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Article Type: Original Article
Synergy between thymol, eugenol, berberine, cinnamaldehyde and streptomycin against planktonic and biofilm-associated food-borne pathogens
Qianjin Liu, Hong Niu, Wuxia Zhang, Haibo Mu, Chunli Sun, Jinyou Duan*
College of science, Northwest A&F University, Yangling 712100, Shaanxi, China
Correspondence: Jinyou Duan, Ph.D Tel.: +86 29 87092226; E-mail: [email protected]
Significance and Impact of the Study: This study has shown the synergistic effect of four components of essential oil (thymol, eugenol, berberine and cinnamaldehyde) combined with streptomycin on planktonic and biofilm-associated
foodborne
pathogens
Listeria
monocytogenes
and
Salmonella
Typhimurium. These findings indicate that combination of specific components of essential oils with streptomycin may provide alternative methods to overcome the problem of 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/lam.12401 This article is protected by copyright. All rights reserved.
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food-borne bacteria both in suspension and in biofilm.
Abstract Essential oils have been found to exert antibacterial, antifungal, spasmolytic, and antiplasmodial activity and therapeutic effect in cancer treatment. In this study, the antibacterial activities of four main essential oils’ components (thymol (Thy), eugenol (Eug), berberine (Ber), cinnamaldehyde (Cin)) were evaluated against two foodborne pathogens, Listeria monocytogenes and Salmonella Typhimurium, either alone or in combination with streptomycin. Chequerboard assay demonstrated that Thy and Cin elicited a synergistic effect with streptomycin against L. monocytogenes, while a synergy existed between Cin or Eug and streptomycin against S. Typhimurium. Further experiments showed that this synergy was sufficient to eradicate biofilms formed by these two bacteria. Thus, our data highlighted that the combinations of specific components from essential oils and streptomycin were useful for the treatment of foodborne pathogens, which might be help prevent the spread of antibiotic resistance through improving antibiotic effectiveness.
Keywords: thymol; eugenol; berberine; cinnamaldehyde; streptomycin; synergistic; bacterial biofilms
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Introduction When bacteria attach to biotic or inert surfaces, they can form biofilm which is considered as a universal survival lifestyle for microbes to protect themselves from antimicrobial attack. Susceptibility tests have demonstrated biofilm bacteria to survive after treatment with antibiotics at hundreds or even thousand times of the minimum inhibitory concentration against planktonic cells (Costerton et al. 1999; Costerton 1999). The failure in the prevention and eradication of microbial biofilms might create a number of serious problems in industrial fluid processing operations (bio-deterioration), food safety (contamination), and public health issues (infectious diseases) (Mittelman 1998; Flemming and Wingender 2010; Van Houdt and Michiels 2010). L. monocytogenes is the causal organism of the serious food-borne illness listeriosis, and may grow as biofilms on food and food-processing equipments that protect it against environmental stress (Møretrø and Langsrud 2004). S. Typhimurium as a typical foodborne pathogen which can form biofilms on food processing facility surfaces (Chae and Schraft 2000; Prouty and Gunn 2003; Ryu and Beuchat 2005). According to the scientific report of European Food Safety Authority (EFSA), Salmonella was identified as the leading causative agent and a total of 108,614 confirmed cases of human salmonellosis including 90 deaths were reported in 2009 (Authority 2011). Essential oils are a rich source of biologically active compounds, which are aromatic and volatile oily liquids obtained from plant materials of different plant parts (flowers, buds, seeds, leaves, twigs, bark, herbs, wood, fruits, roots) (Oussalah et al. 2007). Essential oils are complex mixtures comprising many compounds which are chemically derived from terpenes
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and their oxygenated compounds (Oussalah et al. 2007). Thy is a monocyclic monoterpene compound isolated from Thymus vulgaris, Monarda punctuate or Origanum vulgare spp (Wei et al. 2014). Eug (4-allyl-2-methoxyphenol) is an aromatic molecule found in essential oils and various plants, including cloves, bay leaves and cinnamon leaves (Xu et al. 2013). Ber is an isoquinoline derivative alkaloid that can be isolated from many medicinal herbs including Papaveraceae, Berberidaceae, Fumariaceae, Menispermaceae, Ranunculaceae, Rutaceae, and Annonaceae (Grycová et al. 2007; Chen et al. 2012). Cin is an aromatic α, β-unsatured aldehyde, and the major component in essential oils from some cinnamon species (Singh et al. 2007; Wang et al. 2009). The antimicrobial properties of essential oils have been known for many centuries and, up to now, a large number of essential oils and their constituents have been investigated for their antimicrobial properties against some bacteria and fungi (Smith-Palmer et al. 1998; Hammer et al. 1999; Dorman and Deans 2000; Elgayyar et al. 2001). One of the outstanding antimicrobial properties of many essential oils is that they can be effective even against microbial biofilms. The essential oils from E. camaldulensis and M. spicata significantly retard bacterial biofilm formation by Streptococcus mutans and Streptococcus pyogenes (Rasooli et al. 2009). Oils of Cymbopogon citratus and Syzygium aromaticum have been shown to elicit a promising anti-biofilm activity against Candida albicans (de Andrade et al. 2013). Some studies have explored the combination of synthetic drugs with essential oils with a aim to evaluate and enhance antimicrobial efficacy (Rosato et al. 2007). For example, a synergistic effect was observed between Norfloxacin and essential oil (and its main
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components) from Pelargonium graveolens (Rosato et al. 2008), between gentamicin and the essential oil from Croton zehntneri (Santos et al. 2011), as well as between eugenol (the main component of cloveoilfrom Eugenia aromatica) with antibiotics (Hemaiswarya and Doble 2009). To our knowledge, the anti-biofilm capacity of essential oils, especially of their particular constituents in combination with antibiotics against microbial biofilms remains largely unknown. In the current study, the anti-biofilm activity of four main essential oils’ components (Thy, Eug, Ber, Cin) in combination with the amino glycoside antibiotic streptomycin was explored.
