Methods for detecting resistance l: To determine whether organism resses resistances to agents potentially d for therapy igned to determine extent of acquired istance
Methods for detecting resistance Goal: To determine whether organism expresses resistances to agents potentially used for therapy Designed to determine.
Jan 17, 2016
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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
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)
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
Methods for detecting resistance
Standardization
Inoculum size
Growth medium
Incubation atmosphere, temperature, duration
Antimicrobial concentrations used
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
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.
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
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
Methods for detecting resistance
Directly measure antimicrobial activity
Conventional methodsBroth dilutionAgar dilutionDisk diffusion
E-Test strips
Commercial systems
Special screens and indicator tests
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
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
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
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
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
Conventional methods
Disk diffusion
Zone sizes have been correlated with MICsto establish interpretive criteria
Typically, 12 – 13 disks can be placed oneach plate
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)
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
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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