MIDTERMS

Comprehensive Reviewer: Staphylococcus, Streptococcus & Enterococcus

PART 1: STAPHYLOCOCCUS

General Characteristics

• Gram-positive cocci in clusters ("grape-like")

• Catalase positive (key differentiator from Streptococcus)

• Facultative anaerobes, non-motile

Specimen Collection

• Common specimens: Wound, Blood, Sputum, Urine

• Direct Gram stain done from specimen → look for Gram(+) cocci & neutrophils

Culture Media

MSA Reactions:

• S. aureus → ferments mannitol → yellow (acid, phenol red turns yellow)

• S. epidermidis → no fermentation → stays pink/red (alkaline)

• S. saprophyticus → ferments mannitol → yellow/pink

Macroscopic Characteristics (S. aureus)

• Medium-large, smooth, butyrous, creamy colonies; entire margin

• Pigment: Staphyloxanthin (yellow/golden), not always present

• Narrow zone of β-hemolysis on SBA

Key Differentiation Tests

1. Catalase Test — Staph vs. Strep

• Reaction: 2H₂O₂ --catalase--> 2H₂O + O₂

• (+) bubbling = Staphylococcus; (–) = Streptococcus

2. Coagulase Test — S. aureus vs. CoNS (Coagulase-Negative Staph)

• Principle: Coagulase converts fibrinogen (plasma) → fibrin (clot)

• Slide coagulase: detects bound coagulase (clumping factor) — quick screen

• Tube coagulase: detects free coagulase — gold standard, confirmatory

  • (+) = clot doesn't flow when tube inverted

  • (–) = flows freely

• Latex agglutination (e.g., BBL Staphyloslide): latex coated with fibrinogen & IgG reacts with clumping factor & Protein A → agglutination = rapid detection

3. Modified Oxidase Test — Micrococcus vs. Staphylococcus

• Reagent: 6% tetramethyl-p-phenylenediamine dihydrochloride in DMSO

• (+) = dark blue-purple (cytochrome C) → Micrococcus

• (–) = no color change → Staphylococcus

4. Bacitracin Susceptibility Test — Micrococcus/Rothia vs. Staphylococcus

• 0.04 units bacitracin disk

• Zone >10mm = Susceptible; <10mm or none = Resistant

• Micrococcus & Rothia = Susceptible; Staphylococcus = Resistant

5. Novobiocin Susceptibility Test — S. saprophyticus vs. other CoNS

• 5 μg novobiocin disk (Kirby-Bauer/CLSI)

• Susceptible = zone >16mm; Resistant = zone ≤16mm

• S. saprophyticus = Resistant; other CoNS = Sensitive

6. DNase Test — presumptive ID of S. aureus vs. CoNS (detects thermostable nuclease)

• Methyl Green indicator: clearing = colorless zone

• Toluidine Blue O indicator: clearing = bright pink zone

• Without indicator: flood with 1N HCl → clear zone around colonies within 5 min = (+)

Detection of Antibiotic-Resistant S. aureus (MRSA)

1. Cefoxitin Disk Screen Test

2. Latex agglutination for PBP2a

3. Mueller-Hinton agar + NaCl + 6 µg/mL Oxacillin

4. Chromogenic agar

5. Detection of mecA gene or its product PBP2a

Identification Flowchart

Gram stain (GPC clusters) → Catalase
   (+) Staphylococcus          (–) Streptococcus
        ↓
   Coagulase Test
   (+) S. aureus    (–) CoNS
                       ↓
                  Novobiocin Test
              (S) CoNS    (R) S. saprophyticus
                  ↓
           Mannitol/Trehalose fermentation
           (–) S. epidermidis   (+) other CoNS

Key Differences Table (S. aureus / S. epidermidis / S. saprophyticus)

PART 2: STREPTOCOCCUS & ENTEROCOCCUS

General Characteristics

• Gram-positive cocci in chains or pairs

• Catalase (–), Oxidase (–), non-motile, anaerogenic (no gas), ferment carbohydrates

• Capnophilic (grow better in CO₂-rich, low-O₂ environment)

• Growth enhanced by blood, serum, glucose

• Grow well on SBA & Chocolate agar; do NOT grow on MacConkey (Gram + inhibited)

• Most species = normal microbiota except S. pyogenes

• Notorious pathogens: S. pyogenes, S. pneumoniae, S. agalactiae, Viridans strep, VRE, S. gallolyticus

  • S. agalactiae (Group B) → neonatal sepsis

  • Viridans group → subacute bacterial endocarditis

  • Enterococcus → VRE, nosocomial infections

  • S. gallolyticus → linked to colorectal cancer

Hemolysis Patterns (Smith & Brown Classification)

Lancefield Classification

• Based on C-carbohydrate extraction from cell wall (dilute acetic acid + heat 10 min)

