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Phytomedicine ] (]]]]) ]]]–]]]
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Synergistic interaction of eugenol with antibiotics against
Gram negative bacteria
S. Hemaiswarya, M. Doble
Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India
Abstract
Eugenol, the principal chemical component of clove oil from Eugenia aromatica has been long known for its
analgesic, local anesthetic, anti-inflammatory, and antibacterial effects. The interaction of the eugenol with ten
different hydrophobic and hydrophilic antibiotics was studied against five different Gram negative bacteria. The MIC
of the combination was found to decrease by a factor of 5–1000 with respect to their individual MIC. This synergy is
because of the membrane damaging nature of eugenol, where 1 mM of its concentration is able to damage nearly 50%
of the bacterial membrane. Eugenol was also able to enhance the activities of lysozyme, Triton X-100 and SDS in
damaging the bacterial cell membrane. The hydrophilic antibiotics such as vancomycin and b-lactam antibiotics which
have a marginal activity on these gram negative bacteria exhibit an enhanced antibacterial activity when pretreated
with eugenol. Reduced usage of antibiotics could be employed as a treatment strategy to slow down the onset of
antibiotic resistance as well as decrease its toxicity. Experiments performed with human blood cells indicated that the
concentration of eugenol used for the combination studies were below its cytotoxic values. Pharmacodynamic studies
of the combinations need to be performed to decide on the effective dosage.
r 2009 Published by Elsevier GmbH.
Keywords: Eugenol; Clove oil; Synergy; Antibacterial; Gram negative bacteria; Antibiotics
Introduction
Medicinal plants, particularly their active components, have been a dependable source of therapeutics for
the treatment of various ailments since time immemorial. The rapid propagation of antibiotic resistance has
urged the humankind to use plants as a reliable source
for the discovery of active antimicrobial agents and
possibly, even novel classes of antibiotics. An alternative
approach to the use of new antibacterial compounds is
the use of antibiotic synergists. Synergism in antimicrobial therapy is well known and is used to describe
Corresponding author. Tel.: +91 44 2257 4107;
fax: +91 44 2257 4102.
E-mail address: [email protected] (M. Doble).
supra-additive activity of antibiotics when used in
combinations with other compounds.
Eugenol, the principal chemical component of clove
oil from Eugenia aromatica has been long known for its
analgesic, local anesthetic, anti-inflammatory, and antibacterial effects. It is used in the form of a paste or
mixture as dental cement, filler and restorative material.
It belongs to the class of essential oils that is generally
recognised as safe (GRAS) by the Food and Drug
Administration. It is a known antibacterial agent
against pathogens including Escherichia coli O157:H7,
Listeria monocytogenes (Blaszyk and Holley, 1998),
Campylobacter jejuni, Salmonella enterica, Staphylococcus aureus, Lactobacillus sakei and Helicobacter pyroli
(Friedman et al., 2002; Walsh et al., 2003) and is
reported to act primarily by disrupting the cytoplasmic
0944-7113/$ - see front matter r 2009 Published by Elsevier GmbH.
doi:10.1016/j.phymed.2009.04.006
Please cite this article as: Hemaiswarya, S., Doble, M., Synergistic interaction of eugenol with antibiotics against Gram negative bacteria.
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membrane (Gill and Holley, 2006a). The synergistic
effect of combination of eugenol, carvacrol and cinnamaldehyde has been reported against seven different
bacterial strains (Didry et al. 1993).
The aim of the current study is to examine the
influence of a terpenic compound, eugenol, on the
activity of ten hydrophobic and hydrophilic antibiotics
against five Gram-negative strains. The antibiotics
selected for the study have different mode of action.
water followed by the addition of water. The rest of the
compounds were dissolved in water.
The experiments that were conducted with eugenol
and antibiotics and also the experiments to prove their
mechanism of action are listed in Table 1.
