Multiple drug-susceptibility screening in Mycobacterium bovis: new nucleotide polymorphisms in the embB gene among ethambutol susceptible strains

•We assessed animal Mycobacteriumbovis susceptibility to six antitubercular drugs.•We used the standard method and a simplified, dichotomous REMA(d-REMA) method.•There was good agreement between the d-REMA assay and the reference method.•Four new single-nucleotide polymorphisms were identified in the embB gene.•No association was found between embB genetic profiles and EMB resistance results.

SummaryObjectivesPyrazinamide-resistant Mycobacterium bovis isolates of animal origin were assessed for drug susceptibility to five antituberculosis drugs by the agar based Middlebrook 7H11 method as gold standard as well as by a simplified, dichotomous resazurin microtitre assay (d-REMA).MethodsA total of 53 M. bovis isolates were typed and tested against isoniazid, rifampin, streptomycin, ethambutol, kanamycin and the control drug pyrazinamide. On the basis of the results obtained, pncA and embB genes were PCR-amplified and DNA-sequenced for all isolates.ResultsAll M. bovis isolates, classified into 21 spoligotype/MIRU-VNTR profiles, were resistant to pyrazinamide by both methods, as expected. The pncA gene sequencing confirmed the presence of the resistance-conferring H57D mutation. All strains were found to be susceptible to the other five drugs by the agar based gold standard method. The d-REMA was in agreement with these results for all five drugs, with the exception of 12 isolates, which showed ambiguous and therefore inconclusive results in ethambutol testing. Mutations in the embB gene were observed in all 53 isolates: four new single-nucleotide polymorphisms were identified. No association was found between embB genetic profiles and ethambutol resistance results by the gold standard.ConclusionAll M. bovis isolates were sensitive to the most common antituberculosis drugs used for treatment. There was a good agreement between the d-REMA assay and the agar based reference method. Among ethambutol susceptible isolates, four new embB mutations were found.