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. 2017 Dec 1;83(24):e01814-17.
doi: 10.1128/AEM.01814-17. Print 2017 Dec 15.

Conjugation-Mediated Horizontal Gene Transfer of Clostridium perfringens Plasmids in the Chicken Gastrointestinal Tract Results in the Formation of New Virulent Strains

Jake A Lacey  1   2   3 , Anthony L Keyburn  1   3 , Mark E Ford  1 , Ricardo W Portela  1   4 , Priscilla A Johanesen  1 , Dena Lyras  1 , Robert J Moore  5   2   3   6
Affiliations

Conjugation-Mediated Horizontal Gene Transfer of Clostridium perfringens Plasmids in the Chicken Gastrointestinal Tract Results in the Formation of New Virulent Strains

Jake A Lacey et al. Appl Environ Microbiol. .

Abstract

Clostridium perfringens is a gastrointestinal pathogen capable of causing disease in a variety of hosts. Necrotic enteritis in chickens is caused by C. perfringens strains that produce the pore-forming toxin NetB, the major virulence factor for this disease. Like many other C. perfringens toxins and antibiotic resistance genes, NetB is encoded on a conjugative plasmid. Conjugative transfer of the netB-containing plasmid pJIR3535 has been demonstrated in vitro with a netB-null mutant. This study has investigated the effect of plasmid transfer on disease pathogenesis, with two genetically distinct transconjugants constructed under in vitro conditions, within the intestinal tract of chickens. This study also demonstrates that plasmid transfer can occur naturally in the host gut environment without the need for antibiotic selective pressure to be applied. The demonstration of plasmid transfer within the chicken host may have implications for the progression and pathogenesis of C. perfringens-mediated disease. Such horizontal gene transfer events are likely to be common in the clostridia and may be a key factor in strain evolution, both within animals and in the wider environment.IMPORTANCEClostridium perfringens is a major gastrointestinal pathogen of poultry. C. perfringens strains that express the NetB pore-forming toxin, which is encoded on a conjugative plasmid, cause necrotic enteritis. This study demonstrated that the conjugative transfer of the netB-containing plasmid to two different nonpathogenic strains converted them into disease-causing strains with disease-causing capability similar to that of the donor strain. Plasmid transfer of netB and antibiotic resistance was also demonstrated to occur within the gastrointestinal tract of chickens, with approximately 14% of the isolates recovered comprising three distinct, in vivo-derived, transconjugant types. The demonstration of in vivo plasmid transfer indicates the potential importance of strain plasticity and the contribution of plasmids to strain virulence.

Keywords: Clostridium perfringens; conjugation; in vivo plasmid transfer; necrotic enteritis; pathogenicity; virulence.

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Figures

FIG 1
FIG 1
Phylogenetic tree showing evolutionary relationships of total gene content (gene presence and absence) between strains PBD1 and NE33 before and after the mating procedure. Other strains were included in the comparison to root the tree.
FIG 2
FIG 2
Virulence of C. perfringens strains in the necrotic enteritis disease induction model. The lesion scores of individual 24-day-old broiler chickens challenged with different C. perfringens strains are shown. The horizontal bars represent the average lesion score for each group, and the error bars represent the standard error for each group. Intestinal lesions in the small intestine (duodenum to ileum) were scored as previously reported: 0, no gross lesions; 1, thin or febrile walls; 2, focal necrosis or ulceration (one to five foci); 3, focal necrosis or ulceration (six to 15 foci); 4, focal necrosis or ulceration (16 or more foci); 5, patches of necrosis 2 to 3 cm long; and 6, diffuse necrosis typical of field cases. The results presented are the pooled data from three independent trials, n = 30. The strains tested are as follows: EHE-NE18, BER-NE33, BER-NE33/pNetB, PBD1, and PBD1/pNetB. A one-way ANOVA Kruskal-Wallis test with a Dunn's posttest showed a statistical difference between the recipient strains NE33 and PBD1 to the donor strain EHE-NE18 (***, P < 0.01), a statistical difference between each pNetB transconjugant and the original recipient strain (**, P < 0.05), and no statistical difference between EHE-NE18 and either of the transconjugants.
FIG 3
FIG 3
The effect of rifampin resistance marking on the virulence of C. perfringens strains in the necrotic enteritis disease induction model. The lesion scores of individual 24-day-old broiler chickens challenge with different C. perfringens strains are shown. The horizontal bars represent the average lesion score for each group, and the error bars represent the standard error for each group. Intestinal lesions in the small intestine (duodenum to ileum) were scored as previously reported: 0, no gross lesions; 1, thin or febrile walls; 2, focal necrosis or ulceration (one to five foci); 3, focal necrosis or ulceration (six to 15 foci); 4, focal necrosis or ulceration (16 or more foci); 5, patches of necrosis 2 to 3 cm long; and 6, diffuse necrosis typical of field cases. The results are from two separate trials. The strains tested are as follows: WER-NE36, EHE-NE18, and rifampin-resistant strains WER-NE36 and EHE-NE18, n = 20. A one-way ANOVA Kruskal-Wallis test with a Dunn's posttest showed a statistical difference between the rifampin-marked strains of WER-NE36 and EHE-NE18 from their wild-type forms, with P values of 0.001 for both strains examined.
FIG 4
FIG 4
Virulence of C. perfringens strains in the necrotic enteritis disease induction model with cochallenge. The lesion scores of individual 24-day-old broiler chickens challenge with different C. perfringens strains are shown. The horizontal bars represent the average lesion score for each group, and the error bars represent the standard error for each group. Intestinal lesions in the small intestine (duodenum to ileum) were scored as previously reported: 0, no gross lesions; 1, thin or febrile walls; 2, focal necrosis or ulceration (one to five foci); 3, focal necrosis or ulceration (six to 15 foci); 4, focal necrosis or ulceration (16 or more foci); 5, patches of necrosis 2 to 3 cm long; and 6, diffuse necrosis typical of field cases. The results are from a single trial. The groups tested were as follows: WER-NE36 (RifR), BER-NE33, and groups sequentially challenged, first with WER-NE36 (RifR) followed by BER-NE33 and then BER-NE33 followed by WER-NE36 (RifR) (n = 10). A one-way ANOVA Kruskal-Wallis test with a Dunn's posttest showed a statistical difference between the recipient strains BER-NE33 and cochallenge group BER-NE33/WER-NE36 (***, P < 0.01), and no statistical significance was observed between rifampin-marked WER-NE36 and BER-NE33 or to the WER-NE36/BER-NE33 cochallenge group. Transconjugants were isolated only from the BER-NE33/WER-NE36 cochallenge group.
FIG 5
FIG 5
The distribution of isolated colonies from the in vivo cochallenge model. The antibiotic resistance profile indicates the nature of the colonies as the % of total colonies isolated. In the group challenged with BER-NE33 in the morning and WER-NE36 in the afternoon, 63% of the colonies were the rifampin-marked WER-NE36 donor, 20% of the colonies were BER-NE33, 10% of the colonies isolated were shown to be BER-NE33 transconjugants, and 7% were shown to be WER-NE36 transconjugants.
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