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Chapter 28
Antimicrobial Susceptibility
Testing
Wang Hui
COMMON IN VITRO ANTIMICROBIAL
SUSCEPTIBILITY TESTS
• Frequently used in vitro susceptibility tests
• Infrequently used in vitro susceptibility tests
Frequently used in vitro
susceptibility tests
• Dilution tests
Broth microdilution
Agar
Semiautomated
• Diffusion tests
Disk (Kirby- Bauer)
Fixed gradient (Etest or epsilometer)
Spot tests (e.g.β-lactamase)
Infrequently used in vitro susceptibility
tests
•
•
•
•
•
•
•
Minimum bactericidal concentration (MBC)
Serum bactericidal test (SBT,”Schlichter test”)
Time-kill kinetic assay
Drug synergy test
Disk approximation
“Checkerboard”
Time-kill kinetic assay
Broth Microdilution
Minimum Inhibitory Concentration Test
The broth microdilution minimum inhibitory
concentration (MIC) test provides a quantitative
measurement (in μg/ml) of the lowest
concentration of an antimicrobial agent that
inhibit the growth of a bacterium.
Broth Microdilution
Minimum Inhibitory Concentration Test
•
•
•
•
•
•
•
Selected antimicrobics
Growth Media
Bacteria for Inoculation
Incubated of Inoculated Panels
Reading Panels
Specific Control Strains
Interpretation of MIC results
Specific Control Strains
(1)
(2)
(3)
(4)
(5)
(6)
Enterococcus faecalis, ATCC 29212;
S.aureus, (ATCC) 29213(β-lactamase positive);
Escherichia coli, ATCC 25922; and
Pseudomonas aeruginosa, ATCC 27853.
Escherichia coli, ATCC 35218
H.influenzae, ATCC 49247 or 49766 when testing
an H.influzae isolate; and
(7) Neisseria gonorrhoeae, ATCC 49226 when testing
an isolate of N. gonorrhoeae.
Interpretation of MIC results
• Susceptible,
• Intermediately susceptible, or
• Resistant.
Broth Microdilution
Disk diffusion Method(1)
This method, commonly referred to as the
Kirby-Bauer test, provides a qualitative
measure of the ability of an antimicrobic to
inhibit the growth of a rapidly growing
bacterium.
Disk diffusion Method(2)
Disks containing a given concentration of an
antimicrobic are placed on a confluently
inoculated agar plate and incubated for 16 to
24 hours. At the end of the incubation period,
zones of growth inhibition are measured across
the disk diameter and recorded to the nearest
millimeter.
Disk Diffusion Method(3)
Procedure of the Kirby-Bauer test
1、Growth Media
2、Bacteria for Inoculation
3、Filter Paper Disks Containing of an Antimicrobic
4、Zone Diameter of Inhibition
Advantages and Disadvantages of
Tests
Advantages :
1.easy to substitute one disk for another
2. dependent on a commercial provider for the drug
profiles available
3. easier to spot contamination and low-level resistance
Disadvantages:
1. use only with rapidly growing organism
2. MBC can not be done using agar diffusion techniques
E Test (1)
A new test method has been developed
recently called the E Test, which is a
modification of the disk diffusion test but
provides an MIC result.
E Test(2)
The MIC is read at the point where the zone
intersects the MIC scale on the strip. Studies
show this method to give greater than 95%
agreement with the standardized broth
microdilution method
E test(3)
Susceptibility Testing Of Anaerobic
Bacteria
The recommended control strains are:
• Bacteroides fragilis, ATCC 25285;
• Bacteroides thetaiotaomicron, ATCC 29741;
• Clostridiun perfringens, ATCC 13124;
• Eubacterium lentum , ATCC 430555
The β-lactamase Test
•The Purpose of β-lactamase Test
•Methods of β-lactamase
•Organisms of routinely testing for βlactamase
•D Test
Methods of β-lactamase
1. Acidometric Method
2. Chromogenic Cephalosporin Method
3. Iodometric Method
D Test
At the time of inoculation, a disk containing a low
concentration of an inducing antimicrobic is placed
just outside the zone of inhibition normally
obtained with an enzyme-susceptible antimicrobic
. if induced β-lactamase is produced in the
presence of the inducing agent, the zone will
become flattened at the side adjacent to the
inducing disk.
Resistance Screening Plates
Agar plates containing a specified concentration of
a single antimicrobial agent are frequently used
to screen selected organisms for resistance to the
agent.
