What’s New in Antimicrobial Susceptibility Testing?

Myrna T. Mendoza, M.D.*

(*Head, Section of Infectious Diseases, Department of Medicine, University of the Philippines-Philippine General
Hospital and National Kidney and Transplant Institute; Clinical Microbiology Consultant, VRP Medical Center,
Cardinal Santos Medical Center and National Kidney and Transplant Institute)

(Phil J Microbiol Infect Dis 1998; 27(3):113-115)

Specific antimicrobial treatment for infection is usually guided by the offending pathogen
and its predicted antimicrobial susceptibility pattern. The antimicrobial susceptibility test (AST)
performed on etiologic organism provides valuable information for patient care. The AST results
in most cases can predict the clinical outcome and or response to therapy.
The widely used susceptibility testing methods are the disk diffusion and broth agar
dilution tests. The minimum inhibitory concentration (MIC) of a particular drug to a particular
organism can be quantitatively measured in-vitro through the broth agar or micro/or macro-
dilution test. These testing methods have been standardized and the National Committee of
Clinica1 Laboratory Standards (NCCLS) provides susceptibility test guidelines. Most laboratories
in training tertiary hospitals in the Philippines follow the NCCLS technical guidelines.

ANTIMICROBIAL SUSCEPTIBILITY TESTING METHODS

Microbial susceptibility testing guidelines are based on MIC determinations. These are
correlated with the pharmacological properties of the antimicrobial drug and the clinical results
when the drug is used. In MIC determinations, a bacteriological breakpoint is usually drawn
between susceptible and resistant organisms. This breakpoint has been defined as the minimum
concentration at which the drug is predicted to be clinically effective against the etiologic agents.

Dilution MIC Test

Broth Dilution

In the broth dilution MIC method, various concentrations of an antimicrobial drug are
inoculated with a standard suspension of test bacteria. Following an overnight incubation at 35°C,
the MIC is determined by observing the lowest concentration of the drug that will inhibit visible
growth of the test bacteria. For full range MIC testing, 5- 8 concentrations representing a
therapeutically achievable range for an antimicrobial are usually tested. Recent modifications,
however, provide only 1-3 concentrations of each drug and results are reported.
More tests can be done at the same time by limiting dilution steps. This method can be
done by using the test tube dilutions (macrodilution) or the plastic microdilution tray
(microdilution). The broth microdilution MIC test method has been well standardized for most
fastidious rapidly growing bacteria.

Agar Dilution

Different concentrations of antimicrobial agents are incorporated onto Mueller-Hinton

(MH) agar plates. These are inoculated with an inoculum of test organisms adjusted to match 0.5
McFarland standards. Inoculation is done either by a calibrated loop or an inoculum-replicating
device (replicator). Plates are incubated at 35°C overnight and read by observing the lowest drug
concentration that inhibits visible bacterial growth. This concentration is reported as the MIC.
Simultaneous testing of several isolates is possible with this method. However, this method is
labor intensive and time consuming.
The dilution tests are generally not recommended for routine susceptibility test in the
clinical laboratory

Disk Diffusion Susceptibility Test

This is the standard AST used in most bacteriology laboratories in the country. In the disk
diffusion susceptibility test; a standardized inoculum of the organism is swabbed onto the surface
of a Muller-Hinton agar plate, then filter paper disks impregnated with antimicrobial agents are
placed on the agar. After overnight incubation at 35°C, the diameter of the zone of inhibition (ZI)
of bacterial growth around each disk is measured. The size of the ZI is inversely proportional to
the MIC of the organism. This method is an indirect measure of the susceptibility based on MIC-
zone size correlation. Based on the zones of inhibition a qualitative report of "susceptible,"
“intermediate" or "resistant" can be determined for rapidly growing non-fastidious aerobic
bacteria.
Bauer et al first described this simple method in 1966. The test however, must be
performed under controlled and standardized conditions to provide accurate results.
Recommendations on the standardized technique specifically the appropriate culture media to
use, inoculum size and concentration, disk potency and its proper storage are provided by
NCCLS. Different interpretive charts of ZI are also regularly updated and revised for a better
clinical correlation. A disadvantage is that interpretive charts vary with the organism being tested.
Current revisions include guidelines for the use of the disk diffusion test for some fastidious
aerobic bacteria.

