Glycerol monolaurate inhibits induction of vancomycin resistance in Enterococcus faecalis

Abstract

Glycerol monolaurate (GML) is a surfactant that has been found to inhibit the post-exponential phase activation of virulence factor production and the induction of beta-lactamase in Staphylococcus aureus. It has been suggested that signal transduction is the most probable target for GML (S. J. Projan, S. Brown-Skrobot, P. M. Schlievert, F. Vandenesch, and R. P. Novick, J. Bacteriol. 176:4204-4209, 1994). We found that GML suppresses growth of vancomycin-resistant Enterococcus faecalis on plates with vancomycin and blocks the induction of vancomycin resistance, which involves a membrane-associated signal transduction mechanism, either at or before initiation of transcription. Given the surfactant nature of GML and the results of previous experiments, we suggest that GML blocks signal transduction. In contrast, GML has no effect on the induction of erythromycin-inducible macrolide resistance in S. aureus, which does not involve signal transduction.

FIG. 1

Effect of GML on vancomycin resistance in E. faecalis. Bacteria [JH2-2(pAT80)] were grown in BHI broth until they reached a turbidity of 40 Klett units, centrifuged, washed and resuspended in BHI broth, and diluted and plated on BHI agarose plates with or without GML and vancomycin. Plates were incubated for 34 h at 37°C and then photographed. Plates: 1, no GML, no vancomycin; 2, GML (7.5 μg/ml), no vancomycin; 3, no GML, vancomycin (50 μg/ml); 4, GML (7.5 μg/ml), vancomycin (50 μg/ml).

FIG. 2

Effect of GML on growth of E. faecalis in microtiter plates. Bacteria [JH2-2(pAT80)] were grown in microplate wells (200 μl/well). GML and vancomycin (3 μg/ml) were added as indicated. Cell density (optical density [O.D.]) was monitored at 650 nm. Samples (whole contents of certain wells) were removed 40, 80, 120 min after addition of vancomycin and assayed for CAT activity (Fig. 3). Each cell density point represents the mean of two independent measurements. Experiment 1 had no GML and no vancomycin (⧫). Experiment 2 had no GML and vancomycin at 100 min (⊡). Experiment 3 had GML at 10 μg/ml at 60 min and 15 μg/ml at 120 min and vancomycin at 100 min (◘). Experiment 4 had GML at 20 μg/ml at 60 min and 10 μg/ml at 120 min and vancomycin at 100 min (◊).

FIG. 3

Effect of GML on induction of vancomycin resistance. Samples, taken as indicated in the legend to Fig. 2, were treated with xylene (vortexed for 20 s) and directly assayed for CAT activity with a microplate reader. CAT activity was expressed as V_max per mg of dry weight (milli-optical density unit [mOD]). Numbers 1 to 4 correspond to the experiment numbers explained in the legend to Fig. 2. Each CAT activity value represents the mean of two independent measurements. Bars represent times after vancomycin addition of 40 (▪), 80 (▨), and 120 () min.

FIG. 4

Effect of GML on erythromycin-inducible resistance to clindamycin. Bacteria (RN2442) were spread on plates (5 × 10⁷ cells/plate) containing agar (plate 1) or agarose plus GML (20 μg/ml) (plate 2), and antibiotic discs (erythromycin, 15 μg/disc; clindamycin, 2 μg/disc) were placed 1 cm apart as shown. Plates were incubated at 37°C for 16 h and photographed.