Antimicrobial Susceptibility

Antimicrobial susceptibility tests are used to determine which specific antibiotics a particular bacteria or fungus is sensitive to. Most often, this testing complements a Gram stain and culture, the results of which are obtained much sooner. Antimicrobial susceptibility tests can guide the physician in drug choice and dosage for difficult-to-treat infections. ^[1]

Results are commonly reported as the minimal inhibitory concentration (MIC), which is the lowest concentration of drug that inhibits the growth of the organism. Reports typically contain a quantitative result in µg/mL and a qualitative interpretation. The interpretation usually categorizes each result as susceptible (S), intermediate (I), resistant (R), sensitive-dose dependent (SD), or no interpretation (NI).

The results of these tests have no true normal value. The presumed baseline would be “susceptible.” However, in the era of antibiotic resistance, this is not always true.

A high value means that more drug is needed to affect the organism’s function or replication.

A low value means that less drug is needed to affect the organism’s function or replication.

Minimal inhibitory concentration

The MIC is the lowest concentration of drug that inhibits the growth of the organism. Laboratories investigate this by inoculating the organism isolated from the patient into a series of tubes or cups that contain two-fold dilutions of the drug. After a standardized incubation, the lowest concentration of drug that prevents visible growth of the organism is the MIC.

Susceptibility is individual to the organism tested. By itself, it does not describe presence, type, or location of infection.

Culture and susceptibility should be taken under conditions that minimize contaminants. This may be under sterile conditions in an operating room or just clean conditions in a treatment room, office, or emergency department. Note that sterile conditions do not technically exist in the face of active infection. However, the goal is to collect only the organism that is causing infection.

Only one sample is usually needed just before treatment is initiated. Not all organisms are easy to grow in culture, and laboratories typically take about two days to return results.

Specimens can be sent as a swab or in a sterile collection cup. If the specimen is a liquid, then it is best sent in a sterile collection cup, as the laboratory can perform more tests more readily. This needs no preservative or additional preparation for the laboratory to use it. In some cases, specimens are collected in special containers, depending on the procedure needed to collect it (eg, bronchoalveolar [BAL] traps or pleural fluid Vacutainers). These are best transferred to a standard laboratory specimen cup before being sent to the laboratory.

Blood or other bodily fluids collected with the specimen will not change or invalidate the results.

Culture and susceptibility are often performed together, but not in all cases. They are also not usually included with a Gram stain.

Description

Minimal inhibitory concentration

The MIC is the lowest concentration of drug that inhibits the growth of the organism. It is garnered by inoculating the organism isolated from the patient into a series of tubes or cups that two-fold dilutions of the drug. After a standardized incubation, the lowest concentration of drug that prevents visible growth of the organism is the MIC.

The disk diffusion method is a second method of determining antibiotic sensitivity (see image below). With this technique, disks impregnated with various antibiotics are placed on the surface of an agar plate that has been inoculated with the organism isolated from the patient. The antibiotic diffuses outward from the disk over a standard incubation time, and the diameter of the zone of inhibition is measured. The size of this zone is compared with standards to determine the sensitivity of the organism to the drug.

A novel version of this test involves a quantitative diffusion gradient, or Epsilometer (E-test), and uses an absorbent strip with a known gradient of antibiotic concentrations along its length. When the strip is placed on the surface of an agar plate seeded with a microbe to be tested, antibiotic diffuses into the medium. The lowest concentration that inhibits growth is the MIC.

A study by Webber et al indicated that in the disk diffusion test for antimicrobial susceptibility, a 6-hour incubation time can produce results comparable to a 24-hour incubation period. The concordance between tests with a 6-hour period and those with a 24-hour period was 96.7%, while a 10-hour incubation period had the same concordance rate (96.7%) with a 24-hour period. ^[2]

Minimal bactericidal concentration

For certain infections, it may be important to know the concentration of drug that actually kills the organism rather than just inhibiting its growth. This concentration, called 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 blood agar plate. Any organisms that were inhibited but not killed in the MIC test now have a chance to grow because the drug has been diluted significantly. After a standard incubation, the lowest concentration that has reduced the number of colonies by 99.9% is the MBC. Bactericidal antibiotics usually have an MBC equal or very similar to the MIC, whereas bacteriostatic antibiotics usually have an MBC significantly higher than the MIC.

For hard-to-grow organisms, such as obligate anaerobes, routine susceptibility testing is generally not performed. Fortunately, their sensitivities are often predictable.

Beta-lactamase production

For severe infections caused by capable organisms, such as Staphylococcus aureus and Haemophilus influenzae, it is important to know early whether the organism is producing beta-lactamase. Therefore, rapid assays for the enzyme can determine activity in a few minutes versus around two days for a standard MIC test.

Susceptibility testing of fungal agents

In the era of fungal antibiotic resistance, the need for testing of individual fungal isolates for susceptibility to specific antifungal agents has increased. These methods, which are used to determine the minimal fungicidal concentration (MFC), are similar to the tests for bacterial MIC. In most cases, testing must be specifically requested and may not be available at all laboratories.

Indications/Applications

Antimicrobial susceptibility is an appropriate test whenever a specimen is collected from a suspected infection site. In the face of active infection, this information, along with the Gram stain and culture, allows the physician to select an appropriate antimicrobial agent to treat an infection. The MIC and MBC can also aid in proper dosage.

Considerations

In some cases, the laboratory cannot grow a pathogen, even if the sample was taken from a patient with an active infection. So, a negative culture result may not mean the patient is infection-free. In this case, a second sample may be sent. In addition, it is up to the judgement of the treating physician to choose antibiotics and treatment length when no culture or susceptibility is available.

In simple infections or infections with known pathogens, this test is unnecessary. Also, depending on the access to a lab, the complexity of the infection, or the duration of complete treatment, one may not want to send a sample for culture and susceptibility. For example, if the treatment for a common, uncomplicated UTI lasts 3 days, the results of a culture and susceptibility may not impact treatment as typical time for results are 2-5 days. This may change with new technology on the horizon.

Lastly, tracking the antibiotic susceptibility for each organism allows a hospital or community to make antibiogram charts. These can guide clinicians to good initial treatment regimens.