Genomic and phenotypic studies among Clostridioides difficile isolates show a high prevalence of clade 2 and great diversity in clinical isolates from Mexican adults and children with healthcare-associated diarrhea.

Clostridioides difficile is the most common cause of healthcare-associated infection worldwide. Its pathogenesis is mainly due to the production of toxins A, B, and CDT, whose genetic variants may be associated with disease severity. We studied genetic diversity in C. difficile isolates from hospitalized adults and children using different gene and genome typing methods and investigated their association with in vitro expression of toxins. Whole-genome was sequenced in 39 toxigenic C. difficile isolates and used for multilocus sequence typing (MLST), tcdA, and tcdB typing sequence type, and for phylogenetic analysis. Strains were grown in broth media, and expression of toxin genes was measured by real-time PCR and cytotoxicity was determined in culture assays. We found that clustering after genome-wide phylogeny followed clade classification, although different clusters were identified within each clade. The toxin profile tcdA+/tcdB+/cdtA+/cdtB+ and clade 2/ST1 were the most prevalent among isolates from children and adults. Isolates presented two TcdA and three TcdB subtypes, of which A2 and B2 were dominant. Toxin gene expression or cytotoxicity were not associated with genotyping or toxin subtypes. In conclusion, genomic and phenotypic analysis shows high diversity among C. difficile isolates from patients with healthcare-associated-diarrhea. ImportanceClostridioides difficile is a toxin-producing bacterial pathogen associated with healthcare, and different genetic or phenotypic typing have been proposed for classification. We extensively studied cytotoxicity, expression of toxins, whole genome phylogeny, and toxin typing in clinical C. difficile isolates. Most isolates presented a tcdA+ tcdB+cdtA+ cdtB+ pattern, with high diversity in cytotoxicity, and clade 2/ST1 was the most prevalent. However, they all had the same TcdA2/TcdB2 toxin subtype. Advances in genomics and bioinformatics tools offer the opportunity to understand the virulence of C. difficile better and find markers with better clinical use.