Gram-Positive Bacteria Laboratory Identification Algorithm

Gram-Positive Bacterial Identification Algorithm

Gram Stain & First-Look Morphology

• Gram-positive organisms retain crystal-violet → appear purple/blue under light microscopy.
• Initial branching in the algorithm is based on cell shape:
– Cocci (spherical)
– Bacilli (rod-shaped)
– Branching Filaments (appear “fungus-like” on Gram stain)

Oxygen Requirement Sub-branch

• Aerobic / Facultative Aerobic – capable of growth in the presence of O₂.
• Anaerobic – optimal or exclusive growth in low- or no-O₂ environments.

Bacilli Branch

• Aerobic/FACULTATIVE:
– Listeria (short, tumbling motility at room temp; neonatal sepsis, meningitis).
– Bacillus (spore formers; B. anthracis, B. cereus).
• Anaerobic:
– Clostridium (obligate anaerobes, spore forming; tetanus, botulism, gas gangrene, colitis).
– Cutibacterium (formerly Propionibacterium; acne, shunt infections).
– Corynebacterium (club-shaped, tellurite agar; diphtheria).
• Branching filamentous rods:
– Nocardia – Aerobic, weakly acid-fast (partial mycolic acids), opportunistic pneumonia, brain abscesses, cutaneous infection.
– Actinomyces – Anaerobic, not acid-fast; oral/facial “lumpy jaw,” sulfur granules.

Cocci Branch – Catalase Test (H₂O₂ → H₂O + O₂)

• Catalase-positive: Staphylococcus (grow in grape-like clusters).
• Catalase-negative: Streptococcus & Enterococcus (grow in pairs/chains).

Staphylococcus Arm

1. Coagulase Test (converts fibrinogen → fibrin clot)
   • Positive ⇒ Staphylococcus aureus (gold colonies, complete \beta-hemolysis).
   • Negative ⇒ CoNS (coagulase-negative staphylococci).
2. Novobiocin Sensitivity (disk diffusion) for CoNS discrimination:
   • Sensitive – S. epidermidis (prosthetic-device & catheter infections; biofilms).
   • Resistant – S. saprophyticus (UTI in sexually active ♀).

Streptococcus / Enterococcus Arm

1. Blood-Agar Hemolysis Pattern
   • \beta-Hemolysis (complete, clear zone)
   – Group A Streptococcus (pyogenes): Bacitracin S, PYR +, causes pharyngitis, rheumatic fever, necrotizing fasciitis.
   – Group B Streptococcus (agalactiae): Bacitracin R, PYR –, neonatal meningitis, sepsis, pneumonia.
   • \alpha-Hemolysis (partial, green)
   – Streptococcus pneumoniae: encapsulated, bile soluble, Optochin S; lobar pneumonia, meningitis, otitis media.
   – Viridans group (S. mutans, S. mitis): no capsule, Optochin R; dental caries, endocarditis on damaged valves.
   • \gamma-Hemolysis (none) / possible \alpha
   – Enterococcus (E. faecalis, E. faecium): growth in 6.5\% NaCl, PYR +; UTI, subacute endocarditis, biliary infection, VRE nosocomial pathogen.
   – Streptococcus gallolyticus (formerly S. bovis biotype I): associated with colon cancer & endocarditis; nonenterococcus.

2. Additional Key Tests
   • Bacitracin Sensitivity – rapid screen for Group A (S) vs Group B (R).
   • PYR (pyrrolidonyl arylamidase) – Group A strep & Enterococcus are PYR +.
   • Optochin Sensitivity & Bile Solubility – confirm S. pneumoniae.
   • Growth in 6.5 % NaCl broth – hallmark of Enterococcus spp.

Summary of Diagnostic Pivot Points (Bold on Algorithm)

• Catalase (+ vs –) – separates Staph vs Strep/Enterococcus.
• Coagulase – pinpoints S. aureus.
• Hemolysis pattern – triages Streptococci.
• Bacitracin/Optochin/Novobiocin disks, PYR, salt tolerance, bile solubility – fine-tune species ID.
• Important pathogens are designated in bold italics on the original sheet.

Linking to Disease & Therapy (Clinical Pearls)

• S. aureus – toxin-mediated diseases (TSS, scalded-skin), MSSA vs MRSA (altered \mathrm{PBP2a}).
• S. epidermidis – treat biofilm infections with rifampin + vancomycin.
• Group A Strep – early antibiotics prevent acute rheumatic fever but not PSGN.
• Group B Strep – intrapartum penicillin prophylaxis if maternal vaginal culture positive.
• Enterococcus – intrinsic cephalosporin resistance; VRE carries vanA/vanB (changes D-Ala-D-Ala → D-Ala-D-Lac).
• Nocardia – sulfonamide sensitive; mimic TB radiographically.
• Actinomyces – high-dose penicillin G; drains with sulfur granules.

Ethical & Practical Considerations

• Rapid, inexpensive bedside tests (catalase, coagulase, PYR) enable early targeted therapy and antimicrobial stewardship.
• Differentiation of colonizer vs pathogen (e.g., CoNS vs S. aureus) prevents overtreatment.
• Infection-control implications: MRSA & VRE require contact precautions.

Important note from transcript: Enterococcus may appear \alpha- or \gamma-hemolytic (highlighting need for confirmatory tests).

Mnemonic Aids
• “SHy ESE at NoN’s – S. Saprophyticus R, Epidermidis S to Novobiocin.
• “B-Babies, A-Adults” – Group B affects newborns; Group A affects older kids/adults.