S.aureus :oxacillin
E.faecium and E.fecalis :vancomycin
S.pneumoniae :penicillin
Detection Of Extended-Spectrum
β-Lactamase-Producing Isolates
Certain enteric Gram-negative bacilli,
primarily E.coli and Klebsiella spp.,
demonstrating resistance to various extendedspectrum cephalosporins.
ESBLs(1)
If reduced activity is detected by either diskdiffusion or MIC, a confirmation test is to be
performed that involves testing the isolate’s
relative susceptibility to both ceftazidime and
cefotaxime and without the addition of clavulanic
acid. If either the zone of inhibition increases by
>5mm or the MIC decreases <3 two-fold dilutions
when clavulanic acid is added, the isolate is
declared an ESBL-producing strain and it is
reported as resistant to all cephalosporins and
monobactams.
ESBLs(2)
ESBLs(3)
ESBLs(4)
ESBLs(5)
ESBLs(6)
ESBLs(7)
ESBLs(8)
Other Tests To Detect Drug-Inactivating
Enzymes
H. influenzae produce choramphenicol
acetyltransferase
Semi-automated Susceptibility
Testing
• Systems Composed of Major Components
• Testing Errors
• Advantages and Disadvantages of Test
INFREQUENTLY USED
ANTIMICROBIC SUSCEPTIBILITY
TEST METHODS
•
•
•
•
The Minimum Bactericidal Concentration Test
The Serum Bactericidal Test
Time-Kill Kinetics Test
Checkerboard Microdilution Test
The Minimum Bactericidal
Concentration Test
Certain organisms may be inhibited by low
concentration of an antimicrobic but require
much higher concentrations to be killed. This
can be determined by the minimum
bactericidal concentration (MBC)
The Serum Bactericidal Test
The serum bactericidal test(SBT) measures the
level of cidal antimicrobial activity in patient
serum directed against the patient`s isolate
.The result is expressed as the highest serum
dilution that retains the ability to kill 99.9% of
the inoculum .
Time-Kill Kinetics Test
The time-kill kinetic assay measures the rate of
killing while being exposed to a single drug
concentration, usually one that is
physiologically achievable at standard dosage.
The time-kill kinetic assay is currently thought
to be the best method to study in vitro drug
synergy .
Checkerboard Microdilution Test
A standard 96-well microtiter tray is loaded
horizontally with increasing concentrations of
drug A, and vertically with increasing
concentrations of drug B so that as one scans
the plate from lower left to upper right, both
drugs in each well are present in increasing
concentrations that span the therapeutic range
from easily achievable (susceptible) to
unachievable (resistant).
REFERENCES
• 1.Snydman DR, Cuchural GJ Jr, McDermont L, et al. Correlation of
various in vitro testing methods with clinical outcomes in patients with
Bacteroides fragilis group infections treated with cefoxitin:a retrospective
analysis. Antimicrob Agents Chemother 1992; 36: 504-544.
• 2.Clinical and Laboratory Standards Institute. Performance Standards for
Antimicrobial Disk Susceptibility Tests.9th ed. Standard M2-A9. Villanova,
PA: National Committee for Clinical Laboratory Standards, 2006.
• 3.National Committee for Clinical Laboratory Standards. Methods for
Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow
Aerobically.2nd ed. Standard M6-A2. Villanova, PA: National Committee
for Clinical Laboratory Standards, 1990.
• 4.Hindler JA, Swenson JM. Susceptibility testing of fastidious bacteria. In:
Murray PR, Baron EJ, Pfaller MA, Tenover FC, and Yollken RH.eds.
Manual of Clinical Microbiology.7th ed. Washington D.C.: American
Society for Microbiology, 1999: 1544-1554.
REFERENCES
• 5.Berke I, Tierno PM. Comparison of efficacy and cost-effectiveness of
BIOMIC VIDEO and vitek antimicrobial susceptibility test system for use
in the clinical microbiology laboratory. J Clin Microbiol 1996; 34: 19801984.
• 6.Livermore DM.β-lactamases in laboratory and clinical resistance. Clin
Microbiol Rev, 1995; 8:557-584.
• 7. Blaser J. Interactions of antimicrobial combinations in vitro: the
relativity of synergism. Scand J Infect Dis 1991; 74(suppl):71-79.
8.Van der Auwera P. Synergistic, additive, and antagonistic activity of
antimirobial agents: how to measure? What are the clinical consequences?
Applications to the new fluoroquinolones. Infect Dis Newsl 1992; 11:49-54.