast stain, positively stained acid-fast bacilli
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Test your knowledge on foodborne pathogens and microscopy techniques in microbiology. This quiz covers various aspects, including enterotoxin production, spirochetes observation, motility characteristics, and mycobacterial behaviors. Challenge yourself with these important microbiological concepts!
