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Antimicrobial activity of Mentha piperita essential oil in combination with silver ions
Accepted Manuscript Title: Antimicrobial activity of Mentha piperita essential oil in combination with silver ions Author: Aijaz Ahmad Amber Khan Neha Samber Nikhat Manzoor PII: DOI: Reference:
S2213-7130(14)00022-4 http://dx.doi.org/doi:10.1016/j.synres.2014.11.001 SYNRES 13
To appear in: Received date: Revised date: Accepted date:
21-4-2014 3-11-2014 4-11-2014
Please cite this article as: Ahmad A, Khan A, Samber N, Manzoor N, Antimicrobial activity of Mentha piperita essential oil in combination with silver ions, Synergy Res. (2014), http://dx.doi.org/10.1016/j.synres.2014.11.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Antimicrobial activity of Mentha piperita essential oil in combination with silver ions Aijaz Ahmad§, Amber Khan†, Neha Samber and Nikhat Manzoor*
Present address: Department of Pharmaceutical Sciences, Tshwane University of Technology,
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§
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Medical Mycology Lab, Department of Biosciences, Jamia Millia Islamia, New Delhi-110025
Arcadia Campus, Pretoria, SA-0001
Present address: Department of Pharmacy and Pharmacology, Faculty of Health Sciences,
an
†
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Medical School, University of Witwatersrand, Johannesburg, South Africa
*Corresponding author
Mailing address: Department of Biosciences, Jamia Millia Islamia, New Delhi-110025, India Phone: +91-11-2698-1717- Ext. 3410. Fax: +91-11-2698-0229 E-mail address: [email protected] “Correspondence and Reprints”
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Abstract Peppermint (Mentha piperita L.) is time-honored for its medicinal properties and its antimicrobial characteristics are well established and supported in the literature. In the present
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study the composition and in vitro antimicrobial activity of Mentha piperita essential oil (MpEO) alone and in combination with silver ions (Ag+) against the cultures of Escherichia coli,
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Staphylococcus aureus and Candida albicans is highlighted. The nature of the interaction was
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studied from fractional inhibitory concentration indices (FICIs) for MpEO plus Ag+, calculated from chequerboard microdilution assays. On Gas Chromatography-Mass Spectrophotometry
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(GC-MS) analysis, MpEO showed a high content of menthol (34.82%). FICI values depicted a high synergism of MpEO with Ag+ against C. albicans (∑FIC =0.48) and E. coli (0.40), while as
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indifferent effect against S. aureus (0.95). No antagonistic activity was seen in the strains tested in the present study. Combinational activity was further confirmed by disc diffusion and time kill
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curve assays. From these results we suggest that MpEO with Ag+ have great potential as
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antimicrobial agents required to achieve an effective reduction in opportunistic pathogenic microorganisms.
Keywords: Mentha piperita essential oil; Silver ions; Synergy; Escherichia coli; Staphylococcus aureus; Candida albicans
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1.1 Introduction There is a steady increase in the incidence of antimicrobial resistance worldwide. Resistance has particularly spread in pathogens causing nosocomial infections, but also in organisms causing
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community acquired infections. Besides the known pathogens, drug resistance has been observed in opportunistic microorganisms [1]. Resistance to antimicrobial agents has resulted in morbidity
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and mortality from treatment failures and increased health care costs. There is little doubt that
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emergent antibiotic resistance is a serious global problem highlighting the need for novel antimicrobial agents and therapies. Several studies have demonstrated that natural extracts and
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essential oils have significant antibiotic properties [2]. Mentha Piperita essential oil (MpEO) has high antimicrobial activity due to its components the major ones being α-terpinene, isomenthone,
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trans-carveol, pipertitinone oxide and β-caryophyllene [3]. Since ancient times, silver ions have been known to be effective against a broad range of microorganisms. These ions are used to
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control bacterial growth in a variety of medical applications, including dental work, catheters,
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and the healing of burn wounds. The antimicrobial property of Ag has been investigated more extensively than any other inorganic antimicrobial compound. Also micromolar concentrations of silver have no harmful effects on humans [4].
In view of the lack of new classes of drugs and molecular targets, drug combination therapy might be considered a viable strategy, considering the multiplicity of microbial targets against which current agents are effective [5]. Drug interactions are described as synergistic, indifferent, additive or antagonistic. An advantage of using a combination is the synergistic effect, in which the antimicrobial activity is greater than the individual contribution of each agent. In the present study, we have evaluated the in vitro antimicrobial activity of MpEO against three different
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opportunistic pathogens Candida albicans, Escherichia coli and Staphylococcus aureus. We have also explored the possibility of synergistic interaction between MpEO and Ag+ against these three microbes using chequerboard microdilution assay, disc diffusion assay and time kill
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curves.
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1.2 Materials and Methods
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All media constituents were obtained from HiMedia (India). Extra pure grade of Mentha piperita essential oil (ISO 9001:2008) was purchased from Mohan Perfumery Co., Tilak Bazar, Delhi,
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India. DMSO and AgNO3 (99.99%) was purchased from Sigma Aldrich (USA). All inorganic chemicals were of analytical grade and were procured from E. Merck (India). MpEO was
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dissolved in 1% DMSO and AgNO3 was dissolved in sterile distilled water to make up the
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1.2.1 Strains and media
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desired concentrations.
Stock cultures of the bacteria and fungi were maintained on nutrient agar and Yeast extractPeptone-Dextrose (YPD) agar slants and stored at 4°C. Staphylococcus aureus MTCC 902, Escherichia coli MTCC 443 and Candida albicans ATCC 90028 were grown and sub-cultured in Mueller-Hinton broth, Luria-Bertani broth and YPD media respectively at 37ºC in orbital shaker at 200×rpm (REMI CIS 24 BL).
1.2.2 Gas Chromatography-Mass Spectrophotometry analysis Analysis of MpEO was carried out by GC-MS as described previously [6]. Briefly, Analysis of MpEO was carried out by GC-MS using a Shimadzu 2010 gas chromatograph fitted with an AB-
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Wax column. Helium was used as the carrier gas. Sample (0.1mL) was injected in the splitless mode. The chemical components from the MpEO were identified by comparing the mass spectra from the total ion chromatogram, and retention indices using NIST and Mass Finder GC-MS
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libraries.