Results and discussion
Antimicrobial activity of essential oils’ components and streptomycin against L. monocytogenes The MIC of streptomycin and four main essential oils’ components (Thy, Eug, Ber, Cin) obtained by the broth dilution method are shown in Table 1. Streptomycin was active against L. monocytogenes at the concentration of 8 µg ml-1. This finding is similar to the MIC obtained by other authors. Zhang et al (2013) reported that the
streptomycin against L.
monocytogenes with the MIC values 8 µg ml-1. Regarding the MIC values for the four essential oils’ components (Thy, Eug, Ber, Cin), it can be seen that L. monocytogenes was more sensitive to Cin (512 µg ml-1). These results are in agreement with those obtained by Babu et al (2011) which observed a high sensitivity of L. monocytogenes to Cin essential oil. The strong microbicidal activity of Cin might be ascribed to the high electrophilic properties
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of the carbonyl group adjacent to the double bound that make this compound particularly reactive with nucleophiles, such as protein sulfhydryl and amino groups of the microorganism (Neri et al. 2006). Eug and Thy had the same antimicrobial activity against L. monocytogenes with both presenting MIC of 1024 µg ml-1. Ber was active only at very high concentration against L. monocytogenes (8192 µg ml-1), it was same as previously reported that Ber was ineffective as an antibacterial because it was readily extruded by pathogen (Hsieh et al. 1998). Synergistic effects resulting from the combination of antibiotics with different plant extracts have been studied by a number of researchers (Betoni et al. 2006; Esimone et al. 2006; Hemaiswarya et al. 2008; Klein et al. 2013), but little attention has been paid previously to the synergistic effects of an individual natural essentials’ components (Thy, Eug, Ber, Cin) and streptomycin. In this study, the effect of four specific essential oils’ components (Thy, Eug, Ber, Cin) in combination with streptomycin on the L. monocytogenes was detected by chequerboard assay. The results are presented in Table 2. Both Thy and Cin were found to present synergistic effect with streptomycin against L. monocytogenes (Fractional Inhibitory Concentration, FIC index < 0.5). While the remaining combinations between Eug or Ber and streptomycin showed additive effect in the chequerboard assay (FIC index 0.5 – 1.0). Previous studies have also reported antibiofilm activity of essential oil and their components and their synergy with antibiotics against bacterial biofilms built by Pseudomonas putida and E. coli (Kavanaugh and Ribbeck 2012; Khan and Ahmad 2012; Kerekes et al. 2013). Koraichi Saad et al (2011) observed that Thy could inhibit Pseudomonas aeruginosa adherence and biofilmformation. Amalaradjou et al (2011) reported
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that Cin effectively prevented uropathogenic E.coli biofilm on plates and catheters. However, there is very limited information about the effect of these synergistic combinations streptomycin and Cin, streptomycin and Thy on L. monocytogenes in biofilm cultures. This rendered us to ask whether these synergistic combinations were also effective in breaking down biofilms formed by L. monocytogenes. As expected, the synergistic combinations between Cin or Thy with streptomycin had stronger anti-biofilm activities (Fig 1A) than the individual components. These synergistic combinations could also cause a reduction of live bacteria, compared with that of the control (Fig 1B). Also, images from fluorescence microscopy (Fig 1C) evidenced that the architecture of L. monocytogenes biofilms exposed to the synergistic combinations displayed very few scattered cell aggregates, in which there were much less viable cells than exposed to the individual components, these results may be regarded as the effect of essential oils’ components. Previous studies have indicated that essential oil could inhibit quorum sensing (QS) system and influence the expressions of intercellular adhesion gene and biofilm-related gene in biofilm (Zhou et al. 2013). So when antibiotics treat bacteria biofilms combined with essential oil, essential oil may damage biofilms and make antibiotics easily access to biofilm bacteria.
