MEDICINE: INFECTIOUS DISEASES


These include the provision that there is no approved new animal drug that is labeled for such use and that contains the same active ingredient, which is in the required dosage form and concentration, except where a veterinarian finds, within the context of a valid veterinarian-client-patient relationship, that the ap- proved new animal drug is clinically ineffective for its intended use.8 Two important determinants of clini- cal efficacy of antibiotics are pharmacokinetics, includ- ing bioavailability following administration by routes other than intravenous, and susceptibility of com- monly encountered bacteria to the antimicrobial drug or combination in question.1,9 Several studies have shown that TMP-SDZ is well absorbed after oral ad- ministration to horses and has a pharmacokinetic pro- file that is consistent with efficacy following oral dosing at 12-hour intervals.4–7,10 To date, there is a paucity of data regarding the comparative in vitro susceptibility of S. zooepidemicus and other equine- origin bacterial pathogens to TMP-SDZ and TMP- SMZ. The objective of this study was therefore to generate such data for S. zooepidemicus and other common equine pathogens.


2. Methods


Antimicrobial susceptibility testing was performed on equine-origin isolates of S. zooepidemicus (n  282), S. equi (n  55), C. pseudotuberculosis (n  96), and A. equuli (n46). The Actinobacillus spp. tested included A. equuli subsp. equuli (n  14),A. equuli subsp. hemolyticus (n  14), A. equuli subsp. hemolyticus biovar 1 (n  14), and A. equuli subsp. hemolyticus biovar 2 (n  5). These bacteria had been collected between 1986 and 2016 from adult horses and foals with clinical disease, and had been stored as frozen stabilates at 80°C in skim milk or on glass beads.a The identity of each bacterial isolate was confirmed based on colony morphology, Gram-staining characteristics, biochemical charac- teristics, and results of genetic testing using the matrix-assisted laser desorption ionization–time of flight mass spectrometry system. Susceptibility testing was performed using the broth microdilution procedureb, following Clinical Laboratory Standards Institute protocols.11 Briefly, one bacterial colony was inoculated into brain heart infusion broth and incubated for 4 hours at 35°C. A small amount of this inoculated broth was then added to 0.85% NaCl solution to achieve a 0.5 McFarland Standard con- centration, as measured using a nephelometer. Ten microliters of this suspension were added to Mueller Hinton broth, and platesb were inoculated with 100 L of the Mueller Hinton broth in each well. The following bacterial strains were run weekly as controls in accordance with the standard quality control procedures in place at the Veterinary Medical Teaching Hospital Microbiology Laboratory (MDL): Staphylococcus aureus America Type Cul- ture Collection (ATCC) 29213, Enterococcus faecalis ATCC 29212, E. coli ATCC 25922, E. coli ATCC 35218, and Pseudomonas aeruginosa ATCC 27853.


SensititreTM platesb were custom made for the


MDL by the manufacturer. The range of TMP-SDZ or TMP-SMZ concentrations tested was 0.12/2.4 g/mL to 8/152g/mL for each antimicrobial combi- nation. The minimum inhibitory concentration (MIC) was recorded as the lowest concentration of antimicrobial drug combination (TMP-SDZ or TMP- SMZ) that inhibited visible growth of bacteria. An isolate was considered to be susceptible to TMP- SDZ or TMP-SMZ if its MIC value was  2/38 g/ mL, as recommended by the Clinical Laboratory Standards Institute.11 The respective concentra- tions at which 50% (MIC50) and 90% (MIC90) of isolates of a particular bacterial species were sus- ceptible to TMP-SDZ and TMP-SMZ, were also de- termined. In order to create numerical data that could be analyzed, only the concentration of TMP in the fixed ratio combination was used. Concentra- tions that were at or below the lower limit of quan- titation of the MIC test (i.e.,  0.12 g/mL) were ascribed a value of 0.12 g/mL to facilitate statisti- cal analysis using the Wilcoxon signed-rank test for paired data. A P-value of  .05 was used to ascribe statistical significance to differences between groups (TMP-SDZ vs TMP-SMZ).


3. Results


Overall Findings The MIC values for both TMP-SDZ and TMP-SMZ against isolates of S. zooepidemicus, S. equi, C. pseu- dotuberculosis, and A. equuli are shown in Table 1. Of the 479 isolates tested, only 1 (0.21%), a S. zooepi- demicus isolate, was resistant to TMP-SDZ. The same isolate was also resistant to TMP-SMZ.


S. equi subsp. zooepidemicus


Of the 282 S. zooepidemicus isolates tested, 281 (99.6%) were susceptible to both TMP-SDZ and TMP-SMZ (MIC  2.0/38 g/mL), whereas 1 (0.4%) isolate was resistant to both drug combinations (MIC  8/152 g/mL). With the exception of the resistant isolate, all S. zooepidemicus isolates were highly susceptible, with MIC values ranging be- tween 0.12/2.4 g/mL and 1/19 g/mL for both TMP-SDZ and TMP-SMZ (Table 1). One hundred thirty-four of the 282 S. zooepidemicus isolates (47.5%) had an MIC value that was the same for both TMP-SDZ and TMP-SMZ, whereas 148 isolates (52.5%) had an MIC value for TMP-SDZ that was one concentration higher than for TMP-SMZ. In other words, 52.5% of isolates were one dilution less susceptible to TMP-SDZ than to TMP-SMZ. Statis- tical analysis showed this difference to be highly


significant (P.0001). The MIC50 values for TMP- SDZ and TMP-SMZ were 0.25/4.75 g/mL and


0.12/2.4 g/mL, respectively. The MIC90 value for both drug combinations was 0.25/4.75 g/mL.


S. equi subsp. equi Of the 55 S. equi isolates, all (100%) were highly sus- ceptible to both drug combinations (MIC range of


AAEP PROCEEDINGS  Vol. 66  2020 433


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