Phylogenomic analysis of gastroenteritis-associated Clostridium perfringens in England and Wales over a 7-year period indicates distribution of clonal toxigenic strains in multiple outbreaks and extensive involvement of enterotoxin-encoding (CPE) plasmids

Abstract

Clostridium perfringens is a major enteric pathogen known to cause gastroenteritis in human adults. Although major outbreak cases are frequently reported, only limited whole-genome sequencing (WGS) based studies have been performed to understand the genomic epidemiology and virulence gene content of outbreak-associated C. perfringens strains. We performed phylogenomic analysis on 109 C. perfringens isolates (human and food) obtained from disease cases in England and Wales between 2011 and 2017. Initial findings highlighted the enhanced discriminatory power of WGS in profiling outbreak C. perfringens strains, when compared to the current Public Health England referencing laboratory technique of fluorescent amplified fragment length polymorphism analysis. Further analysis identified that isogenic C. perfringens strains were associated with nine distinct care-home-associated outbreaks over the course of a 5-year interval, indicating a potential common source linked to these outbreaks or transmission over time and space. As expected, the enterotoxin cpe gene was encoded in all but 4 isolates (96.3 %; 105/109), with virulence plasmids encoding cpe (particularly pCPF5603 and pCPF4969 plasmids) extensively distributed (82.6 %; 90/109). Genes encoding accessory virulence factors, such as beta-2 toxin, were commonly detected (46.7 %; 51/109), and genes encoding phage proteins were also frequently identified. Overall, this large-scale genomic study of gastroenteritis-associated C. perfringens suggested that three major cpe-encoding (toxinotype F) genotypes underlie these outbreaks: strains carrying (1) pCPF5603 plasmid, (2) pCPF4969 plasmid and (3) chromosomal-cpe strains. Our findings substantially expanded our knowledge on type F C. perfringens involved in human-associated gastroenteritis, with further studies required to fully probe the dissemination and regional reservoirs of this enteric pathogen, which may help devise effective prevention strategies to reduce the food-poisoning disease burden in vulnerable patients, such as the elderly.

Keywords: Clostridium perfringens; food poisoning; gastroenteritis; genomic epidemiology; outbreaks; phylogenomics.

Fig. 1.

Population structure and statistics of the 109 C. perfringens genomes in this study. (a) Population structure of 109 C. perfringens isolates analysed in this study. Mid-point rooted maximum-likelihood phylogeny inferred from 73 882 SNPs identified in 110 diarrhoea-associated C. perfringens isolates (including NCTC 8239). The colour-coded rings indicate the cohort-specific origins of the isolates. Cluster VIII (green ring; clusters determined via hierBAPS clustering analysis) consists of primarily isolates obtained from multiple CH-associated outbreaks. Historical FP isolate NCTC 8239 was used as a reference genome as indicated in the figure. Bootstrap values are represented in the tree. Branch lengths are indicative of the estimated nucleotide substitution per site (SNPs). (b) Temporal distribution of all 109 C. perfringens genomes included in this study (2011–2017). (c) Contig count distribution of C. perfringens genome assemblies in this study. More than 70 % of the total assemblies are <200 contigs.

Fig. 2.

Phylogenomic analysis of CH-associated C. perfringens isolates. (a) Mid-point rooted maximum-likelihood phylogeny inferred from 64 560 SNPs (in core gene alignment) identified in 35 CH C. perfringens isolates. The colour strips indicate outbreaks (1), location of outbreaks (2) and fAFLP types (3) corresponding to the isolates. Branch lengths are indicative of the estimated SNP distance. Lineages and sub-lineages were determined via hierBAPS (level 1 and 2) clustering analysis. NCTC 8239 was used as a reference genome in this tree. Bootstrapping values are represented on the tree. (b) Unrooted maximum-likelihood tree (inferred from 191 SNPs in 18 genomes) of a sub-lineage IVc showing SNP distances between 18 North-East England derived isolates of individual outbreaks (labelled with locations, years and SNP range in the outbreaks; branches are colour-coded corresponding to individual outbreaks). SNPs between branches are indicated in red. (c) Pairwise within-outbreak core-SNP distance between isolates. (d) Pairwise outside-sub-lineage (IVb vs IVc) SNP comparison between isolates. Data were analysed using the Mann-Whitney test; ****, P<0.0001.

Fig. 3.

Phylogenomic analysis of FP C. perfringens isolates. (a) Mid-point rooted maximum-likelihood phylogeny of FP C. perfringens inferred from 70 613 SNPs (in core gene alignment) identified in 75 individual isolates. Lineages were determined via hierBAPS clustering analysis. Bootstrap values are represented in the tree. (b) Unrooted maximum-likelihood tree inferred from 2505 SNPs in lineage VII (31 isolates). Four distinct clusters were identified in individual outbreaks comprising genetically similar strains (labelled with locations, years and SNP range within the outbreaks; branches are colour-coded corresponding to outbreak labels). (c) Pairwise SNP distance comparison in-between isolates within outbreaks. (d) Pairwise SNP comparisons of within-major-lineage isolates in-between individual outbreaks. Lineage I, outbreaks 1,4,13 and 14; lineage IV, outbreaks 3 and 7; lineage VII, outbreaks 2, 7 and 10. Data were analysed using the Kruskal-Wallis test; ****, P<0.0001.

Fig. 4.

Virulence profiles of C. perfringens isolates including virulence plasmids. Binary heatmaps displaying the presence and absence of toxin genes, AMR genes, plasmids, plasmid-related sequences and tcp conjugative loci in: (a) CH isolates, (b) FP isolates. Outbreaks are colour-coded according to the colour system in previous figures. Coloured cells represent the presence of genes and white cells represent the absence of genes.

Fig. 5.

Investigations of predicted plasmids carried by CH and FP isolates. (a) Comparison of reference plasmids pCPF4969 and pCPF5603 with annotated features. CDS, Coding sequence. (b) Genomic comparison of pCPF5603 reference plasmid and predicted cpe plasmids from three CH isolates. (c) Plasmid comparison between pCPF4969 plasmid and FP isolate predicted cpe plasmids. (d) cpe regions (Tn5565) extracted computationally from FP lineage I representative isolate genomes. (e) A computationally extracted 11 kbp region of NCTC 8239 that encodes Tn5565 (including cpe and flanking IS1470 elements) compared to the predicted cpe region from PH004.