Accepted Article

Received Date : 17-Sep-2013 Revised Date : 03-Jan-2014 Accepted Date : 07-Jan-2014 Article type

: Original Article

Membrane disruption and anti-quorum sensing effects of synergistic interaction between Lavandula angustifolia (lavender oil) in combination with antibiotic against plasmid-conferred multi-drug resistant Escherichia coli Polly Soo Xi Yapa, Thiba Krishnanb, Beow Chin Yiapc, Cai Ping Hud, Kok-Gan Chanb, Swee Hua Erin Limc,*

a

School of Postgraduate Studies and Research, International Medical University, No. 126, Jalan Jalil Perkasa 19, Bukit Jalil, 57000 Kuala Lumpur, Malaysia b Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia c School of Pharmacy, Department of Life Sciences, International Medical University, No. 126, Jalan Jalil Perkasa 19, Bukit Jalil, 57000 Kuala Lumpur, Malaysia d School of Health Sciences, Department of Chinese Medicine, International Medical University, No. 126, Jalan Jalil Perkasa 19, Bukit Jalil, 57000 Kuala Lumpur, Malaysia

*Corresponding author. Email: [email protected]

Abstract Aim: The aim of this study was to investigate the mode of action of the lavender essential oil

(LV) on antimicrobial activity against multi-drug resistant Escherichia coli J53 R1 when used singly and in combination with piperacillin. Method and Results: In the time-kill analysis, a complete killing of bacteria was observed based on colony counts within 4 h when LV was combined with piperacillin during exposure at This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jam.12444 This article is protected by copyright. All rights reserved.

Accepted Article

determined FIC concentrations. Analysis of the membrane permeabilizing effects of LV on treated cultures through their stability against sodium dodecyl sulfate revealed that the LV played a role in disrupting the bacterial cell membrane. The finding is further supported by scanning electron microscopy analysis and zeta potential measurement. In addition, reduction in light production expression of Escherichia coli [pSB1075] by the LV showed the presence of potential Quorum Sensing (QS) inhibitors. Conclusions: These results indicated that the LV has the potential to reverse bacterial resistance to piperacillin in E. coli J53 R1. It may operate via two mechanisms: alteration of outer membrane permeability and inhibition of bacterial QS. Significance and Impact of the Study: These findings offer a novel approach to develop a new option of phytopharmaceuticals against multi-drug resistant E. coli.

Keywords: Lavandula angustifolia, essential oil, chemical composition, antibacterial activity, membrane permeability, light production, quorum sensing

Running headline: Mode of action of lavender EO

Introduction The threats of pathogenic infections have almost been eradicated with the introduction of antibiotic treatment regimes. Unfortunately, the repetitive consumption and increasing doses of antibiotics has led to the manifestation of drug tolerance and resistance in microorganisms due to the natural selection process. As a result of this phenomenon, new antibiotics would need to be developed in order to overcome this tolerance/resistance. Natural products are viewed as a

This article is protected by copyright. All rights reserved.

Accepted Article

privileged group of structures which have evolved to interact with a wide variety of protein targets for specific purposes. Many attempts have been made to investigate the potential role of plant extracts and essential oils for their efficacy to combat the problems of antibiotic resistance in bacteria. Essential oils (EOs) are products of the secondary metabolism of aromatic plants. They are highly complex volatile compounds which consist of about twenty to sixty components in various concentrations. It has been long acknowledged that the essential oil of Lavandula angustifolia exhibits various biological activities including antimicrobial, antimutagenic, antiinflammatory and analgesic properties (Hajhashemi et al. 2003; D'Auria et al. 2005; Evandri et al. 2005; Fabio et al. 2007).

In our previous study, we investigated the in vitro effectiveness of the association between betalactam antibiotics and essential oils against plasmid-conferred multi-drug resistant bacteria. The results obtained highlighted the occurrence of a pronounced synergistic relationship between piperacillin and lavender oil (LV) against bacteria encoding a plasmid for beta-lactamase TEM1, with a fractional inhibitory index (FIC) of 0.26 (Yap et al. 2013). The preference for combination therapy is rationalised by the fact that many diseases have a complex pathophysiology. In addition, compared to the synthetic products, there are many advantages to using natural products. The antimicrobial compounds produced possess fewer adverse effects such as better patient tolerance, and is relatively inexpensive, has wide public acceptance due to their traditional applications besides being renewable and better biodegradability. Although the in vitro antimicrobial activity of LV have been extensively reported, very little is known about its mode of action. The composition, structure as well as functional groups of the essential oils play an important role in determining their antimicrobial activity. Considering that a vast range of

This article is protected by copyright. All rights reserved.

Accepted Article

different groups of chemical compounds are present in one EO, it is most likely that antibacterial activities will not be attributed to one specific mechanism or component. Hence, there may be several targets in a cell which result in the potentiating influence. Since understanding its mode of action will have implications for its spectrum of activity which will aid in combating the phenomenon of the developing of antibiotic resistance in microorganisms, the objectives of this study were to examine the mode of action of LV when used alone or in combination with an antibiotic and its components against model bacteria.

Materials and method Lavender essential oil (LV) and antibiotic The lavender essential oil (Lavandula angustifolia) used in all assays was purchased from Aroma Trading Ltd. (Milton Keynes, UK). Piperacillin (Sigma Aldrich, St Louis, MO) was dissolved in its solvent as described in the Clinical and Laboratory Standards Institute (CLSI) M100-S21 guidelines.