Results
Minimum inhibitory concentrations
Material and methods
Bacterial strains
The strains used in this study were Escherichia coli
(NCIM 2931), Enterobacter aerogenes (NCIM 5139),
Proteus vulgaris (NCIM 2813), Salmonella typhimurium
(NCIM 2501), Pseudomonas aeruginosa (NCIM 5029).
They are specifically grown in Ca supplemented Muller
Hinton Broth (Himedia, India) at an optimal growth
temperature of 37 1C with aeration for 16–18 h. The
Minimum Inhibitory Concentration (MIC) of antibiotics and eugenol is determined with 105 CFU/ml of
colonies by adjusting the optical density at 600 nm and
growing them on appropriate agar plates.
Antimicrobial compounds
The antibiotics selected for the study were penicillin,
ampicillin, oxacillin, erythromycin, polymyxin B sulphate, tetracycline, chloramphenicol, vancomycin and
rifampin which were obtained from Himedia (India) and
norfloxacin and eugenol were purchased from Sigma
(Bangalore, India). Each compound is dissolved in its
respective solvents viz., erythromycin and chloramphenicol were dissolved in 95% ethanol; rifampin and
eugenol in Dimethylsulphoxide (DMSO); norfloxacin
was initially dissolved in minimal amount of alkaline
Table 1.
As can be seen from Table 2, all the antibiotics tested
were active against these Gram negative bacteria with
MICs ranging in micromolar concentrations except in
the case of penicillin, oxacillin, vancomycin and
erythromycin which were active only at very high
concentration. The first three antibiotics were not
specific against Gram negative bacteria, since they are
designed to be active against Gram positive bacteria,
particularly against the methicillin resistant strains of S.
aureus (MRSA). The MIC value of eugenol against all
the bacteria is in the range of 20 mM.
Interaction of eugenol with antibiotics
The results were plotted as isobolograms for determining the antagonistic, additive, or synergistic effect of
eugenol in combination with antibiotic. The graph is
represented with the MIC of eugenol in the x-axis. A
ratio of the MIC of the antibiotic in combination
experiments to the MIC of the antibiotic alone is
represented in the Y-axis. A straight line that connects
the ratio 1 and the MIC of eugenol indicates the line of
additivity. The MIC of the combination is plotted in this
x, y plot and any point on this line indicates additivity
and points which are well below the line (below the 95%
confidence band) indicates synergistic effect of the drugeugenol combination. Antagonism is represented by
points above the line of additivity.
Experiments conducted with eugenol and antibiotics.
Experiment conducted
Assay done
Reference
MIC determination
Checkerboard testing
Membrane damaging effect of
eugenol
Microdilution method
Microdilution method
LIVE/DEADs BacLight Bacterial Viability test kit
Uptake of nitrocefin by HB101 cells transformed with
pBR322
Bacteriolysis
CLSI (1993)
CLSI (1993)
Hilliard et al. (1999)
Angus et al. (1982)
Hydrophobicity of the cell surface
Partitioning of the cells into p-xylene
Hemolysis
Human red blood cells
Chapman and
Georgopapadajou (1988)
Nishida et al. (2007)
Helander et al. (1997)
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MIC values of the combination of eugenol and
the ten antibiotics against the five microorganisms
are represented graphically in Fig. 1. The concentration of antibiotics in the combination is 5–1000
fold lower than when used alone indicating that
the combination is synergistic in nature. The concave
curves representing the MIC of the combination
Table 2.
were well below the line of additivity in all the
cases.
Membrane damaging effect of eugenol
The Baclight reagent distinguishes the intact and
damaged cell membrane based on the permeability of
MIC of antibiotics alone against Gram negative bacteria. (MIC of eugenol when used alone ¼ 20 mM).
