Genomic investigations in animal enteric disease-associated Clostridium perfringens

The anaerobic bacterium Clostridium perfringens is commonly found in the intestinal tract of humans and animals. However, there are marked differences in virulence between isolates and toxinotypes, which largely depend on various virulence factors produced by these strains. Studying C. perfringens genomes has been limited by fragmented assemblies from short-read sequencing and incomplete clinical metadata. Here, we present a high-quality collection of 236 isolates from animal hosts that underwent detailed pathomorphological examination. From 220 of them, genomes were generated by PacBio long-read sequencing, enabling comprehensive, structural level analysis of their virulome, plasmids, conjugative elements and biosynthetic gene clusters (BGCs). One hundred forty isolates were collected from animals with signs of C. perfringens-associated enteric disease, 32 from animals with no signs of C. perfringens-associated disease and 64 from healthy animals. An additional 383 publicly available C. perfringens complete or draft genomes were included for comparative analyses. We discovered 2 previously undescribed pore-forming toxin (PFT) homologues and 11 novel haemolysin and aerolysin-type PFT variants. Both findings expand the known spectrum of the C. perfringens virulome. Moreover, we defined two novel putative plasmid conjugative loci in a collection of 888 here assembled and circularized plasmids. They may facilitate HGT, supporting the dissemination of virulence and metabolic traits. We predicted 414 BGCs that were frequently toxinotype specific and encoded centrally for a bacteriocin that could help their carriers to outcompete other bacteria in shared environments. Furthermore, comparative analysis of C. perfringens plasmids revealed three distinct clusters based on their conjugation system. Altogether, these findings significantly expand the landscape of animal-associated C. perfringens through high-quality genome data and highlight novel virulence-associated features that provide a foundation for future studies of this important pathogen.