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Antibiotic_Sensitivity_Testing_Presentation.pptx
- 1. Antibiotic Sensitivity Testing(AST) • Methods, Interpretation, and Quality Control • Source: Isenberg – Clinical Microbiology Procedures Handbook
- 2. Introduction • Antibiotic SensitivityTesting (AST) determines bacterial response to antibiotics. • Purpose: • • Guide effective antibiotic therapy • • Detect resistance mechanisms • • Support infection control and surveillance
- 3. Major Methods ofAST • 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
- 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 • QCStrains: 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 vitroresults 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.