Analysis of the complete genome sequences of Clostridium perfringens strains harbouring the binary enterotoxin BEC gene and comparative genomics of pCP13-like family plasmids

Background BEC-producing Clostridium perfringens is a causative agent of foodborne gastroenteritis. It was first reported in 2014, and since then, several isolates have been identified in Japan and the United Kingdom. The novel binary ADP-ribosylating toxin BEC, which consists of two components (BECa and BECb), is encoded on a plasmid that is similar to pCP13 and harbours a conjugation locus, called Pcp, encoding homologous proteins of the type 4 secretion system. Despite the high in vitro conjugation frequency of pCP13, its dissemination and that of related plasmids, including bec -harbouring plasmids, in the natural environment have not been characterised. This lack of knowledge has limited our understanding of the genomic epidemiology of bec -harbouring C. perfringens strains. Results In this study, we determined the complete genome sequences of five bec -harbouring C. perfringens strains isolated from 2009 to 2019. Each isolate contains a ~ 3.36 Mbp chromosome and 1–3 plasmids of either the pCW3-like family, pCP13-like family, or an unknown family, and the bec -encoding region in all five isolates was located on a ~ 54 kbp pCP13-like plasmid. Phylogenetic and SNP analyses of these complete genome sequences and the 211 assembled C. perfringens genomes in GenBank showed that although these bec -harbouring strains were split into two phylogenetic clades, the sequences of the bec -encoding plasmids were nearly identical (>99.81%), with a significantly smaller SNP accumulation rate than that of their chromosomes. Given that the Pcp locus is conserved in these pCP13-like plasmids, we propose a mechanism in which the plasmids were disseminated by horizontal gene transfer. Data mining showed that strains carrying pCP13-like family plasmids were unexpectedly common (58/216 strains) and widely disseminated among the various C. perfringens clades. Although these plasmids possess a conserved Pcp locus, their ‘accessory regions’ can accommodate a wide variety of genes, including virulence-associated genes, such as becA / becB and cbp2 . These results suggest that this family of plasmids can integrate various foreign genes and is transmissible among C. perfringens strains. Conclusion This study demonstrates the potential significance of pCP13-like plasmids, including bec -encoding plasmids, for the characterisation and monitoring of the dissemination of pathogenic C. perfringens strains.