Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.
PLoS Genet. 2012 Jun;8(6):e1002804. doi: 10.1371/journal.pgen.1002804. Epub 2012 Jun 28.
Enterococcus faecium has become a nosocomial pathogen of major importance, causing infections that are difficult to treat owing to its multi-drug resistance. In particular, resistance to the β-lactam antibiotic ampicillin has become ubiquitous among clinical isolates. Mutations in the low-affinity penicillin binding protein PBP5 have previously been shown to be important for ampicillin resistance in E. faecium, but the existence of additional resistance determinants has been suggested. Here, we constructed a high-density transposon mutant library in E. faecium and developed a transposon mutant tracking approach termed Microarray-based Transposon Mapping (M-TraM), leading to the identification of a compendium of E. faecium genes that contribute to ampicillin resistance. These genes are part of the core genome of E. faecium, indicating a high potential for E. faecium to evolve towards β-lactam resistance. To validate the M-TraM results, we adapted a Cre-lox recombination system to construct targeted, markerless mutants in E. faecium. We confirmed the role of four genes in ampicillin resistance by the generation of targeted mutants and further characterized these mutants regarding their resistance to lysozyme. The results revealed that ddcP, a gene predicted to encode a low-molecular-weight penicillin binding protein with D-alanyl-D-alanine carboxypeptidase activity, was essential for high-level ampicillin resistance. Furthermore, deletion of ddcP sensitized E. faecium to lysozyme and abolished membrane-associated D,D-carboxypeptidase activity. This study has led to the development of a broadly applicable platform for functional genomic-based studies in E. faecium, and it provides a new perspective on the genetic basis of ampicillin resistance in this organism.
屎肠球菌已成为一种重要的医院病原体，由于其多重耐药性，导致感染难以治疗。特别是，临床分离株对β-内酰胺类抗生素氨苄西林的耐药性已普遍存在。先前已表明，低亲和力青霉素结合蛋白 PBP5 的突变对于屎肠球菌的氨苄西林耐药性很重要，但也存在其他耐药决定因素。在这里，我们在屎肠球菌中构建了高密度转座子突变体文库，并开发了一种转座子突变体跟踪方法，称为基于微阵列的转座子图谱（M-TraM），从而确定了屎肠球菌中有助于氨苄西林耐药性的基因汇编。这些基因是屎肠球菌核心基因组的一部分，表明屎肠球菌有很高的潜力进化为β-内酰胺耐药性。为了验证 M-TraM 的结果，我们适应了 Cre-lox 重组系统，在屎肠球菌中构建了靶向、无标记突变体。我们通过构建靶向突变体证实了四个基因在氨苄西林耐药性中的作用，并进一步研究了这些突变体对溶菌酶的耐药性。结果表明，ddcP 基因编码一种低分子量青霉素结合蛋白，具有 D-丙氨酰-D-丙氨酸羧肽酶活性，是高水平氨苄西林耐药性所必需的。此外，ddcP 的缺失使屎肠球菌对溶菌酶敏感，并消除了膜相关的 D，D-羧肽酶活性。这项研究导致了一种在屎肠球菌中进行基于功能基因组的广泛应用平台的开发，并为该生物体中氨苄西林耐药性的遗传基础提供了新的视角。
