Virulence plasmid pINV as a genetic signature for phylogeny.

is a major health burden in low- and middle-income countries, where it is a leading cause of mortality associated with diarrhoea in children, and shows an increasing incidence among travellers and men having sex with men. Like all spp., has evolved from commensal following the acquisition of a large plasmid pINV, which contains genes essential for virulence. Current sequence typing schemes of are based on combinations of chromosomal genetic loci, since pINV-encoded virulence genes are often lost during growth in the laboratory, making these elements inappropriate for sequence typing. By performing comparative analysis of pINVs from strains isolated from different geographical regions and belonging to different serotypes, we found that in contrast to plasmid-encoded virulence genes, plasmid maintenance genes are highly stable pINV-encoded elements. For the first time, to our knowledge, we have developed a plasmid multilocus sequence typing (pMLST) method based on different combinations of alleles of the and toxin-antitoxin (TA) systems, and the partitioning system. This enables typing of pINV plasmids into distinct 'virulence sequence types' (vSTs). Furthermore, the phylogenies of vST alleles and bacterial host core genomes suggests an intimate co-evolution of pINV with the chromosome of its bacterial host, consistent with previous findings. This work demonstrates the potential of plasmid maintenance loci as genetic characteristics to study as well as to trace the molecular phylogenesis of pINV and the phylogenetic relationship of this plasmid with its bacterial host.