Genome sequencing and molecular typing of Clostridium chauvoei

High quality circular genome sequences were generated for the Clostridium chauvoei type strain DSM 7528T (ATCC 10092T) and a field strain 12S0467 isolated in Germany. Comparative genome analysis of the two strains revealed few inversions and translocations in local collinear blocks, indicating a conserved genome with only a small number of accessory genes. Significant homology for C. chauvoei was observed with C. septicum. The species genome shows a large number of genes, the products of which are involved in proteolysis, cleavage of glycosidic linkages (sialidases) and metal ion transportation. Triplicates of fliC were identified in each of the two circular genomes. Sporulation and germination process related genes were homologous to those of the Clostridia cluster I species with highest homology to C. septicum, but novel variations in regulatory genes were also identified. A comparative genomic study was carried out with a total of 64 C. chauvoei strains. The strain collection mostly included strains of European origin whereas few strains were of exotic origin. Pan-genome analysis of the species based on the available C. chauvoei strains shows that the species has an open pan-genome structure. New gene acquisition was observed to be very limited. This probably indicates that the species is undergoing replication only in very isolated areas e.g. inside the host tissue, so that the chances of acquiring foreign genetic material are limited. The predicted prophage identified in the genomes was similar for all strains, indicating that the genomes are immune to any new phage type. A CRISPR type I-B system was identified in all genomes which may contribute to comprehensive phage immunity. There was an obvious correlation of strains from the same region/farm i.e. displaying the same repeat numbers/spacer sequences. The strains originating from outside Europe showed a unique spacer matrix composition. Homologous recombination plays an important role in the evolution of some bacterial pathogens. This study found only a limited number of possible recombination events contributing towards the evolution of C. chauvoei. Maximum Likelihood phylogenetic analysis based on the core genome (Parsnp) identified associated isolates with strong bootstrap support values (100%). These values were highly prominent for strains originating from the same farm/outbreak/animal. Reference genome and alignment free analysis methods (kSNP 3) based on pan-genome SNPs (SNP, Single Nucleotide Polymorphism) were found to be useful for initial clustering of isolates. The strains were clustered based on unique SNPs. The number of allele specific SNPs which are unique for each strain was higher for strains from exotic origin. Read mapping and SNP calling (Snippy) based on a related reference genome (12S0467) was able to provide novel insights to understand the general, outbreak and within-host microevolution of a pathogen. The median pairwise SNP difference value was higher for C. chauvoei strains from Bavaria and Austria as compared to strains from Lower Saxony. The diversity of geographically related strains could therefore be indicative of other environmental factors or herd management influencing strain microevolution. Nonsynonymous SNPs contributed significantly to the observed microevolution of the pathogen. Based on the strain variability observed for strains from the same host and strains from the same outbreak, this study also proved the possibility of genetically different C. chauvoei populations in one host. A very recent study, based on genome sequence data, has pointed out the applicability of strain differentiation based on CRISPR spacer sequences for C. chauvoei. The current study similarly proved the applicability of typing approaches for this pathogen based on CRISPR elements and core genome MLST. The CRISPR spacer diversity was found to be inadequate to differentiate strains of European origin. However core genome MLST was able to differentiate the C. chauvoei strains within Europe and Germany and thus proved to be a valuable tool for strain differentiation. The current study applied the advantage of long read based sequencing technology to generate high quality complete genome reference sequences for a field strain from Germany and the type strain of C. chauvoei. The phylogenetic positioning of the species within the genus was evaluated and the close relatedness to C. septicum was confirmed. Several genome analysis software tools provided novel insights in the genome content and composition of the pathogen. Comparative genome sequence analysis based on 64 strains showed limited horizontal gene transfer and novel gene acquisition. Strain typing based on core genome MLST analysed used the first time for this species could differentiate strains even at farm level.