Novel Multidrug-Resistant Enterococcal Mobile Linear Plasmid pELF1 Encoding vanA and vanM Gene Clusters From a Japanese Vancomycin-Resistant Enterococci Isolate
Novel Multidrug-Resistant Enterococcal Mobile Linear Plasmid pELF1 Encoding vanA and vanM Gene Clusters From a Japanese Vancomycin-Resistant Enterococci Isolate
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DOI：
10.3389/fmicb.2019.02568
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发表时间：
2019-11-13
影响因子：
5.2
通讯作者：
Tomita, Haruyoshi
中科院分区：
文献类型：
--
作者：
Hashimoto, Yusuke;Taniguchi, Makoto;Tomita, Haruyoshi
万古霉素耐药肠球菌在临床上是一种棘手的病原体，因为治疗选择很少。在日本鉴定了一株VanA/VanM型万古霉素耐药屎肠球菌临床分离株。该菌株命名为AA 708，含有5个质粒，其中一个质粒在琼脂糖凝胶电泳期间不经S1核酸酶处理而迁移，这表明线性拓扑结构。我们将此质粒命名为pELF 1。全基因组测序（WGS）分析表明，pELF 1基因全长143，316 bp，含有vanA和vanM基因簇。此外，mfold分析和WGS数据表明，pELF 1的左端可能形成发夹结构，而不是其右端。pELF 1质粒不被λ核酸外切酶消化，表明末端蛋白结合到质粒的5 ′末端，类似于链霉菌线性质粒。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果也与核酸外切酶检测结果一致。在阻滞试验中，含有蛋白酶K未处理的pELF 1右端的DNA似乎没有像蛋白酶K处理的pELF 1那样移动，这表明末端蛋白可能附着在pELF 1的右端。回文序列在pELF 1的右末端序列形成发夹结构;然而，与链霉菌属（Streptomyces spp.）并不高。pELF 1具有两种不同的末端结构。偶联实验表明，pELF 1可转移到E. faecalis、粪肠球菌E. faecium、E. casseliflavus和E. hills。这些接合子不仅对万古霉素有较高的耐药性，而且对链霉素、卡那霉素和红霉素也有较高的耐药性。这些结果表明，pELF 1具有赋予肠球菌属的多药耐药性的能力。同时，这可能导致临床危害。
Vancomycin-resistant enterococci are troublesome pathogens in clinical settings because of few treatment options. A VanA/VanM-type vancomycin-resistant Enterococcus faecium clinical isolate was identified in Japan. This strain, named AA708, harbored five plasmids, one of which migrated during agarose gel electrophoresis without S1 nuclease treatment, which is indicative of a linear topology. We named this plasmid pELF1. Whole genome sequencing (WGS) analysis of the AA708 strain revealed that the complete sequence of pELF1 was 143,316 bp long and harbored both the vanA and vanM gene clusters. Furthermore, mfold analysis and WGS data show that the left end of pELF1 presumably forms a hairpin structure, unlike its right end. The pELF1 plasmid was not digested by lambda exonuclease, indicating that terminal proteins were bound to the 5 ' end of the plasmid, similar to the Streptomyces linear plasmids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results were also consistent with the exonuclease assay results. In retardation assays, DNAs containing the right end of proteinase K-untreated pELF1 did not appear to move as well as the proteinase K-treated pELF1, suggesting that terminal proteins might be attached to the right end of pELF1. Palindromic sequences formed hairpin structures at the right terminal sequence of pELF1; however, sequence similarity with the well-known linear plasmids of Streptomyces spp. was not high. pELF1 was unique as it possessed two different terminal structures. Conjugation experiments revealed that pELF1 could be transferred to E. faecalis, E. faecium, E. casseliflavus, and E. hirae. These transconjugants exhibited not only high resistance levels to vancomycin, but also resistance to streptomycin, kanamycin, and erythromycin. These results indicate that pELF1 has the ability to confer multidrug resistance to Enterococcus spp. simultaneously, which might lead to clinical hazards.