Genetic basis for daptomycin resistance in enterococci

The emergence of multidrug resistant enterococci as leading causes of hospital acquired infection is an important public health concern. Little is known about the genetic mechanisms by which enterococci adapt to strong selective pressures, including the use of antibiotics. The lipopeptide antibiotic daptomycin is approved to treat Gram-positive bacterial infections including those caused by enterococci. Since its introduction, resistance to daptomycin by strains of Enterococcus faecalis and E. faecium has been reported, but is still rare. We evolved daptomycin resistant strains of the multidrug resistant E. faecalis strain V583. Based on the availability of a fully closed genome sequence for V583, we used whole genome resequencing to identify the mutations that became fixed over small time scales (~ 2 weeks) upon serial passage in the presence of daptomycin. By comparison of the genome sequences of the three adapted strains to parental V583, we identified seven candidate daptomycin resistance genes and three different mutational paths to daptomycin resistance in E. faecalis. Mutations in one of the seven candidate genes, encoding a putative cardiolipin synthase (EF0631), were found in each of the adapted E. faecalis V583 strains, as well as in E. faecalis and E. faecium daptomycin resistant clinical isolates. Alleles of EF0631 from daptomycin resistant strains are dominant in trans, and confer daptomycin resistance upon a susceptible host. These results demonstrate a mechanism of enterococcal daptomycin resistance that is genetically distinct from that occurring in staphylococci, and indicate that enterococci possessing alternate EF0631 alleles are selected for during daptomycin therapy. However, our analysis of E. faecalis clinical isolates indicates that resistance pathways independent from mutant forms of EF0631 also exist.多重耐药肠球菌（multidrug resistant enterococci）作为医院获得性感染的主要致病菌，已成为一项重大公共卫生关切。目前学界对肠球菌适应强选择压力（如抗生素使用）的遗传机制尚不清楚。脂肽类抗生素达托霉素（lipopeptide antibiotic daptomycin）已获批用于治疗革兰氏阳性菌感染，其中包括肠球菌引发的感染。自其投入临床应用以来，已有粪肠球菌（Enterococcus faecalis）和屎肠球菌（E. faecium）菌株对达托霉素产生耐药性的报道，但此类耐药病例仍较为罕见。我们以多重耐药粪肠球菌V583菌株为亲本，通过连续传代诱导获得了达托霉素耐药菌株。鉴于V583已获得全闭合基因组序列，我们采用全基因组重测序（whole genome resequencing）技术，鉴定了在达托霉素选择压力下连续传代、约2周的短时间尺度内固定的突变。将3株适应株的基因组序列与亲本V583进行比对，我们在粪肠球菌中鉴定出7个候选达托霉素耐药基因，以及3种不同的达托霉素耐药突变通路。7个候选基因中的一个——编码推定的心磷脂合酶（putative cardiolipin synthase）的EF0631基因——所发生的突变，在所有适应的粪肠球菌V583菌株中均被检出，同时在临床分离的达托霉素耐药粪肠球菌和屎肠球菌菌株中也存在此类突变。达托霉素耐药菌株携带的EF0631等位基因具有反式（trans）显性效应，可使易感宿主菌株获得达托霉素耐药性。本研究结果揭示了一种与葡萄球菌（staphylococci）中已报道的达托霉素耐药机制存在遗传差异的肠球菌达托霉素耐药通路，并表明在达托霉素治疗过程中，携带变异EF0631等位基因的肠球菌会被正向筛选富集。不过，我们对粪肠球菌临床分离株的分析显示，不依赖EF0631突变体的达托霉素耐药通路同样存在。