Antimicrobial activity essential oils’ components and streptomycin against S. Typhimurium The antimicrobial activity of the tested essential oils and streptomycin against S. Typhimurium is presented in Table 1. Thy was more active against the S. Typhimurium bacterium showing a MIC value =256 µg ml-1 compared to its activity against L.
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monocytogenes, the same was also evident for Ber. These observations are in agreement with previous study which show S. Typhimurium is more sensitive to Thy than L. monocytogenes (Cosentino et al. 1999). However, Eug and Cin appeared more active against L. monocytogenes. These observations are consistent with previous observations. Mith et al (2014) observed that Cin was more effective against L. monocytogenes than S. Typhimurium. Generally, Gram-positive bacteria are more sensitive to essential oils than Gram-negative bacteria (Smith-Palmer et al. 1998; Lopez et al. 2005). However, two essential oils’ components in this study did not completely follow the trend described above. This suggests that there might be some particular anti-Gram-negative substances in these two components. The results of chequerboard assay are presented in Table 2. Cin with streptomycin were found to have the similar synergistic effect against both S. Typhimurium and L. monocytogenes However, eugenol showed synergistic action with streptomycin against S. Typhimurium but not against L. monocytogenes. These observation are consistent with the descriptions in the literature. Eug showed potent synergistic activity with antibiotics against Gram negative bacteria. There was nearly a 5–1000 fold decrease in the MIC of the antibiotics tested (Gallucci et al. 2006). The remaining combinations streptomycin and Thy, streptomycin and Ber showed additive effect in the chequerboard assay. The synergistic combinations also demonstrated higher anti-biofilm activity towards S. Typhimurium (Fig 2). Obviously, the synergistic combinations streptomycin and Cin, streptomycin and Eug had a pronounced effect in the decrease of biofilm mass (Fig 2A) and live bacteria (Fig 2B) than streptomycin, Cin or Eug treatment alone. These findings were also evidenced by the visualization of biofilms by scanning electron microscopy (Fig 2C), in which the architecture
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of S. Typhimurium biofilms exposed to these synergistic combinations for 24 h displayed fewer scattered cell aggregates when compared to that of individual reagents. The tested four essential oils’ components synergisms with antibiotics have been presented in previous literatures. Palaniappan and Holley (2010) observed that treatment with low concentrations of carvacrol and Thy enhanced bacterial susceptibility to ampicillin, penicillin, tetracycline and also demonstrated the synergy between the Cin and ampicillin. The combination of Ber with oxacillin produced a large synergistic effect against methicillin-resistant Staphylococcus aureus (Yu et al. 2005). A notable study by Gallucci et al (2006) described synergistic in vitro interactions between Eug and penicillin against E. coli and methicillin-resistant Staphylococcus aureus. To our knowledge, this is the first report of synergism between streptomycin and Cin or Eug on S. Typhimurium biofilm.
Essential oils facilitated antibiotic access to biofilm bacteria There are many factors accounting for the resistance of biofilm bacteria to antibiotics (Davies 2003). One factor that is generally perceived to play a role in antibiotic resistance is the inability of the antibiotic to penetrate into biofilms, thereby reducing antibiotic available to interact with biofilm bacteria. Given that essential oils can penetrate and damage biofilms (Nuryastuti et al. 2009). It rendered us to see whether essential oils could facilitate streptomycin entry into biofilms. The results of immunofluorescence analysis are shown in Fig 3. L. monocytogenes or S. Typhimurium exposed to streptomycin alone exhibited a weak green fluorescence. In contrast, green fluorescence was observed in biofilms formed by both these bacteria after treatment
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with the combined of components of essential oils with streptomycin (Thy and streptomycin, Cin and streptomycin, Eug and streptomycin). These findings clearly show that essential oils facilitate streptomycin access into the biofilms formed by these two bacteria. These observations are in agreement with previous study. Jabra-Rizk et al (2006) tested the ability of farnesol to act in synergy with gentamicin by facilitating gentamicin entry into staphylococcal biofilms.
Materials and methods
Bacterial strains L. monocytogenes (CMCC 54004) and S. Typhimurium (SL1344) were generous gifts received from Prof. Xiaodong Xia (College of Food Science and Engineering, Northwest A&F University). The strains were cultivated in tryptic soy broth (TSB, Oxoid, Basingstoke, Hampshire, UK) at 37 °C for 12 h. Following incubation, dilution of the overnight culture with TSB matched to the 0.4 McFarland turbidity standard (approximately 108 cfu ml-1) was used for experimental procedures.
