Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

The antibacterial mechanism of carvacrol and thymol

against Escherichia coli
J. Xu1, F. Zhou1, B.-P. Ji1, R.-S. Pei1 and N. Xu2
1 College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China
2 State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

Keywords
carvacrol, flow cytometry, membrane
permeability, membrane potential, thymol.
Correspondence
Feng Zhou, College of Food Science and
Nutritional Engineering, China Agricultural
University, 17 Qinghua Donglu, Haidian
District, Beijing, China.
E-mail: Xujing_BB@163.com
2008 ⁄ 0263: received 15 February 2008;
revised 16 April 2008 and accepted 23 April
2008
doi:10.1111/j.1472-765X.2008.02407.x
Abstract
Aims: To investigate the antibacterial mechanism of carvacrol and thymol
against Escherichia coli.
Methods and Results: The time-kill curve results showed that carvacrol and
thymol at 200 mg l)1 could inhibit the growth of E. coli. Flow cytometry and
fluorescent dyes were used to explore the effect of two components on mem-
brane permeability and membrane potential. In membrane permeability experi-
ment, the mean fluorescence intensity of cells treated with 200 mg l)1 carvacrol
or thymol were lower than nonexposed cells. The ratio of red to green fluores-
cence intensity of DiOC2(3) reflected the change of membrane potential. Car-
vacrol and thymol at 200 mg l)1 caused the ratio of red ⁄ green decreasing from
0Æ42 of control to 0Æ08 and 0Æ07, respectively.
Conclusions: Carvacrol and thymol had desired antimicrobial effect on E. coli.
The antibacterial effects were attributed to their ability to permeabilize and
depolarize the cytoplasmic membrane.
Significance and Impact of the Study: This study showed the potential use of
flow cytometry as a suitable method to investigate the mode of antibacterial
action of essential oil components.

of EO components against E. coli. In previous studies,

Introduction
Food poisoning caused by Escherichia coli is an emergent
problem in both developing and developed countries. The
addition of synthetic preservatives could inhibit the
growth of E. coli, but consumers have growing concern
about their safety. Some naturally derived antibacterials,
such as essential oils, show desired antimicrobial activity.
Among them, the most-effective one is oregano essential
oil (EO) (Lambert et al. 2001). Carvacrol and thymol, the
major components of oregano EO, are legally registered
flavourings and foodstuffs in the Council of Europe
(2000).
The antimicrobial properties of carvacrol and thymol
are reported in previous studies (Lambert et al. 2001;
Valero and Francés 2006), but not much was known
about their mode of action against micro-organisms (Burt
2004). In our research, fluorescent staining combined
with flow cytometry was used to explore the mechanism
Novo et al. (2000) used flow cytometry to analyse the
effect of amoxicillin on membrane potential, membrane
permeability and bacterial counts of Staphylococcus aureus
and Micrococcus luteus. Breeuwer and Abee (2000)
reviewed the use of fluorescent dyes combined with flow
cytometry to assess cell viability.
The cytoplasmic membrane was an important cellular
target for EO components owing to their hydrophobic
structure (Burt 2004); therefore, fluorescent dyes of 5(6)-
carboxyfouorescein diacetate (cFDA) and 3,3¢-diethyloxa-
carbocyanine iodide (DiOC2(3)) combined with flow
cytometry were used to analyse the effect of two compo-
nents on membrane permeability and membrane poten-
tial. cFDA is a nonfluorescent precursor, which can be
taken up by viable cell, and the diacetate groups are
hydrolysed intracellularly by nonspecific esterase, produc-
ing a highly fluorescent fluorophore carboxyfluorescein
(cF) (Petit et al. 1993). cF is more negatively charged at

