Characterization of Monolaurin Resistance in Enterococcus faecalis

Abstract

There is increasing concern regarding the presence of vancomycin-resistant enterococci in domestically farmed animals, which may act as reservoirs and vehicles of transmission for drug-resistant enterococci to humans, resulting in serious infections. In order to assess the potential for the use of monolaurin as a food preservative, it is important to understand both its target and potential mechanisms of resistance. A Tn917 mutant library of Enterococcus faecalis AR01/DGVS was screened for resistance (MIC, >100 g/ml) to monolaurin. Three mutants were identified as resistant to monolaurin and were designated DGRM2, DGRM5, and DGRM12. The gene interrupted in all three mutants was identified as traB, which encodes an E. faecalis pheromone shutdown protein and whose complementation in trans restored monolaurin sensitivity in all three mutants. DGRM2 was selected for further characterization. E. faecalis DGRM2 showed increased resistance to gentamicin and chloramphenicol (inhibitors of protein synthesis), while no difference in the MIC was observed with the cell wall-active antibiotics penicillin and vancomycin. E. faecalis AR01/DGVS and DGRM2 were shown to have similar rates (30% cell lysis after 4 h) of cell autolytic activity when activated by monolaurin. Differences in cell surface hydrophobicity were observed between the wild type and the mutant, with the cell surface of the parent strain being significantly more hydrophobic. Analysis of the cell wall structure of DGRM2 by transmission electron microscopy revealed an increase in the apparent cell wall thickness and contraction of its cytoplasm. Taken together, these results suggest that the increased resistance of DGRM2 was due to a change in cell surface hydrophobicity, consequently limiting the diffusion of monolaurin to a potential target in the cytoplasmic membrane and/or cytoplasm of E. faecalis.

Substantiation in Enterococcus faecalis of Dose-Dependent Resistance and Cross-Resistance to Pore-Forming Antimicrobial Peptides by Use of a Polydiacetylene-Based Colorimetric Assay

“…The increased rigidity of bacterial cell membranes may prevent the insertion of alamethicin into the phospholipid bilayer. These findings are supported by the previous studies showing that the presence of monolaurin changed the cellular saturated-to-unsaturated fatty acid ratio from 1.4 to 4.35 (7). The decrease in the S/U ratio indicated the increased fluid nature of the cell membrane of Alm r variant Efv4.2.…”

Comparison of antibacterial effect of seven 1-monoglycerides on food-borne pathogens or spoilage bacteria

“…Electron microscopy studies observed morphological changes of bacteria growing in the presence of MAGs showing damage of the cell membrane under the influence of MAG (Bergsson et al 1998(Bergsson et al , 1999. Dufour et al (2007) discovered that in mutant strains of Ent. faecalis, the resistance to monolaurin is caused by the changes in the cell surface and thus MAG cannot penetrate the cell and reach the place where it could act.…”

“…Although the mechanism for the antibacterial activity of fatty acids and their derivatives has not been fully recognized, it was found that they cause damage to the cell membrane used as a permeable barrier and inhibit the transport of amino acids to the cell (Dufour et al 2007;Thormar and Hilmarsson 2007). Another hypothesis is based on the penetration of fatty acids with a short and medium chain in a non-dissociated form into bacterial cells and their dissociation inside the cells, which leads to acidification of the cell contents (Sado Kamdem et al 2008).…”

Antimicrobial Effects of 1-Monocaprylin and 1-Monocaproin Through <i>in vitro</i> Growth Inhibition and Molecular Docking Studies

“…The precise mode of the action against microorganisms is not yet clear and postulated several hypotheses. One hypothesis is based on the amphiphilic character of MAG molecule which aids in their penetration and incorporation into cytoplasmic membrane and subsequently disrupts the cell permeability and prevents the transportation of the nutrients (Nair et al, 2004;Dufour et al, 2007). An alternative hypothesis postulates the penetration of fatty acids and dissociated in the inner cell environment, thereby increasing the intracellular acidity (Sun et al, 1998).…”