Bone

This section focuses on the colony morphology and identification of major pathogens associated with bone infections (osteomyelitis). The organisms covered here include Staphylococcus aureus, beta-hemolytic streptococci, and Kingella spp

Staphylococcus aureus

Colony Morphology
- Agar Plate
  - Blood Agar
    - Size: Medium to large (2-4 mm in diameter)
    - Shape: Round, circular
    - Elevation: Slightly raised
    - Color: Typically golden-yellow or creamy-white, but can sometimes appear white
    - Texture: Buttery, smooth, and opaque
    - Hemolysis: Beta-hemolytic (complete clearing of the blood agar around the colonies)
  - Mannitol Salt Agar (MSA)
    - Appearance: Colonies appear yellow on MSA because S. aureus can ferment mannitol, producing acid and changing the pH indicator (phenol red) in the media
    - Background Color: The agar surrounding the colonies will also turn yellow
- Gram Stain
  - Gram-positive cocci
  - Arranged in clusters, resembling “grape-like” clusters
Identification Methods
- 1. Presumptive Identification
  - Gram Stain: Gram-positive cocci in clusters
  - Catalase Test: Positive (bubbles are produced when hydrogen peroxide is added)
- 2. Definitive Identification
  - Coagulase Test
    - Principle: Detects the enzyme coagulase, which clots plasma
    - Procedure: Inoculate a small amount of S. aureus into rabbit plasma containing EDTA (to prevent clotting). Incubate at 35-37°C
    - Interpretation
      - Positive: Clot formation within a specified time (e.g., 4 hours) indicates S. aureus
      - Negative: No clot formation
  - Latex Agglutination Test (for Protein A)
    - Principle: Detects Protein A, a cell wall component specific to S. aureus
    - Procedure: A latex reagent coated with IgG antibodies is used to test for Protein A
    - Interpretation: Agglutination (clumping) indicates the presence of S. aureus
  - Chromogenic Media (e.g., CHROMagar Staph aureus)
    - Principle: Uses chromogenic substrates to differentiate S. aureus from other staphylococci
    - Appearance: S. aureus colonies appear as a specific color (e.g., mauve or blue)
  - Molecular Methods
    - PCR (Polymerase Chain Reaction): Can be used to detect specific genes (e.g., mecA gene for methicillin resistance)
    - MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry): Provides a rapid and accurate identification of microorganisms based on their protein profiles

Beta-Hemolytic Streptococci

Colony Morphology
- Agar Plate
  - Blood Agar
    - Size: Small to medium (0.5-2 mm in diameter)
    - Shape: Circular, convex
    - Color: Usually translucent or gray-white
    - Texture: Smooth, sometimes mucoid
    - Hemolysis: Beta-hemolytic (complete clearing of the blood agar around the colonies)
      - The degree of hemolysis can vary depending on the species (e.g., Streptococcus pyogenes typically exhibits a more pronounced beta-hemolysis)
- Gram Stain
  - Gram-positive cocci
  - Arranged in chains or pairs
Identification Methods
- 1. Presumptive Identification
  - Gram Stain: Gram-positive cocci in chains
  - Catalase Test: Negative (no bubbles are produced)
  - Beta-hemolysis on Blood Agar
- 2. Definitive Identification
  - Lancefield Grouping (Serological Testing)
    - Principle: Identifies the specific Lancefield group based on the presence of a specific carbohydrate antigen on the cell wall
    - Procedure: Uses serological reagents (e.g., latex agglutination kits) that react with the specific Lancefield group antigen
    - Interpretation: Agglutination indicates the presence of the corresponding Lancefield group
    - Common Beta-Hemolytic Streptococci and Their Lancefield Group
      - Streptococcus pyogenes (Group A): Causes strep throat, skin infections (e.g., impetigo, cellulitis), and other invasive infections
      - Streptococcus agalactiae (Group B): Commonly associated with neonatal infections and infections in pregnant women
      - Groups C, F, and G: Can also cause skin infections, less common than Group A
  - Bacitracin Susceptibility (for Group A Streptococcus)
    - Principle: Group A Streptococcus is typically susceptible to bacitracin
    - Procedure: A bacitracin disk is placed on a blood agar plate inoculated with the organism
    - Interpretation
      - Susceptible: Zone of inhibition (no growth) around the disk indicates presumptive identification of Group A Streptococcus
      - Resistant: No zone of inhibition
  - PYR Test (for Group A Streptococcus)
    - Principle: Detects the enzyme L-pyrrolidonyl arylamidase
    - Procedure: Add PYR reagent to the bacterial isolate on a filter paper disk
    - Interpretation
      - Positive: Red color change indicates presumptive identification of Group A Streptococcus
      - Negative: No color change
  - SXT Susceptibility (for Group B Streptococcus)
    - Principle: Group B Streptococcus is typically resistant to SXT (trimethoprim-sulfamethoxazole)
    - Procedure: An SXT disk is placed on a blood agar plate inoculated with the organism
    - Interpretation
      - Resistant: Zone of inhibition (no growth) around the disk indicates presumptive identification of Group B Streptococcus
      - Susceptible: No zone of inhibition
  - Molecular Methods
    - PCR: Can be used to detect specific genes (e.g., emm gene for S. pyogenes)