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174 Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 47 (2008) 174–179
J. Xu et al.
physiological pH, and thus less likely to leak from the
cells (Breeuwer and Abee 2000). Hence, cells which have
an intact cytoplasmic membrane and esterase activity are
able to accumulate the fluorescent probe within them,
resulting in higher fluorescent intensities. DiOC2(3) is a
kind of cationic carbocyanine dye. It can accumulate on
membrane with electron gradient and can emit two
kinds of fluorescence. The ratio of red to green fluores-
cence intensity provides a measurement of membrane
potential that is independent of cell size (Novo et al.
2000). Carbonyl cyanide 3-chlorophenylhydrazone
(CCCP), a proton ionophore, was employed to verify
whether DiOC2(3) could reflect membrane potential
(Novo et al. 1999).
The purpose of this study was to use flow cytometry to
investigate the effect of carvacrol and thymol on mem-
brane permeability and membrane potential of E. coli.
Materials and methods
Bacterial strains and growing conditions
Escherichia coli AS1Æ90 (obtained from China General
Microbiological Culture Collection Center) was main-
tained on Mueller Hinton agar (MHA) slants at 4C.
Inocula were prepared by 16-h culture in Mueller Hinton
broth (MHB) at 37C. The 16-h culture was diluted with
MHB to achieve an inoculum of approximate
1 · 107 CFU ml)1.
Preparation of reagents
Carvacrol and thymol were obtained from Sigma (St.
Louis, MO, USA). The components were diluted with
MHB to a concentration of 800 mg l)1 and stored at 4C.
cFDA, DiOC2(3) and CCCP were purchased from
Sigma (St Louis, MO, USA) and prepared as a stock solu-
tion at 10, 3 mmol l)1 and 500 lmol l)1, respectively, in
dimethyl sulfoxide (DMSO). All the solutions were kept
in a dark place at )20C before using.
The study of time-kill curve
The two components were diluted with MHB to
achieve concentrations ranging from 100 to 800 mg l)1.
Inocula were added into 20-ml tube containing 15 ml
MHB as the medium to achieve an initial inoculum of
approximate 1 · 106 CFU ml)1. All samples were main-
tained at 37C. After 0, 0Æ5, 1Æ0, 1Æ5, 2Æ0, 4Æ0, 6Æ0, 8Æ0,
10Æ0 and 12Æ0 h from the time of incubation, 0Æ5-ml
portion was removed from each tube for colony count-
ing. At least two replications were performed for each
sample.
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Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 47 (2008) 174–179
Antibacterial mechanism of carvacrol and thymol against E. coli
cFDA staining
The concentrations of EO components used in membrane
permeability assessment were 100 and 200 mg l)1. Inocula
were added into each tube containing 15-ml medium to
achieve an initial inoculum of approximate
1 · 106 CFU ml)1. The cell suspension without the addi-
tion of EO components was used as control. All samples
were incubated at 37C for 2 h. Then, 1-ml suspension
from each tube was transferred into 1Æ5 ml centrifugal
tube. The cells were washed twice by centrifugation at
10 000 g for 10 min in phosphate-buffered saline (PBS,
pH 7Æ4), and resuspended by PBS containing
0Æ5 mmol l)1 ethylenediamine tetraacetic acid (EDTA),
which could increase the permeability of membrane to
probes. Ten microlitres of 10 mmol l)1 cFDA was added
to each tube followed by incubation at 40C for 10 min
(Budde and Rasch 2001). After incubation, the samples
were washed and resuspended in PBS and kept on ice in
the dark until the flow cytometric analysis was per-
formed.
DiOC2(3) staining
The concentration of carvacrol and thymol used in mem-
brane potential assessment was 200 mg l)1. The pretreat-
ment was the same to cFDA staining. After washing
twice, 10 ll of 500 lmol l)1 CCCP was added to one
tube without the addition of any EO components to
reduce the membrane potential to 0. Ten microlitres of
3 mmol l)1 DiOC2(3) was added to each tube followed
by incubation at room temperature for 15–30 min before
flow cytometric analysis.
Cytometric analysis
Escherichia coli without treatment of EO components and
fluorescent dyes was used to locate bacterial populations
in the forward and side scatter channels. The flow rate
was corresponded to 100–500 cells per s. The mean fluo-
rescence intensity (MFI) was defined as the ratio between
the bacterial population and fluorescent standard beads
using absolute values of the fluorescence intensities.
Carboxyfluorescein was excited at 488 nm and detected
at 520 nm (Nguefack et al. 2004). Fluorescence histogram
overlay of cF was achieved by Windows Multiple Docu-
ment Interface (WinMDI version 2Æ9; Joe Trotler, San
Diego, CA).
DiOC2(3) was excited at 488 nm. Its red and green
fluorescence were detected through 610-, 19-nm band-
width and 530-, 20-nm bandwidth band-pass filter,
respectively (Novo et al. 2000). The fluorescence ratios of
red ⁄ green were achieved by Cellquest software.
175
Antibacterial mechanism of carvacrol and thymol against E. coli J. Xu et al.

(a) (b)
10 10

8 8
LogN (cfu ml–1)

LogN (cfu ml–1)

6 6

4 4
Figure 1 Time- and concentration-dependent
2 2 effect of carvacrol (a) and thymol (b) on Esc-
herichia coli. Samples were incubated at 37C
0 0 for 12 h in Mueller Hinton broth treated with
0 2 4 6 8 10 12 0 2 4 6 8 10 12
0 (h), 100 ( ), 200 ( ) and 400 mg l)1 (d)
Time (h) Time (h) essential oil components.

