Epsilon-toxin plasmids of Clostridium perfringens type D are conjugative

Isolates of Clostridium perfringens type D produce the potent epsilon-toxin (a CDC/USDA overlap class B select agent) and are responsible for several economically significant enterotoxemias of domestic livestock. It is well established that the epsilon-toxin structural gene, etx, is encoded on large plasmids. We have now shown that at least two of these plasmids are conjugative. The etx gene on these plasmids was insertionally inactivated using a chloramphenicol resistance cassette to phenotypically tag the plasmid. High frequency conjugative transfer of these tagged plasmids into the C. perfringens type A strain JIR325 was demonstrated and the resultant transconjugants were shown to act as donors in subsequent mating experiments. We also demonstrated the transfer of unmarked native epsilon-toxin plasmids into strain JIR325, by exploiting the high transfer frequency. The transconjugants isolated from these experiments expressed functional epsilon-toxin since their supernatants caused cytopathic effects on MDCK cells and were toxic in mice. Using the widely accepted multiplex PCR approach to toxin genotyping, these type A-derived transconjugants were now genotypically type D. These findings have significant implications for the C. perfringens typing system since it is based on the toxin profile of each strain. Our study demonstrates the fluid nature of these toxinotypes and their dependence upon the presence or absence of toxin plasmids, some of which have for the first time been shown to be conjugative.