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.

Importance: The anaerobic bacterium Clostridium perfringens can 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 the netB gene 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 from C. 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.

FIG 1

PCR analysis of transconjugants. DNA from the strains indicated was subjected to PCR analysis for the presence of the netB, catP, and tetA(P) genes, and the resultant products were separated by agarose gel electrophoresis. (A) PCR analysis of primary transconjugants. Lane 1, HyperLadder I (Bioline). The genomic DNA templates for the PCRs are as follows: lane 2, JIR12298 (JIR4394 transconjugant carrying the ΔnetB::catP plasmid pJIR3536); lane 3, JIR12295 (JIR325 transconjugant carrying the Tc^r plasmid pJIR3537); lane 4: JIR12293 (JIR325 transconjugant carrying pJIR3536); lane 5, JIR12290 (JIR325 transconjugant carrying pJIR3536 and the Tc^r plasmid pJIR3537); lane 6, JIR12231 (EHE-NE18 ΔnetB::catP carrying pJIR3536 and pJIR3537 and other plasmids); lane 7, EHE-NE18 (wild-type strain with the original netB plasmid pJIR3535 and pJIR3537). (B) PCR analysis of plasmid DNA isolated from the CW504-based secondary transconjugants. The templates are as follows: lane 1, CW504 (plasmid-free recipient); lane 2, JIR12308-derived pJIR3536 and pJIR3844, which was subsequently detected in this study; lane 3, pJIR3537 from JIR12309; lane 4, pCW3 from JIR4; lane 5, HyperLadder I (Bioline).

FIG 2

Analysis of C. perfringens plasmid DNA purified from CW504 derivatives. (A) Agarose gel electrophoresis of purified undigested plasmid DNA. Lane 1, plasmids from JIR12308, which includes pJIR3536 and pJIR3844; lane 2, HindIII-digested λcI857 DNA; lane 3, negative control, plasmid DNA preparation from the plasmid-free strain CW504; lane 4, pJIR3537 from JIR12309; lane 5, positive control, pCW3 from JIR4. (B) Agarose gel electrophoresis of purified plasmid DNA digested with the restriction endonucleases indicated. Lanes 1 and 5, JIR12308 (pJIR3536 and pJIR3844, which was detected subsequently in this study); lanes 2 and 6, JIR12309 (pJIR3537); lanes 3 and 7, JIR4 (pCW3); lane 4, HindIII-digested λcI857 DNA.

FIG 3

Southern hybridization analysis of purified plasmid DNA. Undigested plasmid DNA was separated by agarose gel electrophoresis, transferred to nitrocellulose, and blotted with the probes indicated. Lane 1, CW504 (plasmid-free negative control); lane 2, pJIR3536; lane 3, pJIR3537; lane 4, pCW3; lane 5, pJIR418 (catP positive control); lane 6, DIG-labeled HindIII-digested λcI857 DNA markers.

FIG 4

Comparative genetic maps of plasmids from the ΔnetB::catP derivative of C. perfringens strain EHE-NE18. The four plasmids pJIR3536, pJIR3844, pJIR3537, and pJIR3843 are shown, and pCW3 is included for comparison. Open reading frames (ORFs) are represented by arrows. Yellow depicts genes that are likely to be involved in plasmid replication and maintenance, orange depicts genes from the tcp conjugation locus, dark blue depicts gene regions that encode proteins of unknown function but which are common to all known conjugative C. perfringens plasmids, light blue depicts genes present on pJIR3536, dark green depicts the catP insertion that replaces the majority of the netB gene, light green depicts genes present on pJIR3844, pink depicts genes that are present on pJIR3537 and pCW3, and purple depicts genes present on pJIR3843.

FIG 5

A de Bruijn graph resulting from the assembly of sequences derived from pIIR3536 and pJIR3844. Represented is a two-dimensional layout of the de Bruijn graph (43) formed by 127,164 30-mers obtained from Illumina sequencing of plasmid DNA from JIR12308, which carries pJIR3536 and pJIR3844. The k-mers have been arranged so that Euclidean distances in the diagram correspond as closely as possible to the shortest path through the graph. The pJIR3536 (netB) and pJIR3844 (cbp2) plasmids are paths through this diagram. The locations of the netB, cpb2, and tcp genes are indicated.