Antimicrobial compounds Four components from natural essential oils (thymol (Thy), eugenol (Eug), berberine (Ber), cinnamaldehyde (Cin)) were purchased from Ziyi reagent (Shanghai, China). Stock solutions of essential oils at concentration of 100 mg ml-1 were prepared in dimethylsulfoxide (DMSO) except Thy whose stock solution was prepared in ethanol.
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Streptomycin was USP grade, purchased from solarbio (Beijing, China), and was dissolved in sterile water to a final concentration of 10 mg ml-1 and stored at 4°C until use.
Minimum inhibitory concentration assay The minimum inhibitory concentrations (MIC) were determined for the four components (Thy, Eug, Ber, Cin) of the natural essential oils and also streptomycin by the broth dilution method, according to standards of the CLSI (Clinical Laboratory Standardization Institute) (Schwalbe et al. 2007). All assays were carried out in triplicate. The antibacterial activities were examined after incubation at 37°C for 24 h. MIC was determined as the lowest concentration of test samples that resulted in a complete inhibition of visible growth in the broth.
Chequerboard assay The synergistic antimicrobial effects of one of the following four essential oils’ components (Thy, Eug, Ber, Cin) in combination with streptomycin were assessed by the checkerboard test as previously described (Cha et al. 2007). Serial twofold dilutions of the antimicrobial compounds were prepared in TSB that the final concentrations of both drugs ranged from 1/16 to 4 times the MIC for streptomycin and from 1/256 to 4 times the MIC for four essential oils’ components. 50 µl of the streptomycin was added to the rows of a 96-well microtitre plate in diminishing concentrations, and 50 µl of each four essential oils’ components solution was added to the columns in diminishing concentrations. The wells were then inoculated with 100 µl of L. monocytogenes or S. Typhimurium suspension
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containing 105 CFU. Columns 12 served as controls containing TSB and inoculum alone. After 24 h of incubation at 37°C, the MIC was determined as the minimal concentration at which there was not visible growth. The fractional inhibitory concentration index (FICI) is the sum of the FIC of each of the drugs, which in turn is defined as the MIC of each drug when it is used in combination divided by the MIC of the drug when it is used alone.
Biofilm formation assay One hundred microlitres, approximately 108 cfu of each bacterial suspension which was prepared in TSB were added to individual wells of a sterile flat-bottomed 96-well polystyrene microtitre plates (Corning, NY). The microtitre plates were covered and incubated at 37°C for 24 h to allow cell attachment and biofilm formation. Then, the supernatant containing non-adhered cells was removed with pipette and the plate was washed (without shaking) three times using 100 µl 0.9% (w/v) NaCl. Existing biofilms were incubated at 37°C in 90 µl TSB supplemented with 10 µl compounds (Essential oils or streptomycin alone or their combination) for 24 h, and each treatment included 6 wells. Biofilms incubated with TSB were only used as control. Biofilm mass (Crystal violet staining assay) and live bacteria (MTT assay) were evaluated. All assays were performed 3 times with similar results. Error bars represent SD.
Crystal violet staining assay Biofilm formation was quantified in terms of biomass after 24 h of incubation at 37°C. At the sampling time, culture medium from each well was gently removed with pipette and the
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plates were washed three times with 0.9% NaCl to get rid of any loosely attached bacterial cells. Subsequently, attached strongly biofilm bacteria to the wells were fixed with 100 µl of absolute methanol for 15 min. The plates were air dried at room temperature for 10 min. The biofilm was then stained by adding 100 µl of crystal violet (1%, w/v) for 20 min (RT). Excess crystal violet was removed by rinsing the wells with sterile distilled water for three times. Stained material was then solubilised in 150 µl of glacial acetic acid. The level (OD) of the crystal violet present in the destaining solution was measured at 595 nm.
Methyl Tetrazolium (MTT) assay After incubation of the bacteria with antimicrobial compounds, the viable cells were determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium-bromide (MTT) assay (Mosmann 1983). The MTT salt (Sigma Aldrich, UK) was dissolved in sterile phosphate buffered saline (PBS) to get a final concentration of 5 mg ml-1. After washing the plates as in the crystal violet staining assay, 100 µl of TSB and 10 µl of MTT solution (5 mg ml-1) were pipetted into each well and incubated for 1 h at 37°C under sterile conditions. The insoluble purple formazan (obtained by enzymatic hydrolysis of MTT by the dehydrogenase found in living cells) was further dissolved in 100 µl methyl-sulfoxide (DMSO). The absorbance of each well was then measured at 570 nm using the microplate reader (Perlong, Beijing, China).