E-test (PDM Epsilometer AB Biodisk)

AB Biodisk has recently introduced a new quantitative antimicrobial susceptibility test

method. The test provides an Epsilometer test (E-test) device for direct quantification of
antimicrobial susceptibility of microorganisms. This method combines the ease of disk diffusion
and accuracy of the MIC broth dilution techniques. The E-test utilizes a rectangular plastic strip
device that contains predefined, continuous exponential gradient of antibiotic concentrations that
correspond to MIC dilutions. This plastic test device has a reading and interpretive scale
corresponding to 2-fold MIC dilutions indicated on the surface of the strip. In this method, the
strip is applied onto the surface of an inoculated MH agar plate. The drug on the strip is
immediately released and diffused in the agar. After 24 hours of incubation, an elliptical zone of
inhibition of bacterial growth is seen around the test strip. The zone edge intersects the plastic
strip at a specific level corresponding to the inhibitory concentration of the drug that inhibits the
microorganism (See Figure). This concentration is a direct measure of the susceptibility of the
microorganism to the particular test drug. Basically a diffusion technique, the test agar and
inoculum suspension concentration are similar to the disk diffusion technique. The antibiotic
gradient strip is stable, giving reproducible inhibitory concentrations. The high correlation of the
E-test results with the broth or agar MIC dilution tests has been shown by several studies since
1988.
There are also supportive evidences that E-test is applicable to fastidious microorganisms
such as S. pneumoniae and H. influenzae. It can also be used for epidemiologic surveillance of
clinically important resistant organisms such as MRSA and multiply-resistant nosocomial
organisms.
This new method of AST, which is based on dilution and diffusion tests, will give direct
quantification of the MIC without the need of interpretive charts. E-test is now acknowledged and
accepted worldwide because it can be easily adapted in the routine bacteriology laboratory and
can provide more accurate and reliable AST results. This is going to be particularly helpful in
patients with life-threatening infections such as infective endocarditis, meningitis and sepsis.
Since it is not inexpensive, it is recommended locally to confirm questionable or unusual AST
results based on disk diffusion tests.

E-test plate showing susceptibility test result of an isolate of Pseudomonas aeruginosa:

Piperacillin-tazobactam 2 ug (S), Gentamicin 2 ug (S), Aztreonam 1.5 ug (S),
Amikacin 4 ug (S) and Ciprofloxacin 0.225 ug (S).

REFERENCES

1. Wood GL, Washington JA. Antibacterial Susceptibility Tests: Dilution and Disk Diffusion Methods. In Murray P.R (ed.):
Manual of Clinical Microbiology. Washington DC: ASM Press 1995; 1327-1341.
2. National Committee for Clinical Laboratory Standards, 1996. Performance Standards for Antimicrobial Susceptibility
Testing. M100-S5. Villanova, Pa.
3. Macias E, Mason E, Ocera H, La Rocco M. Comparison of E. test with standard broth microdilution for determining
antibiotic susceptibilities of penicillin-resistant strains of S. pneumoniae. J Clin Microbiol 1994; 32:430-432.
4. Kiska D, Kerr A, Jones M, et al. Comparison of antimicrobial susceptibility. Methods for detection of penicillin resistant S.
pneumoniae. J Clin Microbiol 1995; 33:229-232.
5. Tenover F, Baker CN, Smenson J. Evaluation of commercial methods for determining antimicrobial susceptibility of S.
pneumoniae. J Clin Microbiol 1994; 34:10-14.
6. Jorgensen JH, Homell AW, Maher L. Quantitative antimicrobial susceptibility testing of Hemophilus influenzae and S.
pneumoniae by using the E-test 1991; 29:109-114.