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

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_CTn1-3) 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_CTn2 and 023_CTn3 from the genome of RT023 reference strain CD305 and the transfer of 023_CTn3 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".

Plasmids can transfer to Clostridium difficile CD37 and 630Δerm both by a DNase resistant conjugation-like mechanism and a DNase sensitive mechanism.

Broad host range conjugative plasmids that replicate in Escherichia coli have been widely used to mobilise smaller replicons, bearing their cognate origin of transfer (oriT) into a variety of organisms that are less tractable genetically, such as Clostridium (Clostridioides) difficile. In this work we demonstrated that the oriT region of pMTL9301 (derived from RK2) is not required for transfer between E. coli and C. difficile strains 630Δerm and CD37 and that this oriT-independent transfer is abolished in the presence of DNase when CD37 is the recipient. Transfer to the 630Δerm strain is DNase resistant even without an obvious oriT, when E. coli CA434 is used as a donor and is sensitive to DNase when E. coli HB101 is the donor.