Antimicrobial Susceptibility Testing

Antimicrobial Susceptibility Testing

Antimicrobial Susceptibility Testing

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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.

Kirby-Bauer plate with zones of inhibition

⭐ 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. E-test strips showing zones of inhibition and MIC values

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. D-test for inducible clindamycin resistance

    ⭐ 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.
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Practice Questions: Antimicrobial Susceptibility Testing

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4 Bacterial Growth Environments_____ - environment w/ O2Obligate Anaerobes - environment w/o O2Facultative Anaerobes - either w/ or w/o O2Intracellular bacteria

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4 Bacterial Growth Environments_____ - environment w/ O2Obligate Anaerobes - environment w/o O2Facultative Anaerobes - either w/ or w/o O2Intracellular bacteria

Obligate Aerobes

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