Antibiotic_Sensitivity_Testing_Presentation.pptx

Antibiotic_Sensitivity_Testing_Presentation.pptx

1. Antibiotic Sensitivity Testing (AST) Methods, Interpretation, and Quality Control Source: Isenberg – Clinical Microbiology Procedures Handbook

2. Introduction Antibiotic Sensitivity Testing (AST) determines bacterial response to antibiotics. Purpose: • Guide effective antibiotic therapy • Detect resistance mechanisms • Support infection control and surveillance

3. Major Methods of AST 1. Disk Diffusion (Kirby–Bauer Method) 2. Broth Dilution (Macro & Micro) 3. E-test (Gradient diffusion) 4. Time-Kill Assay 5. Checkerboard Synergy Testing 6. Automated Systems (e.g., VITEK, BACTEC)

4. Disk Diffusion (Kirby–Bauer Method) Principle: Antibiotic diffuses from disk into agar; inhibits bacterial growth. Medium: Mueller–Hinton Agar (pH 7.2–7.4, 4 mm depth) Steps: • Prepare 0.5 McFarland inoculum • Swab agar plate evenly • Place antibiotic disks • Incubate 16–18 hrs at 35°C • Measure inhibition zones and interpret using CLSI standards

5. Broth Dilution Method (MIC) Types: Macrodilution and Microdilution Principle: Determines lowest antibiotic concentration preventing visible growth (MIC) MBC: Lowest concentration killing ≥99.9% bacteria Results guide therapeutic dosing

6. E-test (Gradient Diffusion) Combines principles of diffusion and dilution. Plastic strip with predefined antibiotic gradient on inoculated agar. MIC read at point where elliptical zone intersects strip.

7. Time-Kill Assay Purpose: Study bactericidal activity over time. Method: Count viable cells at intervals after antibiotic exposure. Interpretation: • 3-log₁₀ CFU/mL reduction = bactericidal • Synergy = ≥2-log₁₀ CFU/mL reduction vs single agent

8. Checkerboard Synergy Testing Used for evaluating antibiotic combinations. Principle: Two drugs tested in 2D dilution matrix. Result expressed as FIC Index (FICI): • FICI ≤ 0.5 → Synergy • FICI > 4.0 → Antagonism

9. Interpretation & Reporting Follow CLSI M7-A6 standards. Interpret MIC or zone size using breakpoints. Report categories: • S – Susceptible • I – Intermediate • R – Resistant Confirm atypical resistance (e.g., ESBL, MRSA, VRE).

10. Quality Control QC Strains: E. coli ATCC 25922, S. aureus ATCC 25923, etc. Check media pH, disk potency, and inoculum density. Maintain QC logs and investigate deviations.

11. Limitations In vitro results may not always predict in vivo outcomes. Resistant subpopulations may be missed. Fastidious organisms may need special methods.

12. References Isenberg HD. Clinical Microbiology Procedures Handbook, ASM Press. NCCLS/CLSI Standards: M2-A8, M7-A6. Jorgensen JH & Ferraro MJ, Clin Infect Dis., 2000.