Beyond difficile: three novel toxin B-producing Clostridioides species from human patients with diarrhea

Clostridioides difficile infection (CDI), primarily mediated by toxin B (TcdB), is a leading cause of healthcare-associated and community-acquired diarrhea worldwide. Although this genus exhibits substantial genomic diversity, functional studies have focused mainly on a limited set of isolates, leaving other genomospecies understudied in this regard. Here, we characterized five isolates derived from five distinct patients with suspected CDI cases that tested negative for the tcdC PaLoc marker. Integrated genomic, proteomic, and phenotypic analyses confirm that these strains constitute three novel toxin-producing species and their distinct carbon utilization profiles, exoproteomes, and spore protein repertoires suggest niche specialization within the human gut ecosystem. These species harbour monotoxin PaLoc architectures encoding TcdB7 or TcdB11, located on either chromosomal or extrachromosomal elements, which frequently co-occur with binary toxin loci. Functional assays confirmed secretion of active TcdB, cytotoxicity in mammalian cells, and moderate enterotoxicity in a murine ileal loop model, although virulence was attenuated relative to C. difficile R20291 from Clade 2. Collectively, these findings expand the taxonomy of toxigenic Clostridioides, highlight the dynamic evolution of TcdB-mediated pathogenicity, and emphasize the importance of refining diagnostic workflows and surveillance strategies to address emerging diarrheal diseases. The three novel species were designated as Clostridioides cryptodifficilis sp. nov., Clostridioides divergens sp. nov., and Clostridioides subdifficilis sp. nov.