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Fluorescence microscopy Static biofilms were grown on glass coverslips which were submerged with 1 ml of sterile TSB in 24-well plates, After 24 h of incubation at 37°C, the medium was carefully removed and the biofilm was gently washed three times with PBS. Existing biofilms were incubated at 37°C in TSB supplemented with antimicrobial compounds for 24 h as indicated. Biofilms were fixed in 4% paraformaldehyde solution for 30 min at room temperature. After washed with 2 ml PBS, 5-(4, 6-dichlorotriazinyl) aminofluorescein (5-DTAF) was added and incubated with shaking for 2 h at room temperature. The slides were washed 3 times in PBS and inverted onto a microscopic slide. Biofilms were imaged through the following excitation and emission wavelengths: 488 nm excitation and 505 to 530 nm emission detection range for 5-DTAF.
Scanning electron microscopy (SEM) A modified SEM method was used to analyze the biofilm morphology (Kim et al. 2007). Static biofilms were fixed in 2.5% glutaraldehyde at 4°C for 24 h. Cells were rinsed with 0.1 M PBS three times with 10 min at intervals. The cultures were then dehydrated in a gradient alcohol concentration (50%, 70%, 80%, 90% and 100%) for 10 min at each concentration. The specimen was left in 100% alcohol to prevent it from drying and mounted onto an aluminum stub with carbon tape, sputter coated with gold before examination.
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Immunofluorescence The immunofluorescence analysis was used to investigate the streptomycin concentration within the bacterial biofilm. In short, biofilms on glass coverslips were fixed in 4% paraformaldehyde. After treatment with 0.25% Triton X-100 and blocking with 1% bovine serum albumin (BSA) in PBS, cover slips were incubated with a polyclonal antibody for streptomycin (rabbit anti-streptomyicn ployclone. Abcam) at 4 °C overnight, and then incubated with a second Dylight 488-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Inc) for 1 h at room temperature. Finally microscopic slides were observed under the fluorescence microscope. Immunoreactivity was quantified by using Image Pro Plus (version 5.0, Media Cybernetics, Silver Spring, MD, USA).
Statistical analysis All tests were performed at least in triplicate. All graphical evaluations were made using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA). Differences between means of biofilm mass (A595nm) and live bacteria (A570nm) were considered significant when p ≤ 0.05.
Acknowledgments This work was supported by the “Interdisciplinary Cooperation Team” Program for Science and Technology Innovation of the Chinese Academy of Sciences, Science Technology Research and Development Program of Shaanxi Province (No.2012K02-06).
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Conflict of Interest No conflict of interest declared References Amalaradjou, M.A.R., Narayanan, A. and Venkitanarayanan, K. (2011) Trans-cinnamaldehyde decreases attachment and invasion of uropathogenic Escherichia coli in urinary tract epithelial cells by modulating virulence gene expression. J Urol 185, 1526-1531. Authority, E. (2011) The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2009. EFSa J 9, 2090. Babu, A.J., Sundari, A.R., Indumathi, J., Srujan, R. and Sravanthi, M. (2011) Study on the antimicrobial activity and minimum inhibitory concentration of essential oils of spices. Vet World 4, 311-316. Betoni, J.E.C., Mantovani, R.P., Barbosa, L.N., Di Stasi, L.C. and Fernandes Junior, A. (2006) Synergism between plant extract and antimicrobial drugs used on Staphylococcus aureus diseases. Mem Ins Oswaldo Cruz 101, 387-390. Cha, J.-D., Jeong, M.-R., Jeong, S.-I. and Lee, K.-Y. (2007) Antibacterial activity of sophoraflavanone G isolated from the roots of Sophora flavescens. J Microbiol Biotechnol 17, 858-864. Chae, M.S. and Schraft, H. (2000) Comparative evaluation of adhesion and biofilm formation of different Listeria monocytogenes strains. Int J Food Microbiol 62, 103-111. Chen, X.-W., Di, Y.M., Zhang, J., Zhou, Z.-W., Li, C.G. and Zhou, S.