Zoonotic

This section focuses on the colony morphology and identification of major pathogens associated with zoonotic infections that can affect the skin, soft tissues, and bones. These include Bacillus anthracis, Brucella spp., Francisella tularensis, and Yersinia spp.

Important Note: These organisms are potential bioterrorism agents and/or pose significant laboratory safety risks. All procedures must be performed in a BSL-2 or BSL-3 laboratory with appropriate personal protective equipment (PPE) and protocols. Consultation with a laboratory supervisor, infectious disease specialist, and/or public health officials is crucial

Bacillus anthracis

Colony Morphology
- Agar Plate
  - Blood Agar
    - Size: Medium to large (2-5 mm in diameter)
    - Shape: Irregular, often with a “medusa head” appearance (curled, comma-shaped outgrowths)
    - Color: Grayish-white
    - Texture: Non-hemolytic, “ground glass” appearance (opaque and granular)
    - Hemolysis: Non-hemolytic (initially), but may show some hemolysis after prolonged incubation
- Gram Stain
  - Gram-positive rods
  - Large, rectangular rods with a “bamboo cane” appearance
  - Spores are typically present (oval, central, and non-swelling)
Identification Methods
- 1. Presumptive Identification
  - Gram Stain: Gram-positive rods with spores
  - Colony Morphology: Large, irregular colonies with a “medusa head” appearance on blood agar
  - Catalase Test: Positive
  - Motility: Non-motile
- 2. Definitive Identification
  - Gamma Phage Lysis
    - Principle: Susceptible to lysis by a specific bacteriophage
    - Procedure: Streak the isolate on a blood agar plate and apply a drop of gamma phage to the surface. Incubate and observe for lysis (clearing) of the bacterial growth
    - Interpretation: Lysis indicates Bacillus anthracis
  - PCR (Polymerase Chain Reaction)
    - Principle: Detects specific genes associated with Bacillus anthracis virulence factors (e.g., the cap gene for capsule production and the genes for the anthrax toxins)
    - Procedure: Nucleic acid extraction from the isolate and PCR amplification using specific primers
    - Interpretation: Amplification indicates Bacillus anthracis
  - ELISA (Enzyme-Linked Immunosorbent Assay)
    - Principle: Detects the presence of Bacillus anthracis antigens
    - Procedure: A sample is tested for the presence of anthrax antigens
    - Interpretation: Positive result indicates Bacillus anthracis
  - Immunofluorescence (Direct and Indirect)
    - Principle: Detects Bacillus anthracis using labeled antibodies
    - Procedure: Use specific antibodies to look for antigen present in a sample
    - Interpretation: Positive result indicates Bacillus anthracis

Brucella spp.

Colony Morphology
- Agar Plate
  - Blood Agar
    - Incubation: Requires prolonged incubation (up to 14 days) in a CO2-enriched atmosphere (5-10% CO2)
    - Size: Small (0.5-1 mm in diameter)
    - Shape: Circular, convex
    - Color: Translucent, non-pigmented
    - Texture: Smooth, glistening
    - Hemolysis: Non-hemolytic
- Gram Stain
  - Gram-negative coccobacilli or short rods
  - Often appear as small, faint, and pleomorphic
Identification Methods
- 1. Presumptive Identification
  - Gram Stain: Gram-negative coccobacilli
  - Colony Morphology: Small, translucent colonies on blood agar after prolonged incubation in CO2
  - Oxidase Test: Positive
  - Catalase Test: Positive
- 2. Definitive Identification
  - Biochemical Tests
    - Urease Test: Positive (variable, but most species are positive)
    - H2S Production: Variable
    - CO2 Requirement: Requires CO2 for growth
  - Serological Tests
    - Serum Agglutination Test (SAT): Detects antibodies to Brucella spp. in patient serum
    - ELISA: Detects antibodies to Brucella spp. in patient serum
    - Complement Fixation Test (CFT): Detects antibodies to Brucella spp. in patient serum
  - Molecular Methods
    - PCR: Detects Brucella spp. DNA in the sample. May also be used for species identification
  - Culture Confirmation
    - Principle: Growth in culture is the definitive method for identification, but it is slow and requires special media and incubation conditions
    - Procedure: Culture the sample on blood agar and other enriched media (e.g., trypticase soy agar with 5% sheep blood). Incubate in a CO2-enriched atmosphere for up to 14 days
    - Interpretation: Growth of small, translucent colonies is suggestive of Brucella spp. Further testing (biochemical tests, serology, or molecular methods) is needed for definitive identification

