Antimicrobial Susceptibility Testing

AST Foundations - Sizing Up The Bugs

• Purpose: Guides therapy, tracks resistance.
• MIC (Minimum Inhibitory Concentration): Lowest drug level preventing visible growth. 📌 MIC 'Inhibits'.

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

• MBC (Minimum Bactericidal Concentration): Lowest drug level killing 99.9% of bacteria. 📌 MBC 'Kills'.
• Breakpoints (CLSI/EUCAST): Standardized values defining S/I/R categories.

Diffusion & Dilution - Classic AST Tactics

1. Kirby-Bauer Disk Diffusion Test:

• Medium: Mueller-Hinton Agar (MHA).
• Inoculum: Standardized to 0.5 McFarland turbidity. 📌 'King Bauer's MHArena for 0.5 McChampions'.
• Procedure: Antibiotic-impregnated discs placed on inoculated MHA.
• Incubation: 35°C for 16-20h (aerobic, ambient air).
• Result: Measure zone of inhibition diameter (mm); interpret as Susceptible (S), Intermediate (I), or Resistant (R) using CLSI/EUCAST guidelines.

2. Dilution Methods (Determine Minimum Inhibitory Concentration - MIC):

• Broth Dilution (Microdilution & Macrodilution):
  • Serial twofold dilutions of antibiotic in broth (e.g., Mueller-Hinton Broth).
  • Inoculated with standardized bacterial suspension.
  • MIC: Lowest antibiotic concentration showing no visible growth after incubation (35°C, 16-20h).
• Agar Dilution:
  • Antibiotic incorporated into agar medium at various concentrations.
  • Reference method, less common in routine labs.

⭐ Mueller-Hinton agar is the recommended medium for routine susceptibility testing of non-fastidious bacteria by the Kirby-Bauer method.

Comparison: Disk Diffusion vs. Broth Dilution

Gradient & Automation - Modern AST Upgrades

• E-test (Epsilometer test):
  • Uses a strip with a predefined, continuous antibiotic gradient.
  • Placed on an inoculated agar plate, forms an elliptical zone of inhibition.
  • MIC is read directly where the inhibition ellipse edge intersects the strip.
• Automated Systems (e.g., VITEK, BD Phoenix, MicroScan):
  • Principle: Turbidimetry (measures growth by ↑ turbidity) or colorimetry (detects metabolic activity via color change).
  • Advantages: Rapid results (e.g., 4-18 hrs), standardization, data management, high throughput.
  • Disadvantages: High cost, specific consumables, may miss certain resistance mechanisms (e.g., inducible resistance).

⭐ The E-test is particularly useful for determining MICs of fastidious organisms and for drugs not available in disk format for Kirby-Bauer.

Resistance Spotlight - Unmasking Superbugs

• Key Resistance Mechanisms & Detection:

mCIM positive result (Carbapenemase)

⭐ The presence of ESBLs is confirmed by demonstrating synergy between an indicator cephalosporin and clavulanic acid.

• Carbapenemase Detection Algorithm (Phenotypic):

High‑Yield Points - ⚡ Biggest Takeaways

• Kirby-Bauer disc diffusion measures zones of inhibition to determine susceptibility.
• MIC (Minimum Inhibitory Concentration) is the lowest antibiotic concentration preventing visible bacterial growth.
• MBC (Minimum Bactericidal Concentration) is the lowest antibiotic concentration killing 99.9% of bacteria.
• E-test (epsilometer test) determines MIC using a strip with an antibiotic gradient.
• CLSI guidelines standardize AST methods and interpretation criteria.
• Key resistance mechanisms detected include ESBLs, MRSA, and VRE.
• Automation (e.g., VITEK, MicroScan) provides rapid and standardized AST results for high-throughput labs.