Major genetic discontinuity and novel toxigenic species in Clostridioides difficile taxonomy

Abstract

<i>Clostridioides difficile</i> infection (CDI) remains an urgent global One Health threat. The genetic heterogeneity seen across <i>C. difficile</i> underscores its wide ecological versatility and has driven the significant changes in CDI epidemiology seen in the last 20 years. We analysed an international collection of over 12,000 <i>C. difficile</i> genomes spanning the eight currently defined phylogenetic clades. Through whole-genome average nucleotide identity, and pangenomic and Bayesian analyses, we identified major taxonomic incoherence with clear species boundaries for each of the recently described cryptic clades CI-III. The emergence of these three novel genomospecies predates clades C1-5 by millions of years, rewriting the global population structure of <i>C. difficile</i> specifically and taxonomy of the <i>Peptostreptococcaceae</i> in general. These genomospecies all show unique and highly divergent toxin gene architecture, advancing our understanding of the evolution of <i>C. difficile</i> and close relatives. Beyond the taxonomic ramifications, this work may impact the diagnosis of CDI.