Antibiotics
MIC in mM
Ampicillin
Penicillin
Oxacillin
Erythromycin
Norfloxacin
Chloramphenicol
Polymyxin B
Tetracycline
Vancomycin
Rifampin
E. coli
E. aerogenes
P. vulgaris
P. aeruginosa
S. typhimurium
Log P
0.040
0.028
5
5
0.0025
0.008
0.002
0.002
0.3125
0.039
0.625
5
5
5
0.005
0.0039
0.005
0.005
0.3125
0.0195
0.156
0.156
0.625
5
0.005
0.0098
0.005
0.005
0.3125
0.0024
5
5
5
0.16
0.0025
0.63
0.002
0.078
2.5
0.78
0.0195
0.0195
5
5
0.005
0.0098
0.005
0.005
0.3125
0.0049
0.4
1.5
2.4
3.06
2.1
2.11
4.86
0.3
3.1
2.7
1
Ratio of polymyxin B amount
in combination/alone
Ratio of ampicillin amount
in combination/alone
1
0.8
0.6
0.4
0.2
0
0
5
10
15
0.8
0.6
0.4
0.2
0
20
0
5
10
Eugenol (mM)
15
20
0
5
10
Eugenol (mM)
15
20
Eugenol (mM)
1
Ratio of chloramphenicol
amount in combination/alone
Ratio of oxacillin amount in
combination/alone
3
0.8
0.6
0.4
0.2
0
0
5
10
Eugenol (mM)
15
20
1
0.8
0.6
0.4
0.2
0
Fig. 1. Interaction of eugenol with antibiotics ampicillin (A), polymyxin (B), oxacillin (C), chloramphenicol (D), penicillin (E),
erythromycin (F), vancomycin (G), rifampicin (H), norfloxacin (I), tetracycline (K) in decreasing MIC against E. coli (B), P.
aeruginosa ( ), E. aerogenes (n), S. typhimurium (&) and P. vulgaris (K). Dotted lines represent the line of additivity.
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1
Ratio of erythromycin amount in
combination/alone
Ratio of penicillin amount in
combination/alone
4
0.8
0.6
0.4
0.2
0
0
5
10
15
20
1
0.8
0.6
0.4
0.2
0
0
5
Ratio of rifampicin amount in
combination/alone
Ratio of vancomycin amount in
combination/alone
1
0.8
0.6
0.4
0.2
0
5
10
Eugenol (mM)
15
20
0.8
0.6
0.4
0.2
0
15
20
1
0.8
0.6
0.4
0.2
0
0
5
10
Eugenol (mM)
15
20
0
5
10
Eugenol (mM)
15
20
1
1
Ratio of tetracycline amount in
combination/alone
Ratio of norfloxacin amount in
combination/alone
0
10
Eugenol (mM)
Eugenol (mM)
0
5
10
Eugenol (mM)
15
20
0.8
0.6
0.4
0.2
0
Fig. 1. (Continued)
the two fluorescent dyes. A significant reduction of
fluorescence emission at the green wavelength
(SYTO-9) was observed for the bacteria treated with
eugenol when compared to the control sample. The red
dye (Propidium iodide) penetrated into the damaged
membrane of the bacteria, thereby reducing the intensity
of the green dye. Even at a eugenol concentration
of 1 mM the cell membrane damage was 50%
and it increased to 80% at 10 mM concentration
(Fig. 2).
Enhancement of nitrocefin uptake by eugenol
Nitrocefin is normally excluded by the outer cell
membrane, but if it is able to pass this barrier, then it
will be cleaved by the enzyme, b-lactamase, which is
localized within the periplasmic space (Angus et al.
1982). This cleavage will result in a colour change from
yellow to red. Increase in the conversion of nitrocefin
was noticed in cells pretreated with eugenol than in the
control (untreated cells) (Fig. 3) which could be
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5
Hydrophobicity of the cell surface (Fig. 5)
Measurement of partioning of cells between buffer
and p-xylene is an indication of its surface hydrophobicity. A decrease in absorbance at 600 nm was observed
when compared to that of the control indicating that
more cells partition to the organic solvent. The
hydrophobicity of the cell surface increased in the order
E. coli 4E. aerogenes 4P. aeruginosa 4S. typhimurium
4P. vulgaris.
