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Prevalence and Genetic Diversity of Toxin Genes in Clinical Isolates of Clostridium perfringens: Coexistence of Alpha-Toxin Variant and Binary Enterotoxin Genes (bec/cpile).

ABSTRACT: Clostridium perfringens (C. perfringens) is responsible for food-borne gastroenteritis and other infectious diseases, and toxins produced by this bacterium play a key role in pathogenesis. Although various toxins have been described for C. perfringens isolates from humans and animals, prevalence of individual toxins among clinical isolates has not yet been well explored. In the present study, a total of 798 C. perfringens clinical isolates were investigated for prevalence of eight toxin genes and their genetic diversity by PCR, nucleotide sequencing, and phylogenetic analysis. Besides the alpha-toxin gene (plc) present in all the isolates, the most common toxin gene was cpe (enterotoxin) (34.2%), followed by cpb2 (beta2 toxin) (1.4%), netB (NetB) (0.3%), and bec/cpile (binary enterotoxin BEC/CPILE) (0.1%), while beta-, epsilon-, and iota-toxin genes were not detected. Genetic analysis of toxin genes indicated a high level of conservation of plc, cpe, and netB. In contrast, cpb2 was revealed to be considerably divergent, containing at least two lineages. Alpha-toxin among 46 isolates was classified into ten sequence types, among which common types were distinct from those reported for avian isolates. A single isolate with bec/cpile harbored a plc variant containing an insertion of 834-bp sequence, suggesting its putative origin from chickens.

SUBMITTER: Matsuda A

PROVIDER: S-EPMC6628447 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Prevalence and Genetic Diversity of Toxin Genes in Clinical Isolates of Clostridium perfringens: Coexistence of Alpha-Toxin Variant and Binary Enterotoxin Genes (bec/cpile).

Matsuda Asami A Aung Meiji Soe MS Urushibara Noriko N Kawaguchiya Mitsuyo M Sumi Ayako A Nakamura Mayumi M Horino Yuka Y Ito Masahiko M Habadera Satoshi S Kobayashi Nobumichi N

Toxins 20190606 6

Clostridium perfringens (C. perfringens) is responsible for food-borne gastroenteritis and other infectious diseases, and toxins produced by this bacterium play a key role in pathogenesis. Although various toxins have been described for C. perfringens isolates from humans and animals, prevalence of individual toxins among clinical isolates has not yet been well explored. In the present study, a total of 798 C. perfringens clinical isolates were investigated for preval ...[more]

PMID: 31174364

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Project description:BackgroundBEC-producing Clostridium perfringens is a causative agent of foodborne gastroenteritis. It was first reported in 2014, and since then, several isolates have been identified in Japan and the United Kingdom. The novel binary ADP-ribosylating toxin BEC, which consists of two components (BECa and BECb), is encoded on a plasmid that is similar to pCP13 and harbours a conjugation locus, called Pcp, encoding homologous proteins of the type 4 secretion system. Despite the high in vitro conjugation frequency of pCP13, its dissemination and that of related plasmids, including bec-harbouring plasmids, in the natural environment have not been characterised. This lack of knowledge has limited our understanding of the genomic epidemiology of bec-harbouring C. perfringens strains.ResultsIn this study, we determined the complete genome sequences of five bec-harbouring C. perfringens strains isolated from 2009 to 2019. Each isolate contains a ~ 3.36 Mbp chromosome and 1-3 plasmids of either the pCW3-like family, pCP13-like family, or an unknown family, and the bec-encoding region in all five isolates was located on a ~ 54 kbp pCP13-like plasmid. Phylogenetic and SNP analyses of these complete genome sequences and the 211 assembled C. perfringens genomes in GenBank showed that although these bec-harbouring strains were split into two phylogenetic clades, the sequences of the bec-encoding plasmids were nearly identical (>99.81%), with a significantly smaller SNP accumulation rate than that of their chromosomes. Given that the Pcp locus is conserved in these pCP13-like plasmids, we propose a mechanism in which the plasmids were disseminated by horizontal gene transfer. Data mining showed that strains carrying pCP13-like family plasmids were unexpectedly common (58/216 strains) and widely disseminated among the various C. perfringens clades. Although these plasmids possess a conserved Pcp locus, their 'accessory regions' can accommodate a wide variety of genes, including virulence-associated genes, such as becA/becB and cbp2. These results suggest that this family of plasmids can integrate various foreign genes and is transmissible among C. perfringens strains.ConclusionThis study demonstrates the potential significance of pCP13-like plasmids, including bec-encoding plasmids, for the characterisation and monitoring of the dissemination of pathogenic C. perfringens strains.

Project description:Clostridium perfringens is a widespread pathogen, but the precise quantification of this subdominant gut microbe remains difficult due to its low fecal count (particularly in asymptomatic subjects) and also due to the presence of abundant polymerase-inhibitory substances in human feces. Also, information on the intestinal carriage of toxigenic C. perfringens strains in healthy subjects is sparse. Therefore, we developed a sensitive quantitative real-time PCR assays for quantification of C. perfringens in human feces by targeting its ?-toxin and enterotoxin genes. To validate the assays, we finally observed the occurrence of ?-toxigenic and enterotoxigenic C. perfringens in the fecal microbiota of healthy Japanese infants and young adults.The plc-specific qPCR assay was newly validated, while primers for 16S rRNA and cpe genes were retrieved from literature. The assays were validated for specificity and sensitivity in pre-inoculated fecal samples, and were finally applied to quantify C. perfringens in stool samples from apparently healthy infants (n 124) and young adults (n 221).The qPCR assays were highly specific and sensitive, with a minimum detection limit of 10(3) bacterial cells/g feces. Alpha-toxigenic C. perfringens was detected in 36% infants and 33% adults, with counts ranging widely (10(3)-10(7) bacterial cells/g). Intriguingly, the mean count of ?-toxigenic C. perfringens was significantly higher in infants (6.0±1.5 log10 bacterial cells/g), as compared to that in adults (4.8±1.2). Moreover, the prevalence of enterotoxigenic C. perfringens was also found to be significantly higher in infants, as compared to that in adults. The mean enterotoxigenic C. perfringens count was 5.9±1.9 and 4.8±0.8 log10 bacterial cells/g in infants and adults, respectively.These data indicate that some healthy infants and young adults carry ?-toxigenic and enterotoxigenic C. perfringens at significant levels, and may be predisposed to related diseases. Thus, high fecal carriage of toxigenic C. perfringens in healthy children warrants further investigation on its potential sources and clinical significance in these subjects. In summary, we present a novel qPCR assay for sensitive and accurate quantification of ?-toxigenic and enterotoxigenic C. perfringens in human feces, which should facilitate prospective studies of the gut microbiota.

Dataset Information

Prevalence and Genetic Diversity of Toxin Genes in Clinical Isolates of Clostridium perfringens: Coexistence of Alpha-Toxin Variant and Binary Enterotoxin Genes (bec/cpile).

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Prevalence and Genetic Diversity of Toxin Genes in Clinical Isolates of <i>Clostridium perfringens</i>: Coexistence of Alpha-Toxin Variant and Binary Enterotoxin Genes (<i>bec</i>/<i>cpile</i>).

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