Taylorella equigenitalis is the causative agent of contagious equine metritis, an internationally regulated sexuallytransmitted infection in horses, which is of great concern as it usually results in temporary infertility. Taylorella asinigenitalis, the second member of the genus, is mainly found in donkeys and is considered non-pathogenic, although a first natural outbreak was reported in 2019 in the United Arab Emirates. Multilocus sequence typing (MLST) is currently used to study the epidemiology of Taylorella spp. but, while highly transposable and reproducible, it only focuses on < 0.5 % of the genome (seven genes). We therefore aimed to develop a robust core genome MLST (cgMLST) based on the analysis of 370 T. equigenitalis and 68 T. asinigenitalis genomes belonging to 46 and 18 sequence types (STs), respectively. Typing results based on 1333 loci (84.0 % of the genome) from T. equigenitalis genomes and 1255 loci (80.3 %) from T. asinigenitalis genomes showed that the discriminatory power of both species-specific cgMLSTs was greater than that of MLST, with 368 and 68 distinct core genome STs (cgSTs), respectively. Clustering was congruent between the cgMLST and MLST methods, with few inconsistencies for T. equigenitalis. Maximum allelic distance between epidemiologically-related strains was used to define cgMLST clustering thresholds, set at <= 15 and 20 allelic distances for T. equigenitalis and T. asinigenitalis, respectively. These parameters grouped the cgSTs into 47 and 11 clonal groups (CGs), respectively. Overall, the cgMLST method outperformed conventional MLST in distinguishing clonal strains from epidemiologically-linked strains, supporting the hypothesis that typing based on a few housekeeping genes does not always accurately reflect genomic relatedness between strains, and making cgMLST more suitable for outbreak investigations.

Clostridioides difficile is an anaerobic, spore-forming entero-pathogen, able to persist in the environment and to cause diarrhea in humans and animals. We took advantage of all necropsies of Equidae in Normandie, France, from 2019 to 2021 (n=100), to systematically recover the digestive content in animal caecum and, if of reduced consistency and possibly diarrheal, in any intestinal segment. 27 Equidae were positive for C. difficile and 25 provided 34 isolates forming the first equine strain collection in France (CloDifEqui). Strains were toxigenic in 20 Equidae, including one co-colonized by a non-toxigenic strain. Toxigenic isolates displayed different toxin profiles and belonged to 8 ribotypes (WEBRIBO): 078, 126 (TcdA TcdB and CDT toxins), 005, 012, 020, 181, AI-53 (TcdA and TcdB), 017 (TcdB). Non-toxigenic isolates were of ribotypes 009, 035 and 439. The predominant ribotypes were 017 (n=5 animals, N=8 isolates) and 009 (n=5, N=7). In two premises, a few Equidae shared the same ribotype, either 009 or 020, suggesting two potential transmission clusters. C. difficile infection was shown by free toxin detection in 4 animals displaying both post-mortem signs of diarrhea (colon watery content) and toxigenic isolates. These isolates belonged to ribotype 126 (n=1) or 017 (n=3) and were the least susceptible to vancomycin. The infection by ribotype 017 was responsible for animal death in two cases. Finally, French Equidae represented a previously underestimated reservoir of C. difficile and ribotype 017 was highly virulent in these animals, both preoccupying findings in a One Health perspective. ### Competing Interest Statement The authors have declared no competing interest.

In 2018, a T. asinigenitalis strain (MCE663) was isolated in a Persian onager tested for contagious equine metritis (CEM) in a United Kingdom (UK) zoo. This bacterium had never been reported in the UK and Multilocus Sequence Typing described a new atypically divergent ST (ST60). Although the causative agent of CEM is the bacterium Taylorella equigenitalis, a first natural outbreak of endometritis caused by T. asinigenitalis ST70 was reported in 2019, putting its pathogenic potential into question. In this context, we aimed to further sequence the T. asinigenitalis MCE663 genome and characterize the strain using phenotypical and genetic approaches. Results showed that it gathered all identification characteristics of T. asinigenitalis with smaller colonies and it was susceptible to all tested antibiotics. Genome-level phylogeny showed that the genome MCE663 formed a distinct phylogroup, and only shared ≈ 96.1% of average nucleotide identity (ANI) with the three published T. asinigenitalis genomes, which together shared ≈ 98.3% ANI. According to current cut-offs consensus for species and subspecies delineation (95% and 98%, respectively), our results support the first insights of a sublineage delineation within the T. asinigenitalis species.

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.