Gas Chromatography-Mass Spectrometry (GC-MS) analysis The GC-MS analysis was performed on an Agilent GC-MS, 7890A GC System with a triple-axis detector (5975C MSD) using an HP-5MS column (30 m × 250 μm × 0.25 μm). The MS

conditions were as follows: Helium was used as the carrier gas. The sample was injected with an autoinjector (Agilent Technologies 7693 Autosampler). The temperature of the injector was set at 250 °C, and the oven column was temperature-programmed from 60 °C (5 min) to 220 °C at a rate of 4 °C min-1 for 10 min and 240 °C at a rate of 1 °C min-1 for 5 min. The column flow was

This article is protected by copyright. All rights reserved.

Accepted Article

1 ml min-1 with a split ratio of 40:1. MS data was acquired in EI mode with scan range 30 -450 m/z. The temperature of the MS source and MS quad was set at 250 °C and 150 °C, respectively. Compounds were identified by comparison of the mass spectra with those in NIST libraries. The percentage (relative) of the identified compounds was computed from their GC peak area. In this study, only those components present in the oils in amounts higher than 0.1% were taken into consideration.

Bacterial strain and growth conditions Unless stated otherwise, the bacteria strain used was Escherichia coli J53 R1 carrying plasmid encoding for beta-lactamase TEM-1(Alvarez et al. 2004) and was a kind gift from George A. Jacoby (Lahey Clinic, MA, USA). E. coli was cultured in Mueller-Hinton broth (MHB; Oxoid, Cambridge, UK) and all test conditions were carried out according to the CLSI M07-A8 guidelines.

Time-kill assay For the time-kill assay, a standard inoculum of approximately 105 cfu ml-1 was used and then

incubated at 37°C with shaking at 5 g (200 rpm). The concentrations tested in MHB (supplemented with 0.5% Tween 80 to enhance the oil solubility) were determined in our previous work (Yap et al. 2013). Tween 80 (K & H Chemicals, UK) stock solution in distilled

water was prepared to achieve 10% (v/v) solution and filter sterilised. Subsequently, the EOs or antibiotics were then added into the medium supplemented with Tween 80. The final concentrations of drugs in each flask were the following: control (without treatment); LV (0.5% v/v); piperacillin (128 µg ml-1); LV (0.5% v/v) in combination with piperacillin (128 µg ml-1).

This article is protected by copyright. All rights reserved.

Accepted Article

The final volume of each flask was 10mL. Directly after addition of the inoculum and after incubation, 100 µl of samples was removed for viable counting every 4 h until 24 h as a preliminary experiment (data not shown). Due to the rapid rate of killing which occurred for LV, sampling time was shortened to 5, 10, 30, 60, 120, 180, 240 min. Samples were serially diluted with 0.9% (w/v) sodium chloride, then spread on Mueller-Hinton agar (MHA) and incubated at

37°C for 24 h. The experiment was performed in triplicates.

Outer membrane (OM) permeability To examine the function of EO in permeating OM barriers when used singly and in combination, the methods of Hemaiswarya and Doble (2009) and Marri et al. (1996) were employed (Marri et al. 1996; Hemaiswarya and Doble 2009). Overnight bacterial culture was harvested and washed with PBS (pH 7.4). The culture was then adjusted to OD = 0.3 at 625nm and exposed to antibiotic and/or EO treatment. The treatment time for lavender-piperacillin was 5 min as determined by the results from the time-kill analysis. After that, the cells were washed to remove the treatment and used for the assay. For each treated sample, the culture was divided into two portions with a final volume of 10mL each. Subsequently, one of the portions was added with sodium dodecyl sulphate (SDS) and one without. The SDS was used at the final concentration of 0.1% (w/v). It functioned as a membrane permeabilising probe. Cell death caused by sudden influx of SDS was determined by measuring the decrease of optical density (OD) various intervals of time (0, 5, 10, 30, 60 min) using a UV-Vis spectrophotometer (Shimadzu Corp., Japan) (Eumkeb et al. 2012). The experiment was performed in triplicates.

This article is protected by copyright. All rights reserved.

Accepted Article

Bacterial surface charge – zeta potential Cell surface charge was evaluated and expressed as zeta potential. The zeta potential of bacterial suspensions after contact with LV and piperacillin alone and in combination, was determined using a Nano Zetasizer (Malvern Instruments, UK). Cell suspension in phosphate buffered saline (PBS) (pH 7.4), without treatment, was used as control. The zeta potential was obtained by measuring the electrophoretic mobility of the cells. The experiment was repeated in triplicates.

Scanning electron microscopy Scanning electron microscope (SEM) observations were carried out on cells after 16 h incubation in MHB at 37°C. Cells were treated with LV and piperacillin alone and in combination for 5 min. Then, the cells were harvested and the pellet was washed with PBS (pH 7.4). The samples were then fixed with 4% glutaraldehyde and 1% osmium tetroxide. The samples were further dehydrated using sequential exposure to different concentrations of acetone ranging from 35 to 100%. After dehydration, the samples were subjected to critical point drying and finally, the samples were sputter-coated with gold, followed by SEM observations (JOEL JSM-6400, Japan).

Biosensors and Growth Conditions Biosensors used in this study are listed in Table 1. All the strains were cultured in Luria Bertani (LB) broth (1% peptone, 0.5% yeast extract, 0.5% NaCl, per 100 ml distilled water) with shaking (6 g). The strains were routinely cultured at 37 °C and supplemented with antibiotic.