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1.2.3 Antimicrobial susceptibility test
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The minimal inhibitory concentration (MIC) was defined as the lowest concentration of the test compound that causes inhibition of visible growth (turbidity). MIC was determined in vitro in
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liquid medium by the macrobroth dilution method as per the guidelines of CLSI reference document M27-A3 [7] for fungi, document NCCLS/CLSI M11-A6 [8] for gram negative M100-S15 [9] for gram positive bacteria. Positive control
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bacteria, and document CLSI
ciprofloxacin for bacteria and fluconazole for yeasts and negative vehicle control (1% DMSO)
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were also included in every set of experiments. To determine the minimal lethal concentration
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(MLC) values after reading the corresponding MIC values, 20 µl samples from all optically clear tubes (complete growth inhibition) plus the last tube showing growth were sub-cultured on agar plates. The plates were incubated at 37ºC for a minimum of 3 days, until growth was clearly visible in the control samples, and MLC values were determined as the lowest concentration of the test compounds for which there was no visible growth.
Checkerboard microdilution assay
Drug interaction was studied using the checkerboard microdilution assay in 96-well microtitre plates as described previously [10, 11]. The initial concentration of cell suspension in the medium was 1×103 CFU mL-1, with the following concentration ranges of test compounds: C.
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albicans, 0.00625-0.2 mg/mL MpEO and 0.000075-0.0006 mg/mL Ag+; S. aureus, 0.05-0.8 mg/mL MpEO and 0.0006-0.0192 mg/mL Ag+; and E. coli, 0.1-6.4 mg/mL MpEO and 0.000075-
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0.0006 mg/mL Ag+.
To assess the interaction of the combination of MpEO with Ag+ the data obtained
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spectrophotometrically was further analysed using fractional inhibitory concentration index
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(FICI) defined as: FICI = FICA + FICB = MICA in combination/MICA tested alone + MICB in combination/MICB tested alone, where MICA and MICB are the MICs of compounds A and B
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respectively. Synergy and antagonism were defined by a FICI of ≤0.5 and >4, respectively. A
1.2.4 Disc diffusion assays
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FICI result of >0.5 but ≤4 was considered as a result of indifference.
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The assay was performed as discussed previously [12]. Briefly, strains were inoculated into
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liquid media and grown overnight at 37°C. Cells were then washed three times with distilled water and approximately 1×105 cells/ml were inoculated into half-strength molten agar media at 42ºC and poured into 100mm diameter Petri-plates. After the top layer had solidified; sterile paper discs (4mm) were impregnated with drugs alone, and in combination, and placed on the agar surface. The concentration range for MpEO and Ag+ when applied alone was 0.16–20.48 mg/mL and 0.00094–0.015 mg/mL respectively while it was 0.0125–0.4 mg/mL and 0.00015– 0.0024 mg/mL when applied in combination, were spotted on the discs in a 10 μL volume. To avoid the evaporation of the volatile essential oils, immediately after pouring the essential oils on discs plates were sealed using parafilm. After incubation at 37ºC for 24h and 48h, the size and pattern of the growth inhibition zone around the disc on agar were evaluated.
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1.2.5 Time–kill curves Time kill studies were performed according to the standard protocol [13]. Briefly, the cell
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suspension of 5×106 cfu/mL was diluted 1:10 in media to give a final inoculum concentration of 5×105 cfu/mL. Final concentrations of MpEO and AgNO3 were ¼ MIC values for each strain.
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Cultures (5 mL final volume) were incubated at 37ºC with agitation (200 rpm). At pre-
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determined time points (0, 2, 4, 8, 12 and 24 h) 100 µL aliquots were removed and transferred to Eppendorf tubes, centrifuged (3900 g at 4ºC for 1 min) and rinsed twice with 0.9 mL of sterile
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distilled water to obtain compound-free cells. Pellets were suspended in 100 mL of sterile distilled water and serially diluted. 20 µL was spread onto SDA plates and incubated at 37ºC for
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2. Results
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the numbers of cfu/mL.
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24h (for yeast) and 48 h (for bacteria) or until the colonies were seen on the plates, to determine
2.1 Gas Chromatography-Mass Spectrometry (GC-MS) analysis GC-MS analysis of MpEO was done to confirm the specific chemotype. Nineteen compounds representing >95% oil were identified and the major compounds have been highlighted in Table 1. As expected the major compounds identified were menthol (34.82%), carvone (19.54%) and menthone (9.10%).
2.2 Antimicrobial activity The MIC and MLC values obtained for both MpEO and Ag+ as well as for the positive controls against all the three microbial species are shown in Table 1. Evaluation of MIC/MLC showed
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that both MpEO as well as Ag+ was active in vitro against all the tested microorganisms. The order of sensitivity to MpEO (MIC; MLC) was C. albicans (0.08 mg/mL; 0.16 mg/mL) > S. aureus (0.32 mg/mL; 0.64 mg/mL) > E. coli (5.12 mg/mL; 20.48 mg/mL). Sensitivity to Ag+ was
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observed in the order C. albicans (0.00047 mg/mL; 0.00094 % mg/mL) > E. coli (0.00093 mg/mL; 0.0019 mg/mL) > S. aureus (0.0075 mg/mL; 0.015 mg/mL). These findings validate our
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previous results (3) that MpEO is fungicidal at relatively low MIC values. C. albicans is highly
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sensitive to both MpEO and Ag+, but a difference in sensitivity was observed with the bacterial
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strains, E. coli being more sensitive to Ag+ while S. aureus was sensitive to MpEO.
2.3 Susceptibility in combination with silver
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The FIC indices of MpEO combined with Ag+ against all the tested microorganisms were calculated using the checkerboard microtiter assay. The data, shown in Table 2, indicates
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significant synergistic combined effects between MpEO and Ag+ against C. albicans and E. coli,
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while indifferent interactions were observed against S. aureus.
In vitro activity of MpEO alone and in combination with Ag+ was determined by agar disc diffusion assay (Figure 1). The antimicrobial activity of both MpEO and Ag+ increased with their increasing concentrations. Results depict silver ions showed potent inhibitory effects against C. albicans and E. coli than S. aureus, however, MpEO showed lower sensitivity towards E. coli. Large and clear zones of inhibitions (ZOI) around discs impregnated with MpEO along with Ag+, clearly demonstrate that MpEO showed a powerful fungicidal effect when combined with Ag+. The mean diameters of the ZOI for Ag+ and MpEO against C. albicans, E. coli and S. aureus were 18 & 16mm, 16 & 12 mm and 18 & 17, respectively. The ZOI increased to 24 and
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21 mm around the discs impregnated with MpEO plus Ag+ against C. albicans and E. coli, respectively. However, slight increase (21mm) was observed when S. aureus cells were treated with both the agents in combinations. In addition, the combination of MpEO and Ag+ yielded
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significantly clearer and larger zones than those of either alone. The control disc impregnated
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with the solvent DMSO, showed no antimicrobial activity.
