Methods for detecting resistance

Methods for detecting resistance

Goal: To determine whether organismexpresses resistances to agents potentiallyused for therapy

Designed to determine extent of acquiredresistance


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)


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


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


Growth mediaMueller-Hinton Agar

Incubation conditions

Temperature: 35°C

Atmosphere: room air (most)5 – 10% CO2 (fastidious)


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



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


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


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


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


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


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


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

Methods for detecting resistance

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