Molecular characterisation of theChlamydia pecorumplasmid from porcine, ovine, bovine, and koala strains indicates plasmid-strain co-evolution

Background. Highly stable, evolutionarily conserved, small, non-integrative plasmids are commonly found in members of the Chlamydiaceae and, in some species, these plasmids have been strongly linked to virulence. To date, evidence for such a plasmid in Chlamydia pecorum has been ambiguous. In a recent comparative genomic study of porcine, ovine, bovine, and koala C. pecorum isolates, we identified plasmids (p Cpec ) in a pig and three koala strains, respectively. Screening of further porcine, ovine, bovine, and koala C. pecorum isolates for pCpec showed that p Cpec is common, but not ubiquitous in C. pecorum from all of the infected hosts. Methods. We used a combination of (i) bioinformatic mining of previously sequenced C. pecorum genome data sets and (ii) pCpec PCR-amplicon sequencing to characterise a further 17 novel p Cpec s in C. pecorum isolates obtained from livestock, including pigs, sheep, and cattle, as well as those from koala. Results and Discussion. This analysis revealed that pCpec is conserved with all eight coding domain sequences (CDSs) present in isolates from each of the hosts studied. Sequence alignments revealed that the 21 p Cpec s show 99% nucleotide sequence identity, with 83 single nucleotide polymorphisms (SNPs) shown to differentiate all of the plasmids analysed in this study. SNPs were found to be mostly synonymous and were distributed evenly across all eight p Cpec CDSs as well as in the intergenic regions. Although conserved, analyses of the 21 p Cpec sequences resolved plasmids into 12 distinct genotypes, with five shared between p Cpec s from different isolates, and the remaining seven genotypes being unique to a single p Cpec . Phylogenetic analysis revealed congruency and co-evolution of p Cpec s with their cognate chromosome, further supporting polyphyletic origin of the koala C. pecorum . This study provides further understanding of the complex epidemiology of this pathogen in livestock and koala hosts and paves the way for studies to evaluate the function of this putative C. pecorum virulence factor.