Susceptibility testing in veterinary medicine: what you can and can't conclude from S, I, and R (Proceedings)


"Susceptible" and "Resistant" are thrown around in the fields of microbiology, medicine, public health, and epidemiology with great frequency. Unfortunately, these classifications are often used in a manner inconsistent with their correct application.

"Susceptible" and "Resistant" are thrown around in the fields of microbiology, medicine, public health, and epidemiology with great frequency. Unfortunately, these classifications are often used in a manner inconsistent with their correct application. To understand the application of veterinary susceptibility testing interpretive criteria, it is necessary to understand some key definitions and how these criteria are developed.

Veterinary breakpoints have been developed by the Veterinary Antimicrobial Susceptibility Testing (VAST) Subcommittee of the Clinical and Laboratory Standards Institute (CLSI), formerly the National Committee for Clinical and Laboratory Standards (NCCLS). The CLSI process consists of tripartite participation by academia, government, and industry (pharma, manufacturers, and private labs). In the consensus process, all parties have an opportunity to review and comment on the documents. The CLSI Area Committee on Microbiology consists of the Antimicrobial Susceptibility Testing (AST) Subcommittee (human pathogens) and the VAST Subcommittee (veterinary pathogens).

This presentation seeks to clarify the definitions of breakpoints, the relationship between serial dilution and disk diffusion breakpoints, and the inputs for CLSI-approved interpretive criteria. This proceedings article draws heavily from two CLSI publications with recognition of the efforts and contributions of the CLSI VAST Subcommittee, members of other CLSI committees that have provided valuable guidance and input, and the CLSI staff.

     • CLSI publication M31-A3 - Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Approved Standard - Third Edition.

     • CLSI publication M37-A3 – Development of In Vitro Susceptibility Testing Criteria and Quality Control Parameters for Veterinary Antimicrobial Agents; Approved Guideline – Third Edition.

     • In addition, the CLSI VAST Subcommittee has developed documents M42 and M49. These relate to antimicrobial disk susceptibility testing and broth dilution susceptibility testing of bacteria isolated from aquatic organisms, respectively.

Copies of the current editions may be obtained from CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA. The CLSI office may be reached at 610-688-0100, (fax) 610-699-0700, or on the web at In this paper, quotations from CLSI are presented in boxes or in bullet point format. Information from M31-A3 and M37-A3 are presented for purposes of general information. Only the official documents should be relied upon for guidance.

A common source of confusion is the difference between an MIC (Minimum Inhibitory Concentration), an MBC (Minimum Bactericidal Concentration) and a breakpoint. An MBC is the lowest dilution where the culture has been completely sterilized. It is not routinely determined. Treatment decisions are made related to MICs, and more specifically, the breakpoint MICs. An MIC (as defined in M31-A3) is the lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism in an agar or broth dilution susceptibility test. A breakpoint (also as defined in M31-A3) is the MIC or zone diameter value used to indicate susceptible, intermediate, and resistant. Breakpoints approved by the CLSI/VAST Subcommittee include both serial dilution interpretive breakpoints and disk diffusion interpretive zone diameters that are correlated back to the serial dilution breakpoints.

A standard consists of specific, essential requirements for materials, methods, or practices for voluntary use in unmodified form. A guideline provides criteria for a general operating practice, procedure, or material which may be used as written or modified by the user to fit specific needs.

Methods for bacterial susceptibility testing - Microwell dilution method:

This system uses a plate with wells that contain different concentrations of the selected antimicrobials (or a series of tubes). Ideally we would have a well for each antimicrobial at 1:2 dilution intervals to accurately evaluate the minimal inhibitory concentration (MIC) of the compound for each pathogen. However, practical consideration of cost often mandates focusing on the susceptible and intermediate breakpoint dilutions. For example, the CLSI/VAST approved breakpoints for ceftiofur and bovine respiratory disease are 2, 4, and 8 μg/ml (for S, I, and R, respectively). Breakpoint testing would only test against the 2 and 4 μg/ml concentrations and are interpreted in this manner.

     • A pathogen growing in neither of the wells would be considered susceptible

     • A pathogen growing only in the 2 μg/ml well would be considered intermediately susceptible

     • A pathogen growing in both wells would be considered resistant

Some labs are using an extended-range susceptibility testing plate with additional dilutions beyond just the susceptible and intermediate breakpoints. In these labs, you may receive a report with the S, I, or R interpretation along with the actual MIC.

Methods for bacterial susceptibility testing - Kirby-Bauer ("disk diffusion")

A paper disk containing the antimicrobial is placed on an agar plate that has been inoculated with the pathogen. The plate is incubated and the zones of inhibition (absence of any visible bacterial growth) are measured surrounding the disks. The diameter of the zone is correlated back to serial-dilution concentrations used to set "susceptible", "intermediate susceptibility", and "resistant" classifications for pathogens. This technique is obviously heavily dependent on quality control. Depth and contents of the agar, inoculum concentration, growth conditions, and antimicrobial contents of the disks must be closely controlled.

The size of the zone of inhibition is dependent on the MIC of the organism, the rate of diffusion of the antimicrobial in the agar (related to depth and composition), duration of incubation, inoculum concentration, and temperature of incubation. To assure that a laboratory is producing valid results, they must periodically test QC (quality control) organisms with known zone diameters to confirm they are adhering to standardized methods.

