Necrotic Enteritis-Derived Clostridium perfringens Strain with Three Closely Related Independently Conjugative Toxin and Antibiotic Resistance Plasmids

Abstract

The pathogenesis of avian necrotic enteritis involves NetB, a pore-forming toxin produced by virulent avian isolates of Clostridium perfringens type A. To determine the location and mobility of the netB structural gene, we examined a derivative of the tetracycline-resistant necrotic enteritis strain EHE-NE18, in which netB was insertionally inactivated by the chloramphenicol and thiamphenicol resistance gene catP. 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 the netB gene 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 from C. 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 the netB gene and other potential virulence genes, and (iii) a 70-kb plasmid that carried the cpb2 gene, which encodes a different pore-forming toxin, beta2 toxin.

Toxin Plasmids of Clostridium perfringens

“…Similarly, the netB-derived plasmids pJIR3536 and pNetB-NE10 share ϳ35 kb of conserved backbone (Fig. 2) with pCPF5603 and pCW3 (13,19,235,236). As mentioned above, some type E iota-toxin-encoding plasmids share substantial similarity with pCPF5603 (18), while others more closely resemble pCPF4969 (23).…”

“…A recent study (19) determined that NetB is encoded on a large conjugative plasmid in the type A avian necrotic enteritis strain EHE-NE18, which also carries two other large plasmids. High-throughput sequencing identified three closely related conjugative plasmids in this strain, including (i) the 82-kb plasmid pJIR3535, which encodes the netB gene and other potential virulence genes (Fig.…”

“…For example, no C. perfringens isolate has been found to carry both iap/ibp genes and the cpb or etx gene, suggesting fundamental plasmid incompatibility issues. However, some toxin plasmid combinations can be stably maintained in a single C. perfringens cell; e.g., some chicken necrotic enteritis strains can carry three related plasmids, including two different toxin plasmids, while type B isolates carry their cpb and etx genes on separate plasmids (13,16,19). In this regard, it is notable that the cpbcarrying plasmids and etx-carrying plasmids in type B strains are much less diverse than the cpb-carrying plasmids in type C strains or the etx-carrying plasmids of type D strains (15)(16)(17), further suggesting that only certain plasmid combinations can be stably maintained in the same C. perfringens cell.…”

“…It has now become clear that many important C. perfringens toxins are encoded by large plasmids (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). Other recent studies, described later in this review, have provided important insights into the diversity of C. perfringens toxin plasmids, the critical importance of these plasmids for pathogenesis, and the ability of toxin plasmids to transfer among C. perfringens strains.…”

Clostridium perfringens extracellular toxins and enzymes: 20 and counting

“…With a few important exceptions, these toxins are encoded on large conjugative plasmids 4,10,[12][13][14][15][16][17][18] , which allows for potential toxin gene transfer between different C. perfringens strains in the gastrointestinal tract and may prolong disease 10 . C. perfringens utilises chromosomally encoded toxins, such as alpha-toxin and perfringolysin O, during human histotoxic infections or human food poisoning (C. perfringens enterotoxin, CPE) 3 .…”