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Objectives: Study of the rifampicin resistance of Rhodococcus equi strains isolated from French horses over a 20-year period., Methods: Rifampicin susceptibility was tested by disk diffusion (DD) and broth macrodilution methods, and rpoB gene sequencing and MLST were performed on 40 R. equi strains, 50.0% of which were non-susceptible to rifampicin., Results: Consistency of results was observed between rifampicin susceptibility testing and rpoB sequencing. Strains non-susceptible to rifampicin by DD had a substitution at one of the sites (Asp516, His526 and Ser531) frequently encountered and conferring rifampicin resistance. High-level resistance was correlated with His526Asp or Ser531Leu substitutions; low-level resistance was correlated with Asp516Tyr substitution, a novel substitution for R. equi. Strains susceptible to rifampicin by DD showed no substitution in the three sites, except for two strains carrying, respectively, the His526Asn and Asp516Val substitutions (previously correlated with low-level rifampicin resistance). Both strains were isolated from an animal from which ten other strains were also isolated and found to be rifampicin-non-susceptible by DD. MLST showed the presence of 10 STs (including the novel ST43), but no association was observed with rifampicin resistance., Conclusions: This study confirms that certain substitutions in RpoB are more likely to confer high- or low-level rifampicin resistance, describes a new substitution conferring rifampicin resistance in R. equi and suggests non-clonal dissemination of rifampicin-resistant strains in France. Standard DD may miss strains with a low-level rifampicin-resistant substitution; further studies are needed to remedy the absence of R. equi-specific clinical breakpoints., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)