Li Weiwei, Hu Jiamin, Li Ling, Zhang Mengge, Cui Qingyu, Ma Yanan, Su Hainan, Zhang Xuhua, Xu Hai, Wang Mingyu
State Key Laboratory of Microbial Technology, Microbial Technology Institute, Shandong University, Qingdao, China.
Division of Science and Technology, Ludong University, Yantai, China.
Front Microbiol. 2022 Jun 21;13:896916. doi: 10.3389/fmicb.2022.896916. eCollection 2022.
Daptomycin (DAP), a last-resort antibiotic for treating Gram-positive bacterial infection, has been widely used in the treatment of vancomycin-resistant enterococci (VRE). Resistance to both daptomycin and vancomycin leads to difficulties in controlling infections of enterococci. A clinical multidrug-resistant EF332 strain that shows resistance to both daptomycin and vancomycin was identified, for which resistance mechanisms were investigated in this work. Whole-genome sequencing and comparative genomic analysis were performed by third-generation PacBio sequencing, showing that EF332 contains four plasmids, including a new multidrug-resistant pEF332-2 plasmid. Two vancomycin resistance-conferring gene clusters and were found on this plasmid, making it the second reported vancomycin-resistant plasmid containing both clusters. New mutations in chromosomal genes and that, respectively, encode cardiolipin synthase and glycerophosphoryl diester phosphodiesterase were identified. Their potential roles in leading to daptomycin resistance were further investigated. Through molecular cloning and phenotypic screening, two-dimensional thin-layer chromatography, fluorescence surface charge test, and analysis of cardiolipin distribution patterns, we found that mutations in decrease surface negative charges of the cell membrane (CM) and led to redistribution of lipids of CM. Both events contribute to the DAP resistance of EF332. Mutation in leads to changes in CM phospholipid compositions, but cannot confer DAP resistance. Neither mutation could result in changes in cellular septa. Therefore, we conclude that the daptomycin resistance of EF332 is conferred by new mutations. This work reports the genetic basis for vancomycin and daptomycin resistance of a multidrug-resistant strain, with the finding of new mutations of that leads to daptomycin resistance.
达托霉素（DAP）是治疗革兰氏阳性菌感染的一种最后手段抗生素，已广泛用于治疗耐万古霉素肠球菌（VRE）。对达托霉素和万古霉素均耐药会导致控制肠球菌感染困难。本研究鉴定出一株对达托霉素和万古霉素均耐药的临床多重耐药EF332菌株，并对其耐药机制进行了研究。通过第三代PacBio测序进行全基因组测序和比较基因组分析，结果表明EF332含有四个质粒，包括一个新的多重耐药pEF332 - 2质粒。在该质粒上发现了两个赋予万古霉素耐药性的基因簇，这使其成为第二个报道的同时含有这两个基因簇的耐万古霉素质粒。还鉴定出分别编码心磷脂合酶和甘油磷酸二酯磷酸二酯酶的染色体基因和中的新突变。进一步研究了它们在导致达托霉素耐药性方面的潜在作用。通过分子克隆和表型筛选、二维薄层色谱、荧光表面电荷测试以及心磷脂分布模式分析，我们发现中的突变降低了细胞膜（CM）的表面负电荷，并导致CM脂质重新分布。这两个事件都促成了EF332对DAP的耐药性。中的突变导致CM磷脂组成发生变化，但不能赋予DAP耐药性。这两种突变均不会导致细胞隔膜发生变化。因此，我们得出结论，EF332对达托霉素的耐药性是由新的突变赋予的。本研究报道了一株多重耐药菌株对万古霉素和达托霉素耐药的遗传基础，并发现了导致达托霉素耐药的新突变。