Anti-QS Assay: Quantification of Light Production from E. coli [pSB401] and E. coli [pSB1075] Light production was quantified using a Tecan Microplate Reader (Infinite M200Pro, Switzerland). The HSLs used were purchased from Cayman Chemical (Ann Arbor, MI, USA).

This article is protected by copyright. All rights reserved.

Accepted Article

Stock solutions for the 3-oxo-C6-HSL, (0.5 μg ml-1) and 3-oxo-C12-HSL (10 μg ml-1) were prepared in acetonitrile and stored at −20 °C. The EO was prepared as described in the time-kill assay. Briefly, an overnight culture of E. coli biosensors cells was diluted to an OD600 nm of 0.1. Then, 200 μl of E. coli biosensors cells with essential oil were added into the well of Greiner 96well microtitre plate. For E. coli [pSB401] and E. coli [pSB1075], 2 μl of 3-oxo-C6-HSL (0.005 μg ml-1) and 2 μl of 3-oxo-C12-HSL (0.1 μg ml-1) were supplemented, respectively. The light

production and OD495 nm were determined every 30 min for 24 h by the Microplate Reader.

Production of light is given as relative light units (RLU) per unit of optical density at 495 nm, which accounted for the influence of increased growth on the total light production (Winzer et al. 2000). Reduction of total light production in E. coli [pSB401] and E. coli [pSB1075] suggested

anti-QS properties of the essential oil. Biosensor cells treated with Tween 80, which was used to enhance the oil solubility alone, were used as negative control.

Statistical analysis All results represent the average of three independent experiments. The data was presented as mean ± standard deviation (SD) and analysed by one-way analysis of variance (ANOVA) and Student’s t-test. The P < 0.05 was considered as significant, calculated using the GraphPad Prism 5 statistical software.

Results Gas Chromatography-Mass Spectrometry (GC-MS) analysis Chemical compositions study of Lavandula angustifolia oil was elucidated by GC/MS analysis and is shown on Table 2. The major constituents (>1.0 %) of the oil were linalyl anthranilate

This article is protected by copyright. All rights reserved.

Accepted Article

(38.42%), linalool (34.56%), β-caryophyllene (4.81%), isoborneol (2.57%), cis-beta-farnesene (2.11%),

trans-β-ocimene

(1.24%),

3-octanone

(1.22%),

hexyl butyrate

(1.08%)

and

caryophyllene oxide (1.04%).

Time-kill assay The time-kill curves for E. coli J53 R1 at different treatments with LV and piperacillin alone or in combination are shown in Figure 1. Time-kill analysis showed synergistic interaction between LV and piperacillin. In the time-kill assay for synergy, an interpretation of ‘synergy’ required a ≥2 log10 decrease in cfu ml-1 by the drug combination when compared with the most active single drug and a ≥2 log10 decrease in the cfu ml-1 below the starting inoculum (Lorian 2005). Synergy

was detected at 4 h whereby the number of viable cells was reduced by more than 2 log factors by the drug combination when compared with LV treatment alone. In contrast, piperacillin alone at sub-concentration did not show effected reduction in the first 2 h and there was a rebound in cell numbers at the following time points. LV alone at sub-concentration also did not show a complete killing profile within the time of study as compared to the combination of LV and piperacillin.

Outer membrane (OM) permeability SDS was used as a permeabilising probe in this experiment. Table 3 shows that piperacillin alone did not alter the OM permeability through the influx of SDS into the cell, thus the OD reading was not decreased. In contrast, LV alone and the combination of piperacillin and LV, altered the OM permeability. The differences in absorbance between pre- and post-treatment of the combinations with SDS were significant at P < 0.05 (at time starting from 5 min). The effect of

This article is protected by copyright. All rights reserved.

Accepted Article

LV alone was also significant (P < 0.05) but was less than those of piperacillin plus LV. Sublethal concentrations of piperacillin did not increase OM permeability significantly (P > 0.05). Although SDS readily dissolves the cytoplasmic membrane of Gram-negative bacteria, the treatment of E. coli with 0.1% SDS did not have any significant lytic effect as shown in the

control without pre-treatment.

Bacterial surface charge – zeta potential The surface charge of cells is frequently determined based on their zeta potential, which is calculated from the mobility of cells in the presence of an electrophorectic force under defined pH and salt concentrations. The E. coli tested had an original negative surface charge of -15mV. Generally, all the treatments reduce the negative charges on the cell surface. LV and piperacillin combination resulted in the most negatively charged bacterial surface, followed by LV alone and then piperacillin alone (Figure 2).

Scanning electron microscopy The morphological changes of E. coli J53 R1 when exposed to 0.5% (v/v) of LV, 128µg ml-1 of

piperacillin and 0.5% (v/v) of LV plus 128 µg ml-1 of piperacillin were examined. The scanning electron micrograph revealed that normal cells grown in MHB in the absence of LV and piperacillin were typical rod shape with smooth surface (Figure 3A). Wrinkled surfaces were noted on cells with post-treatment with LV alone (Figure 3B). Cells treated with LV plus piperacillin showed shrinkage of cell surfaces and several destructive openings on their cell envelopes and many cells showed irregularities from the original rod shape (Figure 3D).

This article is protected by copyright. All rights reserved.