-F. (2012) Interaction of herbal compounds with biological targets: a case study with berberine. The Sci World J 2012. Cosentino, S., Tuberoso, C., Pisano, B., Satta, M., Mascia, V., Arzedi, E. and Palmas, F. (1999) In‐vitro antimicrobial activity and chemical composition of Sardinian thymus essential oils. Lett Appl Microbiol 29, 130-135. Costerton, J., Stewart, P.S. and Greenberg, E. (1999) Bacterial biofilms: a common cause of persistent infections. Science 284, 1318-1322. Costerton, J.W. (1999) Introduction to biofilm. International journal of antimicrobial agents 11, 217-221; discussion 237-219. Davies, D. (2003) Understanding biofilm resistance to antibacterial agents. Nat Rev Drug Discov 2, 114-122. de Andrade, F.B., Midena, R.Z., Koga-Ito, C.Y. and Duarte, M.A.H. (2013) Conventional and natural products against oral infections. Dorman, H. and Deans, S. (2000) Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J Appl Microbiol 88, 308-316. Elgayyar, M., Draughon, F., Golden, D. and Mount, J. (2001) Antimicrobial activity of essential oils from plants against selected pathogenic and saprophytic microorganisms. J Food Prot 64, 1019-1024. Esimone, C., Iroha, I., Ibezim, E., Okeh, C. and Okpana, E. (2006) In vitro evaluation of the interaction between tea extracts and penicillin G against Staphylococcus aureus. Afr J Biotechnol 5. Flemming, H.-C. and Wingender, J. (2010) The biofilm matrix. Nat Rev Microbiol 8, 623-633. Gallucci, N., Casero, C., Oliva, M., Zygadlo, J. and Demo, M. (2006) Interaction between terpenes and penicillin on bacterial strains resistant to beta-lactam antibiotics. Mol Med Chem 10, 30-32. Grycová, L., Dostál, J. and Marek, R. (2007) Quaternary protoberberine alkaloids. Phytochemistry 68, 150-175. Hammer, K.A., Carson, C. and Riley, T. (1999) Antimicrobial activity of essential oils and other plant extracts. J Appl Microbiol 86, 985-990.
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Rasooli, I., Shayegh, S. and Astaneh, S. (2009) The effect of Mentha spicata and Eucalyptus camaldulensis essential oils on dental biofilm. Int J Dent Hyg 7, 196-203. Rosato, A., Vitali, C., De Laurentis, N., Armenise, D. and Antonietta Milillo, M. (2007) Antibacterial effect of some essential oils administered alone or in combination with Norfloxacin. Phytomedicine 14, 727-732. Rosato, A., Vitali, C., Gallo, D., Balenzano, L. and Mallamaci, R. (2008) The inhibition of Candida species by selected essential oils and their synergism with amphotericin B. Phytomedicine 15, 635-638. Ryu, J.-H. and Beuchat, L.R. (2005) Biofilm formation by Escherichia coli O157: H7 on stainless steel: effect of exopolysaccharide and curli production on its resistance to chlorine. Appl Environ Microbiol 71, 247-254. Santos, N., Coutinho, H., Viana, G., Rodrigues, F.F. and Costa, J.G. (2011) Chemical characterization and synergistic antibiotic activity of volatile compounds from the essential oil of Vanillosmopsis arborea. Med Chem Res 20, 637-641. Schwalbe, R., Steele-Moore, L. and Goodwin, A.C. (2007) Antimicrobial susceptibility testing protocols: Crc Press. Singh, G., Maurya, S., DeLampasona, M. and Catalan, C.A. (2007) A comparison of chemical, antioxidant and antimicrobial studies of cinnamon leaf and bark volatile oils, oleoresins and their constituents. Food Chem Toxicol 45, 1650-1661. Smith-Palmer, A., Stewart, J. and Fyfe, L. (1998) Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens. Lett Appl Microbiol 26, 118-122. Van Houdt, R. and Michiels, C.W. (2010) Biofilm formation and the food industry, a focus on the bacterial outer surface. J Appl Microbiol 109, 1117-1131. Wang, R., Wang, R. and Yang, B. (2009) Extraction of essential oils from five cinnamon leaves and identification of their volatile compound compositions. Innov Food Sci Emerg 10, 289-292. Wei, Z., Zhou, E., Guo, C., Fu, Y., Yu, Y., Li, Y., Yao, M., Zhang, N. and Yang, Z. (2014) Thymol inhibits Staphylococcus aureus internalization into bovine mammary epithelial cells by inhibiting NF-κB activation. Microb pathogenesis 71, 15-19. Xu, J.-S., Li, Y., Cao, X. and Cui, Y. (2013) The effect of eugenol on the cariogenic properties of Streptococcus mutans and dental caries development in rats. Exp Ther Med 5, 1667-1670. Yu, H.-H., Kim, K.-J., Cha, J.-D., Kim, H.-K., Lee, Y.-E., Choi, N.-Y. and You, Y.-O. (2005) Antimicrobial activity of berberine alone and in combination with ampicillin or oxacillin against methicillin-resistant Staphylococcus aureus. J Med Food 8, 454-461. Zhang, A., Mu, H., Zhang, W., Cui, G., Zhu, J. and Duan, J. (2013) Chitosan Coupling Makes Microbial Biofilms Susceptible to Antibiotics. Sci Rep 3. Zhou, L., Zheng, H., Tang, Y., Yu, W. and Gong, Q. (2013) Eugenol inhibits quorum sensing at sub-inhibitory concentrations. Biotechnol Lett 35, 631-637.
