Necrotic enteritis-derived Clostridium perfringens strain with three closely related independently conjugative toxin and antibiotic resistance plasmids.
Necrotic enteritis-derived Clostridium perfringens strain with three closely related independently conjugative toxin and antibiotic resistance plasmids.
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作者：
Bannam TL;Yan XX;Harrison PF;Seemann T;Keyburn AL;Stubenrauch C;Weeramantri LH;Cheung JK;McClane BA;Boyce JD;Moore RJ;Rood JI
禽类坏死性肠炎的发病机制涉及NetB，这是一种由A型产气荚膜梭状芽胞杆菌禽类分离株产生的成孔毒素。为了确定netB结构基因的位置和移动性，我们检测了耐四环素坏死性肠炎菌株EHE-NE18的衍生物，其中netB被氯霉素和甲砜霉素耐药基因catP插入失活。四环素和甲砜霉素抗性都可以在平板交配中一起或单独转移至受体菌株。单独的转接合子可以在随后的交配中充当供体，这证明四环素抗性决定簇和netB基因存在于不同的接合元件上。从转接合子中分离出大质粒并通过高通量测序进行分析。对所得数据的分析表明，原始菌株中实际上存在三个大的接合质粒，每个质粒都有自己的毒素或抗生素抗性位点。每个质粒都包含一个高度保守的 40 kb 区域，其中包括与来自产气荚膜梭菌的 47 kb 接合四环素抗性质粒 pCW3 密切相关的质粒复制和转移区域。这些质粒如下：(i) 与 pCW3 非常相似的接合型 49-kb 四环素抗性质粒，(ii) 包含 netB 基因和其他潜在毒力基因的接合型 82-kb 质粒，以及 (iii) 携带 cpb2 基因的 70-kb 质粒，该基因编码不同的成孔毒素，β2 毒素。厌氧细菌产气荚膜梭菌可引起禽类胃肠道疾病，称为坏死性肠炎。尽管质粒编码的成孔毒素 NetB 是一个重要的毒力因子，但疾病的发病机制尚不清楚。在这项工作中，我们证明携带 netB 基因的质粒是接合性的，并且具有 40 kb 的区域，该区域与来自产气荚膜梭菌的每个测序接合质粒中发现的复制和转移区域非常相似。我们还表明，该菌株含有两个额外的大质粒，它们也是接合的并携带类似的 40 kb 区域。这些质粒之一编码β2毒素，另一个编码四环素抗性。据我们所知，这是首次报道携带三种密切相关但不同的独立接合质粒的细菌菌株。这些结果对于我们了解病原菌中毒力和抗生素抗性基因的传播具有重要意义。
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. 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.