Methods for detecting resistance

1. Methods for detecting resistance Goal: To determine whether organism expresses resistances to agents potentially used for therapy Designed to determine extent of acquired resistance

2. Methods for detecting resistance • Goals of standardization • Optimize growth conditions • Maintain integrity of antimicrobial agent • Maintain reproducibility and consistency Standards set by: Clinical Laboratory Standards Institute (CLSI)

4. Methods for detecting resistance Standardization Limits: In no way mimic in vivo environment Results 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

5. Methods for detecting resistance Standardization Inoculum size Growth medium Incubation atmosphere, temperature, duration Antimicrobial concentrations used

6. Methods for detecting resistance Inoculum preparation Standardized inoculum size using turbidity standard McFarland standard: 0.5 McFarland = 1.5 x 108 CFU/mL Adjust by eye or using instrument Growth media Mueller-Hinton Agar

7. Methods for detecting resistance Incubation conditions Temperature: 35°C Atmosphere: room air (most) 5 – 10% CO2 (fastidious) Incubation time GNR: 16 – 18 hrs. GPC: 24 hrs.

8. Methods for detecting resistance 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

9. Methods for detecting resistance Directly measure the activity of one or more antimicrobial agents against an isolate Directly measure the presence of a specific resistance mechanism in an isolate Measure complex interactions between agent and organism Detect specific genes which confer resistance

10. Methods for detecting resistance Directly measure antimicrobial activity Conventional methods Broth dilution Agar dilution Disk diffusion E-Test strips Commercial systems Special screens and indicator tests

11. Conventional methods Inoculum preparation for manual methods Pure culture, 4 – 5 isolated colonies, 16 – 24 hrs old GNR: inoculated into broth and incubated until reaching log phase GPC: suspended in broth or saline and tested directly

12. 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 to visibly inhibit growth

13. 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

14. 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

15. Conventional methods Disk diffusion (Kirby-Bauer) Surface of agar plate seeded with lawn of test organism Inoculum: swab from 0.5 McFarland Disks containing known conc. of agent placed on surface of plate Measure diameter of zone of inhibition

16. Conventional methods Disk diffusion Zone sizes have been correlated with MICs to establish interpretive criteria Typically, 12 – 13 disks can be placed on each plate

17. Conventional methods Antibiotic 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)

18. Interpretive categories Susceptible: agent may be appropriate for therapy; resistance is absent or clinically insignificant Intermediate: agent may be useful if conc. at site of infection; may not be as useful as susceptible agent; serves as safety margin for variability in testing Resistant: agent may not be appropriate for therapy; inhibitable dose not acheivable or organism possesses resistance mechanism

19. Automated systems Manual preparation of isolate suspension Manual – completely automated inoculation Automated incubation, reading of results Automated interpretation and data management