Amanda T. Harrington, PhD, D(ABMM)
Associate Professor, Pathology
Director, Clinical Microbiology Laboratory
Loyola University Medical Center
ANTIMICROBIAL
SUSCEPTIBILITY TESTING
OVERVIEW
Determination of antimicrobial susceptibilities of significant
bacterial isolates is one of the principal functions of the
clinical microbiology laboratory
Provide technically accurate information
Report in easily interpreted format
Results help guide clinician in selection of most appropriate
agent
Trend is to direct clinician toward most narrow spectrum, least
expensive agent to which pathogen should respond
Purpose is to ‘predict outcome of treatment’ with
antimicrobial agents tested= in vitro testing
ANTIMICROBIAL SUSCEPTIBILITY
TESTING
When should a laboratory perform susceptibility testing?
With pathogens identified from a clinically relevant infection
With pathogens for which standardized methods are available
With pathogens where resistance is known to be a clinical problem
ANTIMICROBIAL SUSCEPTIBILITY
TESTING
When should a laboratory not routinely perform susceptibility
testing?
For organisms that are not typically considered pathogens (i.e.
normal flora)
For organisms that are not clinically relevant or in relevant quantity
from the source of recovery/specimen type
“Work up everything”
ANTIMICROBIAL SUSCEPTIBILITY
TESTING
Methods currently available
Disk diffusion
Broth Microdilution
E-test
Automated instruments
ANTIMICROBIAL SUSCEPTIBILITY
TESTING
DISK DIFFUSION
DISK DIFFUSION METHOD
Also known as the Kirby-Bauer Method
Medium
Mueller Hinton agar for most bacteria
Batch to batch reproducibility
Low in sulfonamide and tetracycline inhibitors
Years of clinical experience
Supplemented with 5% sheep blood for streptococci
Other media for fastidious bacteria
Haemophilus Test Media
Supplemented GC agar base
DISK DIFFUSION METHOD
Inoculum
Diluted at a standard concentration=Adjust to 0.5 McFarland
standard (1x108 CFU/ml)
Evenly seeded throughout the plate with the isolate of interest
Commercially prepared disks, each of which are pre-impregnated
with a standard concentration of a particular antibiotic, are then
evenly dispensed and lightly pressed onto the agar surface.
The test antibiotic immediately begins to diffuse outward from the
disks, creating a gradient of antibiotic concentration in the agar
such that the highest concentration is found close to the disk with
decreasing concentrations further away from the disk.
After an overnight incubation, the bacterial growth around each
disc is observed.
DISK DIFFUSION METHOD
Incubation
35o C.
Ambient air with exceptions
Streptococci: CO2
Haemophilus: CO2
16-18 hours with exceptions
Staphylococci and enterococci: 24 hours
S.pneumoniae: 20-24 hours
DISK DIFFUSION METHOD
Measure diameter of zone with caliper
Reflected light with exceptions
Staphylococci and enterococci examined by transmitted light for inner
colonies
HOW TO READ DISK DIFFUSION METHOD
Reflected Light vs. Transmitted Light
The zone around an antibiotic disk that has no growth is
referred to as “the zone of inhibition”
approximates the minimum antibiotic concentration sufficient to
prevent growth of the test isolate.
This zone is then measured in mm and compared to a
standard interpretation chart used to categorize the isolate as
susceptible, intermediately susceptible, or resistant.
INTERPRETATION OF DISK DIFFUSION
Inner colonies
Resistant subpopulation
Mixed culture
High-frequency mutants
Ignore swarming of Proteus
80% endpoint with
sulfonamides
CONSIDERATIONS
Advantages
Flexibility in antibiotic selection
Low cost
Simple to perform
No special equipment necessary
Most established and best proven method
Continues to be updated through CLSI
Qualitative results easily interpreted by clinician
DISK DIFFUSION METHOD
Disadvantages
Not standardized for all organisms
Qualitative results only (S/I/R)
Not rapid (results available in 16-24 hours)
Time consuming since read and entered manually
Is often media dependent
DISK DIFFUSION METHOD
BROTH MICRODILUTION
AND ETEST
Minimum inhibitory concentration (MIC) is defined as the
lowest concentration of antimicrobial agent required to inhibit
growth of the organism.
It cannot be determined from this testing whether the
concentration is bacteriocidal (i.e. the organism has been
killed).
MIC
Microtitre trays with two-fold
dilutions of antibiotics are
inoculated with a
standardized inoculum of the
bacteria and incubated under
standardized conditions.
After incubation, the MIC is
recorded as the lowest
concentration of
antimicrobial agent with no
visible growth.
