Analysis of Clostridium difficile Ribotype 023 strains reveals the recent emergence of a highly related virulent clade with unique characteristics. C. difficile RT023

Clostridium difficile is the leading cause of nosocomial acquired human diarrhoea worldwide and can broadly be divided into five clades, of which clade 3 is the least characterised and consists predominantly of PCR ribotype (RT) 023 strains. In this study, analysis of the clinical presentation of RT023 infections in the Netherlands show severe infections akin to those seen with “hypervirulent” strains from clades 2 and 5. We present the full genome sequence of strain CD305 as the reference for clade 3 and analyse Whole Genome Sequencing (WGS) of a further 79 previously uncharacterised RT023 strains of diverse European origin. Phylogenetic analysis revealed minor genetic divergence, suggesting the recent emergence of the clade. Genomic analyses reveal distinctive surface, toxin and mobile element characteristics within this clade, including glycosylation of the S-layer protein matrix which was confirmed phenotypically. We show that a recently characterised trehalose metabolism system is distinct among clade 3 strains and highlight potential genetic disparities which produce false-negative results with esculin agar detection for strains from this clade. This implies that the newly characterised clade 3 strains may be under reported, which, given their recent emergence and adaptable phenotype suggests they should be more highly prioritised.