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Clostridium perfringens is a causative agent of food-borne gastroenteritis for whichC. perfringens enterotoxin (CPE) has been considered an essential factor. Recently, we experienced two outbreaks of food-borne gastroenteritis in which non-CPE producers ofC. perfringens were strongly suspected to be the cause. Here, we report a novel enterotoxin produced byC. perfringens isolates, BEC (binary enterotoxin ofC. perfringens ). Culture supernatants of theC. perfringens strains showed fluid-accumulating activity in rabbit ileal loop and suckling mouse assays. Purification of the enterotoxic substance in the supernatants and high-throughput sequencing of genomic DNA of the strains revealed BEC, composed of BECa and BECb. BECa and BECb displayed limited amino acid sequence similarity to other binary toxin family members, such as theC. perfringens iota toxin. ThebecAB genes were located on 54.5-kb pCP13-like plasmids. Recombinant BECb (rBECb) alone had fluid-accumulating activity in the suckling mouse assay. Although rBECa alone did not show enterotoxic activity, rBECa enhanced the enterotoxicity of rBECb when simultaneously administered in suckling mice. The entertoxicity of the mutant in which thebecB gene was disrupted was dramatically decreased compared to that of the parental strain. rBECa showed an ADP-ribosylating activity on purified actin. Although we have not directly evaluated whether BECb delivers BECa into cells, rounding of Vero cells occurred only when cells were treated with both rBECa and rBECb. These results suggest that BEC is a novel enterotoxin ofC. perfringens distinct from CPE, and that BEC-producingC. perfringens strains can be causative agents of acute gastroenteritis in humans. Additionally, the presence ofbecAB on nearly identical plasmids in distinct lineages ofC. perfringens isolates suggests the involvement of horizontal gene transfer in the acquisition of the toxin genes.

BEC, a Novel Enterotoxin of Clostridium perfringens Found in Human Clinical Isolates from Acute Gastroenteritis Outbreaks