left, which indicated that fluorescence intensity decreased

Results
to some extent. Carvacrol 100, 200 mg l)1 and thymol
100, 200 mg l)1 deceased the MFI to 32Æ12, 22Æ97 and
Time-kill curve course of Escherichia coli treated with EO
28Æ55, 19Æ97, respectively. When the concentration of the
components
two components was increased to 400 mg l)1, viable cells
The time-kill data of two components were displayed in were not sufficient for flow cytometric analysis after 2-h
Fig. 1a,b. At lower concentration, the components yielded incubation.
smaller effect on the viability of E. coli. One hundred mil-
ligram per litre of carvacrol and thymol could not inhibit
Effect of EO components on membrane potential
the growth of E. coli. The number of viable cells
decreased gradually with the increased concentration of With the addition of carvacrol, thymol and CCCP, there
two components. In the first 6 h, 200 mg l)1 carvacrol was a decrease in red fluorescence accompanying a
and thymol caused a sharp decrease of the number of via- slightly increase in green fluorescence, resulting in a
ble cells, with 5Æ96 and 5Æ99 reduction in log CFU ml)1, decreased fluorescent ratio of red ⁄ green. The fluorescent
respectively. After 6-h incubation, the number of viable ratio of the control suspension was 0Æ42. Carvacrol and
cells did not change significantly. When the concentration thymol at the concentration of 200 mg l)1 decreased the
increased to 400 mg l)1, E. coli cells decreased to unde- fluorescent ratio from 0Æ42 to 0Æ08 and 0Æ07, respectively
tectable level after 6-h exposure to carvacrol and thymol. (Fig. 3). Carvacrol and thymol had almost the same effect
When the concentration of two components increased to on membrane potential. A lower ratio was found for cells
800 mg l)1, no growth was found in tested samples treated with CCCP.
within half an hour (data not show).
Discussion
Effect of EO components on membrane permeability
According to the antibacterial activity of each component,
As can be seen in Fig. 2a,b, two components showed a carvacrol and thymol possessed inhibitory effects against
concentration-dependent ability to decrease the fluores- the growth of E. coli at a similar concentration. This con-
cence intensity of cF. The MFI value of the control sus- clusion coincided with the results of Helander et al.
pension was 36Æ24. Compared with control, fluorescence (1998), who found that E. coli was inhibited equally by
histograms of cells treated with components shift towards carvacrol and thymol. However, Friedman et al. (2002)
128

128

(a) (b)

0 0 Figure 2 Fluorescence histogram overlay of

Escherichia coli following exposure to
Events

Events

1 1
2 carvacrol (a) and thymol (b) stained with
2
5(6)-carboxyfluorescein diacetate. The
concentrations of essential oil components
used in this study were 0 (0), 100 (1) and
0

100 101 102 103 104 100 101 102 103 104 200 mg l)1 (2).

