Antimicrobial Susceptibility Testing

AST Fundamentals - Test Tube Tales

• Purpose: Guides therapy, tracks resistance.
• Core Terms:
  • MIC: Minimum Inhibitory Concentration; lowest drug level inhibiting visible growth.
  • MBC: Minimum Bactericidal Concentration; lowest drug level killing 99.9% of bacteria.
  • Breakpoint: MIC threshold defining Susceptible (S), Intermediate (I), Resistant (R).
• Breakpoint Utility:
  • Clinical: Predicts treatment success.
  • Epidemiological: Monitors resistance trends.

⭐ MIC is the lowest concentration of an antimicrobial that inhibits visible growth of a microorganism after overnight incubation.

Diffusion Methods - Zone Rangers

• Kirby-Bauer (Disk Diffusion) Test:
  • Medium: Mueller-Hinton Agar (MHA), depth 4mm.
  • Inoculum: Standardized to 0.5 McFarland turbidity.
  • Incubation: 35°C for 16-18 hours (aerobic).
  • Disks: Antibiotic-impregnated paper disks.
  • Result: Measure zone of inhibition (ZOI) diameter.
• Zone Interpretation:
  • Compare ZOI diameter to standards (CLSI/EUCAST).
  • Categories: Susceptible (S), Intermediate (I), Resistant (R).
• Factors Affecting Zone Size:
  • Inoculum density (↑ density → ↓ zone).
  • Agar depth (↓ depth → ↑ zone).
  • Temperature & time of incubation.
  • Drug diffusion rate.
  • Bacterial growth rate.

⭐ Mueller-Hinton Agar (MHA) is the standard medium for Kirby-Bauer testing due to its defined composition and minimal inhibition of common antimicrobials like sulfonamides and trimethoprim.

Dilution & Gradient Methods - Potion Potency

• Broth Dilution (Micro/Macro):
  • Serial dilutions of antimicrobial in broth.
  • Minimum Inhibitory Concentration (MIC): Lowest concentration inhibiting visible bacterial growth.
  • Minimum Bactericidal Concentration (MBC): Lowest concentration killing ≥99.9% of bacteria (determined by subculturing from clear MIC tubes).

  ⭐ MBC is the lowest concentration of an antimicrobial that kills ≥99.9% of the initial inoculum.

• Agar Dilution:
  • Antimicrobial incorporated into agar medium at various concentrations.
  • MIC: Lowest concentration preventing visible colony formation.
• E-test (Epsilometer test):
  • Plastic strip with a predefined antimicrobial gradient on an inoculated agar plate.
  • MIC: Read where the elliptical zone of inhibition intersects the calibrated strip.

Resistance & Special Tests - Superbug Sleuthing

• MRSA: Screen: Cefoxitin disk (zone ≤ 21mm for S. aureus). Confirm: mecA gene PCR.

• ESBLs: Screen with indicator cephalosporins (e.g., Cefotaxime, Ceftazidime). Confirm: DDST (keyhole effect) or Combination disk test (drug + Clavulanate vs. drug alone, zone diameter difference ≥ 5mm).

• AmpC β-lactamases: Often resistant to Cefoxitin. Detection challenging; use inhibitor-based tests (e.g., with cloxacillin) or AmpC disk test.

• Carbapenemases: Modified Carbapenem Inactivation Method (mCIM): Test isolate inactivates meropenem. Carba NP test: Color change. Hodge test (historical).

• Inducible Clindamycin Resistance: D-test.

  ⭐ A positive D-test (flattening of clindamycin zone ["D" shape] adjacent to an erythromycin disk) indicates inducible erm gene-mediated resistance to clindamycin, predicting potential clinical failure despite in vitro susceptibility to clindamycin alone.

High‑Yield Points - ⚡ Biggest Takeaways

• Kirby-Bauer (disk diffusion) assesses susceptibility via zone of inhibition (ZOI) diameter.
• MIC is the lowest drug level preventing visible bacterial growth in vitro.
• MBC is the lowest drug level killing 99.9% of the initial bacterial inoculum.
• E-test uses an antibiotic gradient strip for direct MIC value determination.
• Broth dilution quantitatively determines MIC; can also find MBC.
• Interpret AST results (S, I, R) using current CLSI/EUCAST guidelines.
• Detection of MRSA, VRE, ESBLs requires specialized AST methods and criteria.