Antimicrobial therapy: interpreting susceptibility results (Proceedings)

The design of antimicrobial regimens is addressed in the next section in these proceedings ("Antimicrobial Therapy: Regimen Design"), but the concepts within regimen design related to determining the concentration of drug required to inhibit growth of bacterial pathogens deserve a more thorough discussion. Antimicrobial susceptibility testing must not be viewed as a black box into which a veterinarian places a clinical sample of an infected site and receives a "yes" or "no" from the diagnostic laboratory.

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. "Susceptible" suggests that the pathogen's growth is likely to be inhibited and clinical success is likely with labeled doses of the antimicrobial drug, and "resistant" suggest that the concentration of antimicrobial drug required to inhibit the pathogen is not achievable in the patient. Problems with interpretation of laboratory or published results 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 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.

Breakpoints are developed through a multi-step process in which data are gathered 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.

A few breakpoints have been validated for cattle, as listed below. The majority of the breakpoints for cattle have been validated for respiratory disease, so interpreting results from other pathogens should be done with caution. (These breakpoints are published by CLSI in "Performance Standards for Antimicrobial Disk and Dilution Susceptibility Test for Bacteria Isolated from Animals; Approved Standard – Second Edition," M31-A3, 2007.)

Breakpoints approved for cattle include

     • Ceftiofur (respiratory disease, mastitis)

     • Danofloxacin (respiratory disease)

     • Enrofloxacin (respiratory disease)

     • Florfenicol (respiratory disease)

     • Penicillin/novobiocin (mastitis)

     • Pirlimycin (mastitis)

     • Tetracycline as class representative (respiratory disease)

     • *Breakpoints for tetracycline and BRD are based on PK data of oxytetracycline at 20 mg/kg IM once. Criteria are only applicable to the injectable formulation.

     • Tilmicosin (respiratory disease)

     • Tulathromycin (respiratory disease)

Breakpoints for other drugs used in cattle but not developed in cattle (mostly based on human PK data, efficacy data, and population MIC data) include:

     • Ampicillin

     • Clindamycin (class representative for lincosamides)

     • Penicillin

     • Suflasoxasole (class representative for sulfonamides)

Other important points

1. A result of "S" or "susceptible" from a diagnostic lab for antimicrobial drugs with no cattle breakpoints should be used with care. See the lists above for validated breakpoints (with associated sites of infection). Also, recognize that a drug with a validated breakpoint for one site of infection (and pathogen) is not necessarily applicable to all other pathogens or sites of infection.

2. Susceptibility test results for enteric pathogens are essentially meaningless, since there are NO validated breakpoints for any pathogens or antimicrobials. There may be clinical trial data that show therapeutic efficacy for a particular pathogen with a particular drug, but there are few or no published clinical efficacy data that correlate clinical efficacy, dose, and drug concentration at the site of infection. Culture results may be useful for identifying pathogens, but susceptibility test results are not useful.

3. Veterinarians are encouraged to do as much susceptibility testing as possible, whether you test individual cases (e.g., non-responsive cases, recurring cases, valuable animals, etc.) or whether you attempt to periodically gather data in your practice for the purposes of collecting your own population data. Continual sampling of pathogen populations will allow you (and other clinicians if the data are summarized and published by a diagnostic laboratory) to select appropriate empirical therapy in the absence of or prior to availability of results from testing.

References available on request from the author