Effect of eugenol on hemolysis (Fig. 6)
OD at 500 nm
Fig. 2. Effect of 1 ( ), 2.5 ( ), 5 ( ) and 10 mM ( ) of eugenol
on the damage caused to membrane of Gram negative bacteria
(organisms are placed in the increasing order of hydrophobicity).
0.4
0.38
0.36
0.34
0.32
0.3
0.28
0.26
0.24
0.22
0.2
0.18
Eugenol caused only 5.2% hemolysis at concentrations of 2.5 mM showing its low cytotoxic activity. At 5
and 10 mM nearly 49 and 73% hemolysis was observed
respectively. In the combination experiments against
the Gram negative bacteria eugenol at concentrations less than 2.5 mM was used which leads to minimal
hemolysis. So these studies indicate that eugenol
below its cytotoxic concentration is able to induce
synergistic effect with antibiotics against Gram negative
bacteria.
Discussion
0
100
200
300
400
500
600
Time in secs
Fig. 3. Effect of eugenol on the uptake of nitrocefin into E. coli
HB101 cells transformed with pBR322. (Untreated cells (&),
cells treated with 1 (’), 2.5 (n), 5 (+), and 10 mM (m) of
eugenol).
attributed to the enhanced permeabilising nature of this
essential oil.
Bacteriolysis
To further understand the membrane permeabilising
effect of eugenol, the bacteria was subjected to
pretreatment with different concentrations of eugenol
for 10 min and investigated for its stability against
lysozyme, Triton X-100 or SDS. Decrease in turbidity of
the aqueous bacterial suspension is an indication of the
reduction in live cells. Eugenol at its subinhibitory
concentrations sensitized the microorganisms against
lysozyme (Fig. 4A), Triton X 100 (Fig. 4B) and SDS
(Fig. 4C). The susceptibility of the microorganisms to
lysozyme and detergents is found to be in the order of
E. coli4E. aerogenes4P. aeruginosa4S. typhimurium4
P. vulgaris.
Essential oils have been known to possess antibacterial activity by its action through the disruption of the
cell membrane. Many studies have proved the synergistic action of essential oil fractions from different plants
with synthetic drugs as antifungal agents (Shiota et al.
2000; Shin and Kang 2003) and as antibacterial agents
(Jedlickova et al. 1992). The role of principal components of essential oil fractions in such an interaction is
less known and not explored thoroughly. Eugenol, a
major component of essential oil fractions from plants
like Cinnamomum sp., Eugenia sp. (Chericoni et al.,
2005), showed potent synergistic activity with antibiotics
against Gram negative bacteria. There was nearly a
5–1000 fold decrease in the MIC of the antibiotics
tested. Eugenol was reported to act in synergy with
penicillin against E. coli (strain not specified) with a FIC
(Fractional Inhibitory Concentration) value of 0.16
(Gallucci et al., 2006). Several other terpenes like
citronellol, carvacrol, geraniol, menthol, myrcene and
thymol have also been tested in combination with
penicillin against E. coli and MRSA (Gallucci et al.,
2006). Essential oils and their active components
particularly citronellol, eucalyptol, geraniol, thymol
and triacetin were found to interact synergistically with
norfloxacin against B. subtilis ATCC 6633, B. cereus
ATCC 11778, S. aureus ATCC 6538, S. aureus ATCC
29213 and E. coli ATCC 35218 (Rosato et al., 2007).
