Genomic analysis reveals the presence of hypervirulent and fluoroquinolone-resistant Clostridioides difficile in farmed mussels (Mytilus galloprovincialis) in Slovenia

Clostridioides difficile is one of the leading causes of antibiotic-associated diarrhea. In this study, we characterized 76C. difficile isolates, obtained from three Mediterranean mussel (Mytilus galloprovincialis) farms in Slovenia from November 2014 to October 2015 (sampling period 1) and from January to December 2021 (sampling period 2). The overall isolation rate of C. difficile from all the examined mussels was 59.8 %. A statistically significant trend of seasonal variation was observed, with a higher isolation rate in the colder months of the year (87.9 %; sea temperature ≤ 15 °C) compared with the warmer months (31.8 %; sea temperature > 15 °C). Whole-genome sequencing (WGS) revealed that the isolates belonged to 31 different sequence types (STs), which were associated with three clades (1, 4, and 5) and two cryptic clades (C-II and C-III). Five isolates, which belonged to ST11 (clade 5), harbored all the main toxin genes (A⁺B⁺CDT⁺) and chromosomal mutations conferring fluoroquinolone resistance. Core genome multilocus sequence typing (cgMLST) revealed four clusters of 2-3 isolates, three of which included isolates from different farms, suggesting that clonal C. difficile strains are circulating among the Slovenian mussel farms. The results highlight the presence of hypervirulent strains in mussels; therefore, at-risk population groups should be alerted to the risks associated with consuming shellfish.