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Microbiology

Soft Tissue

This section focuses on the colony morphology and identification of major pathogens associated with soft tissue infections. The organisms covered here include Staphylococcus aureus, beta-hemolytic streptococci, Enterobacteriaceae, and anaerobes

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)

Enterobacteriaceae

  • Colony Morphology
    • Agar Plate
      • Blood Agar
        • Size: Medium to large (1-3 mm in diameter)
        • Shape: Variable, can be round, convex, or irregular
        • Color: Typically gray, opaque, or slightly translucent
        • Texture: Smooth, moist
      • MacConkey Agar
        • Principle: Selective and differential medium
        • Appearance
          • Lactose Fermenters: Colonies appear pink or red (due to acid production from lactose fermentation)
          • Non-Lactose Fermenters: Colonies appear colorless or translucent
      • Eosin Methylene Blue (EMB) Agar
        • Principle: Selective and differential medium
        • Appearance
          • Lactose Fermenters: Colonies appear purple or dark with a metallic green sheen (e.g., Escherichia coli)
          • Non-Lactose Fermenters: Colonies appear colorless or translucent
    • Gram Stain
      • Gram-negative rods
  • Identification Methods
    • 1. Presumptive Identification
      • Gram Stain: Gram-negative rods
      • Colony Morphology: Growth on MacConkey agar (pink or red colonies indicate lactose fermentation)
      • Oxidase Test: Negative (most Enterobacteriaceae are oxidase-negative, Plesiomonas is an exception)
    • 2. Definitive Identification
      • Biochemical Tests
        • Triple Sugar Iron (TSI) Agar: Tests for glucose, lactose, and sucrose fermentation, as well as hydrogen sulfide (H2S) production
        • Citrate Utilization: Determines if the organism can use citrate as a sole carbon source
        • Urease Test: Detects the production of urease, which breaks down urea
        • Indole Production: Tests for the production of indole from tryptophan
        • Motility: Tests for motility
        • Lysine Decarboxylase: Tests for the production of lysine decarboxylase
        • Ornithine Decarboxylase: Tests for the production of ornithine decarboxylase
        • Phenylalanine Deaminase: Tests for the production of phenylalanine deaminase
      • Commercial Identification Systems
        • API 20E/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
      • Serotyping: Can be used to identify specific serotypes of Escherichia coli, Salmonella, and Shigella

Anaerobes

  • Colony Morphology
    • Agar Plate
      • Anaerobic Blood Agar (e.g., Brucella Blood Agar)
        • Incubation: Incubated in an anaerobic environment (e.g., anaerobic chamber or anaerobic jar)
        • Appearance: Colony morphology varies greatly depending on the species
          • Colonies can be small, large, circular, irregular, pigmented, or non-pigmented
          • Many anaerobes have a characteristic odor
          • Some may exhibit fluorescence under UV light
      • Bacteroides Bile Esculin (BBE) Agar
        • Principle: Selective and differential medium for Bacteroides fragilis
        • Appearance: Bacteroides fragilis colonies grow well on BBE agar and hydrolyze esculin, turning the agar black
      • Laked Blood Agar (LBA)
        • Principle: Used to enhance pigment production by Prevotella and Porphyromonas species
        • Appearance: Colonies may exhibit a brick-red fluorescence under UV light
    • Gram Stain
      • Gram-positive cocci, rods, or Gram-negative rods
      • Morphology can vary greatly depending on the species
      • Some may exhibit unusual shapes or arrangements
  • Identification Methods
    • 1. Presumptive Identification
      • Gram Stain: Gram-positive or Gram-negative rods or cocci
      • Colony Morphology: Characteristic odor, growth only under anaerobic conditions
      • Aerotolerance Test: Determines if the organism can grow in the presence of oxygen
    • 2. Definitive Identification
      • Biochemical Tests
        • Catalase Test: Determines if the organism produces catalase
        • Indole Production: Tests for the production of indole
        • Lipase Test: Tests for the production of lipase
        • Antibiotic Susceptibility Testing: Used to determine the susceptibility of the isolate to various antibiotics
        • Gas-Liquid Chromatography (GLC): Used to identify the volatile and nonvolatile fatty acids produced by the organism
      • Commercial Identification Systems
        • Anaerobe Identification Panels: Rapid identification panels for common anaerobes
        • Vitek 2 System (Anaerobe Card): Automated identification system
        • MALDI-TOF MS: Provides a rapid and accurate identification of microorganisms based on their protein profiles

Additional Considerations

  • Mixed Cultures: Soft tissue infections can be polymicrobial, involving multiple organisms
  • 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
  • Anaerobic Culture Techniques: Proper collection, transport, and processing of anaerobic specimens are crucial for accurate results

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
  • Lactose Fermentation: The ability of an organism to utilize lactose, a sugar, as a carbon source
  • Aerotolerance: The ability of an organism to grow in the presence of oxygen
  • Catalase: An enzyme that breaks down hydrogen peroxide
  • Indole Production: The ability of an organism to produce indole
  • BBE Agar: Bacteroides Bile Esculin Agar
  • LBA: Laked Blood Agar
  • AST: Antimicrobial Susceptibility Testing