OBJECTIVES: To analyse characteristics of Clostridioides difficile PCR ribotype 176 clinical isolates from Poland, the Czech Republic and Slovakia with regard to the differences in its epidemiology. METHODS: Antimicrobial susceptibility testing and whole genome sequencing were performed on a selected group of 22 clonally related isolates as determined by multilocus variable-number tandem repeat analysis (n = 509). Heterologous expression and functional analysis of the newly identified methyltransferase were performed. RESULTS: Core genome multilocus sequence typing found 10-37 allele differences. All isolates were resistant to fluoroquinolones (gyrA_p. T82I), aminoglycosides with aac(6')-Ie-aph(2'')-Ia in six isolates. Erythromycin resistance was detected in 21/22 isolates and 15 were also resistant to clindamycin with ermB gene. Fourteen isolates were resistant to rifampicin with rpoB_p. R505K or p. R505K/H502N, and five to imipenem with pbp1_p. P491L and pbp3_p. N537K. PnimBG together with nimB_p. L155I were detected in all isolates but only five were resistant to metronidazole on chocolate agar. The cfrE, vanZ1 and cat-like genes were not associated with linezolid, teicoplanin and chloramphenicol resistance, respectively. The genome comparison identified six transposons carrying antimicrobial resistance genes. The ermB gene was carried by new Tn7808, Tn6189 and Tn6218-like. The aac(6')-Ie-aph(2'')-Ia were carried by Tn6218-like and new Tn7806 together with cfrE gene. New Tn7807 carried a cat-like gene. Tn6110 and new Tn7806 contained an RlmN-type 23S rRNA methyltransferase, designated MrmA, associated with high-level macrolide resistance in isolates without ermB gene. CONCLUSIONS: Multidrug-resistant C. difficile PCR ribotype 176 isolates carry already described and unique transposons. A novel mechanism for erythromycin resistance in C. difficile was identified.

• Keywords
• Clostridioides difficile infection, epidemiology, macrolide resistance methyltransferase, whole genome sequencing,
• MeSH
• Anti-Bacterial Agents * pharmacology   MeSH
• Drug Resistance, Bacterial * MeSH
• Bacterial Proteins genetics   MeSH
• Clostridioides difficile * genetics   drug effects   isolation & purification   classification   MeSH
• Genomic Islands * MeSH
• Clostridium Infections * microbiology   epidemiology   MeSH
• Humans MeSH
• Methyltransferases genetics   MeSH
• Microbial Sensitivity Tests MeSH
• Drug Resistance, Multiple, Bacterial * genetics   MeSH
• Multilocus Sequence Typing MeSH
• Ribotyping MeSH
• Whole Genome Sequencing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic epidemiology   MeSH
• Poland epidemiology   MeSH
• Names of Substances
• Anti-Bacterial Agents * MeSH
• Bacterial Proteins MeSH
• Methyltransferases MeSH

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.

• Keywords
• MLST, antimicrobial resistance, erm(B), plasmid-bound metronidazole resistance, ribotyping, surface water, wastewater treatment plant,
• MeSH
• Anti-Bacterial Agents pharmacology   therapeutic use   MeSH
• Drug Resistance, Bacterial genetics   MeSH
• Clostridioides difficile * genetics   MeSH
• Clostridioides MeSH
• Clostridium Infections * veterinary   MeSH
• Humans MeSH
• Metronidazole pharmacology   therapeutic use   MeSH
• Microbial Sensitivity Tests MeSH
• Wastewater MeSH
• Plasmids genetics   MeSH
• Rivers MeSH
• Water MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Metronidazole MeSH
• Waste Water MeSH
• Water MeSH

Clostridioides difficile is the most common pathogen responsible for hospital-acquired diarrhea. This complication of antibiotic treatment mainly endangers the health of elder patients. Preventing the development of C. difficile infections (CDI) is still a challenge that needs to be addressed. In our study, the results of 872 C. difficile positive stool samples were used to describe the epidemiological situation affected by a change in the prescription of fluoroquinolone antibiotics. In a total, 93 of strains were typed by polymerase chain reaction (PCR) and capillary gel electrophoresis. Between years 2014 and 2018 the decline in the fluoroquinolones consumption was 69.3 defined daily dose (DDD) per 1000 patient-days (from 103.3 to 34.0), in same period CDI incidence declined by 1.3 cases per 10,000 patient-bed days (from 5.6 to 4.3). Results of epidemiologic and statistical analysis shows that decline in fluoroquinolones consumption has significant influence on CDI incidence and prevalence of hypervirulent strains. In the University Hospital Hradec Králové properly managed antibiotic stewardship policy has reduced CDI incidence by 23.2% and lowered rate of hypervirulent ribotypes 001 and 176.

• Keywords
• C. difficile infections, Clostridioides difficile, antibiotic stewardship, capillary electrophoresis ribotyping, fluoroquinolones,
• Publication type
• Journal Article MeSH