genome-wide identification of ampicillin resistance determinants in enterococcus faecium全基因组的氨苄青霉素抗性因素识别肠球菌都有效.pdfVIP

Genome-Wide Identification of Ampicillin Resistance Determinants in Enterococcus faecium Xinglin Zhang, Fernanda L. Paganelli, Damien Bierschenk, Annemarie Kuipers, Marc J. M. Bonten, Rob J. L. Willems, Willem van Schaik* Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands 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 b-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 b-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.