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2.4 Time kill curves
The synergism observed using checkerboard and disc-diffusion assays was further confirmed by
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time-kill curves. As shown in figure 2, MpEO and Ag+ at their respective ¼ MIC values, did not affect the growth of tested microorganisms. MpEO alone at lower concentrations had a weak
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antimicrobial effect. In contrast, the combination of MpEO with Ag+ showed potent antimicrobial activity. In the case of C. albicans, after 12h of incubation, the MpEO -Ag+
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combination yielded a 4.5-log-CFU/ml decrease compared with an MpEO treated alone (Figure
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2A). The decrease for E. coli after 12h was observed to be 8.1-log in viable counts compared with the number of CFU/ml produced in MpEO treatment (Figure 2B). However, the MpEO Ag+ combination decreases to only <2-log-CFU/ml compared with the number of CFU produced by MpEO alone at 12 h in case of S. aureus, which mirrors the FICI values derived from the chequerboard assay results.
3. Discussion
Mentha piperita has been used as a folk medicine in tropical and subtropical regions and is known to possess pharmacological activity, including antimicrobial and disinfectant properties. Metals have been described to be effective antimicrobial agents, and the efficacy of Ag+ as a
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disinfectant has been known for centuries [14]. Drug combination is most widely used for the treatment of many dreadful diseases. The main aim of drug combination therapy is to achieve synergy, dose and toxicity reduction, and to minimize or delay the induction of drug resistance
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[15].
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Even though there are number of studies to describe the chemical composition of MpEO, GC-
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MS analysis was done to confirm the chemotypic variations. The major compound identified was menthol which is in accordance with the previous findings. The MIC values for the MpEO
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against E. coli are comparatively greater than C. albicans and S. aureus. As already demonstrated, our results also showed that the activity of the agents against each microbe varies
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due to structural differences between the microorganisms. In addition the permeability differences due to the hydrophobic (MpEO) and hydrophilic (Ag+) nature of agents across the
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cell membrane may influence the extent of the antimicrobial activity. The presence of the
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lipopolysaccharides in the outer membrane of the gram-negative bacteria (E. coli) may hinder the penetration of the hydrophobic MpEO into the cells. Thus the MIC values of the MpEO were higher in E. coli in comparison to S. aureus and C. albicans. The order of sensitivity to the Ag+ was C. albicans>E. coli>S. aureus, which is in consistent with the previous studies that the hydrophilic agents are transported into the cell passively via the channels rather than having to diffuse simply through the cell wall matrix [16]. In addition it is also hypothesized that the positively charged Ag+ are trapped by the negatively charged peptidoglycans present in the cell membrane of S. aureus, thus restricting their entry into the cell to reach their target.
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The results of synergistic action of MpEO with Ag+ demonstrated the potential of Ag+ to enhance the antimicrobial activity of MpEO and, thus, support previous findings on synergistic action between natural products and metal ions [17]. In the present work, we have focused on the
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combinations of the MpEO with Ag+ at sub-MIC values and observed that combined agents showed an increase in antimicrobial activities as measured by FICI. The results showed the
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synergistic interactions in E. coli (FICI=0.40) and C. albicans (FICI=0.48), but the FICI for the
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combination of MpEO and Ag+ against S. aureus were within the indifferent range (FICI=0.95). Essential oils are known for impairing membrane structures and functions and can bind to
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proteins and sterols and bring about structural changes in the cell wall and membrane, leading to cell distortion and death [18]. Silver ions have been reported to block DNA replication when
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DNA is in its condensed form and can also deactivate vital enzymes of the cell. For their synergistic antimicrobial activities, we hypothesize that MpEO might interact with the cell
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membranes forming pores, thus altering the membrane integrity and reducing the restricted
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penetration of Ag+ into the cells. The dual killing antimicrobial activity of MpEO and Ag+ give rise to the observed enhanced antimicrobial activity of MpEO and Ag+ at the sub-MIC values.
The in vitro hemolytic assay is a possible screening tool for gauging in vivo toxicity to host cells. Several studies have previously reported that Ag+ has no or least cytoxicity and hemolytic activity over a different range of concentrations which exceeds the antimicrobial concentrations by a factor of several times [19]. MpEO has also been reported to possess negligible hemolytic activity [20] and some recent studies have even shown the antihemolytic effects of M. piperita [21].
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4. Conclusion To our knowledge, this is the first attempt to study synergistic interaction between MpEO and Ag+. MPEO may prove to be a potent phytotherapeutic and/or combination agent with Ag+.
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These results suggest that MpEO and Ag+ when used in combination may offer an opportunity to produce novel combinations acting synergistically to control important infectious pathogens.
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Since the MpEO concentration effective in vitro is achievable in vivo, the combination of this
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agent with Ag+ represents an attractive prospect for the development of new management strategies for opportunistic pathogenic microorganisms, and should be investigated further on in
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vivo models.
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Acknowledgements
This work was supported by Indian Council of Medical Research (ICMR), New Delhi (Grant No.
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59/24/2008/BMS/TRM [2008-04780]).
References
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[2] Avato P, Raffo F, Guglielmi G, Vitali C, Rosato A. Extracts from St. John’s Worth and their antimicrobial activity. Phytother Res 2004; 18:230–2. [3] Saharkhiz MJ, Motamedi M, Zomorodian K, Pakshir K, Miri R, Hemyari K. Chemical Composition, Antifungal and Antibiofilm Activities of the Essential Oil of Mentha piperita L. ISRN Pharm. 2012; 2012: 718645.
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[4] Berger TJ, Spadaro JA, Chapin SE, Becker RO. Electrically Generated Silver Ions: Quantitative Effects on Bacterial and Mammalian Cells. Antimicrob Agents Chemother 1976; 9(2):357–8.
invasive fungal infections. Clin. Microbiol. Rev 2005; 18:163-94.
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[5] Mukherjee PK, Sheehan DJ, Hitchcock CA, Ghannoum MA. Combination treatment of
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[6] Khan A, Ahmad A, Manzoor N, Khan LA. Antifungal Activities of Ocimum sanctum
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Essential Oil and its Lead Molecules. Nat Prod Commun 2010; 5 (2):345–9.
[7] CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts.
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Approved Standard M27-A3, third ed. Clinical and Laboratory Standards Institute, Wayne, PA, 2008.
