Whole-genome analyses of Enterococcus faecium isolates with diverse daptomycin MICs

Abstract

Daptomycin (DAP) is a lipopeptide antibiotic frequently used as a "last-resort" antibiotic against vancomycin-resistant Enterococcus faecium (VRE). However, an important limitation for DAP therapy against VRE is the emergence of resistance during therapy. Mutations in regulatory systems involved in cell envelope homeostasis are postulated to be important mediators of DAP resistance in E. faecium. Thus, in order to gain insights into the genetic bases of DAP resistance in E. faecium, we investigated the presence of changes in 43 predicted proteins previously associated with DAP resistance in enterococci and staphylococci using the genomes of 19 E. faecium with different DAP MICs (range, 3 to 48 μg/ml). Bodipy-DAP (BDP-DAP) binding to the cell membrane assays and time-kill curves (DAP alone and with ampicillin) were performed. Genetic changes involving two major pathways were identified: (i) LiaFSR, a regulatory system associated with the cell envelope stress response, and (ii) YycFGHIJ, a system involved in the regulation of cell wall homeostasis. Thr120 → Ala and Trp73 → Cys substitutions in LiaS and LiaR, respectively, were the most common changes identified. DAP bactericidal activity was abolished in the presence of liaFSR or yycFGHIJ mutations regardless of the DAP MIC and was restored in the presence of ampicillin, but only in representatives of the LiaFSR pathway. Reduced binding of BDP-DAP to the cell surface was the predominant finding correlating with resistance in isolates with DAP MICs above the susceptibility breakpoint. Our findings suggest that genotypic information may be crucial to predict response to DAP plus β-lactam combinations and continue to question the DAP breakpoint of 4 μg/ml.

FIG 1

Amino acid changes in 43 predicted proteins associated with DAP resistance in E. faecium isolates. Black squares indicate the presence of amino acid changes. Cls, cardiolipin synthase; GdpD, glycerophosphodiester phosphodiesterase; Cfa, cyclopropane-fatty-acyl-phospholipid synthase; PTS-EIIA, phosphotransferase system, phosphoenolpyruvate-dependent sugar EIIA 2; SulP, sulfate transporter; XpaC, 5-bromo-4-chloroindolyl phosphate hydrolysis protein; RrmA, rRNA (guanine-N1-)-methyltransferase A; PTS-IIA, PTS system fructose IIA component; PspC, phage shock protein C; LepB, signal peptidase I; Aad, aldehyde-alcohol dehydrogenase; NrdR, nicotinamide mononucleotide transporter; PTS IID, mannose/fructose/sorbose transporter subunit IID; EzrA, septation ring formation regulator; RpeS-5S, ribosomal protein S5; BrnQ, branched-chain amino acid transport protein; RpoN, RNA polymerase sigma factor 54; TelA, telurite resistance protein; MprF, lysylphosphatidylglycerol synthetase; PgsA, CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase.

FIG 2

Time-kill assays with DAP (5× the MIC) with or without AMP (64 μg/ml). Bacteria were grown in Mueller-Hinton broth (MHB) supplemented with calcium (50 mg/liter). The E. faecium strains are indicated at the top of each panel. AMP, ampicillin; DAP, daptomycin. The limit of detection was 200 CFU/ml.

FIG 3

BODIPY-labeled DAP (BDP-DAP) staining of E. faecium strains. (A) Fluorescence intensities of representative E. faecium strains. Cells were treated with BDP-DAP and fluorescence was normalized to cell protein content. Intensities were compared to E. faecium DO. Rfu, relative fluorescence units; *, P < 0.05; **, P < 0.001; NS, not significant. (B) BDP-DAP staining of representative E. faecium cells at concentrations of 4 μg/ml and 64 μg/ml. The top images capture bacterial cells under fluorescence microscopy (bars, 1 μm). The bottom images are the same bacterial cell in phase contrast. The mutated pathway is indicated in parentheses.