Eugenol is known to act by disruption of the cell
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120
120
100
100
Relative turbidity (%)
at 4 min (Mean ± SD)
Relative turbidity (%)
at 4 min (Mean ± SD)
6
80
60
40
20
0
Control
1
2.5
5
80
60
40
20
0
10
Control
Concentration of eugenol in mM
1
2.5
5
Concentration of eugenol in mM
10
Relative turbidity (%)
at 4 min (Mean ± SD)
120
100
80
60
40
20
0
Control
1
2.5
5
10
Concentration of eugenol in mM
Microorganisms
Percentage hemolysis of RBCs
vu
lg
m
ur
im
ph
S.
ty
P.
iu
os
in
ug
er
P.
a
og
ae
r
E.
a
en
es
ol
E.
c
ar
is
40
35
30
25
20
15
10
5
0
i
% adherence to xylene
Fig. 4. Effect of eugenol on enhancing the activity of (A) lysozyme, (B) Triton X 100 – 0.1%, (C) SDS – 0.1% against E. coli ( ), E.
aerogenes ( ), P. aeruginosa ( ), S. typhimurium (&), P. vulgaris ( ). *Significant with Po0.05.
90
80
70
60
50
40
30
20
10
0
1
2.5
5
10
Eugenol concentration in mM
Fig. 5. Hydrophobicity of the bacterial cell surface after
treatment with eugenol as determined by their partition to
xylene.
Fig. 6. Effect of eugenol on the hemolysis of human
erythrocytes.
membrane with an increased uptake of propidium
iodide associated with lowered cell viability, rapid
depletion of cellular ATP and its subsequent release at
a concentration of 10 mM. It is reported that 5 and
10 mM of eugenol increased the uptake of propidium
iodide by Escherichia coli O157:H7 to 20 and 100%
respectively over a 10-min period (Gill and Holley,
2006a). It was also observed that eugenol between 5 and
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10 mM concentration inhibited the motility of E. coli
(Gill and Holley, 2006a).
In our study there is loss of 50% of membrane
integrity when the cells where treated with eugenol for
10 min. The interaction of eugenol primarily is with the
cytoplasmic membrane of the bacteria. Due to this
action it is able to enhance the activity of antibiotics
such as ampicillin, penicillin, oxacillin, erythromycin,
norfloxacin, tetracycline, chloramphenicol, vancomycin,
rifampin and polymyxin B. Enhanced performance of
drugs in combination with eugenol can also be due to
the action of the drugs on different targets when
compared to that of the action of eugenol. The
antibiotics ampicillin, penicillin, oxacillin, polymyxin B
and vancomycin are known to affect the cell membrane
by acting on different targets. Norfloxacin, apart from
inhibiting the DNA synthesis at low amounts
(0.25 MIC) interacts with the outer membrane by
removing the divalent cations from LPS-binding sites
thereby damaging the cell membrane (Campos et al.
2006) Eugenol too brings about membrane disruption,
but by inhibiting ATPase (Gill and Holley 2006b).
Erythromycin, tetracycline, chloramphenicol and rifampin target the ribosome of the bacteria. Synergy is
believed to be brought about by a combination of drugs
because they block one or more of different targets in
the metabolic pathway (Hemaiswarya et al. 2008). Only
with exact knowledge of the mechanisms underlying the
synergy effects, it will be possible to develop a new
generation of safe and standardized with high efficacy
(Wagner and Ulrich-Merzenich 2009). In the present
study the interaction of eugenol potentiated the activity
of antibiotics which act on different targets. Eugenol
being a hydrophobic compound with a log P of 2.3
(Fujisawa et al. 2002) is found to be more active against
P. vulgaris which is relatively more hydrophobic than
the other organisms. Though the MIC of eugenol did
not vary much between the organisms, it increases the
susceptibility of the organisms that are more hydrophobic to antibiotics and also to lysozyme and
detergents.
At low concentrations (2.5 mM) eugenol was less
cytotoxic and it was increased at higher concentrations.
He et al. (2007) and Manabe et al. (1987) also showed
increased toxicity at higher concentrations of eugenol
with rat and human erythrocytes. Ten male and 10
female rats which were given 89.7 mg/kg eugenol for 12
weeks showed no adverse effects (Trubeck Laboratories
1958). In another study groups of 10 males and 10
females were fed diets containing 0, 0.1 and 1.0%
eugenol for 19 weeks without any adverse effect on their
growth rate, haematology, organ weights and histology
of the major tissues (Hagan et al. 1967). Intravenous
administration of eugenol at 6.52 M was found to cause
hemorrhagic lung edema in rats (Wright et al. 1995).
