The phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen Bacillus cereus

Abstract

The natural antimicrobial compound carvacrol shows a high preference for hydrophobic phases. The partition coefficients of carvacrol in both octanol-water and liposome-buffer phases were determined (3.64 and 3.26, respectively). Addition of carvacrol to a liposomal suspension resulted in an expansion of the liposomal membrane. Maximum expansion was observed after the addition of 0.50 micromol of carvacrol/mg of L-alpha-phosphatidylethanolamine. Cymene, a biological precursor of carvacrol which lacks a hydroxyl group, was found to have a higher preference for liposomal membranes, thereby causing more expansion. The effect of cymene on the membrane potential was less pronounced than the effect of carvacrol. The pH gradient and ATP pools were not affected by cymene. Measurement of the antimicrobial activities of compounds similar to carvacrol (e.g., thymol, cymene, menthol, and carvacrol methyl ester) showed that the hydroxyl group of this compound and the presence of a system of delocalized electrons are important for the antimicrobial activity of carvacrol. Based on this study, we hypothesize that carvacrol destabilizes the cytoplasmic membrane and, in addition, acts as a proton exchanger, thereby reducing the pH gradient across the cytoplasmic membrane. The resulting collapse of the proton motive force and depletion of the ATP pool eventually lead to cell death.

FIG. 1.

Relationship between the concentrations of carvacrol in the water and octanol phases. Carvacrol was added to the (water-saturated) octanol phase, and both phases were mixed (see Materials and Methods). From the linear part of the curve, indicated by the open symbols, a log P of 3.64 was calculated.

FIG. 2.

Relationship between the concentrations of carvacrol in the buffer phase and in the liposomes (6.25 mg of phospholipid/ml). From the linear part of the curve, indicated by the open symbols, a log P of 3.26 was calculated.

FIG. 3.

Effects of carvacrol (filled circles) and cymene (open circles) on the relief of fluorescence self-quenching of R₁₈-labeled liposomes. Maximum fluorescence (100%) was obtained by the addition of 10% Triton X-100.

FIG. 4.

Effect of carvacrol on the membrane potential of B. cereus in the presence of glucose (glu), nigericin (nig), and different concentrations of carvacrol (cl) or cymene (cy). Carvacrol and cymene were added at 250 s. The membrane potential was monitored with the fluorescent probe DiSC₃ and measured in arbitrary units (a.u.).

FIG. 5.

Effect of cymene (2.0 mM) and carvacrol (1 mM) on the pH gradient across the cytoplasmic membrane. The pH was measured with the fluorescent probe cFDASE (see Materials and Methods) at an external pH of 5.81. The ratio was calculated from the fluorescence at 490 nm (pH dependent) and 440 nm (pH independent). glu, glucose; cy, cymene; cl, carvacrol.

FIG. 6.

Intracellular (filled symbols) and extracellular (open symbols) ATP pools of glucose-energized vegetative cells of B. cereus in the absence (▴, ▵) or in the presence (▪, □) of 1 mM (A) or 2.4 mM (B) cymene. Cymene was added at 5 min (indicated by arrow).

FIG. 7.

Growth of B. cereus in BHI in the presence of different concentrations of thymol, carvacrol, menthol, carvacrol methyl ester, and cymene (30°C). All data are means of triplicate measurements.

FIG. 8.

Schematic overview of the hypothesized activity of carvacrol. Undissociated carvacrol diffuses through the cytoplasmic membrane towards the cytoplasm and dissociates, thereby releasing its proton to the cytoplasm. It then returns undissociated by carrying a potassium ion (or other cation) from the cytoplasm, which is transported through the cytoplasmic membrane to the external environment. A proton is taken up, and carvacrol in the protonated form diffuses again through the cytoplasmic membrane and dissociates by releasing a proton to the cytoplasm.