Accepted Article

Anti-QS Assay: Quantification of Light production from E. coli [pSB401] and E. coli [pSB1075] The essential oil was further tested for possible anti-QS effects using Escherichia coli biosensors

which will show a reduction in light production in the presence of QS inhibitors. As a prerequisite, we have verified that the essential oil did not show bactericidal effect on all biosensor cells as determined by growth curve (Figure 4). In the present experimental condition, increasing concentrations of LV from 0.01%, 0.025% to 0.05% showed inhibition of light production produced by E. coli pSB1075 (Figure 5). However, LV did not inhibit light production in E. coli pSB401. Tween 80 did not show any antimicrobial effects in the performed

assays when applied at these concentrations.

Discussion A total of 33 compounds were identified and presented in Table 2. Only those present in the oils in amount higher than 0.1% are presented here. The GC-MS analysis has shown that the predominant compounds were linalyl anthranilate (38.4%) and linalool (34.6%). For comparison, previous studies which were conducted in Poland on five varieties of lavender oils showed the presence of linalool and linalyl anthranilate to be 23.9-15.8% and 12.3-1.6% respectively. The findings were lower compared to the current study (Adaszynska et al. 2012). Besides that, another study in India reported linalyl acetate (47.56 %) as the major components of the lavender oil which was absent in the present study (Verma et al. 2010).

The beta-lactam antibiotic, piperacillin is known to affect the cell membrane of bacteria by acting on different targets such as transpeptidases and transglycosylases (PBPs) (Walsh 2000). In addition, synergy is expected to be achieved by a combination of drugs because they block one

This article is protected by copyright. All rights reserved.

Accepted Article

or more different targets in the metabolic pathway and in the present study, the mechanism of action of piperacillin could be further expanded due to the combination with LV and this could play a role in potentiating the activity of the said antibiotic. Additional support for this idea collectively originates from work showing that natural product-antibiotic treatment of bacteria induces gross and irreversible bacterial membrane disruption (Hemaiswarya and Doble 2009; Cho et al. 2011). This is particularly pronounced on species producing beta-lactamases causing the antibiotic resistance.

The primary mode of action of LV is through the disruption of the bacterial membrane, thereby increasing non-specific penetration of the antibiotic into the bacteria. Effects at sub-lethal concentration of LV cannot be discounted and can be regarded as a result of the interactions with the bacterial membrane. In Gram-negative bacteria, the passage through the OM is dependent on the chemical nature of the antimicrobial product and is regulated by the presence of hydrophilic porins. Hydrophobic substances are often being excluded outside the cells but it is possible to weaken the OM by molecules that disintegrate the lipopolysaccharide (LPS) layer, generally known as membrane permeabilizers (Vaara 1992; Borges et al. 2013). The combination of LV plus piperacillin significantly altered the OM permeabilization of E. coli compared to the control. This finding is quite similar to previous report that flavonoids isolated from smaller galangal and amoxicillin combinations altered the outer membrane of amoxicillin-resistant E. coli whereby these results may be explained by assuming that the OM barrier is disturbed by LV thus potentiates the antibiotic to be more potent to bind and neutralize LPS (Eumkeb et al. 2011). Sublethal injury of bacterial cell membranes may disrupt their permeability and affect the membrane’s ability to osmoregulate the cell adequately (Gilbert et al. 1991). It is important to

This article is protected by copyright. All rights reserved.

Accepted Article

understand that a disturbed cell membrane system may affect other cellular structures in a cascade type of action such as the lysis of bacterial cell wall, followed by the loss of intracellular dense material (Carson et al. 2002). The hypothesis is further confirmed by the scanning electron micrograph revealing that the overall bacterial cell surface treated with LV is structurally disparate from the control. Such morphological alterations mainly occurred due to the disruption

of membrane structure as evident by the previous findings (Souren et al. 2011; Bajpai et al. 2013; Sharma et al. 2013). In line with the SEM to evaluate the structural alterations, Di Pasqua et al. (2006, 2007) described a strong decrease of the membrane unsaturated fatty acids for the bacteria treated with thymol, carvacrol, limonene, eugenol and cinnamaldehyde (Di Pasqua et al. 2006; Di Pasqua et al. 2007). This result supports the mechanism of action these compounds interacting with the membrane lipid profile and causing the outer cell membrane disruptions appreciable by SEM examination. A study carried out using atomic force miscroscopy (AFM) showed the EO component, carvacol-treated E. coli were completely collapsed onto the slide surface via the 3D rendering of cell surface. Other morphology changes after treatment include presence of vesicles, length reduction and increased roughness (La Storia et al. 2011). Treatment-induced gross cell damage may explain the rapid action of the combination pair observed in the time-kill analysis. The piperacillin-LV treated E. coli underwent a rapid decrease in cell number within the first 1 h and followed by complete elimination at 4 h. Linalyl anthranilate, being the most abundant compound (38.42%) detected, is the ethyl linalool. Generally, limited studies have been performed specifically on isolated linalyl anthranilate compared to linalool. The lack of research effort on this part could be due to linalool being often

found as the most abundant compound present instead of linalyl anthranilate (Adaszynska et al. 2012). Linalool (1,6-octadien-3-ol, 3,7-dimethyl-), a terpenic alcohol, being one of the main

This article is protected by copyright. All rights reserved.

