To investigate a possible Clostridioides difficile reservoir in the Czech Republic, we performed a study in 297 calves from 29 large-scale dairy farms. After enrichment, faecal samples were inoculated onto selective agar for C. difficile. From the 297 samples, 44 C. difficile isolates were cultured (prevalence of 14.8%, 10 farms). The Holstein breed and use of digestate were associated with C. difficile colonisation (p ˂ 0.05). C. difficile isolates belonged to the ribotype/sequence type: RT033/ST11 (n = 37), RT126/ST11 (n = 6) and RT046/ST35 (n = 1). A multiple-locus variable-number tandem-repeat analysis revealed four clonal complexes of RT033 isolates and one clonal complex of RT126 isolates. All isolates were sensitive to amoxicillin, metronidazole and vancomycin. Forty isolates were resistant to ciprofloxacin, twenty-one to clindamycin, seven to erythromycin, seven to tetracycline and six to moxifloxacin. Moxifloxacin resistant isolates revealed an amino-acid substitution Thr82Ile in the GyrA. In conclusion, the calves of Holstein breed from farms using digestate as a product of bio-gas plants are more likely to be colonised by clonally-related C. difficile of ST 11 represented by ribotypes 033 and 126. The identified resistance to moxifloxacin with a Thr82Ile substitution in the GyrA highlights the need for further monitoring by the "One health approach".

Department of Infectious Diseases and Microbiology Faculty of Veterinary Medicine University of Veterinary and Pharmaceutical Sciences 612 42 Brno Czech Republic

Department of Medical Microbiology 2nd Faculty of Medicine and Motol University Hospital Charles University 150 06 Prague Czech Republic

Ruminant and Swine Clinic Faculty of Veterinary Medicine University of Veterinary and Pharmaceutical Sciences 612 42 Brno Czech Republic

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