Isolation and characterization of Clostridioides difficile from wastewater and seafood in Tainan, Taiwan

Clostridiodes difficile (C. difficile) is a gram-positive, spore-forming anaerobic bacillus. C. difficile infections (CDI) can cause antibiotic-associated diarrhea (AAD), toxic megacolon, colonic perforation, and even death. The major virulence factors of C. difficile are three toxins, TcdA (toxin A) and TcdB (toxin B), and the binary toxin CD. In addition, C. difficile spores are highly resistant to environmental stress and is responsible for transmission to human via fecal-oral, food source, and potentially zoonotic routes. Past studies in other nations have indicated that C. difficile can be isolated from various environmental samples as well as from both wild and domesticated animals which might serve as potential reservoirs for human infection. Wastewater treatment plant (WWTP) is considered as an important source of water contamination by multiple intestinal pathogens. Hospital wastewater is also a potential reservoir for infectious pathogens as well. In Taiwan, wastewater is discharged into rivers or recovered by sewage treatment plants after treatment with chloride. Therefore, it is possible that hardy pathogens within wastewater might contaminate seafood that are raised near estuaries. In this study, we were able to isolate C. difficile from multiple water samples from multiple WWTPs, National Cheng Kung University Hospital, and seafood near the Tainan coast. A total of 124 C. difficile isolates were identified in this study. 5 isolates were from WWTPs, 103 from NCKUH, and 16 from seafood source. We performed genotypic analysis, which included multiplex PCR for toxin genes, ribotyping, and MLVA. We also performed phenotypic analysis which included antibiotic susceptibility assays, motility assay, and biofilm formation assay. Interestingly, 49% (61/124)of all total isolates belonged to either the hypervirulent ribotype 027 or 078 lineage which are known to be resistant to fluoroquinolone antibiotics. 53% (55/103) of NCKUH isolates belonged to the hypervirulent RT127 ribotype. Phenotypic analysis suggested that isolates belonging to RT078 lineage exhibited significant lower swimming motility when compared to other toxigenic and non-toxigenic isolates. No significant differences however were observed among all isolates in terms of biofilm formation ability. In the near future, additional water samples from WWTPs and other hospitals in southern Taiwan will be collected for analysis. In summary, for the first time ever in Taiwan, toxigenic C. difficile were isolated from various water source as well as from seafood, indicating that C. difficile is able to persist even after wastewater treatment.