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[8] CLSI/NCCLS. Methods for antimicrobial susceptibility testing of anaerobic bacteria; Approved Standard. 6th ed. M11-A6, 2004.
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[9] Performance Standards for Antimicrobial Susceptibility Testing; Fifteenth Informational
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Supplement, Clinical and Laboratory Standards Institute, M100-S15, 25 (1), 2005. [10] Ahmad A, Khan A, Khan LA, Manzoor N. In vitro synergy of eugenol and methyleugenol with fluconazole against clinical Candida isolates. J Med Microbiol 2010; 59:1178-84.
[11] Ahmad A, Khan A, Manzoor N. Reversal of efflux mediated antifungal resistance underlies synergistic activity of two monoterpenes with fluconazole. Eur J Pharm Sci 2013; 48:80-6.
[12] Khan A, Ahmad A, Xess I, Khan LA, Manzoor N. Anticandidal effect of Ocimum sanctum essential oil and its synergy with fluconazole and ketoconazole. Phytomedicine. 2010; 17(12):921-5.
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[13] National Committee for Clinical Laboratory Standards. Methods for determining bactericidal activity of antimicrobial agents: Approved Standard M26-P, vol.7, no.2. National Committee for Clinical Laboratory Standards, Villanova, Pa, 1987.
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[14] Rivardo F, Martinotti MG, Turner RJ, Ceri H. The activity of silver against Escherichia coli biofilm is increased by a lipopeptide biosurfactant. Can J Microbiol 2010; 56(3):272-
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8.
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[15] Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 2006; 68:621–81.
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[16] Nikaido H. Preventing drug access to targets: cell surface permeability barriers and active efflux in bacteria. Semin Cell Dev Biol 2001; 12(3):215-23.
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[17] Low WL, Martin C, Hill DJ, Kenward MA. Antimicrobial efficacy of silver ions in combination with tea tree oil against Pseudomonas aeruginosa, Staphylococcus aureus
d
and Candida albicans. Int J Antimicrob Agents 2011; 37:162-5.
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[18] Khan A, Ahmad A, Akhtar F, Yousuf S, Xess I, Khan LA et al. Ocimum sanctum essential oil and its active principles exert their antifungal activity by disrupting ergosterol biosynthesis and membrane integrity. Res Microbiol 2010; 161(10):816-23. [19] Golubeva OY, Shamova OV, Orlov DS, Pazina TY, Boldina AS, Kokryakov VN. Study of Antimicrobial and Hemolytic Activities of Silver Nanoparticles Prepared by Chemical Reduction. Glass Physics and Chemistry 2010; 36:628–34. [20] de Freitas MV, Netto Rde C, da Costa Huss JC, de Souza TM, Costa JO, Firmino CB et al. Influence of aqueous crude extracts of medicinal plants on the osmotic stability of human erythrocytes. Toxicol In Vitro 2008; 22(1):219-24.
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[21] Ebrahimzadeh MA, Nabavi SF, Nabavi SM, Esalmi B. Antioxidant and antihemolytic
Table 1: GC-MS Analysis of Mentha piperita essential oil Area% 0.15
1104
0.14
1194
3.32
1466 1496
M
1317
Compound α-pinene β-pinene Limonene
5.16
Eucalyptol
0.32
Cyclohexanol
9.10
Menthone
d
1202
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RRI 1016
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cr
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activity of Mentha piperita. Pharmacologyonline 2010; 1: 744-52.
5.32
Isomenthone
1.64
Menthyl acetate
1572
0.39
Neoiso (ISO) Pulegol
1596
1.05
Caryophyllene
1629
5.44
Neoisomenthol
1641
34.82
1653
0.47
Menthol Pulegone
1674
0.68
Beta-Terpineol
1701
0.16
α-terpineol
1739
0.19
Piperitone
1741
19.54
Carvone
1768
9.54
Anethole
1822
0.11
Geraniol
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1531
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Table 2: Antimicrobial activity of Mentha piperita essential oil alone and in combination with
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Ag+ against E. coli, S. aureus and C. albicans. MIC, MLC and FICI values shown are mean
C. albicans ATCC 90028 MIC
S. aureus MTCC 902
E. coli MTCC 443
MLC
MIC
MLC
MIC
MLC
1.6
3.2
6.4
51.2
204.8
0.0094
0.075
0.15
0.0093
0.019
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Microorganism
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values of three independent experiments.
MpEO
0.8
Ag+
0.0047
MIC in combination (mg/mL)
MpEO
0.125
2.0
4.0
Ag+
0.0015
0.024
0.003
Positive Controls#
0.05
0.0025
0.01
FICI
0.48
0.95
0.40
Ac ce pt e
MIC/MLC Alone (mg/mL)
SYN IND SYN Fluconazole was used as positive control for C. albicans and Ciprofloxacin was used as positive Interpretation
#
control for S. aureus and E. coli IND, Indifference; SYN, Synergy
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Figure Captions
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Figure 1: Disk diffusion assay of C. albicans (A), E. coli (B) and S. aureus (C) isolates showing synergistic inhibition of Ag+ with MpEO. The concentrations for MpEO and Ag+ when applied
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alone were 0.16, 20.48 and 0.64 % v/v and 0.00094, 0.0019 and 0.015 % v/v against C. albicans,
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E. coli and S. aureus, respectively, while for combination it was 0.0125, 0.4 and 0.2 % v/v for MpEO and 0.00015, 0.0003 and 0.0024% w/v for Ag+ against C. albicans, E. coli and S. aureus,
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were spotted on the disks in a 10 μL volume, respectively.
Figure 2: Representative time-kill curves of C. albicans (A), E. coli (B) and S. aureus (C)
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isolates following exposure to ¼ MIC of MpEO (b), ¼ MIC of Ag+ (c) and ¼ MIC of MpEO combined with ¼ MIC of Ag+ (d). Curve (a) represents the control cells without any treatment.
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d
Data represent the mean of three replicates ± standard deviation.
