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Microbiology

Community-Associated

Community-acquired pneumonia (CAP) is a common respiratory infection, and the accurate identification of the causative agents is crucial for guiding appropriate treatment and improving patient outcomes. Understanding the colony morphology and identification methods of the major pathogens involved is essential for effective clinical microbiology

General Principles

  • Pathogen Identification: Accurate identification of the pathogens is critical for proper treatment
  • Specimen Sources: Lower respiratory tract specimens
  • Specimen Processing
    • Sputum: Expectorated from the lower respiratory tract. Quality assessment is important
    • Endotracheal aspirate (ETA): From intubated patients
    • Bronchoalveolar lavage (BAL): Fluid obtained during bronchoscopy
    • Bronchial wash (BW)
    • Bronchial brush (BB)
    • Pleural fluid: Can be present during pneumonia, and is tested to rule out empyema
  • Culture Media
    • Blood Agar (BAP): Used for general growth
    • Chocolate Agar (CHOC): To culture fastidious organisms
    • Selective Media: Used for specific pathogens
    • Buffered Charcoal Yeast Extract (BCYE) Agar: For Legionella
  • Incubation: Incubate cultures at 35-37°C. Legionella require a specific environment
    • CO2 enhanced environment
    • Specific time periods
  • Microscopic Examination: Gram stains are performed
  • Colony Morphology: This is observed, and described
  • Identification
    • Utilizes biochemical testing
    • Commercial systems are used
  • Reporting: Timely reporting is essential

Major Pathogens: Colony Morphology, Gram Stain, and Identification

Streptococcus pneumoniae

  • Source: Sputum, BAL, blood, pleural fluid
  • Colony Morphology
    • BAP: Small, gray, mucoid, glistening colonies. May have a characteristic “draughtsman” appearance (a central raised area and a flattened peripheral edge)
    • Hemolysis: Alpha-hemolytic (greening around the colonies)
  • Gram Stain: Gram-positive, lancet-shaped diplococci (pairs of cocci)
  • Identification
    • Catalase: Negative
    • Optochin Susceptibility: Susceptible (zone of inhibition around the optochin disk)
    • Bile Solubility: Positive (colonies dissolve in bile or a bile salt solution)
    • Pneumococcal Antigen Test: Rapid test to detect pneumococcal capsular antigen

Haemophilus influenzae

  • Source: Sputum, BAL, pleural fluid
  • Colony Morphology
    • CHOC: Small, gray, translucent, slightly mucoid colonies. “Mousy” odor
    • BAP: Will only grow with V factor (NAD) and X factor (hemin)
  • Gram Stain: Gram-negative coccobacilli or pleomorphic rods
  • Identification
    • X and V Factor Requirement: Requires both X factor (hemin) and V factor (NAD) for growth (use of factor strips or a quad plate)
    • Latex Agglutination: (or other rapid antigen tests). For capsular polysaccharide (type b H. influenzae)
    • Commercial Identification Systems

Moraxella catarrhalis

  • Source: Sputum, BAL
  • Colony Morphology
    • BAP: Gray, opaque, smooth, and non-hemolytic colonies
    • Appearance: Often described as “hockey puck” colonies (smooth, easily pushed across the agar surface)
  • Gram Stain: Gram-negative diplococci (kidney bean-shaped)
  • Identification
    • Oxidase: Positive
    • DNase: Positive
    • Butyrate Esterase: Positive
    • Commercial Identification Systems

Legionella pneumophila

  • Source: Sputum, BAL, BW
  • Colony Morphology
    • BCYE Agar: Grayish-white to blue-gray colonies
    • Appearance: Can be somewhat iridescent
  • Gram Stain: Gram-negative rods, but difficult to visualize with Gram stain
  • Identification
    • Culture on BCYE Agar: Enriched medium containing L-cysteine and iron salts
    • Direct Fluorescent Antibody (DFA): Rapid method to detect Legionella antigens in clinical specimens
    • Urine Antigen Test: Rapid test. Useful for diagnosis of L. pneumophila serogroup 1 infection
    • PCR: Molecular testing can also be used

Staphylococcus aureus

  • Source: Sputum, blood, pleural fluid
  • Colony Morphology
    • BAP: Medium to large, circular, opaque, smooth, golden-yellow or cream-colored colonies
    • Hemolysis: Usually beta-hemolytic
  • Gram Stain: Gram-positive cocci in clusters
  • Identification
    • Catalase: Positive
    • Coagulase: Positive

Additional Considerations

  • Patient Demographics: Age, smoking history, and co-morbidities
  • Previous Antibiotic Use: Prior antibiotic use can affect culture results
  • Assessment of Specimen Quality: Evaluate sputum samples
  • Reporting: Provide the results of Gram stain and provide results for the identified organisms

Key Terms

  • Community-Acquired Pneumonia (CAP): Pneumonia acquired outside of a healthcare setting
  • Lower Respiratory Tract: Lungs, bronchi, and bronchioles
  • Sputum: Secretions from the lower respiratory tract
  • Bronchoalveolar Lavage (BAL): Fluid obtained from the lungs via bronchoscope
  • BCYE Agar: Buffered Charcoal Yeast Extract Agar - Used for Legionella
  • BAP: Blood agar plate
  • CHOC: Chocolate agar plate
  • Gram Stain: Staining technique to differentiate bacteria
  • Alpha-Hemolysis: Partial lysis of red blood cells
  • Beta-Hemolysis: Complete lysis of red blood cells
  • Catalase Test: Biochemical test for the enzyme catalase
  • Optochin Susceptibility: Used to differentiate Streptococcus pneumoniae
  • Pneumococcal Antigen Test: Antigen test for S. pneumoniae
  • BCYE Agar: Buffered Charcoal Yeast Extract Agar
  • DFA (Direct Fluorescent Antibody): Test that uses antibodies to identify pathogens
  • PCR: Polymerase Chain Reaction (PCR)
  • Commercial Identification System: A system that uses pre-packaged biochemical tests for identifying microorganisms
  • Pleural Fluid: Fluid from the pleural space
  • Empyema: Pus in the pleural space