This study describes the effect of carvacrol on cells of the food-borne pathogen B. cereus
. Carvacrol acts as a bactericidal compound, with its activity being dependent on the concentration and the time of exposure. Sikkema et al. (23
) found that the accumulation of lipophilic compounds in the cell membrane (tested in liposomes prepared from Escherichia coli
lipids) is proportional to the concentration in the aqueous phase and the membrane aqueous-phase partition coefficient. Enomoto et al. (7
) observed a decrease of Δψ in liposomes during exposure to some fragrance compounds. These hydrophobic compounds dissolve in the membrane, and their activity was closely correlated with the membrane fluidity. Based on these studies, it is expected that more carvacrol dissolves in the membrane at higher concentrations.
Our study has shown that exposure to carvacrol leads to a decrease of the ATPin
concentration. No proportional increase of the ATPout
was observed. Therefore, it is concluded that carvacrol does not enhance the permeability of the membrane for ATP. Consequently, depletion of the internal ATP pool results from a reduced rate of ATP synthesis or increased ATP hydrolysis. A depletion of the ATP pool upon the addition of a lipophilic component has been observed in different studies (13
). In contrast to the present study, Helander et al. (11
) observed a leakage of ATP from cells which were exposed to carvacrol (2 mM). However, this study was carried out with gram-negative bacteria, which have a different cell envelope.
The observation that already-low concentrations of carvacrol (>0.01 mM) cause a decrease of the membrane potential suggests that the membrane becomes more permeable for protons. This conclusion is supported by the observation that exposure of cells to carvacrol also causes dissipation of the proton gradient across the membrane. In accordance with these results, Sikkema et al. (25
) showed an increased proton permeability of liposomal membranes during exposure to tetralin. Similarly, Cartwright et al. (4
) described dissipation of the ΔpH in the presence of ethanol, due to an increased influx of protons.
Analysis of the intracellular and extracellular potassium pools revealed an increased permeability of the cell membrane for K+
upon exposure to carvacrol. K+
is the major cytoplasmic cation of growing bacterial cells, involved in several key functions of bacterial cells. This ion plays a role in the activation of cytoplasmic enzymes, the maintenance of turgor pressure, and possibly the regulation of the cytoplasmic pH (2
). Different studies showed that an efflux of potassium ions is a first indication of membrane damage in bacteria (10
). Δψ depends mainly on the potassium concentration in the cell (2
). Heipieper et al. (9
) showed a significant excretion of K+
to the external environment during exposure of Pseudomonas putida
P8 to phenol. Gradients of solutes across the cytoplasmic membrane which use H+
as the coupling ion can also be affected by a dissipation of the proton motive force.
Although there was no immediate effect of carvacrol on the viability at concentrations of 1 mM and lower, clear effects on different bioenergetic parameters have been observed. Cells can probably cope with very low concentrations of carvacrol. Not only reduction of ATP synthesis by a dissipation of the proton motive force but also other (secondary) effects of carvacrol may result in the bactericidal or bacteriostatic action. For example, an inhibition of several enzymes due to leakage of essential ions, loss of turgor pressure, influence on DNA synthesis, reduced metabolic activities, and other processes in the cell can be a cause of the decreased viability during exposure to carvacrol. A loss of membrane integrity due to disturbance of hydrophobic interactions between lipids and proteins is often an important factor when considering the activity of toxic compounds (24
). It can be concluded that the hydrophobic compound carvacrol interacts with the membranes of B. cereus
by changing their permeability for cations like H+
. The dissipation of ion gradients leads to impairment of essential processes in the cell and finally to cell death.
This study shows that carvacrol has biological effects at concentrations which are relevant for flavoring of foods (e.g., nonalcoholic beverages [0.18 mM/28.54 ppm] and baked goods [15.75 ppm]) (8
). To products associated with outbreaks of B. cereus
(e.g., rice, pasta, and soup), carvacrol could be applied both as an antimicrobial and as a flavoring compound.