The mechanism of resistance to conjugated oligoelectrolytes in enterococcus faecalis
Chilambi, Gayatri Shankar
Date of Issue2018
Interdisciplinary Graduate School
The growing problem of antibiotic resistance in bacteria, along with the dearth of new antibiotics, has led to the search for alternative antimicrobial therapeutic agents. Since the primary reason for the quest for novel antibiotics is to stay one step ahead of the emergence of resistance, knowledge on how bacteria become resistant to a new antimicrobial agent is necessary to facilitate the development of new antimicrobials. Oligophenylenevinylene conjugated oligoelectrolytes (COEs) have π-conjugated backbones and terminal ionic pendant groups. They have a high affinity for the lipid bilayer and spontaneously intercalate into the membrane. The ability of phenylenevinylene COEs to perturb bacterial membranes varies according to the number of aromatic rings and the type of ionic fragment. It was observed that the COEs with pyridinium cationic groups, i.e. COE1-3Py, were more inhibitory than when tetraalkylammonium groups were present (i.e. COE1-3C). In this study, the antibacterial mechanism of these molecules was investigated along with the mechanism of resistance. By exposing Enterococcus faecalis OG1RF to increasing concentrations of COE1-3C or COE1-3Py, we were able to experimentally evolve resistant strains EFC3C and EFC3Py, respectively. Whole genome sequencing of resistant isolates revealed mutations in the liaFSR three component regulatory system. Specifically, EFC3Py had a mutation in liaR, while EFC3C had a mutation in liaF. Fatty acid analysis of EFC3C and EFC3Py revealed an increase in the levels of cis-vaccenic acid and cyclopropane, both of which are associated with increase in membrane order. Transcriptomic analysis of wild type E. faecalis treated with COE1-3C and COE1-3Py indicated that the compounds induced a general stress response along with changes in the expression of membrane transporters. In the EFC3C and EFC3Py strains, gene expression analysis revealed that most of the genes induced or repressed in EFC3C were also altered in EFC3Py. In both mutants, EFC3C and EFC3Py, transporters of the ATP binding cassette superfamily were the most significantly induced or repressed genes compared to the wild type. Overall, the significantly upregulated and downregulated transporter genes were generally involved in transport of ferrichrome, lipoproteins export, amino acids, riboflavin, mannitol/fructose, oligopeptide and choline. In addition, cell envelope response regulon (LiaFSR), dlt operon and penicillin binding proteins (PBPs) were also differentially expressed. The mutations in the liaFSR regulon correlate to the resistance to COEs as confirmed by the deletion and complementation studies. Collectively, the data suggest that the COEs target bacterial membranes and disrupt membrane function by membrane perturbation. The transcriptomic response of E. faecalis to conjugated oligoelectrolytes revealed the mechanisms adopted to tolerate the membrane stress elicited by COEs. Additionally, COE1-3C and COE1-3Py appear to be promising antimicrobials against E. faecalis and that there is a high barrier to the emergence of severely resistant strains constrained by biological limits of membrane adaptation that can occur in E. faecalis.