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Identification of novel Clostridium perfringens type E strains that carry an iota toxin plasmid with a functional enterotoxin gene.

Miyamoto K, Yumine N, Mimura K, Nagahama M, Li J, McClane BA, Akimoto S - PLoS ONE (2011)

Bottom Line: The ∼65 kb plasmid is closely related to the pCPF4969 cpe plasmid of type A isolates.MLST analyses indicated these isolates belong to a unique cluster of C. perfringens.Overall, these isolates carrying a variant functional cpe gene and iota toxin genes represent unique type E strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Wakayama Medical University School of Medicine, Wakayama, Japan. kazuaki@wakayama-med.ac.jp

ABSTRACT
Clostridium perfringens enterotoxin (CPE) is a major virulence factor for human gastrointestinal diseases, such as food poisoning and antibiotic associated diarrhea. The CPE-encoding gene (cpe) can be chromosomal or plasmid-borne. Recent development of conventional PCR cpe-genotyping assays makes it possible to identify cpe location (chromosomal or plasmid) in type A isolates. Initial studies for developing cpe genotyping assays indicated that all cpe-positive strains isolated from sickened patients were typable by cpe-genotypes, but surveys of C. perfringens environmental strains or strains from feces of healthy people suggested that this assay might not be useful for some cpe-carrying type A isolates. In the current study, a pulsed-field gel electrophoresis Southern blot assay showed that four cpe-genotype untypable isolates carried their cpe gene on a plasmid of ∼65 kb. Complete sequence analysis of the ∼65 kb variant cpe-carrying plasmid revealed no intact IS elements and a disrupted cytosine methyltransferase (dcm) gene. More importantly, this plasmid contains a conjugative transfer region, a variant cpe gene and variant iota toxin genes. The toxin genes encoded by this plasmid are expressed based upon the results of RT-PCR assays. The ∼65 kb plasmid is closely related to the pCPF4969 cpe plasmid of type A isolates. MLST analyses indicated these isolates belong to a unique cluster of C. perfringens. Overall, these isolates carrying a variant functional cpe gene and iota toxin genes represent unique type E strains.

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Genetic organization of the variant cpe toxin locus.A. Genetic organization of the toxin locus in pCPPB-1 versus the toxin locus in previously studied C. perfringens type E strain (JGS1987) [16]. Broad bars show ORFs. Long thin bars depict the PCR products as shown in Panel B, amplified with each primer pair (see the text). B. PCR analysis of the toxin locus in C. perfringens isolates carrying the variant cpe gene.
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pone-0020376-g003: Genetic organization of the variant cpe toxin locus.A. Genetic organization of the toxin locus in pCPPB-1 versus the toxin locus in previously studied C. perfringens type E strain (JGS1987) [16]. Broad bars show ORFs. Long thin bars depict the PCR products as shown in Panel B, amplified with each primer pair (see the text). B. PCR analysis of the toxin locus in C. perfringens isolates carrying the variant cpe gene.

Mentions: Surprisingly, this sequencing detected the presence of two ORFs encoding homologues of the iota toxin genes that characterize C. perfringens type E strains. These iota toxin genes of PB-1 strain resided near the variant cpe gene (Figure 3A). The toxin locus in pCPPB-1 has a generally similar organization as the iota toxin plasmid of classical type E strains, except the cpe gene is intact and not disrupted by the nonsense and frame-shift mutations found in other type E strains (Figure 3A) [16].


Identification of novel Clostridium perfringens type E strains that carry an iota toxin plasmid with a functional enterotoxin gene.

Miyamoto K, Yumine N, Mimura K, Nagahama M, Li J, McClane BA, Akimoto S - PLoS ONE (2011)

Genetic organization of the variant cpe toxin locus.A. Genetic organization of the toxin locus in pCPPB-1 versus the toxin locus in previously studied C. perfringens type E strain (JGS1987) [16]. Broad bars show ORFs. Long thin bars depict the PCR products as shown in Panel B, amplified with each primer pair (see the text). B. PCR analysis of the toxin locus in C. perfringens isolates carrying the variant cpe gene.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105049&req=5

pone-0020376-g003: Genetic organization of the variant cpe toxin locus.A. Genetic organization of the toxin locus in pCPPB-1 versus the toxin locus in previously studied C. perfringens type E strain (JGS1987) [16]. Broad bars show ORFs. Long thin bars depict the PCR products as shown in Panel B, amplified with each primer pair (see the text). B. PCR analysis of the toxin locus in C. perfringens isolates carrying the variant cpe gene.
Mentions: Surprisingly, this sequencing detected the presence of two ORFs encoding homologues of the iota toxin genes that characterize C. perfringens type E strains. These iota toxin genes of PB-1 strain resided near the variant cpe gene (Figure 3A). The toxin locus in pCPPB-1 has a generally similar organization as the iota toxin plasmid of classical type E strains, except the cpe gene is intact and not disrupted by the nonsense and frame-shift mutations found in other type E strains (Figure 3A) [16].

Bottom Line: The ∼65 kb plasmid is closely related to the pCPF4969 cpe plasmid of type A isolates.MLST analyses indicated these isolates belong to a unique cluster of C. perfringens.Overall, these isolates carrying a variant functional cpe gene and iota toxin genes represent unique type E strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Wakayama Medical University School of Medicine, Wakayama, Japan. kazuaki@wakayama-med.ac.jp

ABSTRACT
Clostridium perfringens enterotoxin (CPE) is a major virulence factor for human gastrointestinal diseases, such as food poisoning and antibiotic associated diarrhea. The CPE-encoding gene (cpe) can be chromosomal or plasmid-borne. Recent development of conventional PCR cpe-genotyping assays makes it possible to identify cpe location (chromosomal or plasmid) in type A isolates. Initial studies for developing cpe genotyping assays indicated that all cpe-positive strains isolated from sickened patients were typable by cpe-genotypes, but surveys of C. perfringens environmental strains or strains from feces of healthy people suggested that this assay might not be useful for some cpe-carrying type A isolates. In the current study, a pulsed-field gel electrophoresis Southern blot assay showed that four cpe-genotype untypable isolates carried their cpe gene on a plasmid of ∼65 kb. Complete sequence analysis of the ∼65 kb variant cpe-carrying plasmid revealed no intact IS elements and a disrupted cytosine methyltransferase (dcm) gene. More importantly, this plasmid contains a conjugative transfer region, a variant cpe gene and variant iota toxin genes. The toxin genes encoded by this plasmid are expressed based upon the results of RT-PCR assays. The ∼65 kb plasmid is closely related to the pCPF4969 cpe plasmid of type A isolates. MLST analyses indicated these isolates belong to a unique cluster of C. perfringens. Overall, these isolates carrying a variant functional cpe gene and iota toxin genes represent unique type E strains.

Show MeSH
Related in: MedlinePlus