Acquired antimicrobial resistance in equine Rhodococcus equi isolates

CONTEXT: Rhodococcus equi (R. equi) is an important cause of bronchopneumonia in foals. Antimicrobial susceptibility data for equine R. equi isolates are still scarce. The goal of the current research was to determine the antimicrobial susceptibility of recent equine R. equi isolates. MAIN CONCLUSION: Acquired antimicrobial resistance towards macrolides in R. equi isolates from Belgian foals is not very prevalent. The presence of low level acquired resistance towards rifampicin might be clinically relevant and can be attributed to point mutations in the rpoB gene. APPROACH: Antimicrobial susceptibility testing was performed on twenty-three R. equi isolates with the agar dilution assay. using Mueller-Hinton agar supplemented with 5% sheep blood (rifampicin, the macrolides and tetracycline) or 5% lysed horse blood (sulfisoxazole and trimethoprim). Plates were incubated at 35°C for 20-24 hours in an aerobic atmosphere. Since no clinical breakpoints or wild type cut-off values are available for R. equi, acquired resistance was assumed when MIC values showed a bimodal or multimodal distribution or tailing. The two isolates showing acquired resistance towards rifampicin and three at random selected wild type isolates were used for PCR amplification and sequencing of the rpoB gene. RESULTS: The ranges of MIC values were similar to MIC ranges described earlier. The distribution of the MIC values for the macrolides, tetracycline, sulfisoxazole and trimethoprim showed a unimodal distribution. The distribution of the MIC values for rifampicin showed a trimodal distribution with two isolates (8.7%) showing low level acquired resistance. One isolate had an MIC value of 1 µg/ml and one isolate showed an MIC value of 8 µg/ml, while the MIC range of the wild type population was 0.06 – 0.25 µg/ml. The three wild type isolates showed identical rpoB sequences. The isolates showing acquired resistance contained point mutations in the rpoB gene when compared to the wild type isolates at the amino acids His526 and Ser531, respectively. The isolate with MIC 1 µg/ml showed a His526Asn mutation. The isolate with MIC 8 µg/ml showed a Ser531Leu mutation. INTERPRETATION: There was no acquired resistance towards the tested antimicrobial agents according to the microbiological criterion, except for rifampicin. Considering that there are no veterinary clinical breakpoints for R. equi and that the therapeutic result is also strongly dependent on the stage of infection, the lack of acquired resistance does not guarantee a successful therapy. On the other hand, the presence of acquired resistance can indeed hamper the in vivo efficiency of the antimicrobial agent. Even though the current collection of isolates is relatively small, these results suggest that acquired antimicrobial resistance against macrolides is not very common in recent equine isolates obtained in Belgium. The presence of rifampicin resistance in more than 8% of the isolates emphasizes the importance of the combination therapy (rifampicin + macrolide). Even though the clinical importance of low and high level resistance is currently not described, there are serious indications that at least high level resistance is clinically relevant. SIGNIFICANCE OF FINDINGS: The current results acknowledge that the combination therapy (rifampicin + macrolide) remains first choice antimicrobial therapy for treating rhodococcosis in horses in Belgium