Skip to main content

Microbiology

Skin

This section focuses on the colony morphology and identification of major pathogens associated with skin infections, specifically Staphylococcus aureus, beta-hemolytic streptococci, and Pseudomonas aeruginosa

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 (the “aureus” in the name refers to gold), 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)

Pseudomonas aeruginosa

  • A. Colony Morphology
    • Agar Plate
      • Blood Agar
        • Size: Medium to large (2-4 mm in diameter)
        • Shape: Flat, spreading, irregular edges
        • Color: Often exhibits a greenish or bluish pigment (pyocyanin) and/or a yellow pigment (pyoverdin)
        • Texture: Smooth, mucoid, or sometimes rough
        • Odor: Often has a characteristic fruity or grape-like odor
        • Hemolysis: May be beta-hemolytic, especially after prolonged incubation
      • MacConkey Agar
        • Growth: Usually grows well on MacConkey agar
        • Appearance: Non-lactose fermenter, colonies appear non-pigmented (colorless or slightly translucent)
      • Cetrimide Agar
        • Principle: Selective media that enhances the production of pyocyanin
        • Appearance: Promotes the production of the blue-green pigment, making it easier to identify P. aeruginosa
    • Gram Stain
      • Gram-negative rods
      • Often appear as straight or slightly curved rods
  • Identification Methods
    • 1. Presumptive Identification
      • Gram Stain: Gram-negative rods
      • Colony Morphology: Greenish or bluish pigment, fruity odor on blood agar
      • Oxidase Test: Positive (turns the reagent blue)
    • 2. Definitive Identification
      • Oxidase Test: Positive (confirms the initial result)
      • Motility: Motile (using a polar flagellum)
      • Growth at 42°C: P. aeruginosa is able to grow at 42°C, which is helpful in differentiating it from other Pseudomonas species
      • Pigment Production: Production of pyocyanin (blue-green), pyoverdin (yellow-green, fluorescent under UV light), and pyorubin (red-brown)
      • Arginine Dihydrolase: Positive
      • Acetate Utilization: Positive
      • Commercial Identification Systems
        • API 20NE: 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

  • Mixed Cultures: Skin infections can often be polymicrobial. When multiple organisms are isolated, it is important to identify all significant pathogens
  • 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 wound, 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
  • Pyocyanin: A blue-green pigment produced by Pseudomonas aeruginosa
  • Pyoverdin: A yellow-green, fluorescent pigment produced by Pseudomonas aeruginosa
  • Mucoid: Having a slimy or sticky consistency
  • Non-lactose fermenter: An organism that cannot ferment lactose
  • AST: Antimicrobial Susceptibility Testing