Approved CLSI/VAST breakpoints: Status as a CLSI/VAST approved veterinary breakpoint means that the breakpoint has been evaluated in light of the data mentioned above and designed to give a reasonable projection of clinical outcome. Remember, that an approved breakpoint is specific for the animal species, disease, pathogen, drug, and regimen (dose, route, duration and frequency).

When any of these factors are changed, the approved breakpoint may no longer be valid for placing the above combination of factors in a population of animal/disease/pathogen /drug where clinical success is likely or unlikely. For example, none of the breakpoints are approved for predicting clinical efficacy of therapy of enteric diseases. It is very important to know when you are, and when you are not using approved interpretive criteria for susceptibility testing results interpretation.

The CLSI has approved the following veterinary specific breakpoints. These breakpoints are detailed in CLSI M31-A3, including the specific pathogens associated with these breakpoints.

The following breakpoints are included in CLSI M31-A3 as "generic" breakpoints, where the breakpoint was determined on the basis of published pharmacokinetic parameters in the designated species in combination with available target pathogen susceptibility data.

Ampicillin - Horses (respiratory disease) and Dogs (skin and soft tissue infections)

Gentamicin - Horses (enterobacteriaceae, Pseudomonas aeruginosa, Actinobacillus spp.) and Dogs (enterobacteriaceae, Pseudomonas aeruginosa)

Oxytetracycline – Cattle (respiratory disease) and swine (respiratory disease)

For some antimicrobials used in veterinary medicine, the CLSI/VAST Subcommittee has found it necessary to use human-derived breakpoints since no sponsor has brought the information to the subcommittee to develop approved breakpoints. The VAST Subcommittee is working on developing "generic" breakpoints for veterinary labels without approved breakpoints and for extralabel uses. The following antimicrobials have human-derived breakpoint criteria adapted by the NCCLS/VAST Subcommittee. For these antimicrobials, and for extralabel use of antimicrobials with approved veterinary breakpoints, it is necessary to evaluate the susceptibility testing results in light of the MIC breakpoint used and the pharmacokinetics/pharmacodynamics of the animal and pathogen being treated.


amikacin, gentamicin, kanamycin


amoxicillin-clavulanic acid, ticarcillin-clavulanic acid

ampicillin, oxacillin, penicillin, ticarcillin, , imipenem, cefazolin


erythromycin, chloramphenicol, trimethoprim-sulfamethoxazole

rifampin, sulfisoxazole, tetracyclines, vancomycin

Interpreting susceptibility testing for extralabel applications: When the disk diffusion method is used for extralabel applications, not only are the dilution MICs suspect as to clinical application, but there is also the question of if the zone diameter criteria still correlate to the MICs. The take-home message is to know what susceptibility testing situations have veterinary approved breakpoints. For unapproved breakpoints, "susceptible" is probably better than "resistant", as this may place the "S" pathogen in a defined population of zone diameters or MICs, but the "S" result does not necessarily mean that there is an increased chance for clinical success.

What do Susceptible, Intermediate, and Resistant mean? Definitions from M31-A3 are as follows.

     • Susceptible: "A category that implies that an infection due to the isolate may be appropriately treated with the dosage regimen of an antimicrobial agent recommended for that type of infection and infecting species, unless otherwise indicated."

     • Intermediate: "A category that implies that an infection due to the isolate may be appropriately treated in body sites where the drugs are physiologically concentrated or when a high dosage of drug can be used; also indicates a "buffer zone" that should prevent small, uncontrolled, technical factors from causing major discrepancies in interpretations."

     • Resistant: "Resistant isolates are not inhibited by the usually achievable concentrations of the agent with normal dosage schedules and/or fall in the range where specific microbial resistance mechanisms are likely (eg, β-lactamases), and clinical efficacy has not been reliable in treatment studies."

Practitioners commonly receive susceptibility information from both standard dilution tests and disk diffusion tests. How do MICs and zone diameters relate? This subject is also addressed in M31-A3.

"Equivalent MIC Breakpoints: Disk diffusion zone diameters correlate inversely with MICs from standard dilution tests, usually broth microdilution. Table 2** lists the zone diameters and MIC breakpoints used for the interpretive guidelines. Zone diameters and MIC breakpoints are correlated based upon zone-diameter versus MIC regression, population distributions, pharmacokinetics, and clinical efficacy studies (also see CLSI/NCCLS document M23). However, the zone diameters may not correspond precisely to the listed MIC breakpoints due to differences in the methodologies and the original databases. Thus, the information provided in Table 2 cannot be used to convert zone diameters to absolute MIC values..."

**Table 2 discussed in the M31-A3 quotations contains serial dilution and zone diffusion interpretive criteria for antimicrobials used in food animal medicine.

How do susceptibility testing results apply to clinical outcomes in the field? A susceptibility testing result does not guarantee the treatment outcome of the specific animal from which the isolate was collected. The result indicates that the animal is in a population of an animal-drug regimen-pathogen relationship with a characteristic relationship between the probability of the different possible clinical outcomes. There may be failures with "S" isolates and there may be successes with "R" isolates. When these susceptibility testing criteria are applied to situations where clinical and/or pharmacokinetic data have not been correlated to clinical outcome, then this relationship of "S", "I", and "R" to clinical outcome may or may not exist.

How are interpretive criteria developed for Veterinary antimicrobial susceptibility testing? CLSI document M37-A3 "offers guidance for the development of quality control (QC) limits and interpretive criteria for antimicrobial susceptibility testing, performed by disk diffusion and dilution testing with bacteria isolated from animals..." This document should be consulted for an understanding of how veterinary breakpoints are developed.

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