Figure legends: Fig 1 Biofilms formed by L. monocytogenes (Lis) were exposed to 32 µg ml-1 Cin, 128 µg ml-1 Thy or 2 µg ml-1 streptomycin (Str) alone or their combination (Cin+Str and Thy+Str ). Biofilms incubated in TSB containing phosphate-buffered saline were used as control. Biofilm mass (A) and live bacteria (B) were quantified. Lis biofilm architectures after 24 h This article is protected by copyright. All rights reserved.
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treatment were further examined by fluorescence microscopy (C). These experiments were performed three times with similar results each time. Error bars represent SD. Fig 2 S. Typhimurium (Sal) biofilms were exposed to 256 µg ml-1 Eug, 128 µg ml-1 Cin or 1 µg ml-1 Str alone or their combination (Eug+Str and Cin+Str). Biofilms incubated in TSB containing phosphate-buffered saline were used as control. Biofilm mass (A) and live bacteria (B) were quantified and biofilm architectures after 24 h treatment were examined by scanning electron microscopy (C). These experiments were performed three times with similar results each time. Error bars represent SD. Fig 3 Essential oils’ components facilitated antibiotic access to certain biofilm bacteria. Lis biofilms were exposed to Str (2 µg ml-1), Str+Thy (128 µg ml-1) and Str+Cin (32 µg ml-1) for 1 h. Sal biofilms were exposed to Str (1 µg ml-1), Str+Eug (256 µg ml-1) and Str+Cin (128 µg ml-1) for 1 h. Biofilms incubated in TSB were used as control. Str residing in biofilms is examined by immunofluorescence. Immunoreactivity was quantified by using Image Pro Plus. These experiments were performed twice with similar results each time. Fig S1 Schematic diagram showing that the synergistic effect of essential oil’s components and streptomycin against biofilm-associated foodborne pathogens
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Table 1. MIC of four essential oils’ components and streptomycin against food-borne bacteria
MIC(µg ml-1) Bacterial species L. monocytogenes S. Typhimurium
Thy
Cin
Eug
Ber
Str
1024 256
512 1024
1024 2048
8192 2048
8 4
Table 2. Synergistic effect of the four essential oils’ components with streptomycin against food-borne bacteria
Combination Str+Thy Str+Cin Str+Eug Str+Ber
Bacteria strains
FIC
FIC index
Remark
L. monocytogenes
0.25/0.125
0.375
Synergy
S. Typhimurium
0.25/0.50
0.75
Additive
L. monocytogenes
0.25/0.0625
0.3125
Synergy
S. Typhimurium
0.25/0.125
0.375
Synergy
L. monocytogenes
0.25/0.28125
0.53125
Additive
S. Typhimurium
0.25/0.125
0.375
Synergy
L. monocytogenes
0.25/0.50
0.75
Additive
S. Typhimurium
0.25/0.75
1
Additive
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Article Type: Original Article
Synergy between thymol, eugenol, berberine, cinnamaldehyde and streptomycin against planktonic and biofilm-associated food-borne pathogens
Qianjin Liu, Hong Niu, Wuxia Zhang, Haibo Mu, Chunli Sun, Jinyou Duan*
College of science, Northwest A&F University, Yangling 712100, Shaanxi, China
Correspondence: Jinyou Duan, Ph.D Tel.: +86 29 87092226; E-mail: [email protected]
Significance and Impact of the Study: This study has shown the synergistic effect of four components of essential oil (thymol, eugenol, berberine and cinnamaldehyde) combined with streptomycin on planktonic and biofilm-associated
foodborne
pathogens
Listeria
monocytogenes
and
Salmonella
Typhimurium. These findings indicate that combination of specific components of essential oils with streptomycin may provide alternative methods to overcome the problem of 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/lam.12401 This article is protected by copyright. All rights reserved.
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food-borne bacteria both in suspension and in biofilm.
Abstract Essential oils have been found to exert antibacterial, antifungal, spasmolytic, and antiplasmodial activity and therapeutic effect in cancer treatment. In this study, the antibacterial activities of four main essential oils’ components (thymol (Thy), eugenol (Eug), berberine (Ber), cinnamaldehyde (Cin)) were evaluated against two foodborne pathogens, Listeria monocytogenes and Salmonella Typhimurium, either alone or in combination with streptomycin. Chequerboard assay demonstrated that Thy and Cin elicited a synergistic effect with streptomycin against L. monocytogenes, while a synergy existed between Cin or Eug and streptomycin against S. Typhimurium. Further experiments showed that this synergy was sufficient to eradicate biofilms formed by these two bacteria. Thus, our data highlighted that the combinations of specific components from essential oils and streptomycin were useful for the treatment of foodborne pathogens, which might be help prevent the spread of antibiotic resistance through improving antibiotic effectiveness.
