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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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Phylogenetic relationships of the tcpA gene among toxin plasmids and antibiotics resistant gene encoding plasmids of C. perfringens.The phylogenetic tree was constructed by Clustal W analysis based on the nucleotide sequence of tcpA genes (accession numbers; pCPF4969, NC007772; pCPF5603, NC007773; pCP8533etx, AB444205; pCW3, DQ366035; pJGS1987, NZ_ABDW01000012; pATCC3626, ABDV01000020; pJGS1721, NZ_ABOO01000017; pJGS1945, NZ_ABDU01000064; pJIR2774, DQ338473; and pJIR26, DQ338471).
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pone-0020376-g004: Phylogenetic relationships of the tcpA gene among toxin plasmids and antibiotics resistant gene encoding plasmids of C. perfringens.The phylogenetic tree was constructed by Clustal W analysis based on the nucleotide sequence of tcpA genes (accession numbers; pCPF4969, NC007772; pCPF5603, NC007773; pCP8533etx, AB444205; pCW3, DQ366035; pJGS1987, NZ_ABDW01000012; pATCC3626, ABDV01000020; pJGS1721, NZ_ABOO01000017; pJGS1945, NZ_ABDU01000064; pJIR2774, DQ338473; and pJIR26, DQ338471).

Mentions: The putative plasmid replication and conjugative transfer region of pCPPB-1 contains tcp genes (intP, tcpA, tcpC to tcpJ), putative adenine methyltransfease gene (dam), and collagen adhesion protein gene (cna), but not a putative intact cytosine methyltransfease gene (dcm). To investigate the genetic relationship of the pCPPB-1 plasmid with other toxin plasmids, the tcpA gene sequence in PB-1 was compared against the tcpA gene on other toxin plasmids, with the rationale that the TcpA protein is the most divergent of the transfer-related proteins carried on conjugative transferable C. perfringens plasmids [20]. Based on the sequence information obtained for the tcpA gene, pCPPB-1 appears to be more closely related with the iota toxin plasmid in type E strain rather than the type A cpe plasmids (Figure 4).


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)

Phylogenetic relationships of the tcpA gene among toxin plasmids and antibiotics resistant gene encoding plasmids of C. perfringens.The phylogenetic tree was constructed by Clustal W analysis based on the nucleotide sequence of tcpA genes (accession numbers; pCPF4969, NC007772; pCPF5603, NC007773; pCP8533etx, AB444205; pCW3, DQ366035; pJGS1987, NZ_ABDW01000012; pATCC3626, ABDV01000020; pJGS1721, NZ_ABOO01000017; pJGS1945, NZ_ABDU01000064; pJIR2774, DQ338473; and pJIR26, DQ338471).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020376-g004: Phylogenetic relationships of the tcpA gene among toxin plasmids and antibiotics resistant gene encoding plasmids of C. perfringens.The phylogenetic tree was constructed by Clustal W analysis based on the nucleotide sequence of tcpA genes (accession numbers; pCPF4969, NC007772; pCPF5603, NC007773; pCP8533etx, AB444205; pCW3, DQ366035; pJGS1987, NZ_ABDW01000012; pATCC3626, ABDV01000020; pJGS1721, NZ_ABOO01000017; pJGS1945, NZ_ABDU01000064; pJIR2774, DQ338473; and pJIR26, DQ338471).
Mentions: The putative plasmid replication and conjugative transfer region of pCPPB-1 contains tcp genes (intP, tcpA, tcpC to tcpJ), putative adenine methyltransfease gene (dam), and collagen adhesion protein gene (cna), but not a putative intact cytosine methyltransfease gene (dcm). To investigate the genetic relationship of the pCPPB-1 plasmid with other toxin plasmids, the tcpA gene sequence in PB-1 was compared against the tcpA gene on other toxin plasmids, with the rationale that the TcpA protein is the most divergent of the transfer-related proteins carried on conjugative transferable C. perfringens plasmids [20]. Based on the sequence information obtained for the tcpA gene, pCPPB-1 appears to be more closely related with the iota toxin plasmid in type E strain rather than the type A cpe plasmids (Figure 4).

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