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. 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.
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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
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.