Membrane adaptation limitations in Enterococcus faecalis underlie sensitivity and the inability to develop significant resistance to conjugated oligoelectrolytes

Abstract

The growing problem of antibiotic resistant bacteria, along with a dearth of new antibiotics, has redirected attention to the search for alternative antimicrobial agents. Conjugated oligoelectrolytes (COEs) are an emerging class of antimicrobial agents which insert into bacterial cell membranes and are inhibitory against a range of Gram-positive and Gram-negative bacteria. In this study, the extent of COE resistance that Enterococcus faecalis could achieve was studied. Enterococci are able to grow in hostile environments and develop resistance to membrane targeting antibiotics such as daptomycin in clinical settings. Herein we expand our knowledge of the antimicrobial mechanism of action of COEs by developing COE-resistant strains of E. faecalis OG1RF. Evolution studies yielded strains with a moderate 4-16 fold increase in antimicrobial resistance relative to the wild type. The resistant isolates accumulated agent-specific mutations associated with the liaFSR operon, which is a cell envelope-associated stress-response sensing and regulating system. The COE resistant isolates displayed significantly altered membrane fatty acid composition. Subsequent, exogenous supplementation with single fatty acids, which were chosen based on those dominating the fatty acid profiles of the mutants, increased resistance of the wild-type E. faecalis to COEs. In combination, genetic, fatty acid, and uptake studies support the hypothesis that COEs function through insertion into and disruption of membranes and that the mechanism by which this occurs is specific to the disrupting agent. These results were validated by a series of biophysical experiments showing the tendency of COEs to accumulate in and perturb adapted membrane extracts. Collectively, the data support that COEs are promising antimicrobial agents for targeting E. faecalis, and that there is a high barrier to the emergence of severely resistant strains constrained by biological limits of membrane remodeling that can occur in E. faecalis.

Fig. 1. (a) Structures of COE1-3C and COE1-3Py (b) adaptation strategy for in vitro evolution – (i) Enterococcus faecalis OG1RF wild type, (ii) COE1-3C adapted strain and (iii) COE1-3Py adapted strain were used for the characterization studies indicated. Blue and green lines indicate evolution in COE1-3C and COE1-3Py. These strains were subjected to MIC tests, whole genome sequencing to identify mutations associated with the adaptations, fatty acid profile analysis and lastly, lipid extraction to study the biophysical interaction of COEs with lipid bilayers using quartz crystal microbalance-dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS).

Fig. 2. Percentage changes in the three dominant fatty acids (>10% each of the total fatty acid content) determined as a percentage of total membrane content; shown as averages ± standard deviations of five biological replicates of E. faecalis OG1RF wild type, EFC3C and EFC3Py. Significant differences between the mutants EFC3C and EFC3Py compared to the wild type was calculated using two-way ANOVA and Tukey's multiple comparisons test and corrected p-values indicated with *p ≤ 0.0001.

Fig. 3. The relative uptake of 5 μM COEs in E. faecalis OG1RF wild type (WT), EFC3C and EFC3Py determined by measuring the absorbance at the wavelength of maximum absorbance of a 5 μM solution of COEs. Significant differences between the treatments in the respective strains was calculated by t-test and p-values indicated with *p ≤ 0.01.

Fig. 4. QCM-D monitoring of COE interactions with supported lipid bilayers. Resonance frequency (Δf) and energy dissipation (ΔD) signals are presented as functions of time. The baseline signals correspond to fabricated SLBs on silica-coated sensor chips, and 5 μM COE compounds were added starting at t = 5 min under continuous flow conditions. All measurement shifts are relative to equivalent buffer conditions.

Fig. 5. Electrochemical impedance spectroscopy (EIS) measurements using a tethered lipid bilayer membrane platform. Increasing concentrations of compound were added at the indicated time point. Control is standard tBLM lipid composition without bacterial cell lipid extract.