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.
Highlights
Clostridium perfringens is a Gram-positive, spore-forming, anaerobic, rod-shaped bacterium, known as an important causative agent of foodborne and non-foodborne gastroenteritis (Grass et al, 2013)
To improve epidemiologic knowledge about C. perfringens involved in foodborne outbreaks (FBOs), we developed a new real-time PCR typing technique targeting 17 genes encoding 15 virulence factors
Our study shows that FBOs were commonly due to meat and meat product foods (52%, 22/42), which is consistent with the epidemiology of C. perfringens (Wen and McClane, 2004; Grass et al, 2013)
Summary
Clostridium perfringens is a Gram-positive, spore-forming, anaerobic, rod-shaped bacterium, known as an important causative agent of foodborne and non-foodborne gastroenteritis (Grass et al, 2013). C. perfringens is commonly found as a contaminant in meat and poultry products, as well as in vegetables and crops This bacterium is an important pathogen involved in foodborne outbreaks (FBOs) and ranks as the fourth most common causative agent of foodborne illness due to bacterial toxins in France and Europe, with more than 1,500 human cases each year (EFSA, 2016). The virulence factors of C. perfringens can be classified functionally as membranedamaging enzymes, pore-forming toxins, intracellular toxins, and hydrolytic enzymes (Revitt-Mills et al, 2015) Genes encoding these virulence factors may be located on the chromosome, and on the large plasmid (Freedman et al, 2016). Partial genome sequencing of the canine isolate revealed three novel putative toxin genes encoding proteins related to the
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