Francisella tularensis

Colony Morphology
- Agar Plate
  - Blood Agar
    - Incubation: Requires enriched media, like chocolate agar or cysteine-glucose blood agar
    - Size: Small (0.5-1 mm in diameter)
    - Shape: Circular, convex
    - Color: Grayish-white, translucent
    - Texture: Smooth, mucoid
    - Hemolysis: Non-hemolytic
- Gram Stain
  - Gram-negative coccobacilli or short rods
  - Very small and faint, making them difficult to visualize
Identification Methods
- 1. Presumptive Identification
  - Gram Stain: Gram-negative coccobacilli
  - Colony Morphology: Small, grayish colonies on enriched media (e.g., chocolate agar)
  - Culture: Requires enriched media with cysteine
- 2. Definitive Identification
  - Serological Tests
    - Serum Agglutination Test (SAT): Detects antibodies to Francisella tularensis in patient serum
    - ELISA: Detects antibodies to Francisella tularensis in patient serum
    - Microagglutination Test (MAT): A more sensitive test than SAT
  - Direct Fluorescent Antibody (DFA) Test
    - Principle: Detects Francisella tularensis antigens in a sample using fluorescently labeled antibodies
    - Procedure: Apply the DFA reagent to a sample and examine under a fluorescent microscope
    - Interpretation: Specific fluorescence indicates Francisella tularensis
  - PCR (Polymerase Chain Reaction)
    - Principle: Detects Francisella tularensis DNA in the sample
    - Procedure: Nucleic acid extraction from the sample and PCR amplification using specific primers
    - Interpretation: Amplification indicates Francisella tularensis

Yersinia spp. (e.g., Yersinia pestis)

Colony Morphology
- Agar Plate
  - Blood Agar
    - Size: Medium (1-2 mm in diameter)
    - Shape: Circular
    - Color: Grayish-white
    - Texture: Smooth, often with a “fried egg” appearance (a raised center with a flatter periphery)
    - Hemolysis: Non-hemolytic
- Gram Stain
  - Gram-negative coccobacilli or short rods
  - Often exhibit a “bipolar staining” appearance (staining more intensely at the ends of the cells)
Identification Methods
- 1. Presumptive Identification
  - Gram Stain: Gram-negative coccobacilli
  - Colony Morphology: Medium-sized, gray colonies with a “fried egg” appearance on blood agar
  - Catalase Test: Positive
  - Oxidase Test: Negative
- 2. Definitive Identification
  - Biochemical Tests
    - Yersinia pestis is typically non-motile at 37°C, but Yersinia enterocolitica and Yersinia pseudotuberculosis are motile at 25°C
    - Urease Test: Negative
    - Glucose Fermentation: Positive
    - Lactose Fermentation: Negative
    - Nitrate Reduction: Positive
  - Serological Tests
    - Serum Agglutination Test (SAT): Detects antibodies to Yersinia pestis in patient serum
    - ELISA: Detects antibodies to Yersinia pestis in patient serum
  - Direct Fluorescent Antibody (DFA) Test
    - Principle: Detects Yersinia pestis antigens in a sample using fluorescently labeled antibodies
    - Procedure: Apply the DFA reagent to a sample and examine under a fluorescent microscope
    - Interpretation: Specific fluorescence indicates Yersinia pestis
  - PCR (Polymerase Chain Reaction)
    - Principle: Detects Yersinia pestis DNA in the sample
    - Procedure: Nucleic acid extraction from the sample and PCR amplification using specific primers
    - Interpretation: Amplification indicates Yersinia pestis
  - Culture Confirmation
    - Principle: Growth in culture is the definitive method for identification, but it is slow and requires special media and incubation conditions
    - Procedure: Culture the sample on blood agar and other enriched media. Incubate at 28-30°C
    - Interpretation: Growth of small, gray colonies is suggestive of Yersinia pestis. Further testing (biochemical tests, serology, or molecular methods) is needed for definitive identification

Additional Considerations

Laboratory Safety: These organisms pose significant laboratory safety risks. All procedures must be performed in a BSL-2 or BSL-3 laboratory with appropriate PPE and protocols
Reporting: Report all suspected cases to the appropriate public health authorities immediately
Clinical Correlation: Always correlate the laboratory findings with the patient’s clinical presentation, including the type of exposure, location, and any other relevant signs and symptoms
Specimen Collection: Follow proper specimen collection guidelines for each suspected infection

Key Terms

Zoonotic: Transmitted from animals to humans
Medusa Head: A colony morphology described as having curled, comma-shaped outgrowths
Bipolar Staining: A staining pattern where the bacteria stain more intensely at the ends of the cells
BSL-2/BSL-3: Biosafety Level 2/3, a set of containment precautions for handling infectious agents
PPE: Personal Protective Equipment
Gamma Phage Lysis: A test for Bacillus anthracis using a specific bacteriophage that lyses the bacteria
SAT: Serum Agglutination Test
DFA: Direct Fluorescent Antibody Test
PCR: Polymerase Chain Reaction
Mucoid: Having a slimy or sticky consistency
Pleomorphic: Having variable shapes
Enriched Media: Media containing additional nutrients that support the growth of fastidious organisms
CO2-Enriched: An environment containing 5-10% carbon dioxide

Bite Wound

Organism Pathogenicity