Accepted Article

components of L. angustifolia essential oil (34.6%), was previously reported to cause increased permeability not only to the negatively charged membranes but also to fungal cells (Alviano et al. 2005; Silva et al. 2011). Due to the nature of the chemical structure, the alcohols possess a strong binding affinity to different molecular structures such as proteins or glycoproteins. Hence, they have great affinities for cell membranes and exhibit high potential to permeate through cell walls, leading to the leakage of cytoplasmic material (Hemaiswarya and Doble 2009; Wang et al. 2012). A report studying green huajiao (Zanthoxylum schinifolium) also suggests that the membrane permeabilizing property leading to antibacterial effect is most likely contributed by the effects of linalool (Diao et al. 2013). Additionally, in the work of Dorman and Deans (2000), linalool was found to inhibit the growth of an array of Gram positive and Gram negative bacteria (Dorman and Deans 2000). Linaool when treated with strong acids or strong catalytic agents, cause complex transformations to take place yielding a range of hydrocarbons (Bedoukian 1951). Metabolism of bacteria often yield acids and alcohols as waste which may potentiate to the

production of hydrocarbons from linaool and contribute to the EO membrane disrupting property. The activity of terpenes, for example, linalool, is concentration dependent. The probable reason in this phenomenon is due to the limited terpene solubility in the vehicle environment and this results in terpene saturation at the site of the action (Cal et al. 2001). Presence of hydrocarbons such as terpenes and aromatics in the EOs play an important role in membrane toxicity as documented in the previous study (Sikkema et al. 1995).

The zeta potential is related to the membrane potential and reflects the metabolic state of the bacteria whereby the values became more negative at higher rates of growth (van der Mei et al. 1993; Tymczyszyn et al. 2007). This method may be exploited to further reveal the membrane

This article is protected by copyright. All rights reserved.

Accepted Article

effects of LV. Bacterial cell surfaces are normally negatively charged under physiological conditions, contributed by the presence of anionic groups such as carboxyl and phosphate in their membranes. However, the magnitude of the charge varies between species and is possibly subject to influence by various culture conditions such as pH and ionic strength (Gilbert et al. 1991; Palmer et al. 2007). In this experiment, it is demonstrated that after LV exposure, the cells became less negatively charged. It is also assumed that accumulation of the essential oil components in the bacterial membrane causes acidifying and protein denaturation thus resulting in the irreversible membrane damage (Borges et al. 2013).

The current study also indicates the anti-quorum sensing ability of LV where it showed promising inhibitory properties for long chain AHL quorum sensing (QS) system. QS is a mechanism that regulates bacterial behaviors such as virulence factors and biofilm using chemical signals. Therefore, anti-QS is a promising field to combat bacterial pathogenicity without posing any selection pressure for the development of resistance (Ryan and Dow 2008). In this study, when the concentration of the oil increased, the inhibitory effects grew significantly stronger in Escherichia coli [pSB1075] but no effect was found in Escherichia coli [pSB401]. Thus, inhibition of Escherichia coli [pSB1075] which carries the lasR receptor gene suggests the presence of anti-QS activity. As the GC/MS analysis showed the presence of linalyl anthranilate (38.42%) and linalool (34.56%) as the major compounds, further experiment is required to determine the possibility of either one or both compounds possessing anti-QS activity. The results of this study indicate that the L. angustifolia essential oil is able to disrupt membrane structures of Gram-negative bacteria. We conclude that the LV exerts its inhibitory effect through

the

permeabilization

of

the

cell

membrane

This article is protected by copyright. All rights reserved.

associated

with

generalized

Accepted Article

membrane disrupting effects. LV has the potential to inhibit las-mediated QS and could play a role in controlling pathogens that use this type of signaling system. Alternatively, it could potentially affect E. coli via the SdiA signal receptor, although additional research is necessary to test this hypothesis (Smith et al. 2011). Given the heterogeneous composition of LV, it seems unlikely that there is only one mode of antimicrobial action. Further research is required to understand fully the mechanisms involved in order to justify the practical applications and possibility of LV as a treatment strategy alongside with the current antibiotics.

Acknowledgement This study was funded by the Fundamental Research Grant Scheme (FRGS) from the Ministry of Higher Education (MOHE), Malaysia under the grant number FRGS/1/2011/SKK/IMU/03/3. The author (Kok-Gan Chan) gratefully acknowledges the University of Malaya for the High Impact Research Grant (HIR/MOHE Grant: A000001-50001). The bacterial strains used in this study were a kind gift from Dr George A. Jacoby.

Conflict of Interests The authors declare that no conflict of interest exists.

References Adaszynska, M., Swarcewicz, M., Dzieciol, M. & Dobrowolska, A. (2012) Comparison of chemical composition and antibacterial activity of lavender varieties from Poland. Nat Prod Res. Alvarez, M., Tran, J. H., Chow, N. & Jacoby, G. A. (2004) Epidemiology of conjugative plasmid-mediated AmpC beta-lactamases in the United States. Antimicrob Agents Chemother 48, 533-7. Alviano, W. S., Mendonca-Filho, R. R., Alviano, D. S., Bizzo, H. R., Souto-Padron, T.,

This article is protected by copyright. All rights reserved.

Accepted Article

Rodrigues, M. L., Bolognese, A. M., Alviano, C. S. & Souza, M. M. (2005) Antimicrobial activity of Croton cajucara Benth linalool-rich essential oil on artificial biofilms and planktonic microorganisms. Oral Microbiol Immunol 20, 101-5. Bajpai, V. K., Sharma, A. & Baek, K., H. (2013) Antibacterial mode of action of Cudrania tricuspidata fruit essential oil, affecting membrane permeability and surface characteristics of food-borne pathogens. Food Control 32, 582-590. Bedoukian, P. Z. 1951. Perfumery synthetics and isolates, New York,, Van Nostrand. Borges, A., Ferreira, C., Saavedra, M. J. & Simoes, M. (2013) Antibacterial Activity and Mode of Action of Ferulic and Gallic Acids Against Pathogenic Bacteria. Microb Drug Resist. Cal, K., Janicki, S. & Sznitowska, M. (2001) In vitro studies on penetration of terpenes from matrix-type transdermal systems through human skin. Int J Pharm 224, 81-8.

