Bio 265 – Clinical Microbiology

Bio 265 – Clinical Microbiology Laboratory Session #6

Instructor: Dr. MP. Di Bonaventura
Contact: mdibonaventura@york.cuny.edu

Session Overview

  • Selected exercises and demonstration tests to differentiate or identify species of Staphylococcus, Streptococcus, and Enterococcus.

Differentiation of Staphylococcus vs. Streptococcus & Enterococcus

Catalase Reaction

  • Staphylococcus: Catalase positive (+)

  • Streptococcus & Enterococcus: Catalase negative (-)

Gram Stain Reaction & Morphology

  • Staphylococcus: Gram positive cocci in clusters

  • Streptococcus: Gram positive cocci in pairs, chains

Staphylococcus Species

General Characteristics

  • Staining and microscopic observations do not allow for differentiation of species.

  • Biochemical or serological tests (e.g., for S. aureus) are necessary to identify species.

Key Species

  • Staphylococcus aureus

  • Staphylococcus epidermidis

  • Staphylococcus saprophyticus

Morphological Characteristics

  • Gram: Positive cocci in clusters

  • Metabolism: Facultative anaerobes

  • Endospore Formation: Non-endospore forming

  • Motility: Non-motile

Clinical Significance of Selected Staphylococcus Species

Staphylococcus aureus

  • Found as a member of the normal microbiota, typically in nostrils.

  • Diseases Caused:

    • Skin infections

    • Pneumonia

    • Food poisoning

    • Toxic shock syndrome

    • Bacteremia

    • Endocarditis

  • Antibiotic Resistance: Exhibits MRSA (Healthcare-associated and Community-associated MRSA) and VRSA.

Staphylococcus epidermidis

  • Member of the normal microbiota of the skin.

  • Diseases: Infections associated with indwelling intravenous catheters.

Staphylococcus saprophyticus

  • Not a member of the normal microbiota.

  • Diseases: Causative agent of urinary tract infections.

Diseases Caused by Staphylococcus aureus

  • Impetigo

  • Staphylococcal Scalded Skin Syndrome (SSSS)

  • Bacteremia

  • Staphylococcal pneumonia (nosocomial)

  • Endocarditis

  • Food poisoning

  • Toxic shock syndrome

Staphylococcal Scalded Skin Syndrome

  • Nature: Toxin-mediated disease.

  • Mechanism: Certain strains produce exfoliatin toxin, which separates skin layers. The toxin circulates through the bloodstream.

  • Management: Patients are placed in isolation to prevent spread and secondary infections.

Demonstration Tests for Staphylococcus Species

Species: S. aureus, S. epidermidis, S. saprophyticus

  1. MSA Plate:

    • Check for mannitol fermentation (positive/negative).

  2. BAP (Blood Agar Plate):

    • Identify which species is hemolytic (produces hemolysins).

    • Note the type of hemolysis.

  3. Coagulase Activity:

    • Identify which species is coagulase positive.

  4. TSA/NOVOBIOCIN Disk:

    • Identify resistance or susceptibility to novobiocin.

Observations

  • Uninoculated vs. After Inoculation and Incubation on MSA:

    • Which species ferments mannitol?

    • Which species does not ferment mannitol?

Hemolysis Activity and Identification of Staphylococcus Species

  • BAP:

    • Observations on hemolytic activity:

    • Identify beta-hemolytic species.

    • Determine which species exhibit no hemolytic activity.

Key Observations and Tests for Staphylococcus aureus

  • Coagulase Activity:

    • S. aureus is coagulase positive.

    • S. epidermidis and S. saprophyticus are coagulase negative.

Streptococci Overview

General Characteristics

  • Gram Stain: Positive cocci in pairs or chains

  • Metabolism: Non-endospore forming, facultative anaerobes, microaerophilic, or aerotolerant anaerobes.

  • Fastidious Nature: Species have specific growth requirements.

  • Hemolytic Activity: Exhibits alpha, beta, or gamma hemolytic activity when cultured on BAP due to hemolysins (streptolysins).

  • Many species are part of normal microbiota in the oral cavity or upper respiratory tract; some are pathogens.

