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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
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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
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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
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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)
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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