Carson, C. F., Mee, B. J. & Riley, T. V. (2002) Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined by time-kill, lysis, leakage, and salt tolerance assays and electron microscopy. Antimicrob Agents Chemother 46, 1914-20. Cho, Y. S., Oh, J. J. & Oh, K. H. (2011) Synergistic anti-bacterial and proteomic effects of epigallocatechin gallate on clinical isolates of imipenem-resistant Klebsiella pneumoniae. Phytomedicine 18, 941-6.

D'auria, F. D., Tecca, M., Strippoli, V., Salvatore, G., Battinelli, L. & Mazzanti, G. (2005) Antifungal activity of Lavandula angustifolia essential oil against Candida albicans yeast and mycelial form. Med Mycol 43, 391-6. Di Pasqua, R., Betts, G., Hoskins, N., Edwards, M., Ercolini, D. & Mauriello, G. (2007) Membrane toxicity of antimicrobial compounds from essential oils. J Agric Food Chem 55, 4863-70. Di Pasqua, R., Hoskins, N., Betts, G. & Mauriello, G. (2006) Changes in membrane fatty acids composition of microbial cells induced by addiction of thymol, carvacrol, limonene, cinnamaldehyde, and eugenol in the growing media. J Agric Food Chem 54, 2745-9.

Diao, W. R., Hu, Q. P., Feng, S. S., Li, W. Q. & Xu, J. G. (2013) Chemical Composition and Antibacterial Activity of the Essential Oil from Green Huajiao ( Zanthoxylum schinifolium ) against Selected Foodborne Pathogens. J Agric Food Chem.

Dorman, H. J. & Deans, S. G. (2000) Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J Appl Microbiol 88, 308-16.

This article is protected by copyright. All rights reserved.

Accepted Article

Eumkeb, G., Siriwong, S., Phitaktim, S., Rojtinnakorn, N. & Sakdarat, S. (2011) Synergistic activity and mode of action of flavonoids isolated from smaller galangal and amoxicillin combinations against amoxicillin-resistant Escherichia coli. J Appl Microbiol 112, 55-64. Evandri, M. G., Battinelli, L., Daniele, C., Mastrangelo, S., Bolle, P. & Mazzanti, G. (2005) The antimutagenic activity of Lavandula angustifolia (lavender) essential oil in the bacterial reverse mutation assay. Food Chem Toxicol 43, 1381-7. Fabio, A., Cermelli, C., Fabio, G., Nicoletti, P. & Quaglio, P. (2007) Screening of the antibacterial effects of a variety of essential oils on microorganisms responsible for respiratory infections. Phytother Res 21, 374-7. Gilbert, P., Evans, D. J., Evans, E., Duguid, I. G. & Brown, M. R. (1991) Surface characteristics and adhesion of Escherichia coli and Staphylococcus epidermidis. J Appl Bacteriol 71, 72-7. Hajhashemi, V., Ghannadi, A. & Sharif, B. (2003) Anti-inflammatory and analgesic properties of the leaf extracts and essential oil of Lavandula angustifolia Mill. J Ethnopharmacol 89, 67-71. Hemaiswarya, S. & Doble, M. (2009) Synergistic interaction of eugenol with antibiotics against Gram negative bacteria. Phytomedicine 16, 997-1005.

La Storia, A., Ercolini, D., Marinello, F., Di Pasqua, R., Villani, F. & Mauriello, G. (2011) Atomic force microscopy analysis shows surface structure changes in carvacrol-treated bacterial cells. Res Microbiol 162, 164-72. Lorian, V. (ed.) 2005. Antibiotics in Laboratory Medicine. Marri, L., Dallai, R. & Marchini, D. (1996) The novel antibacterial peptide ceratotoxin A alters permeability of the inner and outer membrane of Escherichia coli K-12. Curr Microbiol 33, 40-3. Palmer, J., Flint, S. & Brooks, J. (2007) Bacterial cell attachment, the beginning of a biofilm. J Ind Microbiol Biotechnol 34, 577-88. Ryan, R. P. & Dow, J. M. (2008) Diffusible signals and interspecies communication in bacteria. Microbiology 154, 1845-58. Sharma, A., Bajpai, V. K. & Baek, K., H. (2013) Determination of Antibacterial Mode of Action of Allium sativum Essential Oil against Foodborne Pathogens Using Membrane Permeability and Surface Characteristic Parameters. Journal of Food Safety 33, 197-208. Sikkema, J., De Bont, J. A. & Poolman, B. (1995) Mechanisms of membrane toxicity of hydrocarbons. Microbiol Rev 59, 201-22.

Silva, F., Ferreira, S., Queiroz, J. A. & Domingues, F. C. (2011) Coriander (Coriandrum sativum L.) essential oil: its antibacterial activity and mode of action evaluated by flow cytometry. J Med Microbiol 60, 1479-86.

This article is protected by copyright. All rights reserved.