Intravenous treatment of rats with eugenol (1–10 mg/kg)
7
induced dose-dependent hypotension and bradycardia,
which occurred independently (Lahlou et al. 2004).
The primary mechanism of action of eugenol is
through the disruption of the bacterial membrane,
thereby increasing non-specific permeability of the
antibiotics. Other secondary effects at sublethal concentrations cannot be discounted and can be expected as
a consequence of the interactions with the bacterial
membrane. The current study also indicates the ability
of eugenol to sensitise the bacterial cells towards a
heterogeneous group of antibiotics which underlines the
non-specific and general nature of its activity. Antibiotics like penicillin, vancomycin and oxacillin, which
are non specific against Gram negative bacteria, when
pretreated with eugenol become more effective. This
finding can lead to new treatment strategies and could
also pave the way in the reduction of the amount of
antibiotics required for treatment, particularly against
Gram negative bacteria. A reduction in the antibiotic
amount could also lead to reduction in the toxicity and
side effects to the patients.
The applications of the above studies of these
compounds in the in vivo systems or in clinical studies
require the pharmacokinetic and pharmacodynamic
parameters. The interaction of drugs leading to either
synergism or antagonism is not indicative of their
pharmacodynamic efficacy (Den Hollander et al.
1998). Antimicrobials exhibit two primary kinds of
killing activity, namely concentration-dependent and
time dependent. Compounds belonging to the class of
aminoglycosides, fluoroquinolones, ketolides result in
former killing. Here increase in drug concentration leads
to greater rate and extent of microbial death. The PK/PD
parameters that correlate with clinical outcomes for
these agents can be derived from one of the two ratios:
the peak unbound serum concentration (Cmax) to the
MIC [Cmax/MIC] or the 24-h area under the unbound
serum concentration curve (AUC024) to the MIC
[AUC024/MIC] (Jacobs 2004). Antibiotics belonging
to b-lactams, macrolides, glycopeptides, tetracycline
exhibit time dependent killing. For these antibiotics
the best predictor of clinical outcome is the duration of
time the concentration at the site of infection is above
the MIC [t(time)4MIC] (Jacobs 2004). In our study, we
have used ten different antibiotics, where the efficacy of
penicillin, oxacillin, ampicillin, erythromycin and norfloxacin will largely be determined by Cmax/MIC and
AUC024/MIC. The pharmacodynamics of rifampin
and polymyxin B are closely linked to the AUC024/MIC.
Experiments carried out with non-neutropenic and
neutropenic mice with b-lactam antibiotics indicated that
percentage survival was reasonably with log [Cmax/MIC],
t/MIC and log [AUC024/MIC] (Soriano et al. 1996).
Concentration of eugenol in blood and plasma peaked
rapidly (Cmax of 0.123 and 0.27 mg/ml respectively)
following oral administration (40 mg/kg body weight)
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in male Sprague-Dawley rats. The mean T1/2 value in
plasma and blood were 14 and 18.3 h respectively. Since
eugenol has a relative long elimination half life time and
repeated daily administration may lead to a certain
amount of accumulation (Guénette et al. 2007). This
means it may be possible to reduce subsequent dosage of
eugenol. Based on the reports it may be extrapolated
that an oral dose of 0.3 g/kg body weight to achieve the
necessary synergy with the antibiotics. Under in vitro
conditions involving combination studies determination
of these parameters is difficult, since one compound may
alter the parameters of the other. But it is reported that
the PK/PD of calcium channel antagonist feldopine
were essentially unaltered by menthol (Gelal et al. 2004).
The PK/PD values for the drugs and eugenol may be
different when used in combination and it may not be
prudent to extrapolate the individual drug data to
combination therapy.
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Phytomedicine (2009), doi:10.1016/j.phymed.2009.04.006