Probing Genomic Aspects of the Multi-Host Pathogen Clostridium perfringens Reveals Significant Pangenome Diversity, and a Diverse Array of Virulence Factors

Abstract

Clostridium perfringens is an important cause of animal and human infections, however information about the genetic makeup of this pathogenic bacterium is currently limited. In this study, we sought to understand and characterise the genomic variation, pangenomic diversity, and key virulence traits of 56 C. perfringens strains which included 51 public, and 5 newly sequenced and annotated genomes using Whole Genome Sequencing. Our investigation revealed that C. perfringens has an "open" pangenome comprising 11667 genes and 12.6% of core genes, identified as the most divergent single-species Gram-positive bacterial pangenome currently reported. Our computational analyses also defined C. perfringens phylogeny (16S rRNA gene) in relation to some 25 Clostridium species, with C. baratii and C. sardiniense determined to be the closest relatives. Profiling virulence-associated factors confirmed presence of well-characterised C. perfringens-associated exotoxins genes including α-toxin (plc), enterotoxin (cpe), and Perfringolysin O (pfo or pfoA), although interestingly there did not appear to be a close correlation with encoded toxin type and disease phenotype. Furthermore, genomic analysis indicated significant horizontal gene transfer events as defined by presence of prophage genomes, and notably absence of CRISPR defence systems in >70% (40/56) of the strains. In relation to antimicrobial resistance mechanisms, tetracycline resistance genes (tet) and anti-defensins genes (mprF) were consistently detected in silico (tet: 75%; mprF: 100%). However, pre-antibiotic era strain genomes did not encode for tet, thus implying antimicrobial selective pressures in C. perfringens evolutionary history over the past 80 years. This study provides new genomic understanding of this genetically divergent multi-host bacterium, and further expands our knowledge on this medically and veterinary important pathogen.

Keywords: Clostridial infection; Clostridium perfringens; antimicrobial resistance; exotoxins; genomics; pangenome; whole genome sequencing.

Figure 1

16S rRNA phylogeny of representative Clostridium species (25) and C. perfringens. (A) Maximum-likelihood phylogenetic tree of C. perfringens strains and representative Clostridium species (25) based on 16S rRNA genes with aLRT branch support values displayed. (B) Neighbour-joining phylogeny based on 16S rRNA genes supported by 1000 bootstrap replicates based on 1585 sites. Clusters 1 and 2 were assigned for description purposes. Branch support values >70 are shown on the nodes.

Figure 2

Visualisation and statistics of C. perfringens pangenome. (A) Core and accessory genes statistics. (B) Frequency bar graphs of number of identical genes against number of genomes. (C) Number of new genes and unique genes along pangenome computation. (D) Number of conserved genes and total genes along pangenome calculation. (E) Linearised pangenomic view of 56 C. perfringens isolates. Green cells represent syntenies aligned next to Neighbour-Joining core-genome based phylogenetic tree.

Figure 3

Genome comparison and core-genome phylogeny of C. perfringens. Core-genome based Neighbour-Joining (NJ) phylogeny of 56 isolates. Branch support values >70 (based on 1,000 bootstrap replicates) are shown on the nodes. Categories of metadata were displayed for comparison. MJR7757A is assigned to Clade 4 although it is distant from the rest of JFP isolates.

Figure 4

Functional annotation (COG) of C. perfringens core and accessory genomes. (A) Core genome functional annotation. (B) Accessory genome functional annotation. The smaller-scale grey-background charts show the percentage of gene count of each functional class in both core and accessory genomes corresponding to the functional categories. Non-matching genes (unclassified) are not included in the statistics.

Figure 5

Virulence-associated potentials in C. perfringens genomes and plasmids. Heat-map visualising exotoxins, antimicrobial resistance, prophage profiles, and CRISPR counts of 56 C. perfringens genomes. Presence of genes are indicated by cell colours: grey (absence); red, blue, and yellow (presence). Purple-labelled isolates indicate historical isolates (pre-antibiotic era).