Clostridioides difficile is one of the most important human pathogens. The identification of its possible sources is important for the understanding of C. difficile infection (CDI) epidemiology. A total of 16 water samples from wastewater and surface water in South Moravia in the Czech Republic and 82 samples of fish and gulls were collected between May and July 2019. C. difficile isolates were cultured by direct plating and after enrichment on chromogenic media. Susceptibility testing to eight antimicrobials was performed by Etest. C. difficile isolates were characterized by ribotyping, multilocus sequence typing, multilocus tandem repeats analysis, and toxin gene detection. Samples from fish and gulls were C. difficile negative; a total of 15 C. difficile isolates from 8 out of 16 water samples were cultured (6 out of 14 surface water samples yielded 6 isolates, and 2 out of 2 wastewater samples yielded 9 isolates). Direct plating was culture positive in 6 out of 16 samples (12 isolates), and enrichment culture was positive in an additional 2 out of 16 samples (3 isolates). Twelve different ribotyping profiles and 14 sequence types of clades 1, 4, and 5 were identified. Five isolates did not carry genes for toxins, and eight isolates carried genes for toxins A and B; the remaining two isolates (RT078) carried the genes for toxins A, B, and binary. All C. difficile isolates were susceptible to amoxicillin, moxifloxacin, tetracycline, and vancomycin and resistant to ciprofloxacin. A high level of erythromycin resistance (>256 mg/L) was detected in eight isolates. Clindamycin resistance was found in 14 isolates, 6 of which showed a high level of resistance (>256 mg/L) and carried ermB. Surprisingly, one isolate (RT010, ST15) showed resistance to metronidazole (12 mg/L) with the presence of the plasmid pCD-METRO. In conclusion, a diverse spectrum of C. difficile strains was found in wastewater and surface water. A recently discovered plasmid-bound resistance to metronidazole was detected in C. difficile from the surface water sample. IMPORTANCE The combination of direct plating and culture after enrichment was used in order to gain a spectrum of C. difficile ribotypes present in the water samples. Toxigenic C. difficile ribotypes detected in surface water and in wastewater treatment plants overlapped with those derived from patients with CDI and/or animals. Importantly, a recently discovered plasmid-mediated resistance to metronidazole, a drug used for the treatment of CDI, was detected in C. difficile from river water.

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

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

Department of Zoology Fisheries Hydrobiology and Apiculture Faculty of AgriSciences Mendel University Brno Czech Republic

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