Methods for detecting resistance
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Methods for detecting resistance
Goal: To determine whether organismexpresses resistances to agents potentiallyused for therapy
Designed to determine extent of acquiredresistance
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Methods for detecting resistance
Goals of standardization
1. Optimize growth conditions
2. Maintain integrity of antimicrobial agent
3. Maintain reproducibility and consistency
Standards set by:Clinical Laboratory Standards Institute (CLSI)
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Methods for detecting resistance
Standardization
Limits:In no way mimic in vivo environmentResults cannot predict outcome because of:
- diffusion in tissue and host cells- serum protein binding- drug interactions- host immune status and underlying illness- virulence of organism- site and severity of infection
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Methods for detecting resistance
Standardization
Inoculum size
Growth medium
Incubation atmosphere, temperature, duration
Antimicrobial concentrations used
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Inoculum preparationStandardized inoculum size using turbidity
standard
McFarland standard:0.5 McFarland = 1.5 x 108 CFU/mL
Adjust by eye or using instrument
Methods for detecting resistance
Growth mediaMueller-Hinton Agar
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Incubation conditions
Temperature: 35°C
Atmosphere: room air (most)5 – 10% CO2 (fastidious)
Methods for detecting resistance
Incubation time
GNR: 16 – 18 hrs.
GPC: 24 hrs.
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Selection of antimicrobial agents
Organism identification or group
Acquired resistance patterns of local flora
Testing method used
Site of infection
Formulary – the list of antibiotics available at the facility
Methods for detecting resistance
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Methods for detecting resistance
Directly measure the activity of one or moreantimicrobial agents against an isolate
Directly measure the presence of a specificresistance mechanism in an isolate
Measure complex interactions betweenagent and organism
Detect specific genes which confer resistance
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Methods for detecting resistance
Directly measure antimicrobial activity
Conventional methodsBroth dilutionAgar dilutionDisk diffusion
E-Test strips
Commercial systems
Special screens and indicator tests
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Conventional methods
Inoculum preparation for manual methods
Pure culture, 4 – 5 isolated colonies,16 – 24 hrs old
GNR: inoculated into broth and incubateduntil reaching log phase
GPC: suspended in broth or saline andtested directly
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Conventional methods
Broth dilution
Various concentrations of agent in broth
Range varies for each drug
Typically tested at doubling dilutions
Minimum inhibitory concentration (MIC):lowest concentration required tovisibly inhibit growth
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Conventional methods
Broth dilution
Microdilution: testing volume 0.05 – 0.1 mL
Macrodilution: testing volume >1.0 mL
Final concentration of organism:5 x 105 CFU/mL
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Conventional methods
Agar dilution
Doubling dilution is incorporated into agar
Multiple isolates tested on each plate
Final amount of organism spotted:1 x 104 CFU
Visually examine for growth, determine MIC
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Conventional methods
Disk diffusion (Kirby-Bauer)
Surface of agar plate seeded with lawn oftest organismInoculum: swab from 0.5 McFarland
Disks containing known conc. of agent placedon surface of plate
Measure diameter of zone of inhibition
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Conventional methods
Disk diffusion
Zone sizes have been correlated with MICsto establish interpretive criteria
Typically, 12 – 13 disks can be placed oneach plate
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Conventional methodsAntibiotic gradient diffusion
Agent is applied in gradient to a test strip
Plate is seeded with organism as in KB
Agent diffuses away from strip to inhibit growth
Etest (AB BIODISK, Sweden)
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Interpretive categories
Susceptible: agent may be appropriate fortherapy; resistance is absent or clinicallyinsignificant
Intermediate: agent may be useful if conc.at site of infection; may not be as usefulas susceptible agent; serves as safetymargin for variability in testing
Resistant: agent may not be appropriate fortherapy; inhibitable dose not acheivable ororganism possesses resistance mechanism
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Automated systems
Manual preparation of isolate suspension
Manual – completely automated inoculation
Automated incubation, reading of results
Automated interpretation and data management
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