ABSTRACTThe pathogenesis of avian necrotic enteritis involves NetB, a pore-forming toxin produced by virulent avian isolates ofClostridium perfringenstype A. To determine the location and mobility of thenetBstructural gene, we examined a derivative of the tetracycline-resistant necrotic enteritis strain EHE-NE18, in whichnetBwas insertionally inactivated by the chloramphenicol and thiamphenicol resistance genecatP. Both tetracycline and thiamphenicol resistance could be transferred either together or separately to a recipient strain in plate matings. The separate transconjugants could act as donors in subsequent matings, which demonstrated that the tetracycline resistance determinant and thenetBgene were present on different conjugative elements. Large plasmids were isolated from the transconjugants and analyzed by high-throughput sequencing. Analysis of the resultant data indicated that there were actually three large conjugative plasmids present in the original strain, each with its own toxin or antibiotic resistance locus. Each plasmid contained a highly conserved 40-kb region that included plasmid replication and transfer regions that were closely related to the 47-kb conjugative tetracycline resistance plasmid pCW3 fromC. perfringens. The plasmids were as follows: (i) a conjugative 49-kb tetracycline resistance plasmid that was very similar to pCW3, (ii) a conjugative 82-kb plasmid that contained thenetBgene and other potential virulence genes, and (iii) a 70-kb plasmid that carried thecpb2gene, which encodes a different pore-forming toxin, beta2 toxin.IMPORTANCEThe anaerobic bacteriumClostridium perfringenscan cause an avian gastrointestinal disease known as necrotic enteritis. Disease pathogenesis is not well understood, although the plasmid-encoded pore-forming toxin NetB, is an important virulence factor. In this work, we have shown that the plasmid that carries thenetBgene is conjugative and has a 40-kb region that is very similar to replication and transfer regions found within each of the sequenced conjugative plasmids fromC. perfringens. We also showed that this strain contained two additional large plasmids that were also conjugative and carried a similar 40-kb region. One of these plasmids encoded beta2 toxin, and the other encoded tetracycline resistance. To our knowledge, this is the first report of a bacterial strain that carries three closely related but different independently conjugative plasmids. These results have significant implications for our understanding of the transmission of virulence and antibiotic resistance genes in pathogenic bacteria.
Association of a Penicillin Resistance Gene with a Tetracycline Resistance Plasmid (PTP-2) in Staphylococcus aureus