Review: Interpreting Antimicrobial Susceptibility Results

Susceptibility and Resistance

These are related terms to describe the same concept: the concentration of antimicrobial required to inhibit growth of an isolate of bacteria as it relates to the likelihood of clinical success.  "Susceptible" and "resistant" are qualitative terms used to simplify the results of tests of growth inhibition.  Problems with interpretation can occur when the definitions underlying the qualitative terms are not universally agreed upon or when different thresholds are used by the individual that performs the test and the individual that uses the results of the test.

Differentiating inherent and acquired resistance is important from a clinical and epidemiological standpoint, since the concern for resistance associated with treatment failure is generally speaking due to bacteria acquiring resistance genes from other bacteria, rather than a gene that has always been present in a particular bacterial species.  Acquired resistance is most often generated by the acquisition of new bacterial DNA by various mechanisms, including transfer of plasmids (extra-chromosomal DNA) between a resistant and a susceptible organism, or transfer of an integron or other type of moveable gene between a resistant and a susceptible organism.   Transfer of resistance can occur between bacteria of the same species, but also between bacteria of different species and even of different genera.  The significance of this transfer is that the bacteria containing these resistance genes may be selectively targeted for survival by the presence of an antimicrobial, and the resistance DNA is then carried on the next generation or is transferred to other bacteria.

Determining susceptibility or resistance

Since we have described resistance as requiring more antimicrobial than can be delivered to the infection site, we need a way to determine how much of a particular antimicrobial is required to inhibit the growth of a particular organism.  We are all familiar with susceptibility testing as performed by most clinical microbiology labs, but it is useful to briefly review these tests.

The two major types of susceptibility testing performed by veterinary diagnostic laboratories are disk diffusion and broth microdilution.  Disk diffusion testing uses paper disks containing known quantities of antimicrobials, and the zone around which no growth of bacteria occurs correlates with a particular range of antimicrobial concentrations.  The correlation between zone and MIC is a qualitative rather than a quantitative one: zones of inhibition do not linearly correspond to minimum inhibitory concentrations of antimicrobial.

Broth microdilution testing is used to characterize the quantity of antimicrobial required to inhibit bacterial growth.  Varying concentrations of antimicrobial are mixed with the broth used to grow bacterial isolates, and the lowest concentration which demonstrates no growth is the MIC.  This type of testing is usual performed with 96-well plates so multiple drugs can be tested or even more than one isolate can be tested on a plate.  Concentrations of antimicrobial which are clustered around the breakpoint are generally selected for testing, with the idea that these concentrations are also actually clinical achievable in the animal.

What is a breakpoint and how are they determined?

The purpose of the breakpoint is to provide a cutoff or threshold for categorizing organisms with different phenotypes.  The phenotypes practicing veterinarians are interested in identifying are those that might be difficult to eliminate in clinical infections because they require increased concentrations of antimicrobials to inhibit growth.  However, other groups may be interested in other cutoffs, such as epidemiologic cutoffs, whereby populations of organisms could be evaluated over time to watch for significant changes in susceptibility.  Different cutoffs might also be used to evaluate whether the majority of isolates remain wild-type, or whether acquired resistance is penetrating a population.  The breakpoints discussed in this document are clinical breakpoints, related to predicting clinical outcome of antimicrobial therapy for bacterial disease.

Through a multi-step process, data are gathered and/or generated which allow the Clinical Laboratory Standards Institute Veterinary Antimicrobial Susceptibility Testing Subcommittee (CLSI VAST) to select appropriate breakpoints.  (The CLSI is an international, interdisciplinary, nonprofit, standards-developing, and educational organization that promotes the development and use of voluntary consensus standards and guidelines within the health care community.) These data include pharmacokinetics of the drug in question, pharmacodynamics of the drug (how does it work best), and MIC data for at least 100 different isolates of the bacterial species for which the breakpoints will be valid.

For a description of how tilmicosin breakpoints for porcine respiratory disease were determined, click here, and ceftiofur breakpoints for porcine respiratory disease, click here.  A complete review of how breakpoints for human antimicrobials are determined can be found here.

References

Clinical Laboratory Standards Institute, "Performance Standards for Antimicrobial Disk and Dilution Susceptibility Test for Bacteria Isolated from Animals; Approved Standard - Second Edition," M31-A2, 2005.