Antibiogram An antibiogram describes the susceptibility of a clinical bacterial isolate to a variety of antibiotics. These results are the most important factor in determining the choice of antibiotic to treat a patient’s infection. Other factors such as the patient’s renal function and hypersensitivity profile must also be considered in choosing the antibiotic.
Two types of tests are used to establish the antibiogram: (1) the tube dilution test that determines the minimal inhibitory concentration (MIC) and (2) the disk diffusion (Kirby-Bauer) test that determines the diameter of the zone of inhibition (see following discussion and Figures 1 and 2).
Fig1. Determination of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). Top: The patient’s organism is added to tubes containing decreasing amounts of the antibiotic. After incubation overnight, growth of the bacteria is observed visually (dark orange color indicates growth). The lowest concentration of drug that inhibits growth (i.e., 3.1 μg/mL) is the MIC. However, at this point, it is not known whether the bacteria have been killed or whether the drug has only inhibited their growth. Bottom: To determine whether that concentration of drug is bactericidal (i.e., to determine its MBC), an aliquot (0.1 mL) from the tubes is plated on an agar plate that does not contain any drug. The concentration of drug that inhibits at least 99.9% of the bacterial colonies (i.e., 6.2 μg/mL) is the MBC.
Fig2. Antibiotic sensitivity testing. A. Kirby-Bauer test. This is a qualitative test. A zone of inhibition surrounds an antibiotic containing disk. A zone of a certain diameter or greater indicates that the organism is sensitive to that particular antibiotic. Organisms that are resistant may grow all the way up to the disk. B. The Etest is a quantitative test that uses a strip impregnated with a predefined gradient of a particular antibiotic. It is applied to the surface of an agar plate inoculated with the strain of interest. The antimicrobial is released into the agar to form a continuous gradient around the strip. Once the bacteria grow, a symmetrical inhibition ellipse appears centered along the strip. The MIC is read where the edge of the inhibition ellipse intersects the side of the strip, in this case 1 μg/mL. (Reproduced with permission from Ryan KJ. Sherris & Ryan’s Medical Microbiology, 8e. New York, NY: McGraw Hill; 2022.)
Minimal Inhibitory Concentration (MIC)
For many infections, the results of sensitivity testing are important in the choice of antibiotic and are commonly reported as the MIC, which is defined as the lowest concentration of drug that inhibits the growth of the clinical isolate. The MIC is determined by inoculating the purified isolate into a series of tubes containing twofold dilutions of the drug (see Figure 1). After incubation, the lowest concentration of the drug that prevents visible growth of the organism is the MIC. This indicates a precise concentration of the drug to guide the choice of both the drug and the dose.
A second method of determining antibiotic sensitivity is the disk diffusion method, where disks impregnated with various antibiotics are placed on the surface of an agar plate that has been inoculated with the patient isolate (see Figure2). After incubation, during which time the antibiotic diffuses outward from the disk, the diameter of the zone of inhibition is determined. The size of the zone of inhibition is compared with standards to determine the sensitivity of the organism to the drug.
Minimal Bactericidal Concentration
For certain infections, such as endocarditis, it is important to know the concentration of drug that actually kills the organ ism rather than the concentration that merely inhibits growth. This concentration, the minimal bactericidal concentration (MBC), is determined by taking a small sample (0.01 or 0.1 mL) from the tubes used for the MIC assay and spreading it over the surface of a drug-free blood agar plate (see Figure 1). Any organisms that were inhibited but not killed now have a chance to grow because the drug has been diluted significantly. After incubation, the lowest concentration that has reduced the number of colonies by 99.9%, compared with the drug-free control, is the MBC. Bactericidal drugs usually have an MBC equal or very similar to the MIC, whereas bacteriostatic drugs usually have an MBC significantly higher than the MIC.
β-Lactamase Production
For severe infections caused by certain organisms, such as S. aureus and Haemophilus influenzae, it is important to know as soon as possible whether the organism isolated from the patient is producing β-lactamase. For this purpose, rapid assays testing for the enzyme can yield an answer in a few minutes, as opposed to an MIC test or a disk diffusion test, both of which take 18 hours.
A commonly used procedure is the chromogenic β-lactam method, in which a colored β-lactam drug is added to a suspension of the organisms. If β-lactamase is made, hydrolysis of the β-lactam ring causes the drug to turn a different color in 2 to 10 minutes. Disks impregnated with a chromogenic β-lactam can also be used.
