title: Genome-wide identification of ampicillin resistance determinants in Enterococcus faecium.
abstract: 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.
authors: Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors: Xinglin
authors: X
authors: Zhang
authors: Fernanda L
authors: FL
authors: Paganelli
authors: Damien
authors: D
authors: Bierschenk
authors: Annemarie
authors: A
authors: Kuipers
authors: Marc J M
authors: MJ
authors: Bonten
authors: Rob J L
authors: RJ
authors: Willems
authors: Willem
authors: W
authors: van Schaik
mesh_terms: D000667:Ampicillin / Q000494:pharmacology; D000668:Ampicillin Resistance / Q000235:genetics*; D049109:Cell Proliferation / Q000187:drug effects; D003428:Cross Infection / Q000235:genetics / Q000382:microbiology; D004251:DNA Transposable Elements / Q000235:genetics; D016984:Enterococcus faecium / Q000235:genetics / Q000472:pathogenicity; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D016680:Genome, Bacterial; D006801:Humans; D009113:Muramidase / Q000494:pharmacology; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D046915:Penicillin-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D046915:Penicillin-Binding Proteins; D000667:Ampicillin; D009113:Muramidase; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase