Genetic characterization of type A enterotoxigenic Clostridium perfringens strains

Clostridium perfringens type A, is both a ubiquitous environmental bacterium and a major cause of human gastrointestinal disease, which usually involves strains producing C. perfringens enterotoxin (CPE). The gene (cpe) encoding this toxin can be carried on the chromosome or a large plasmid. Interestingly, strains carrying cpe on the chromosome and strains carrying cpe on a plasmid often exhibit different biological characteristics, such as resistance properties against heat. In this study, we investigated the genetic properties of C. perfringens by PCR-surveying 21 housekeeping genes and genes on representative plasmids and then confirmed those results by Southern blot assay (SB) of five genes. Furthermore, sequencing analysis of eight housekeeping genes and multilocus sequence typing (MLST) analysis were also performed. Fifty-eight C. perfringens strains were examined, including isolates from: food poisoning cases, human gastrointestinal disease cases, foods in Japan or the USA, or feces of healthy humans. In the PCR survey, eight of eleven housekeeping genes amplified positive reactions in all strains tested. However, by PCR survey and SB assay, one representative virulence gene, pfoA, was not detected in any strains carrying cpe on the chromosome. Genes involved in conjugative transfer of the cpe plasmid were also absent from almost all chromosomal cpe strains. MLST showed that, regardless of their geographic origin, date of isolation, or isolation source, chromosomal cpe isolates, i) assemble into one definitive cluster ii) lack pfoA and iii) lack a plasmid related to the cpe plasmid. Similarly, independent of their origin, strains carrying a cpe plasmid also appear to be related, but are more variable than chromosomal cpe strains, possibly because of the instability of cpe-borne plasmid(s) and/or the conjugative transfer of cpe-plasmid(s) into unrelated C. perfringens strains. © 2009 Deguchi et al

Primers used in this study.

<p>Primers used in this study.</p

<i>Clostridium perfringens</i> strains used in this study.

<p><i>Clostridium perfringens</i> strains used in this study.</p

The results of Southern blot assay with seven genes.

<p>NT: not tested</p

PCR assay for the <i>pfoA</i> region in <i>pfoA</i>-negative, <i>cpe</i>-positive strains.

<p>(A) Schematic representation of the <i>pfoA</i> region in <i>C. perfringens</i>. Genetic organization of the <i>pfoA</i> region is shown for <i>pfoA</i>-positive, <i>cpe</i>-negative ATCC13124 and <i>pfoA</i>-negative, <i>cpe</i>-positive SM101 strains (accession number: CP000312, and NC008261). The arrows depict the position of primers for the <i>pfoA</i> genotyping assay. The black bars show the predictive PCR products. (B) PCR results of <i>cpe</i>-positive, <i>pfoA</i>-negative strains investigated by the <i>pfoA</i> PCR genotyping assay. An ∼2,375 bp PCR product was obtained by <i>pfoA</i>-positive, <i>cpe</i>-negative ATCC13124 strain. An ∼280 bp PCR products was obtained from <i>pfoA</i>-negative, <i>cpe</i>-positive strains.</p

The results of PCR survey of the representative genes.

<p>The results of PCR survey of the representative genes.</p

Southern blot assay of chromosomal- or plasmid-<i>cpe</i> strains.

<p>DNA digested with <i>Pst</i>I from <i>cpe</i>-positive and <i>cpe</i>-negative (M-08 and MR2-12) type A strains was subjected to 1% agarose electrophoresis prior to Southern blotting and hybridization with a DIG-labeled, <i>pfoA</i>-, <i>virS</i>-, or <i>eno</i>-specific probe.</p

Phylogenetic relationships among 58 <i>cpe</i>-positive or <i>cpe</i>-negative <i>C. perfringens</i> strains.

<p>The phylogenetic tree was constructed by Clustal W analysis based on the concatenated nucleotide sequence of eight housekeeping genes. The phylogenetic clusters are given on the right.</p