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Ag+
Ag+
Control
MPEO + Ag+
MPEO
MPEO + Ag+
MPEO
B
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A
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Control
Ag+
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d
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Control
MPEO + Ag+
MPEO
C
Figure 1
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6
S. aureus
4
b
b c
c 3 2
6 d
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a
log CFU/ml
5
4
cr
2
1
d
0 2
4 Tim e (h)
8
12
0
2
4 8 Tim e (h)
12
24
B
an
A
us
0 0
a
8
a
E. coli
8
6
c
d
log CFU/mL
b
M
10
Ac ce pt e
log CFU/mL
C. albicans
4
2
d
0
0
2
4
8
12
24
Tim e (h)
C
Figure 2
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S2213-7130(14)00022-4 http://dx.doi.org/doi:10.1016/j.synres.2014.11.001 SYNRES 13
To appear in: Received date: Revised date: Accepted date:
21-4-2014 3-11-2014 4-11-2014
Please cite this article as: Ahmad A, Khan A, Samber N, Manzoor N, Antimicrobial activity of Mentha piperita essential oil in combination with silver ions, Synergy Res. (2014), http://dx.doi.org/10.1016/j.synres.2014.11.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Antimicrobial activity of Mentha piperita essential oil in combination with silver ions Aijaz Ahmad§, Amber Khan†, Neha Samber and Nikhat Manzoor*
Present address: Department of Pharmaceutical Sciences, Tshwane University of Technology,
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§
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Medical Mycology Lab, Department of Biosciences, Jamia Millia Islamia, New Delhi-110025
Arcadia Campus, Pretoria, SA-0001
Present address: Department of Pharmacy and Pharmacology, Faculty of Health Sciences,
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†
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Medical School, University of Witwatersrand, Johannesburg, South Africa
*Corresponding author
Mailing address: Department of Biosciences, Jamia Millia Islamia, New Delhi-110025, India Phone: +91-11-2698-1717- Ext. 3410. Fax: +91-11-2698-0229 E-mail address: [email protected] “Correspondence and Reprints”
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Abstract Peppermint (Mentha piperita L.) is time-honored for its medicinal properties and its antimicrobial characteristics are well established and supported in the literature. In the present
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study the composition and in vitro antimicrobial activity of Mentha piperita essential oil (MpEO) alone and in combination with silver ions (Ag+) against the cultures of Escherichia coli,
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Staphylococcus aureus and Candida albicans is highlighted. The nature of the interaction was
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studied from fractional inhibitory concentration indices (FICIs) for MpEO plus Ag+, calculated from chequerboard microdilution assays. On Gas Chromatography-Mass Spectrophotometry
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(GC-MS) analysis, MpEO showed a high content of menthol (34.82%). FICI values depicted a high synergism of MpEO with Ag+ against C. albicans (∑FIC =0.48) and E. coli (0.40), while as
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indifferent effect against S. aureus (0.95). No antagonistic activity was seen in the strains tested in the present study. Combinational activity was further confirmed by disc diffusion and time kill
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curve assays. From these results we suggest that MpEO with Ag+ have great potential as
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antimicrobial agents required to achieve an effective reduction in opportunistic pathogenic microorganisms.
Keywords: Mentha piperita essential oil; Silver ions; Synergy; Escherichia coli; Staphylococcus aureus; Candida albicans
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1.1 Introduction There is a steady increase in the incidence of antimicrobial resistance worldwide. Resistance has particularly spread in pathogens causing nosocomial infections, but also in organisms causing
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community acquired infections. Besides the known pathogens, drug resistance has been observed in opportunistic microorganisms [1]. Resistance to antimicrobial agents has resulted in morbidity
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and mortality from treatment failures and increased health care costs. There is little doubt that
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emergent antibiotic resistance is a serious global problem highlighting the need for novel antimicrobial agents and therapies. Several studies have demonstrated that natural extracts and
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essential oils have significant antibiotic properties [2]. Mentha Piperita essential oil (MpEO) has high antimicrobial activity due to its components the major ones being α-terpinene, isomenthone,
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trans-carveol, pipertitinone oxide and β-caryophyllene [3]. Since ancient times, silver ions have been known to be effective against a broad range of microorganisms. These ions are used to
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control bacterial growth in a variety of medical applications, including dental work, catheters,
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and the healing of burn wounds. The antimicrobial property of Ag has been investigated more extensively than any other inorganic antimicrobial compound. Also micromolar concentrations of silver have no harmful effects on humans [4].
In view of the lack of new classes of drugs and molecular targets, drug combination therapy might be considered a viable strategy, considering the multiplicity of microbial targets against which current agents are effective [5]. Drug interactions are described as synergistic, indifferent, additive or antagonistic. An advantage of using a combination is the synergistic effect, in which the antimicrobial activity is greater than the individual contribution of each agent. In the present study, we have evaluated the in vitro antimicrobial activity of MpEO against three different
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opportunistic pathogens Candida albicans, Escherichia coli and Staphylococcus aureus. We have also explored the possibility of synergistic interaction between MpEO and Ag+ against these three microbes using chequerboard microdilution assay, disc diffusion assay and time kill
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curves.
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1.2 Materials and Methods
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All media constituents were obtained from HiMedia (India). Extra pure grade of Mentha piperita essential oil (ISO 9001:2008) was purchased from Mohan Perfumery Co., Tilak Bazar, Delhi,
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India. DMSO and AgNO3 (99.99%) was purchased from Sigma Aldrich (USA). All inorganic chemicals were of analytical grade and were procured from E. Merck (India). MpEO was
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dissolved in 1% DMSO and AgNO3 was dissolved in sterile distilled water to make up the
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1.2.1 Strains and media
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desired concentrations.
Stock cultures of the bacteria and fungi were maintained on nutrient agar and Yeast extractPeptone-Dextrose (YPD) agar slants and stored at 4°C. Staphylococcus aureus MTCC 902, Escherichia coli MTCC 443 and Candida albicans ATCC 90028 were grown and sub-cultured in Mueller-Hinton broth, Luria-Bertani broth and YPD media respectively at 37ºC in orbital shaker at 200×rpm (REMI CIS 24 BL).
1.2.2 Gas Chromatography-Mass Spectrophotometry analysis Analysis of MpEO was carried out by GC-MS as described previously [6]. Briefly, Analysis of MpEO was carried out by GC-MS using a Shimadzu 2010 gas chromatograph fitted with an AB-
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Wax column. Helium was used as the carrier gas. Sample (0.1mL) was injected in the splitless mode. The chemical components from the MpEO were identified by comparing the mass spectra from the total ion chromatogram, and retention indices using NIST and Mass Finder GC-MS
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libraries.