Kingella spp.

Colony Morphology
- Agar Plate
  - Blood Agar
    - Size: Small to medium (0.5-1 mm in diameter)
    - Shape: Circular
    - Color: Usually translucent or gray-white
    - Texture: Smooth, often with a “pitted” or “corroded” appearance, which can be best observed with a magnifying glass
    - Hemolysis: Non-hemolytic or weakly beta-hemolytic (small zone of clearing)
  - Chocolate Agar
    - Appearance: Colonies may grow better on chocolate agar
- Gram Stain
  - Gram-negative coccobacilli or short rods
  - Often occur in pairs or short chains
Identification Methods
- 1. Presumptive Identification
  - Gram Stain: Gram-negative coccobacilli
  - Catalase Test: Negative
  - Oxidase Test: Positive
- 2. Definitive Identification
  - Biochemical Tests
    - Glucose Fermentation: Positive
    - Lactose Fermentation: Negative
    - Sucrose Fermentation: Negative
    - Nitrate Reduction: Positive
    - Motility: Non-motile
  - Commercial Identification Systems
    - API NH: A miniaturized biochemical test system
    - Vitek 2 System: Automated identification system
    - MALDI-TOF MS: Provides a rapid and accurate identification of microorganisms based on their protein profiles

Additional Considerations

Polymicrobial Infections: Bone infections can be polymicrobial, involving multiple organisms
Anaerobic Cultures: Anaerobic bacteria are also potential pathogens in bone infections. Therefore, anaerobic cultures should be performed on bone specimens
Antimicrobial Susceptibility Testing: Perform AST on all significant isolates to guide antibiotic therapy
Clinical Correlation: Always correlate the laboratory findings with the patient’s clinical presentation, including the type of bone involved, location, and any other relevant signs and symptoms
Reporting: Report the identified organisms, their colony counts (if quantitative cultures were performed), Gram stain morphology, and antimicrobial susceptibility results (if applicable) to the clinician

Key Terms

Beta-hemolysis: Complete clearing of red blood cells around a bacterial colony on blood agar
Coagulase: An enzyme produced by Staphylococcus aureus that clots plasma
Mannitol Fermentation: The ability of an organism to utilize mannitol, a sugar, as a carbon source
Lancefield Grouping: Serological classification of streptococci based on their cell wall antigens
PYR Test: A rapid test to identify the enzyme L-pyrrolidonyl arylamidase, often used to identify Group A Streptococcus
Oxidase Test: A test to determine if an organism produces cytochrome c oxidase
Pitted/Corroded: A colony morphology describing a colony that appears to have a small depression or “bite” taken out of it
Non-hemolytic: No change to the blood agar around the colony
AST: Antimicrobial Susceptibility Testing

Soft Tissue

Bite Wound