CLS07 - Antimicrobial susceptibility testing

  1. Why is standardization of susceptibility testing important (3)?
    • Optimize bacterial growth conditions to ensure inhibition is properly attributed to antimicrobial agent
    • Optimize conditions for maintaining antimicrobial integrity and activity, ensuring failure to inhibit is truly due to resistance
    • Maintain reproducibility and consistency in the resistance profile of organisms, regardless of the testing laboratory
  2. What is the McFarland Standard?
    • Turbidity standards compared to organism suspension to determine CFUs per milliliter
    • .5 McF std is comparable with 1.5x10^8 CFUs/mL
  3. Why is Antimicrobial susceptibility testing not always indicative of results?
    • AST measures in vitro activity
    • in vivo activity may vary based on site of infection, hardware involved, coniditon of patient, etc
  4. When is AST not needed?
    • Organism is not a likely pathogen
    • multiple organisms w/ no predominant organism (rule of 3)
    • Organism has a predictable susceptibility profile
    • Predictability from another antibiotic (eg same group)
  5. Kirby Bauer (disk diffusion) - Method, interpretations, purpose
    • method: antibiotic paper disk placed on surface of inoculated agar (Mueller-Hinton)
    • Zone of inhibition is measured after overnight incubation
    • *concentration determined by hospital staff
    • inhibition zones are correlated with minimum inhibitory concentrations (MICs) obtained w/ broth dilution tests
    • The zone of inhibition (mm) correlated to standardized results for a viable concentration
    • interpretations: susceptable, intermediate, or resisant (based on established criteria)
    • purpose: determine the drug and concentration of drug to use
  6. Broth dilution - types, method, interpretations
    • macrodilution (>1mL tubes) and microdilution (microtiter tray)
    • *microdilution preferred (multiple drugs, automated, less space)
    • method: organisms are incubated in a standardized dilution of liquid Mueller-Hinton agar
    • trays are examined for growth
    • the MIC is the highest dilution (lowest concentration) of a drug that will completely inhibit growth
    • interpretations: susceptible, intermediate (unable to determine), resistant based on CLSI standards
  7. What are breakpoints? How are they implemented
    • Breakpoints are the specific concentrations that separate or define the interpretations of Kirby Bauer or broth dilution (susceptible, intermediate, and resistant)
    • Determined by FDA and submitted to CLSI for review/publication
    • *NOTE - CLSI determines MIC values
  8. What are the downsides to the "gold standard" agar dilution?
    • A single concentration of antibiotic is incorporated into a single plate (several plates for a single antibiotic)
    • ~32 isolates per plate
    • Plates only useful for 1 week
    • Labor intensive, expensive
  9. E test - method, interpretations
    • method: Plastic strips are impregnated with an antibiotic and placed on a plate with a lawn of growth in a radial fashion
    • interp: The scale on the strips gives the MIC by reading the mark with complete inhibition
  10. Beta-lactamase detection - method, useful for...
    • method: chromogenic cephalosporin (cefinase disk) is used to detect beta-lactamase production
    • useful: detecting resistance in N. gonorrhoeae, Haemophilus influenzae, Staphylococci, Enterocooi, Anaerobes
  11. Molecular methods - usefulness and problems
    • useful: characterize resistance of bacteria
    • clarify equivocal results
    • problems: divergent genes will be missed
    • presence of a single gene does not guarantee resistance
    • genes may be silent
    • may be impractical in clinical setting
  12. bactericidal tests - purpose, methods
    • purpose: determine MBC (minimum bactericidal concentration)
    • method: aliquot of MIC tube is subcultured to determine CFUs
    • MBC is concentration resulting in 99.9% reduction
    • serum bactericidal test uses pt serum at trough and peak concentrations of antimicrobial agent
  13. Synergy testing - purpose, results
    • purpose: synergy testing determines effectiveness of combinations of antimicrobial agents
    • results: Synergy - combination is more effective than single agent
    • indifference - combination is no better or worse than single agent
    • antagonism - combination is significantly less effective than single agent
  14. Special resulting requirements per CLSI
    • Salmonella spp and Shigella spp: do not report 1st and 2nd gen cephalosporins or aminoglycosides
    • MRSA/MRSE: do not report any beta-lactam agent as susceptible
    • Enterococcus spp: do not report cephalosporins, clindamycin, trimethoprim/sulfamethoxazole, aminoglycosides
    • Listeria: do not report cephalosporins
Card Set
CLS07 - Antimicrobial susceptibility testing
CLS07 - Antimicrobial susceptibility testing