BROTH MICRODILUTION TESTING
Use the record sheet for orientation of the plates
Check growth in the positive control wells
The MIC is read as the lowest concentration without visible growth
HOW TO READ BROTH MICRODILUTION
HOW TO READ MICROBROTH DILUTION
HOW TO READ BROTH MICRODILUTION
a. Control wells
b. Antibiotic 1
c. Antibiotic 2
d. Antibiotic 3
= MIC well
Etest® consists of a
predefined gradient of
antibiotic concentrations on
a plastic strip and is used to
determine the Minimum
Inhibitory Concentration
(MIC) of antibiotics
ANTIMICROBIAL GRADIENT DIFFUSION
Read MIC at the point where el l ipse intersects the scale.
I f a MIC value between two two -fold di lutions is seen, always round up to the highest value.
Remember to read the MIC value at complete inhibition of al l growth including isolated colonies.
I f the intersect dif fers on either side of the str ip, read the MIC as the greater value.
HOW TO READ E-TEST
Advantages
Generate quantitative results (MICs)
Allow testing of anaerobic and fastidious species of bacteria
Method of choice when no CLSI guidelines
Disadvantages
Increased cost over disk
Largely manual process
Quantitative data may be “challenging” to interpret for some providers
There are some systematic biases toward higher or lower MICs determined by the Etest when testing certain organism-antimicrobial agent combinations
BROTH MICRODILUTION AND E-TEST
AUTOMATED
INSTRUMENTS
BD Phoenix
Beckman Coulter (used to
be Siemens) MicroScan
WalkAway
bioMeriuex Vitek2
Thermo Scientific
Sensititer ARIS
AUTOMATED COMMERCIAL METHODS
Jorgensen, JH and Ferraro, MJ. Antimicrobial Susceptibility Testing: A Review of General Principles and Contemporary Practices.
Clin Infect Dis. (2009) 49 (11): 1749-1755.
Cartridges or broth
microdilution plates
Manual or semi-automated
inoculation
True or extrapolated MIC
Sensitive optical detection
systems allow detection of
subtle changes in bacterial
growth
Results in 3.5-24 hours
depending on system and
organism
TESTING DEVICES
Example of bacterial growth curves generated with the phenotypic VITEK2 system.
Differences in the slope and plateau levels of the curves generated in the presence or
absence of antibiotics define whether or not a strains will be deemed resistant,
intermediary or susceptible to certain antibiotics. The layout of all current automated
systems allow for the simultaneous analysis of multiple drugs for a single bacterial
species in a single diagnostic tool. Alex van Belkum; Wm. Michael Dunne, Jr. Pathogens: Wanted—Dead or Alive ASM Microbe Dec 2015
Advantages
Generate more rapid results
Include computerized data management system
Can set interpretation rules using software
Interface with LIS
Disadvantages
More expensive
Limited to commercially available panels
Failure to detect some inducible, subtle, or emerging resistance
mechanisms
Instrument interpretation
AUTOMATED INSTRUMENTS
Threshold values established for each pathogen -antibiotic (i .e., bug-drug) combination indicating at what level of antibiotic the isolate should be considered to be sensitive, intermediate or resistant. = Clinical Breakpoints
Guidelines and recommendations for these are continuously updated by organizations worldwide, such as CLSI, EUCAST.
The CLSI zone size and MIC interpretive criteria are established by analysis of 3 kinds of data: (1) microbiologic data, including a comparison of MICs and zone sizes
on a large number of bacterial strains, including those with known mechanisms of resistance that have been defined either phenotypically or genotypically
(2) pharmacokinetic and pharmacodynamic data
(3) clinical studies results (including comparisons of MIC and zone diameter with microbiological eradication and clinical efficacy) obtained during studies prior to FDA approval and marketing of an antibiotic
FDA guidelines are for newer antibiotics
Strict quality control guidelines must be followed
INTERPRETATION OF RESULTS FROM AST
A “susceptible” result indicates that the patient's organism should respond to therapy with that antibiotic using the dosage recommended normally for that type of infection and species
A “resistant” result indicates that the organism should not be inhibited by the concentrations of the antibiotic achieved with the dosages normally used with that drug
An “intermediate” result indicates that a microorganism fal ls into a range of susceptibility in which the MIC approaches or exceeds the level of antibiotic that can ordinarily be achieved and for which cl inical response is l ikely to be less than with a susceptible strain.
Categorical reporting typically provides the cl inician with the information necessary to select appropriate therapy.
Exceptions
if the antibiotic is highly concentrated in a body fluid such as urine, or if higher than normal dosages of the antibiotic can be safely administered
Reporting of MICs could aid a physician is selecting from among a group of similar drugs for therapy
S/I/R
Jorgensen, JH and Ferraro, MJ. Antimicrobial Susceptibility Testing: A Review of General Principles and Contemporary Practices.
Clin Infect Dis. (2009) 49 (11): 1749-1755.
Standardized bacterial inoculum size
Culture conditions (growth medium, pH, cation concentration
Blood and serum supplements and thymidine content)
Incubation conditions (atmosphere, temperature, duration)
Concentration of antimicrobials for testing
FACTORS THAT IMPACT RESULTS
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