Antibiotic Susceptibility Tests - Types and Ways

Antibiotic Evaluation, Antimicrobial Effectiveness In Vitro In Vivo

Mar 26, 2009 Donald Reinhardt

Proper antibiotic susceptibility testing of microbial pathogens isolated from patients is critical. The use of an ineffective antibiotic could lead to a patient's death

Today, if someone gets a bacterial infectious disease the first thought is "what antibiotic will help or work?" The doctor and the patient both desire the best and most effective antibiotic. This is what makes testing and analysis of antibiotics very interesting.

In the 1950's and 60's there were sufficient antibiotics on the medical scene to require a closer look at standardized testing of antibiotics. A better way was sought to accurately and precisely forecast which antibiotic would work and which would not.

Drs. Kirby, Bauer, and Sherris established a defined scientific approach to antibiotic testing (KBS technique) . This greatly improved the accuracy and results of testing. There are several important ways to determine antibiotic susceptibility.

Solid, Liquid and Animal Tests of Antibiotics

When chemicals and antibiotics were first tested they were added to nutrient agar petri dishes (circular glass, or plastic, with larger top glass cover overlapping a bottom reservoir) with bacteria streaked over the surface. The next day a zone of inhibition was looked for as shown in figures below (click to enlarge these figures).
Bacteria can also be tested in test tubes with nutrient broth. Sequential dilutions of the chemical or antibiotic are done first, followed by addition of a measured bacterial inoculum to each tube. After overnight incubation the tubes are analyzed: clear tubes indicate inhibition and cloudy tubes indicate bacteria not inhibited by that concentration or titer of antibiotic. In this way scientists can correlate later how much antibiotic yields what zone size on agar.
The zone sizes produced on plates by dropped antibiotic disks are plotted versus the amount in the disk. Simply, this is a regression analysis graph. It is possible to establish MIC (minimum inhibitory concentration) values in broth, blood or serum in this way. Typically, the more resistant a microbe is the smaller the zone. The more susceptible, the bigger the zone.
Finally, if an antibiotic looks promising, it can be tested in mice or rabbits for toxicity (harmful) and effectiveness.

Measurement of the amount of antibiotic injected, or ingested, is the dose. The antibiotic titer is the MIC = minimum inhibitory concentration ( an actual value attainable in blood or serum). MIC is measured in micrograms or units. MIC can be evaluated in blood and tissue fluids . The higher the MIC the more resistant the bacterium is to that antibiotic.

Standardized Antibiotic Testing

Antibiotic testing should always be done with a pure culture of the organism.

The KBS technique eliminates the variables that affect the zone size and could cause false positives or false negatives.

The agar used is Mueller-Hinton. The depth and pH of the agar, inoculum size and incubation temperature are standardized. The click-on photos below show several things:

different antibiotics have characteristic zone sizes for MIC
the same antibiotic at different concentrations will give different zone diameters
resistant bacteria cause zone size decreases. No zone = complete resistance
regression analyses enable microbiologists to determine MIC vs disk zones
populations of the same species vary from susceptible (S) to intermediate (I) to resistant R)

Recently, the valuable E-test (see photo) allows determination of MIC of a microbe with one disk that forms an antibiotic gradient. This permits rapid, clear medical evaluations of antibiotic effectiveness.

Sources

Kronvall, G. 1982. J. Clin. Microbiol. 16:784-793. Analysis of a single reference strain for determination of gentamicin regression line constants and inhibition zone diameter breakpoints in quality control of disk diffusion antibiotic susceptibility testing.

Virella, G. 1997. Microbiology and Infectious Diseases. 3rd ed. National Medical Series for Independent Study. Williams & Wilkins Co, Baltimore, Md. 575pp

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Antibiotic Disk Zone Measurement in mm	Multi-Disk Antibiotic KBS Plate Test	Antibiotic Zones pH 7.2 vs 6.5
E-Test Compare Values	Relations Between Antibiotic Levels and Zone Size