Accepted Article

Smith, J. L., Fratamico, P. M. & Yan, X. (2011) Eavesdropping by bacteria: the role of SdiA in Escherichia coli and Salmonella enterica serovar Typhimurium quorum sensing. Foodborne Pathog Dis 8, 169-178. Souren, P., Dubey, R. C., Maheswari, D. K. & Sc, K. (2011) Trachyspermum ammi (L.) fruit essential oil influencing on membrane permeability and surface characteristics in inhibiting foodborne pathogens. Food Control 22, 725-731. Tymczyszyn, E. E., Del Rosario Diaz, M., Gomez-Zavaglia, A. & Disalvo, E. A. (2007) Volume recovery, surface properties and membrane integrity of Lactobacillus delbrueckii subsp. bulgaricus dehydrated in the presence of trehalose or sucrose. J Appl Microbiol 103, 2410-9. Vaara, M. (1992) Agents that increase the permeability of the outer membrane. Microbiol Rev 56, 395-411. Van Der Mei, H. C., De Vries, J. & Busscher, H. J. (1993) Hydrophobic and electrostatic cell surface properties of thermophilic dairy streptococci. Appl Environ Microbiol 59, 4305-12. Verma, R. S., Rahman, L. U., Chanotiya, C. S., Verma, R. K., Chauhan, A., Yadav, A., Singh, A. & Yadav, A. K. (2010) Essential oil composition of Lavandula angustifolia Mill. cultivated in the mid hills of Uttarakhand, India. Journal of the Serbian Chemical Society 75, 343.

Walsh, C. (2000) Molecular Mechanisms that Confer Antibacterial Drug Resistance. Nature 406, 775-81. Wang, Y. W., Zeng, W. C., Xu, P. Y., Lan, Y. J., Zhu, R. X., Zhong, K., Huang, Y. N. & Gao, H. (2012) Chemical composition and antimicrobial activity of the essential oil of Kumquat (Fortunella crassifolia Swingle) Peel. Int. J. Mol. Sci. 13, 3382-3393. Winson, M. K., Swift, S., Fish, L., Throup, J. P., Jorgensen, F., Chhabra, S. R., Bycroft, B. W., Williams, P. & Stewart, G. S. (1998) Construction and analysis of luxCDABE-based plasmid sensors for investigating N-acyl homoserine lactone-mediated quorum sensing. FEMS Microbiol Lett 163, 185-92. Winzer, K., Falconer, C., Garber, N. C., Diggle, S. P., Camara, M. & Williams, P. (2000) The Pseudomonas aeruginosa lectins PA-IL and PA-IIL are controlled by quorum sensing and by RpoS. J Bacteriol 182, 6401-11.

Yap, P. S. X., Lim, S. H. E., Hu, C. P. & Yiap, B. C. (2013) Combination of essential oils and antibiotics reduce antibiotic resistance in plasmid-conferred multidrug resistant bacteria. Phytomedicine 20, 710-3.

This article is protected by copyright. All rights reserved.

Accepted Article

Tables Table 1 List of strains/plasmid used. Biosensors Description Escherichia coli luxR luxl’ (Photobacterium fischeri [ATCC 7744])::luxCDABE [pSB401] (Photorhabdus luminescens [ATCC 29999]) fusion; pACYC184derived, TetR, AHL biosensor producing bioluminescence in respond to short chain AHL Escherichia coli lasR lasl’ (P. aeruginosa PAO1)::luxCDABE (P. luminescens [pSB1075] [ATCC 29999]) fusion in pUC18 AmpR, AHL biosensor producing bioluminescence in respond to long chain AHL

Table 2 Chemical composition of Lavandula angustifolia essential oil. Peak

Library/ID

RT

Area %

CAS No.

1

Camphene

7.2971

0.2998

000079-92-5

2

1-Octen-3-ol

8.4443

0.393

003391-86-4

3

3-Octanone

8.7357

1.2237

000106-68-3

4

β-Pinene

8.8956

0.2884

000127-91-3

5

Hexyl acetate

9.7795

0.6383

000142-92-7

6

0-Cymene

10.165

0.2303

000527-84-4

7

L-Limonene

10.3155

0.135

005989-54-8

8

trans-β-Ocimene

10.7292

1.2428

003779-61-1

9

cis-β Ocimene

11.1241

0.4847

003338-55-4

10

cis-Linalool oxide

12.0644

0.8985

005989-33-3

11

trans-Linalool oxide

12.6944

0.6785

023007-29-6

12

Linalool

13.5313 34.5583

000078-70-6

13

1-Octen-3-yl-acetate

13.7476

0.7203

002442-10-6

14

Camphor

14.8383

0.9713

000076-22-2

This article is protected by copyright. All rights reserved.

Source (Winson et al. 1998) (Winson et al. 1998)

Isobutyric acid

15.0452

0.1505

002349-07-7

16

Isoborneol

15.6752

2.5706

010385-78-1

17

1-Terpinen-4-ol

16.0607

0.3243

000562-74-3

18

p-Cymenol

16.3146

0.3176

001197-01-9

19

α-Terpineol

16.5779

0.4131

000098-55-5

20

Hexyl butyrate

16.6719

1.0839

002639-63-6

21

Bornyl formate

17.9037

0.121

007492-41-3

22

Linalyl anthranilate

19.2672 38.4164

007149-26-0

23

Borneol

20.0288

0.1494

005655-61-8

24

Lavandulol, acetate

20.198

0.5925

1000352-62-6

25

Linalyl isobutyrate

22.2291

0.1188

000078-35-3

26

Neryl acetate

22.6334

0.2544

000141-12-8

27

Nerol

23.2823

0.547

000106-25-2

28

Hexyl hexanoate

23.3575

0.192

006378-65-0

29

β-caryophyllene

24.467

4.8089

000087-44-5

30

α-Bergamotene

24.9184

0.4025

017699-05-7

31

α-Caryophyllene

25.492

0.121

006753-98-6

32

cis-β-Farnesene

25.6142

2.1131

028973-97-9

33

Caryophyllene oxide

29.413

1.0366

001139-30-6

Accepted Article

15

*Library/ID – Identification of the compounds based on National Institute of Standards and Technology (NIST) library RT – retention time of the peak Area% - % of peak area

This article is protected by copyright. All rights reserved.