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176 Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 47 (2008) 174–179
J. Xu et al.
Fluorescence ratio (red/green)
0.5
0.4
0.3
0.2
0.1
0 crol and thymol are equal to 3Æ52 and 3Æ28, respectively,
0
l
0
P
tro
20
20
C
on
ol
ol
C
C
ym
cr
va
Th
ar
C
Figure 3 Detection of membrane potential of essential oil compo-
nents against Escherichia coli by flow cytometry. The ratio of red to
green fluorescence intensity of DiOC2(3) was calculated using the
mean fluorescence intensity.
reported that the following order in a microplate assay
was carvacrol > thymol. The discrepancy of antimicrobial
order derived from the different method used in vitro
tests of antibacterial activity like diffusion, dilution and
bioautography methods.
Although many methods were used to evaluate the
antibacterial activity of EO components against food-
related micro-organisms, not much was used to explore
the underlying mechanism. In this study, we employed
fluorescent dyes and flow cytometry to investigate the
antibacterial mechanism of carvacrol and thymol against
E. coli. Compared with some traditional methods used in
the studies of antibacterial mechanism, like spectrofluo-
rometer (Ultee et al. 1999, 2002), scanning electron
microscope (Kwon et al. 2003) and confocal laser scan-
ning microscopy (Gill and Holley 2006), flow cytometry
could locate not only specific population but a single cell
and process data with high efficiency (Vives-Rego et al.
2000).
The use of cFDA staining and flow cytometry to assess
the antibacterial mechanism of carvacrol and thymol
against E. coli showed that the MFI of the cells decreased
upon component addition. When the cytoplasmic mem-
brane was disrupted, more cF followed out, resulting in a
lower MFI. Nguefack et al. (2004) once used cFDA to
study the ability of five EO to permeabilize the cytoplas-
mic membrane of Listeria innocua. The results showed
that the fluorescence intensity of cell exposed to EO
decreased faster than nonexposed cell, indicating that EO
permeabilized the cytoplasmic membrane with the leakage
of cF, which was correlated with the high content of thy-
mol. In Gill and Holley’s study, eugenol and carvacrol
disrupt the cellular membranes of E. coli, Listeria mono-
cytogenes and Lactobacillus sakei at 750 to 1500 mg l)1, a
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Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 47 (2008) 174–179
Antibacterial mechanism of carvacrol and thymol against E. coli
concentration sufficient to have an immediate bactericidal
effect (Gill and Holley 2006).
In our study, carvacrol and thymol at the concentra-
tion of 200 mg l)1 could not kill E. coli completely but
inhibited its growth significantly, and compared with
control, their ability to permeabilize membrane was obvi-
ous. In addition, carvacrol and thymol had almost the
same antibacterial activity on E. coli. The log P of carva-
which means that they have relatively strong hydrophobic
capacities. Carvacrol and thymol are structural isomers
and have a phenolic hydroxyl at a different location on
the phenolic ring. The hydroxyl group increased their
hydrophilic ability, which could help them dissolve in
microbial membrane and impair them (Sikkema et al.
1995). As already demonstrated by Lambert et al. (2001),
our results showed that the relative position of hydroxyl
group on the phenolic ring did not appear to influence
the degree of antibacterial activity.
Membrane potential is another important target that
EO components might work on. In our research,
DiOC2(3) was used to assess membrane potential. The
structure of DiOC2(3) is similar to DiOC6(3), another
cyanine dye, which has been used to investigate membrane
potential during growth of E. coli (Monfort and Baleux
1996) and assess the functional state of the Listeria
membrane (Ratinaud and Revidon 1995). DiOC2(3) has
some advantage over DiOC6(3), because it emits two kinds
of fluorescence: the red one is caused by dyes aggregation,
and is highly dependent on both membrane potential and
the size of cell (0Æ5–5 lm), whereas the green one reflects
particle size, and is relatively independent of membrane
potential. The ratio of red to green fluorescence intensity
provides a measure of membrane potential that is largely
independent of cell size (Novo et al. 1999). DiOC2(3) is
inclined to aggregate on cytoplasmic membrane with high
membrane potential. When membrane potential is affected
by proton ionophore, such as CCCP, the red fluorescence
intensities decrease accompanying a constant or slightly
increased green fluorescence, resulting in a decreased
fluorescent ratio of red ⁄ green (Novo et al. 2000).
The fluorescence ratio of cells treated with 200 mg l)1
carvacrol and thymol was significantly lower than the
control. Carvacrol and thymol disturbed the membrane
integrity, increased membrane permeability and caused
the leakage of protons and potassium, finally leading to
the loss of membrane potential. In Ultee et al.’s study,
carvacrol at 1Æ50 mg l)1 reduced the membrane potential
of Bacillus cereus, and the pH gradient across the cell
membrane was completely dissipated in the presence of
150 mg l)1 or more (Ultee et al. 1999). Loss of membrane
potential was also found on Pseudomonas aeruginosa and
Staphylococcus aureus after treatment with carvacrol-con-
177
Antibacterial mechanism of carvacrol and thymol against E. coli
taining oregano EO (Lambert et al. 2001). The presence
of the hydroxyl group on carvacrol and thymol plays an
important role to depolarize membrane potential. The
structure of cymene was like carvacrol, but lacked the
hydroxyl groups. It also could decrease the membrane
potential; however, higher concentrations were needed to
obtain the same reduction as that obtained with carvacrol
(Ultee et al. 2002). It was shown that the presence of free
hydroxyl group is essential for antimicrobial activity of
carvacrol and that this compound could act as a protono-
phore (Arfa et al. 2006). It gets inserted in cytoplasmic
membrane, changes the membrane physical and chemical
properties and affects both lipid ordering and stability of
bilayer, resulting in an increase of proton passive flux
across the membrane.
In terms of the toxicity of carvacrol and thymol on
mammalian models, researchers had different opinions.
Dušan et al. (2006) observed that a short-time presence of
carvacrol and thymol had no damaging effect on Caco-2
cells. Nofrarias et al. (2006) found that 150 ppm of carva-
crol had no significant influence on growth parameters and
lymphocyte composition of peripheral blood. Stammati
et al. (1999) found that carvacrol and thymol appeared to
have no significant effects in vivo, while in vitro, they exhi-
bit mild to moderate toxic effects at the cellular level. More
safety studies should be carried out before carvacrol and
thymol are used as food preservative.
In conclusion, the results indicate that the mechanism
of the action of carvacrol and thymol is the disruption of
the cytoplasmic membrane, which increases its permeabil-
ity and depolarizes its potential. It is also suggested that
flow cytometry is a potential method to explore the mode
of antibacterial action of EO components. Some of the
other targets, such as enzyme activities, require further
research to gain more insight into the mechanism under-
lying the antibacterial effect of EO components.
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