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1.2.3 Antimicrobial susceptibility test
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The minimal inhibitory concentration (MIC) was defined as the lowest concentration of the test compound that causes inhibition of visible growth (turbidity). MIC was determined in vitro in
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liquid medium by the macrobroth dilution method as per the guidelines of CLSI reference document M27-A3 [7] for fungi, document NCCLS/CLSI M11-A6 [8] for gram negative M100-S15 [9] for gram positive bacteria. Positive control
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bacteria, and document CLSI
ciprofloxacin for bacteria and fluconazole for yeasts and negative vehicle control (1% DMSO)
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were also included in every set of experiments. To determine the minimal lethal concentration
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(MLC) values after reading the corresponding MIC values, 20 µl samples from all optically clear tubes (complete growth inhibition) plus the last tube showing growth were sub-cultured on agar plates. The plates were incubated at 37ºC for a minimum of 3 days, until growth was clearly visible in the control samples, and MLC values were determined as the lowest concentration of the test compounds for which there was no visible growth.
Checkerboard microdilution assay
Drug interaction was studied using the checkerboard microdilution assay in 96-well microtitre plates as described previously [10, 11]. The initial concentration of cell suspension in the medium was 1×103 CFU mL-1, with the following concentration ranges of test compounds: C.
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albicans, 0.00625-0.2 mg/mL MpEO and 0.000075-0.0006 mg/mL Ag+; S. aureus, 0.05-0.8 mg/mL MpEO and 0.0006-0.0192 mg/mL Ag+; and E. coli, 0.1-6.4 mg/mL MpEO and 0.000075-
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0.0006 mg/mL Ag+.
To assess the interaction of the combination of MpEO with Ag+ the data obtained
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spectrophotometrically was further analysed using fractional inhibitory concentration index
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(FICI) defined as: FICI = FICA + FICB = MICA in combination/MICA tested alone + MICB in combination/MICB tested alone, where MICA and MICB are the MICs of compounds A and B
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respectively. Synergy and antagonism were defined by a FICI of ≤0.5 and >4, respectively. A
1.2.4 Disc diffusion assays
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FICI result of >0.5 but ≤4 was considered as a result of indifference.
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The assay was performed as discussed previously [12]. Briefly, strains were inoculated into
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liquid media and grown overnight at 37°C. Cells were then washed three times with distilled water and approximately 1×105 cells/ml were inoculated into half-strength molten agar media at 42ºC and poured into 100mm diameter Petri-plates. After the top layer had solidified; sterile paper discs (4mm) were impregnated with drugs alone, and in combination, and placed on the agar surface. The concentration range for MpEO and Ag+ when applied alone was 0.16–20.48 mg/mL and 0.00094–0.015 mg/mL respectively while it was 0.0125–0.4 mg/mL and 0.00015– 0.0024 mg/mL when applied in combination, were spotted on the discs in a 10 μL volume. To avoid the evaporation of the volatile essential oils, immediately after pouring the essential oils on discs plates were sealed using parafilm. After incubation at 37ºC for 24h and 48h, the size and pattern of the growth inhibition zone around the disc on agar were evaluated.
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1.2.5 Time–kill curves Time kill studies were performed according to the standard protocol [13]. Briefly, the cell
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suspension of 5×106 cfu/mL was diluted 1:10 in media to give a final inoculum concentration of 5×105 cfu/mL. Final concentrations of MpEO and AgNO3 were ¼ MIC values for each strain.
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Cultures (5 mL final volume) were incubated at 37ºC with agitation (200 rpm). At pre-
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determined time points (0, 2, 4, 8, 12 and 24 h) 100 µL aliquots were removed and transferred to Eppendorf tubes, centrifuged (3900 g at 4ºC for 1 min) and rinsed twice with 0.9 mL of sterile
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distilled water to obtain compound-free cells. Pellets were suspended in 100 mL of sterile distilled water and serially diluted. 20 µL was spread onto SDA plates and incubated at 37ºC for
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2. Results
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the numbers of cfu/mL.
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24h (for yeast) and 48 h (for bacteria) or until the colonies were seen on the plates, to determine
2.1 Gas Chromatography-Mass Spectrometry (GC-MS) analysis GC-MS analysis of MpEO was done to confirm the specific chemotype. Nineteen compounds representing >95% oil were identified and the major compounds have been highlighted in Table 1. As expected the major compounds identified were menthol (34.82%), carvone (19.54%) and menthone (9.10%).
2.2 Antimicrobial activity The MIC and MLC values obtained for both MpEO and Ag+ as well as for the positive controls against all the three microbial species are shown in Table 1. Evaluation of MIC/MLC showed
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that both MpEO as well as Ag+ was active in vitro against all the tested microorganisms. The order of sensitivity to MpEO (MIC; MLC) was C. albicans (0.08 mg/mL; 0.16 mg/mL) > S. aureus (0.32 mg/mL; 0.64 mg/mL) > E. coli (5.12 mg/mL; 20.48 mg/mL). Sensitivity to Ag+ was
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observed in the order C. albicans (0.00047 mg/mL; 0.00094 % mg/mL) > E. coli (0.00093 mg/mL; 0.0019 mg/mL) > S. aureus (0.0075 mg/mL; 0.015 mg/mL). These findings validate our
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previous results (3) that MpEO is fungicidal at relatively low MIC values. C. albicans is highly
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sensitive to both MpEO and Ag+, but a difference in sensitivity was observed with the bacterial
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strains, E. coli being more sensitive to Ag+ while S. aureus was sensitive to MpEO.
2.3 Susceptibility in combination with silver
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The FIC indices of MpEO combined with Ag+ against all the tested microorganisms were calculated using the checkerboard microtiter assay. The data, shown in Table 2, indicates
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significant synergistic combined effects between MpEO and Ag+ against C. albicans and E. coli,
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while indifferent interactions were observed against S. aureus.
In vitro activity of MpEO alone and in combination with Ag+ was determined by agar disc diffusion assay (Figure 1). The antimicrobial activity of both MpEO and Ag+ increased with their increasing concentrations. Results depict silver ions showed potent inhibitory effects against C. albicans and E. coli than S. aureus, however, MpEO showed lower sensitivity towards E. coli. Large and clear zones of inhibitions (ZOI) around discs impregnated with MpEO along with Ag+, clearly demonstrate that MpEO showed a powerful fungicidal effect when combined with Ag+. The mean diameters of the ZOI for Ag+ and MpEO against C. albicans, E. coli and S. aureus were 18 & 16mm, 16 & 12 mm and 18 & 17, respectively. The ZOI increased to 24 and
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21 mm around the discs impregnated with MpEO plus Ag+ against C. albicans and E. coli, respectively. However, slight increase (21mm) was observed when S. aureus cells were treated with both the agents in combinations. In addition, the combination of MpEO and Ag+ yielded
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significantly clearer and larger zones than those of either alone. The control disc impregnated
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with the solvent DMSO, showed no antimicrobial activity.