Disease Examples Caused by Streptococcus Species

Streptococcus pyogenes (Group A Streptococcus - GAS)

  • Common Conditions:

    • Streptococcal pharyngitis (Strep throat): Inflammation of the pharynx, fever, red throat with pus patches, tender lymph nodes.

    • Wound infections.

    • Necrotizing fasciitis (flesh eating disease):

    • Rare but severe complication of infection; rapid tissue destruction.

Other Significant Species

  • S. salivarius: Normal oral microbiota.

  • S. agalactiae: Associated with neonatal meningitis and sepsis.

  • S. pneumoniae: Encapsulated; can cause pneumonia or meningitis; sometimes a member of upper respiratory tract normal flora.

Tests for Streptococcus Species

Hemolytic Activity on BAP

  1. Streptococcus salivarius: Specify hemolysis type.

  2. S. agalactiae and S. pyogenes: Specify hemolysis type.

    • Identify how to differentiate species according to hemolysis.

    • Differentiate S. salivarius, S. agalactiae, S. pyogenes.

Beta-hemolytic Streptococci Testing

  • Traditional Tests: Often replaced with serological tests.

    1. BAP/Bacitracin Disk Test:

    • Group A (S. pyogenes): Susceptible to bacitracin.

    • Non-Group A (S. agalactiae): Resistant.

    1. CAMP Test on BAP:

    • Group B (S. agalactiae) positive for CAMP factor; S. pyogenes negative.

Enterococcus Species

Enterococcus faecalis/faecium

  1. Bile Esculin Test Tubes:

    • Enterococcus species are positive for bile esculin test.

      • Observation: Determine medium color when esculin is metabolized.

Streptococcus pneumoniae and the Optochin Test

  • Hemolytic Activity:

    • Identify hemolytic species.

  • Optochin Test:

    • S. pneumoniae sensitive to optochin (disk P) and is soluble in bile.

    • Other alpha-hemolytic streptococci are resistant and insoluble in bile.

Laboratory Experiments

Key Tests

  • Gram Stain: For differentiating Staphylococcus versus Streptococcus or Enterococcus.

  • Catalase Test: For Staphylococcus epidermidis versus Streptococcus salivarius.

    • Enterococcus species provide the same result as Streptococcus species.

Experimental Procedure

  • Streaking Staphylococcus species:

    • MSA Plate

    • TSA Plus Novobiocin Disk (to check resistance/susceptibility).

      • Each plate is divided into three sections, each streaked with one species (S. epidermidis, S. aureus, or S. saprophyticus).

  • BAP and Hemolysis:

    • Another set divided into three parts for streaking with S. epidermidis, S. salivarius, or S. aureus.

Gram Stain Analysis

  • After staining and using a microscope with a 100x oil immersion objective lens, describe observable content of both figures pertaining to Gram stain results.

Analysis of Catalase Test Results

  • Results Interpretation:

    • No foaming: Indicates Streptococcus and Enterococcus species.

    • Foaming: Indicates Staphylococcus species.

Results and Observations During Streaking Experiments

Expected Results

  • MSA Plate:

    • Anticipate growth, mannitol fermentation, and color changes of the medium.

  • TSA Plate With Novobiocin Discs:

    • Evaluate susceptibility/resistance to novobiocin.

  • BAP Hemolysis: For S. epidermidis, S. salivarius, S. aureus, record expected hemolytic activity.

Data Compilation

Fill in the Tables

Table 1: Staphylococcus Species

  • Columns: Gram stain reaction & multicellular arrangement, Catalase, MSA (mannitol fermentation), Hemolysis, Coagulase, Susceptibility to novobiocin.

    • Species:

    • S. aureus

    • S. epidermidis

    • S. saprophyticus

Table 2: Streptococcus & Enterococcus Species

  • Columns: Gram stain, Catalase, Susceptibility to Bacitracin, CAMP test, Susceptibility to Optochin.

    • Species:

    • S. salivarius

    • S. pyogenes

    • S. agalactiae

    • S. pneumoniae

    • E. faecalis