LABORATORY DIAGNOSTIC TESTS AND HOW TO INTERPRET THEM


priate for horses, particularly for orally adminis- tered antimicrobials, which often have reduced absorption and bioavailability in horses as compared to humans. For drugs that do not have recommen- dations for horses specifically, clinicians must rely upon knowledge of pharmacokinetic studies of those drugs in horses whenever available, as well as basic pharmacology and the type of infection being treated. The rationale for this how-to session is the challenges clinicians face in interpreting MIC data as they relate to infections in horses, especially when the lab does not provide horse-specific break- points. The objectives are to help practitioners in interpreting MIC data provided by the microbiology lab, and how to predict susceptibility to antimicro- bials for which there are no equine-specific CLSI or microbiology laboratory guidelines.


2. Materials and Methods


Tests Available for Obtaining Antimicrobial Susceptibility Results


Disk Diffusion (Kirby-Bauer) This is a qualitative test and as such does not pro- vide aMIC value. Instead, the results are reported simply as “susceptible” or “resistant” based on a zone of inhibition of bacterial growth on culture media created by the antimicrobial contained in a paper disk. The disks contain a single concentra- tion of each antibiotic. They are placed on the agar plate, which was previously inoculated with the bacterial isolate being tested. The antimicro- bial contained within the disk diffuses outward concentrically into the agar. After 24 hours of incubation, the zone of inhibition of bacterial growth around each disk is measured. The bac- terial isolate is deemed susceptible, intermediate or resistant based on the diameter of this zone of inhibition. The primary disadvantage of the Kirby-Bauer technique is that it only tests one concentration of each antimicrobial. Because this single concentra- tion is based on the drug disposition in humans, it may not be pertinent to horses as this concentration may be unachievable in horses if the drug is not absorbed well in this species. Therefore, the bacte- rial isolate would be considered resistant in vivo in horses, yet is reported as susceptible based on the in vitro result as it pertains to human concentrations. This would be misleading to equine clinicians, and potentially result in therapeutic failure if the neces- sary concentrations are not achieved in the horse.


Broth Microdilution


This is a quantitative test in which the cultured bacteria are inoculated into wells on a plate contain- ing serial dilutions of the antimicrobials to be tested. For example, the wells may contain serial dilutions of 8, 4, 2, 1, 0.5, and 0.25 g/mL for minocycline. The plates are examined after 24 hours with an


238 2019  Vol. 65  AAEP PROCEEDINGS


analytical instrument that detects bacterial growth. The MIC is the lowest concentration of antimicrobial that inhibits bacterial growth. For example, if growth is present in the wells containing 0.25, 0.5, and 1g/mL ofminocycline, but is inhibited in those with 2g/mL or above, then the MIC of that microbe is 2g/mL. Besides being quantitative and testing a number of different concentrations of a specific antimicrobial, each plate tests a number of different antimicrobial drugs simultaneously.


E-Test


The E-test method uses a strip that has the antimi- crobial impregnated on one side, at varying concen- trations. The strip is then placed on an agar plate that contains the bacteria of question. The MIC is read as the concentration on the strip where the zone of inhibition intersects the strip. In this way, the E-test is quantitative. The disadvantage is that each strip only tests one antimicrobial.


Sample Collection and Laboratory Submission


Samples submitted for culture and susceptibility testing include transtracheal wash aspirates, blood cultures, urine, uterine swabs, peritoneal or pleural fluid, and abscess aspirates, among others. Opti- mally, these samples, except for blood cultures that are submitted in blood culture vials, are submitted in commercial culturette swabs with transport me- dium. Whenever possible, the original sample should be submitted as well. These samples should be refrigerated, whereas blood cultures should be placed in an incubator or at least maintained at room temperature. The samples are submitted to a veterinary microbiology lab for aerobic  anaerobic culture and susceptibility testing. If the culture includes anaerobes, an anaerobic transport medium should also be submitted; these are kept at room temperature until submitted. Urine samples should be collected aseptically and placed in sterile specimen cups.


3. Results


The laboratory provides the results of susceptibility testing as “S” or “R” for each of several, often a dozen or more, antimicrobials. Ideally the actual MIC value will be reported adjacent to the designation “S” or “R” as well, so that the clinician can interpret whether the MIC can be realistically expected to be reached in horses for a long enough period of time to be considered effective against that particular bac- terial isolate. For time-dependent drugs, the time above the MIC (TMIC) is important. This refers to the time that serum (and tissue) concentrations remain above the MIC of the offending microbe. Ideally, the TMIC would encompass the entire dos- ing interval for time-dependent antimicrobials like penicillin or cephalosporins; however, at a mini- mum, the TMIC should be 50–75% of the dosing interval to be considered effective, depending on the type of bacteria being treated.


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