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2.4 Time kill curves
The synergism observed using checkerboard and disc-diffusion assays was further confirmed by
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time-kill curves. As shown in figure 2, MpEO and Ag+ at their respective ¼ MIC values, did not affect the growth of tested microorganisms. MpEO alone at lower concentrations had a weak
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antimicrobial effect. In contrast, the combination of MpEO with Ag+ showed potent antimicrobial activity. In the case of C. albicans, after 12h of incubation, the MpEO -Ag+
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combination yielded a 4.5-log-CFU/ml decrease compared with an MpEO treated alone (Figure
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2A). The decrease for E. coli after 12h was observed to be 8.1-log in viable counts compared with the number of CFU/ml produced in MpEO treatment (Figure 2B). However, the MpEO Ag+ combination decreases to only <2-log-CFU/ml compared with the number of CFU produced by MpEO alone at 12 h in case of S. aureus, which mirrors the FICI values derived from the chequerboard assay results.
3. Discussion
Mentha piperita has been used as a folk medicine in tropical and subtropical regions and is known to possess pharmacological activity, including antimicrobial and disinfectant properties. Metals have been described to be effective antimicrobial agents, and the efficacy of Ag+ as a
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disinfectant has been known for centuries [14]. Drug combination is most widely used for the treatment of many dreadful diseases. The main aim of drug combination therapy is to achieve synergy, dose and toxicity reduction, and to minimize or delay the induction of drug resistance
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[15].
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Even though there are number of studies to describe the chemical composition of MpEO, GC-
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MS analysis was done to confirm the chemotypic variations. The major compound identified was menthol which is in accordance with the previous findings. The MIC values for the MpEO
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against E. coli are comparatively greater than C. albicans and S. aureus. As already demonstrated, our results also showed that the activity of the agents against each microbe varies
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due to structural differences between the microorganisms. In addition the permeability differences due to the hydrophobic (MpEO) and hydrophilic (Ag+) nature of agents across the
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cell membrane may influence the extent of the antimicrobial activity. The presence of the
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lipopolysaccharides in the outer membrane of the gram-negative bacteria (E. coli) may hinder the penetration of the hydrophobic MpEO into the cells. Thus the MIC values of the MpEO were higher in E. coli in comparison to S. aureus and C. albicans. The order of sensitivity to the Ag+ was C. albicans>E. coli>S. aureus, which is in consistent with the previous studies that the hydrophilic agents are transported into the cell passively via the channels rather than having to diffuse simply through the cell wall matrix [16]. In addition it is also hypothesized that the positively charged Ag+ are trapped by the negatively charged peptidoglycans present in the cell membrane of S. aureus, thus restricting their entry into the cell to reach their target.
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The results of synergistic action of MpEO with Ag+ demonstrated the potential of Ag+ to enhance the antimicrobial activity of MpEO and, thus, support previous findings on synergistic action between natural products and metal ions [17]. In the present work, we have focused on the
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combinations of the MpEO with Ag+ at sub-MIC values and observed that combined agents showed an increase in antimicrobial activities as measured by FICI. The results showed the
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synergistic interactions in E. coli (FICI=0.40) and C. albicans (FICI=0.48), but the FICI for the
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combination of MpEO and Ag+ against S. aureus were within the indifferent range (FICI=0.95). Essential oils are known for impairing membrane structures and functions and can bind to
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proteins and sterols and bring about structural changes in the cell wall and membrane, leading to cell distortion and death [18]. Silver ions have been reported to block DNA replication when
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DNA is in its condensed form and can also deactivate vital enzymes of the cell. For their synergistic antimicrobial activities, we hypothesize that MpEO might interact with the cell
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membranes forming pores, thus altering the membrane integrity and reducing the restricted
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penetration of Ag+ into the cells. The dual killing antimicrobial activity of MpEO and Ag+ give rise to the observed enhanced antimicrobial activity of MpEO and Ag+ at the sub-MIC values.
The in vitro hemolytic assay is a possible screening tool for gauging in vivo toxicity to host cells. Several studies have previously reported that Ag+ has no or least cytoxicity and hemolytic activity over a different range of concentrations which exceeds the antimicrobial concentrations by a factor of several times [19]. MpEO has also been reported to possess negligible hemolytic activity [20] and some recent studies have even shown the antihemolytic effects of M. piperita [21].
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4. Conclusion To our knowledge, this is the first attempt to study synergistic interaction between MpEO and Ag+. MPEO may prove to be a potent phytotherapeutic and/or combination agent with Ag+.
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These results suggest that MpEO and Ag+ when used in combination may offer an opportunity to produce novel combinations acting synergistically to control important infectious pathogens.
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Since the MpEO concentration effective in vitro is achievable in vivo, the combination of this
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agent with Ag+ represents an attractive prospect for the development of new management strategies for opportunistic pathogenic microorganisms, and should be investigated further on in
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vivo models.
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Acknowledgements
This work was supported by Indian Council of Medical Research (ICMR), New Delhi (Grant No.
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59/24/2008/BMS/TRM [2008-04780]).
References
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[2] Avato P, Raffo F, Guglielmi G, Vitali C, Rosato A. Extracts from St. John’s Worth and their antimicrobial activity. Phytother Res 2004; 18:230–2. [3] Saharkhiz MJ, Motamedi M, Zomorodian K, Pakshir K, Miri R, Hemyari K. Chemical Composition, Antifungal and Antibiofilm Activities of the Essential Oil of Mentha piperita L. ISRN Pharm. 2012; 2012: 718645.
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[4] Berger TJ, Spadaro JA, Chapin SE, Becker RO. Electrically Generated Silver Ions: Quantitative Effects on Bacterial and Mammalian Cells. Antimicrob Agents Chemother 1976; 9(2):357–8.
invasive fungal infections. Clin. Microbiol. Rev 2005; 18:163-94.
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[5] Mukherjee PK, Sheehan DJ, Hitchcock CA, Ghannoum MA. Combination treatment of
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[6] Khan A, Ahmad A, Manzoor N, Khan LA. Antifungal Activities of Ocimum sanctum
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Essential Oil and its Lead Molecules. Nat Prod Commun 2010; 5 (2):345–9.
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Approved Standard M27-A3, third ed. Clinical and Laboratory Standards Institute, Wayne, PA, 2008.
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[8] CLSI/NCCLS. Methods for antimicrobial susceptibility testing of anaerobic bacteria; Approved Standard. 6th ed. M11-A6, 2004.
