Transcript Testing for Antibacterial Susceptibility Part 2

Methods for detecting resistance
Goal: To determine whether organism
expresses resistances to agents potentially
used for therapy
Designed to determine extent of acquired
resistance
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 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
Methods for detecting resistance
Standardization
Inoculum size
Growth medium
Incubation atmosphere, temperature, duration
Antimicrobial concentrations used
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
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.
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
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
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
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
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
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 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
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
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)
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
Automated systems
Manual preparation of isolate suspension
Manual – completely automated inoculation
Automated incubation, reading of results
Automated interpretation and data management