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The major global pathogen  Clostridium difficile  (recently renamed  Clostridioides difficile ) has large genetic diversity including multiple mobile genetic elements. In this study, whole genome sequencing of 86 strains from the poorly characterised clade 3, predominantly PCR ribotype (RT)023, of  C. difficile  revealed distinctive surface architecture characteristics and a large mobile genetic island. These strains have a unique sortase substrate phenotype compared with well-characterised strains of  C. difficile , and loss of the phage protection protein CwpV. A large genetic insertion (023_CTnT) comprised of three smaller elements (023_CTn 1-3 ) is present in 80/86 strains analysed in this study, with genes common among other bacterial strains in the gut microbiome. Novel cargo regions of 023_CTnT include genes encoding a sortase, putative sortase substrates, lantibiotic ABC transporters and a putative siderophore biosynthetic cluster. We demonstrate the excision of 023_CTnT and sub-elements 023_CTn 2  and 023_CTn 3  from the genome of RT023 reference strain CD305 and the transfer of 023_CTn 3  to a non-toxigenic  C. difficile  strain, which may have implications for the use of non-toxigenic  C. difficile  strains as live attenuated vaccines. Finally, we show that the genes within the island are expressed in a regulated manner in  C. difficile  RT023 strains conferring a distinct “niche adaptation”.

Clostridium difficile clade 3 (RT023) have a modified cell surface and contain a large transposable island with novel cargo