• Most useful for classifying beta-hemolytic strep

Academic/Bergey's Classification (Temperature-based)

Group A Streptococcus (S. pyogenes)

• Highly resistant to drying; recoverable from swabs hours later

• BAP colonies: small, translucent, smooth, well-defined β-hemolysis

• Toxins:

  • Streptolysin O (oxygen-labile) → subsurface hemolysis (anaerobic); highly antigenic → ASO titer test (<200 Todd Units/mL = positive)

  • Streptolysin S (oxygen-stable) → surface hemolysis (aerobic); non-antigenic, not tested

Diseases:

• Pharyngitis/tonsillitis (strep throat) — dx by throat culture or RADT

• Scarlet fever — strawberry tongue

  • Obsolete: Dick's test (erythrogenic toxin), Schultz-Charlton test (blanching)

  • Current: RADT, throat culture (gold standard), PCR, ASO/anti-DNase B titers

Lab Tests for Group A:

Group B Streptococcus (S. agalactiae)

• Causes neonatal sepsis (transmitted via colonized birth canal)

• CDC recommends screening pregnant women at 35–37 weeks gestation

• If resistant to bacitracin → do CAMP test or Hippurate Hydrolysis test

• Profile: GPC chains, small β-hemolysis, hippurate (+), CAMP (+), Group B serotyping

Viridans Streptococci ("Alpha-prime")

• Lack Lancefield antigens

• #1 cause of subacute bacterial endocarditis

• Virulence: capsule, cytolysin, extracellular dextran, adhesins

• Profile: bile insoluble, optochin-resistant, no growth in 6.5% NaCl, LAP (+), PYR (–)

LAP (Leucine Aminopeptidase) Test:

• Substrate: Leucine-β-naphthylamide; Reagent: DMACA

• (+) = red color

• LAP (+): Viridans strep, Enterococci, S. pyogenes, S. agalactiae, S. pneumoniae

• LAP (–): Aerococcus, Leuconostoc

Streptococcus pneumoniae

• Alpha-hemolytic — if alpha-hemolytic colony seen, test for pneumococcus

• All test results are OPPOSITE of Viridans

• Formerly Diplococcus pneumoniae

• Causes lobar pneumonia

• Virulence factor: polysaccharide capsule

• Microscopy: GPC in pairs, oval/lancet-shaped

• Rusty-tinged sputum = classic clue

• Colonies: encapsulated = mucoid/dome-shaped; aged/non-encapsulated = dry, flattened ("draughtsman colonies")

• Optochin test: 5 µg disk, Susceptible ≥14mm

• Neufeld-Quellung (Capsular Swelling) Test — capsule swells with anti-capsular serum; used for detection + serotyping

• Coagglutination test — particle-bound antibody enhances visibility of Ag-Ab agglutination

Enterococcus

• Formerly "Group D enterococci" (all produce group D antigen)

• Normal GI microbiota; not highly pathogenic but frequent nosocomial cause

• Resistant to multiple antimicrobials (e.g., VRE = vancomycin-resistant)

• Most common isolate: E. faecalis

• Infections: UTI, endocarditis, bacteremia, wound infection

• Variable hemolysis

• Profile: Bile esculin (+), PYR (+), grows in 6.5% NaCl (+)

Bile Esculin Test — Group D Strep & Enterococcus vs. others

• Group D strep/Enterococcus hydrolyze esculin + tolerate bile → dark brown/black slant = (+)

• Other Gram(+) inhibited by bile salts

• Incubate 35°C, ambient air, 48 hrs

Isolation Procedure (Throat Swab)

1. Label BAP (name, date, medium, "throat swab")

2. Swab posterior pharynx/tonsil area (avoid tongue)

3. Streak plate technique; stab loop into agar at high-density area (reveals hemolysis better)

4. Incubate inverted at 35°C, 24–48h (candle jar optional for CO₂-rich environment)

5. Examine for α/β hemolysis; Gram stain to confirm GPC chains

6. If β-hemolytic → Bacitracin test; if α-hemolytic → Optochin test; if γ-hemolytic → Esculin hydrolysis test

Summary Table: Biochemical Tests

(S = Susceptible, R = Resistant, V = Variable)

Quick Comparison: Staph vs. Strep (Big Picture)

High-yield exam reminders:

• Catalase splits Staph from Strep

• Coagulase splits S. aureus from CoNS

• Novobiocin splits S. saprophyticus (R) from other CoNS (S) — "saprophyticus is Stubbornly Resistant"

• Bacitracin splits S. pyogenes (S) from other beta-hemolytic strep

• Optochin splits S. pneumoniae (S) from Viridans (R)

• Bile esculin + 6.5% NaCl splits Enterococcus/Group D from others

• CAMP/Hippurate splits S. agalactiae (Group B) from other beta-hemolytic strep