Large-Scale Genomic Analyses and Toxinotyping of Clostridium perfringens Implicated in Foodborne Outbreaks in France

Abstract

Clostridium perfringens is both an ubiquitous environmental bacterium and the fourth most common causative agent of foodborne outbreaks (FBOs) in France and Europe. These outbreaks are known to be caused by C. perfringens enterotoxin (CPE) encoded by the cpe gene. However, additional information on the toxin/virulence gene content of C. perfringens has become available in the last few years. Therefore, to understand the enteropathogenicity of this bacterium, we need to describe the toxin and virulence genes content of strains involved in FBOs. In this study, we used a new real-time PCR typing technique based on a comprehensive set of 17 genes encoding virulence factors. The analysis was performed on a collection of 141 strains involved in 42 FBOs in the Paris region. It was combined with whole genome sequence (WGS) phylogenomic reconstruction, based on the coregenome single nucleotide polymorphisms (SNPs) of 58 isolates, representatives of the identified virulence gene profiles. Two or three different virulence gene profiles were detected in 10 FBOs, demonstrating that C. perfringens FBOs may be associated with heterogeneous strains. cpe- positive strains were isolated in 23 outbreaks, confirming the prominent role of CPE in pathogenicity. However, while C. perfringens was the sole pathogen isolated from the incriminated food, the cpe gene was not detected in strains related to 13 outbreaks. This result indicates either that the standard method was not able to isolate cpe+ strains or that the cpe gene may not be the only determinant of the enterotoxigenic potential of C. perfringens strains. Using phylogenomic reconstruction, we identified two clades distinguishing chromosomal cpe -positive from cpe -negative and plasmid-borne cpe. Important epidemiological information was also garnered from this phylogenomic reconstruction that revealed unexpected links between different outbreaks associated with closely related strains (seven SNP differences) and having common virulence gene profiles. This study provides new insight into the characterization of foodborne C. perfringens and highlights the potential of WGS for the investigation of FBOs.

The Circulation of Type F Clostridium perfringens among Humans, Sewage, and Ruditapes philippinarum (Asari Clams)

“…Analysis by MLST indicated that the isolates with plasmid IS 1151 - cpe from a human, four influents, two effluents, and four asari clams were categorized as an identical clone identified as the asari clam clone, which was clearly different from strains with chromosomal cpe and plasmid IS 1470 -like- cpe isolated from meats. In addition, this result agrees with studies that have shown that strains with chromosomal cpe belonged to a single distinct cluster [ 39 , 40 ] and exhibited different genetic characteristics from those with plasmidal cpe revealed by analysis based on whole genome sequencing [ 41 , 42 ]. Additionally, the asari clam clone was predominant among the isolates from influents (40%), suggesting that the asari clam clone is also predominant among unreported human isolates, effluents (50%), and asari clams (57%) with plasmid IS 1151 - cpe tested in this study.…”

Phylogenomic analysis ofClostridium perfringensidentifies isogenic strains in gastroenteritis outbreaks, and novel virulence-related features

“…This is linked to their phenotypic capacity to withstand high temperatures (via production of a protective small acid soluble protein), and high salt concentrations during the cooking process, in addition to the shorter generation time, when compared to plasmid- cpe carrying strains (68, 91). Previous studies have indicated that these strains commonly assemble into distinct clusters that lack the pfo gene, which we also noted in the FP lineage I data from this study (26, 92–94). Nevertheless, plasmid-borne cpe -carrying strains (pCPF4969 or pCPF5603) have also been associated with previous food poisoning outbreaks, with a previous study indicating that pCPF5603-carrying strains (encoding IS 1151 ) were associated with food poisoning in Japanese nursing homes (7 out of 9 isolates) (71).…”

“…pfo and cpb2 ) may be associated with C. perfringens-associated gastroenteritis. A recent WGS-based study on FP C. perfringens outbreaks in France determined that ~30% of isolates were cpe -negative (13/42) (26), indicating this gene may not be the sole virulence determinant linked to C. perfringens gastroenteritis. Although we observed less cpe -negative strains in our collection in comparison to this study, this may be due to our targeted cpe -positive isolation strategy (standard practice at PHE).…”

“…Recent studies analysing a range of diverse C. perfringens strains (from both animal and human-associated infections) indicates a plastic and divergent pangenome, with a significant proportion of accessory genes predicted to be involved in virulence mechanisms and metabolisms, linked to enhanced host colonisation and disease initiation (3, 25). However, studies describing human outbreak-associated C. perfringens infections are limited, and to date only one recent study (58 isolates) has utilised Whole Genome Sequencing (WGS) data to probe the genomic epidemiology of associated strains (3, 26).…”

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

“…A recent WGS-based study on C. perfringens FP outbreaks in France determined that ~30 % of isolates were cpe- negative (13/42) [24], indicating this gene may not be the sole virulence determinant linked to C. perfringens gastroenteritis.…”