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Clostridioides difficile  toxins (TcdA and TcdB) are major exotoxins responsible for  C. difficile  infection (CDI) associated diseases. The previously reported TcdB variants showed distinct biological features, immunoactivities, and potential pathogenicity in disease progression. Here, we performed global comparisons of amino acid sequences of both TcdA and TcdB from 3,269  C. difficile  genomes and clustered them according to the evolutionary relatedness. We found that TcdB was much diverse and could be divided into eight subtypes, of which four were first described. Further analysis indicates that the  tcdB  gene undergoes accelerated evolution to maximize diversity. By tracing TcdB subtypes back to their original isolates, we found that the distribution of TcdB subtypes was not completely aligned with the phylogeny of  C. difficile . These findings suggest that the  tcdB  genes not only frequently mutate, but also continuously transfer and exchange among  C. difficile  strains.

Subtyping analysis reveals new variants and accelerated evolution of Clostridioides difficile toxin B