Antimicrobial Susceptibility Testing
by Renee A. Watase, M.T. (A.S.C.P.),MBA and Thomas S. Reppun, M.D.
Introduction
The appropriate antimicrobial treatment of infectious diseases depends on many factors. The physician must take into consideration the natural history of the infection and the immune status of the patient. The pharmacological properties of the antibiotics must also be considered – ease of absorption, protein-binding, metabolism, ability to reach the site of infection, and excretion. The clinical laboratory can contribute a number of pieces of information to the process. In this technical bulletin, antimicrobial susceptibility testing will be discussed in detail.
Susceptibility Methods
The susceptibility methods used by clinical laboratories include Kirby-Bauer disc diffusion test, macrotube dilution susceptibility test, and the microtube dilution test. In the Kirby-Bauer test, discs containing antibiotics are placed over an agar plate inoculated with the organism. The size of the zone of inhibition is equated with clinical experience of whether or not the organism is sensitive or resistant to the antibiotic at standard doses. Antibiotic susceptibilities are reported in a qualitative form (Susceptible, Intermediate, or Resistant).
Minimal Inhibitory Concentration
The Minimal Inhibitory Concentration (MIC) is the lowest concentration of the antibiotic (mcg/ml) that will inhibit bacterial growth in vitro. The MIC is correlated with the concentration of the antibiotic achievable in blood. The traditional method of determining the MIC is with a macrotube dilution technique. A standard inoculum is tested against serial dilutions of a particular antibiotic (See Figure). In order to determine the MIC for many antibiotics, many tubes of organisms and antibiotics must be tested – a tedious and expensive process. In the VITEK identification and susceptibility system used by DLS, tiny wells of an automated plastic susceptibility card are injected with standard dilutions of antimicrobial agents by the manufacturer. The laboratory staff adds a standard concentration of the organism to the card and the organism is automatically dispersed to all of the wells. The card is incubated for 2-24 hours, and while it is incubating, it is automatically read for turbidity of growth at hourly intervals. A growth curve for the isolate is calculated for each antibiotic. The algorithm stored in the VITEK system analysis program then accurately calculates the organism’s MIC for each of the different antibiotics included on the plastic card. This method of MIC determination is comparable to the “gold standard” of macrotube dilution MIC testing as described above.
The MIC results of the VITEK microtube system are reported with the traditional qualitative interpretation – susceptible, intermediate, or resistant. In some situations, an organism or an antibiotic cannot be tested in the VITEK system. In these cases, a Kirby-Bauer test is performed and only the qualitative interpretations are reported.
The antibiotic MIC values and interpretations that are reported are those which are included by the manufacturer on the VITEK susceptibility card. The antibiotics on a test card are grouped according to whether the organism being tested is Gram positive or Gram negative. The antibiotics for the Gram negative isolate are also grouped according to whether they will be used in an inpatient or an outpatient setting. The Gram negative organisms can be tested against both inpatient and outpatient drugs if the patient is to be admitted or discharged, and the request is given to the laboratory within 3 days from the time of the report. If there is a need to test a particular antibiotic that is not on a VITEK card, qualitative testing via the Kirby-Bauer method can be requested.
MIC Interpretation
Using the MIC data, the dosage of antibiotics may be refined. The MIC result is used in conjunction with therapeutic dosing information to select the most appropriate antimicrobial agent. Due to the peak and trough fluctuation of serm antibiotic levels, 2-4 times the organism’s MIC result should be targeted for the blood level. Specific measurement of the blood levels is not indicated unless the patient is not responding to the antibiotic or there are conditions which might adversely affect the metabolism or excretion. The clinician must also account for the tissue involved since an appropriate tissue concentration is more difficult to achieve than blood. A higher dose may be necessary to insure adequate tissue coverage.
Examples Of Interpretation
The following examples of the use of MIC results are based on information from various sources, listed in the reference section.
The following examples of the use of MIC results are based on information from various sources, listed in the reference section.
Example 1
Results:
- Source: Blood
- Organism tested: Escherichia coli
- Ampicillin MIC: 2 mcg/ml (sensitive)
Interpretation:
- 2-4 x MIC: 4-8 mcg/ml
- Peak blood level based on IV representative dose: 47 mcg/ml
- Since the 2-4x organism MIC is less than the peak blood level (47 mcg/ml), using the representative IV dose for ampicillin will provide adequate therapy.
Example 2
Results:
- Source: Leg wound
- Organism isolated: Escherichia coli
- Ampicillin MIC: 16 mcg/ml (intermediate)
Interpretation:
- 2-4 x MIC: 32-64 mcg/ml
- Peak blood level based on IV representative dose: 47 mcg/ml
- The 2-4x organism MIC (32-64 mcg/ml) falls within and over the range of the obtainable peak blood level for an IV representative dose. In addition, the site of infection is tissue. Since the concentration of antibiotics in tissue would be less than in blood, a higher dosage or different antibiotic should be considered.
MIC Range Of Results
The range of possible MIC results for a particular antibiotic and organism interpretation is quite variable as noted above. The MIC limits used by the microbiology laboratory for determining the qualitative interpretations of susceptible, intermediate, and resistant are derived from the latest NCCLS (National Committee on Clinical Laboratory Standards) publications.
References:
- Hoeprich, P.D. “Infectious Diseases”, 3rd ed., 1983, pp. 195-201.
- Howard, B.J., et. al., “Clinical and Pathogenic Microbiology”, 1987, pp. 150-151.
- NCCLS; M7-A4: “Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically – Fourth Edition: Approved Standard” (Includes charts M100-S7), 1997.
- Sanford, Jay P., et. al., “Guide to Antomicrobial Therapy”, 26th ed., 1996.
