Reduced Chlorhexidine and Daptomycin Susceptibility in Vancomycin-Resistant Enterococcus faecium after Serial Chlorhexidine Exposure

Abstract

Vancomycin-resistant Enterococcus faecium strains (VREfm) are critical public health concerns because they are among the leading causes of hospital-acquired bloodstream infections. Chlorhexidine (CHX) is a bisbiguanide cationic antiseptic that is routinely used for patient bathing and other infection control practices. VREfm are likely frequently exposed to CHX; however, the long-term effects of CHX exposure have not been studied in enterococci. In this study, we serially exposed VREfm to increasing concentrations of CHX for a period of 21 days in two independent experimental evolution trials. Reduced CHX susceptibility emerged (4-fold shift in CHX MIC). Subpopulations with reduced daptomycin (DAP) susceptibility were detected, which were further analyzed by genome sequencing and lipidomic analysis. Across the trials, we identified adaptive changes in genes with predicted or experimentally confirmed roles in chlorhexidine susceptibility (efrE), global nutritional stress response (relA), nucleotide metabolism (cmk), phosphate acquisition (phoU), and glycolipid biosynthesis (bgsB), among others. Moreover, significant alterations in membrane phospholipids were identified for some populations with reduced DAP susceptibility. Our results are clinically significant because they identify a link between serial subinhibitory CHX exposure and reduced DAP susceptibility. In addition, the CHX-induced genetic and lipidomic changes described in this study offer new insights into the mechanisms underlying the emergence of antibiotic resistance in VREfm.

Keywords: Enterococcus; chlorhexidine; daptomycin.

FIG 1

E. faecium adapts to CHX. In vitro evolution of reduced CHX susceptibility in E. faecium 1,231,410 (E. faecium 410) by serial passaging in increasing concentrations of CHX for a period of 21 days. CHX passage MIC (y axis) for each day of passage (x axis) is shown for two independent experiments (A and B).

FIG 2

Reduced DAP susceptibility in CHX-passaged E. faecium populations A and B. The geometric mean and the geometric standard deviation of the CFU/ml count for n = 3 or 4 independent trials are shown. Strains and populations are described in the text. The dashed line represents limit of detection (10³ CFU/ml). The boxed populations were analyzed by whole-genome sequencing. *, P < 0.05 (one-tailed Student t test).

FIG 3

Agar CHX susceptibility assay. The geometric means and the geometric standard deviations of the CFU/ml are shown for wild-type E. faecium 410 and the ΔefrEF deletion mutant (A), the DAP-A2 mutant and the DAP-A2 ΔefrEF deletion mutant (PB302) (B), and the ΔefrEF deletion mutant transformed with empty pLZ12 vector or the complementation vectors pAH201 or pPB202 (C). The CFU/ml value was determined using BHI or BHI-chloramphenicol agars with or without CHX supplementation at 1/8×, 1/4×, or 1/2× MIC CHX. Data from three independent trials are shown for each condition. The dashed line represents the limit of detection (10³ CFU/ml). *, P < 0.05 value (one-tailed Student t test). Cam, chloramphenicol.