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.