Accepted Article

Table 3 Permeabilization of E. coli J53 R1 by LV and/or piperacillin.

Treatment Time (min) Immediately

5

OD625 ± SD (n=3) 10

30

60

0.32±0.003 0.31±0.011

0.32±0.002 0.32±0.003

Control with 0.1% SDS without 0.1% SDS

0.31±0.002 0.31±0.001

0.31±0.002 0.31±0.006

0.31±0.004 0.31±0.003

LV (0.5 % v/v) with 0.1% SDS 0.28±0.002a without 0.1% SDS 0.28±0.002a

0.27±0.001b 0.23±0.012b 0.22±0.002b 0.22±0.015b 0.28±0.003b 0.28±0.001b 0.29±0.003b 0.29±0.006b

Piperacillin (128 µg ml-1) with 0.1% SDS without 0.1% SDS

0.30±0.002 0.30±0.001

0.29±0.005 0.30±0.001

0.31±0.009 0.31±0.005

0.31±0.002 0.31±0.006

0.31±0.002 0.31±0.005

LV (0.5 % v/v) + Piperacillin (128 µg ml-1) with 0.1% SDS 0.28±0.002a 0.24±0.002b 0.23±0.003b 0.22±0.012b 0.20±0.001b without 0.1% SDS 0.27±0.003a 0.28±0.002b 0.28±0.003b 0.28±0.007b 0.29±0.003b

a

Significant difference when compared to the corresponding control (with or without 0.1% SDS). (P < 0.05) b Significant difference between treated and non-treated with 0.1% SDS groups at the corresponding time points. (P < 0.05) Values are mean OD625 ± S.D of three replicates.

This article is protected by copyright. All rights reserved.

Accepted Article

Figures

Figure 1 Time-kill curves for lavender oil (LV) and piperacillin alone and in combination against E. coli J53 R1; symbol represents: (♦) control without treatment; (●) LV (0.5% v/v); (x) piperacillin (128 µg ml-1); (▲) LV (0.5% v/v) and piperacillin (128 µg ml-1). The bars represent the SD of three replicates.

This article is protected by copyright. All rights reserved.

Accepted Article

***

***

***

Figure 2 Zeta potential values (mV) of suspensions of E. coli when exposed to different treatments. Fill represents: (…) control; (6) LV (0.5% v/v); (‰) piperacillin (128 µg ml-1); („) LV (0.5% v/v) and piperacillin (128 µg ml-1). The mean ± SD for three replicates are illustrated. Data were analysed by one-way analysis of variance with *** P < 0.05 being significant.

This article is protected by copyright. All rights reserved.

Accepted Article

Figure 3 Scanning electron micrographs of E. coli J53 R1. (A) Untreated cells, (B) cells treated with Lavender (0.5 % v/v), (C) cells treated with piperacillin (128 µg ml-1) and (D) cells treated with Lavender (0.5 % v/v) + piperacillin (128 µg ml-1) for 5 min.

This article is protected by copyright. All rights reserved.

0.5

pSB401 T80 0.05% LV 0.01% LV 0.025% LV 0.05%

OD600

0.4

0.3

0

5

10 15 Time [h]

20

B 0.8

pSB1075 T80 0.05% LV 0.01% LV 0.025% LV 0.05%

0.6 OD600

Accepted Article

A

0.4

0.2 0

5

10 15 Time [h]

20

Figure 4 Growth effect of LV at various concentrations on A) E. coli pSB401 and B) E. coli pSB1075. Tween 80 was used as negative control.

This article is protected by copyright. All rights reserved.

RLU/OD495

3.0×10 6

pSB401 T80 0.05% LV 0.01% LV 0.025% LV 0.05%

2.0×10 6

1.0×10 6

0 0

5

10

15

20

Time [h]

E.coli pSB1075 LV Oil 1.5×10 4

RLU/OD495

Accepted Article

E.coli pSB401 LV Oil

pSB1075 T80 0.05% LV 0.01% *** LV 0.025% *** LV 0.05% ***

1.0×10 4

5.0×10 3

0 0

5

10

15

20

25

Time [h]

Figure 5 Light production of E. coli [pSB401] and E. coli [pSB1075] by LV with increasing concentration from 0.01% (U), 0.025% (x) to 0.05% (●) while Tween80 (◊) and E. coli [pSB401/1075] supplemented with 3-oxo-C6-HSL/ 3-oxo-C12-HSL, respectively served as control (▼) was included. The data were presented as RLU OD-1 to account for any differences in growth. Data were analysed by one-way analysis of variance with *** P < 0.05 being significant.

This article is protected by copyright. All rights reserved.

Membrane disruption and anti-quorum sensing effects of synergistic interaction between Lavandula angustifolia (lavender oil) in combination with antibiotic against plasmid-conferred multi-drug-resistant Escherichia coli.

The aim of this study was to investigate the mode of action of the lavender essential oil (LV) on antimicrobial activity against multi-drug-resistant ...
1MB Sizes 0 Downloads 3 Views