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[9] Performance Standards for Antimicrobial Susceptibility Testing; Fifteenth Informational
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Supplement, Clinical and Laboratory Standards Institute, M100-S15, 25 (1), 2005. [10] Ahmad A, Khan A, Khan LA, Manzoor N. In vitro synergy of eugenol and methyleugenol with fluconazole against clinical Candida isolates. J Med Microbiol 2010; 59:1178-84.
[11] Ahmad A, Khan A, Manzoor N. Reversal of efflux mediated antifungal resistance underlies synergistic activity of two monoterpenes with fluconazole. Eur J Pharm Sci 2013; 48:80-6.
[12] Khan A, Ahmad A, Xess I, Khan LA, Manzoor N. Anticandidal effect of Ocimum sanctum essential oil and its synergy with fluconazole and ketoconazole. Phytomedicine. 2010; 17(12):921-5.
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[13] National Committee for Clinical Laboratory Standards. Methods for determining bactericidal activity of antimicrobial agents: Approved Standard M26-P, vol.7, no.2. National Committee for Clinical Laboratory Standards, Villanova, Pa, 1987.
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[14] Rivardo F, Martinotti MG, Turner RJ, Ceri H. The activity of silver against Escherichia coli biofilm is increased by a lipopeptide biosurfactant. Can J Microbiol 2010; 56(3):272-
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[15] Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 2006; 68:621–81.
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[16] Nikaido H. Preventing drug access to targets: cell surface permeability barriers and active efflux in bacteria. Semin Cell Dev Biol 2001; 12(3):215-23.
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[17] Low WL, Martin C, Hill DJ, Kenward MA. Antimicrobial efficacy of silver ions in combination with tea tree oil against Pseudomonas aeruginosa, Staphylococcus aureus
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and Candida albicans. Int J Antimicrob Agents 2011; 37:162-5.
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[18] Khan A, Ahmad A, Akhtar F, Yousuf S, Xess I, Khan LA et al. Ocimum sanctum essential oil and its active principles exert their antifungal activity by disrupting ergosterol biosynthesis and membrane integrity. Res Microbiol 2010; 161(10):816-23. [19] Golubeva OY, Shamova OV, Orlov DS, Pazina TY, Boldina AS, Kokryakov VN. Study of Antimicrobial and Hemolytic Activities of Silver Nanoparticles Prepared by Chemical Reduction. Glass Physics and Chemistry 2010; 36:628–34. [20] de Freitas MV, Netto Rde C, da Costa Huss JC, de Souza TM, Costa JO, Firmino CB et al. Influence of aqueous crude extracts of medicinal plants on the osmotic stability of human erythrocytes. Toxicol In Vitro 2008; 22(1):219-24.
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[21] Ebrahimzadeh MA, Nabavi SF, Nabavi SM, Esalmi B. Antioxidant and antihemolytic
Table 1: GC-MS Analysis of Mentha piperita essential oil Area% 0.15
1104
0.14
1194
3.32
1466 1496
M
1317
Compound α-pinene β-pinene Limonene
5.16
Eucalyptol
0.32
Cyclohexanol
9.10
Menthone
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1202
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RRI 1016
us
cr
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activity of Mentha piperita. Pharmacologyonline 2010; 1: 744-52.
5.32
Isomenthone
1.64
Menthyl acetate
1572
0.39
Neoiso (ISO) Pulegol
1596
1.05
Caryophyllene
1629
5.44
Neoisomenthol
1641
34.82
1653
0.47
Menthol Pulegone
1674
0.68
Beta-Terpineol
1701
0.16
α-terpineol
1739
0.19
Piperitone
1741
19.54
Carvone
1768
9.54
Anethole
1822
0.11
Geraniol
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1531
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Table 2: Antimicrobial activity of Mentha piperita essential oil alone and in combination with
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Ag+ against E. coli, S. aureus and C. albicans. MIC, MLC and FICI values shown are mean
C. albicans ATCC 90028 MIC
S. aureus MTCC 902
E. coli MTCC 443
MLC
MIC
MLC
MIC
MLC
1.6
3.2
6.4
51.2
204.8
0.0094
0.075
0.15
0.0093
0.019
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Microorganism
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values of three independent experiments.
MpEO
0.8
Ag+
0.0047
MIC in combination (mg/mL)
MpEO
0.125
2.0
4.0
Ag+
0.0015
0.024
0.003
Positive Controls#
0.05
0.0025
0.01
FICI
0.48
0.95
0.40
Ac ce pt e
MIC/MLC Alone (mg/mL)
SYN IND SYN Fluconazole was used as positive control for C. albicans and Ciprofloxacin was used as positive Interpretation
#
control for S. aureus and E. coli IND, Indifference; SYN, Synergy
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Figure Captions
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Figure 1: Disk diffusion assay of C. albicans (A), E. coli (B) and S. aureus (C) isolates showing synergistic inhibition of Ag+ with MpEO. The concentrations for MpEO and Ag+ when applied
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alone were 0.16, 20.48 and 0.64 % v/v and 0.00094, 0.0019 and 0.015 % v/v against C. albicans,
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E. coli and S. aureus, respectively, while for combination it was 0.0125, 0.4 and 0.2 % v/v for MpEO and 0.00015, 0.0003 and 0.0024% w/v for Ag+ against C. albicans, E. coli and S. aureus,
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were spotted on the disks in a 10 μL volume, respectively.
Figure 2: Representative time-kill curves of C. albicans (A), E. coli (B) and S. aureus (C)
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isolates following exposure to ¼ MIC of MpEO (b), ¼ MIC of Ag+ (c) and ¼ MIC of MpEO combined with ¼ MIC of Ag+ (d). Curve (a) represents the control cells without any treatment.
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d
Data represent the mean of three replicates ± standard deviation.
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Ag+
Ag+
Control
MPEO + Ag+
MPEO
MPEO + Ag+
MPEO
B
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A
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Control
Ag+
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d
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Control
MPEO + Ag+
MPEO
C
Figure 1
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6
S. aureus
4
b
b c
c 3 2
6 d
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a
log CFU/ml
5
4
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2
1
d
0 2
4 Tim e (h)
8
12
0
2
4 8 Tim e (h)
12
24
B
an
A
us
0 0
a
8
a
E. coli
8
6
c
d
log CFU/mL
b
M
10
Ac ce pt e
log CFU/mL
C. albicans
4
2
d
0
0
2
4
8
12
24
Tim e (h)
C
Figure 2
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d
Ac ce pt e us
an
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