Keywords: thymol; eugenol; berberine; cinnamaldehyde; streptomycin; synergistic; bacterial biofilms
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Introduction When bacteria attach to biotic or inert surfaces, they can form biofilm which is considered as a universal survival lifestyle for microbes to protect themselves from antimicrobial attack. Susceptibility tests have demonstrated biofilm bacteria to survive after treatment with antibiotics at hundreds or even thousand times of the minimum inhibitory concentration against planktonic cells (Costerton et al. 1999; Costerton 1999). The failure in the prevention and eradication of microbial biofilms might create a number of serious problems in industrial fluid processing operations (bio-deterioration), food safety (contamination), and public health issues (infectious diseases) (Mittelman 1998; Flemming and Wingender 2010; Van Houdt and Michiels 2010). L. monocytogenes is the causal organism of the serious food-borne illness listeriosis, and may grow as biofilms on food and food-processing equipments that protect it against environmental stress (Møretrø and Langsrud 2004). S. Typhimurium as a typical foodborne pathogen which can form biofilms on food processing facility surfaces (Chae and Schraft 2000; Prouty and Gunn 2003; Ryu and Beuchat 2005). According to the scientific report of European Food Safety Authority (EFSA), Salmonella was identified as the leading causative agent and a total of 108,614 confirmed cases of human salmonellosis including 90 deaths were reported in 2009 (Authority 2011). Essential oils are a rich source of biologically active compounds, which are aromatic and volatile oily liquids obtained from plant materials of different plant parts (flowers, buds, seeds, leaves, twigs, bark, herbs, wood, fruits, roots) (Oussalah et al. 2007). Essential oils are complex mixtures comprising many compounds which are chemically derived from terpenes
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and their oxygenated compounds (Oussalah et al. 2007). Thy is a monocyclic monoterpene compound isolated from Thymus vulgaris, Monarda punctuate or Origanum vulgare spp (Wei et al. 2014). Eug (4-allyl-2-methoxyphenol) is an aromatic molecule found in essential oils and various plants, including cloves, bay leaves and cinnamon leaves (Xu et al. 2013). Ber is an isoquinoline derivative alkaloid that can be isolated from many medicinal herbs including Papaveraceae, Berberidaceae, Fumariaceae, Menispermaceae, Ranunculaceae, Rutaceae, and Annonaceae (Grycová et al. 2007; Chen et al. 2012). Cin is an aromatic α, β-unsatured aldehyde, and the major component in essential oils from some cinnamon species (Singh et al. 2007; Wang et al. 2009). The antimicrobial properties of essential oils have been known for many centuries and, up to now, a large number of essential oils and their constituents have been investigated for their antimicrobial properties against some bacteria and fungi (Smith-Palmer et al. 1998; Hammer et al. 1999; Dorman and Deans 2000; Elgayyar et al. 2001). One of the outstanding antimicrobial properties of many essential oils is that they can be effective even against microbial biofilms. The essential oils from E. camaldulensis and M. spicata significantly retard bacterial biofilm formation by Streptococcus mutans and Streptococcus pyogenes (Rasooli et al. 2009). Oils of Cymbopogon citratus and Syzygium aromaticum have been shown to elicit a promising anti-biofilm activity against Candida albicans (de Andrade et al. 2013). Some studies have explored the combination of synthetic drugs with essential oils with a aim to evaluate and enhance antimicrobial efficacy (Rosato et al. 2007). For example, a synergistic effect was observed between Norfloxacin and essential oil (and its main
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components) from Pelargonium graveolens (Rosato et al. 2008), between gentamicin and the essential oil from Croton zehntneri (Santos et al. 2011), as well as between eugenol (the main component of cloveoilfrom Eugenia aromatica) with antibiotics (Hemaiswarya and Doble 2009). To our knowledge, the anti-biofilm capacity of essential oils, especially of their particular constituents in combination with antibiotics against microbial biofilms remains largely unknown. In the current study, the anti-biofilm activity of four main essential oils’ components (Thy, Eug, Ber, Cin) in combination with the amino glycoside antibiotic streptomycin was explored.
Results and discussion
Antimicrobial activity of essential oils’ components and streptomycin against L. monocytogenes The MIC of streptomycin and four main essential oils’ components (Thy, Eug, Ber, Cin) obtained by the broth dilution method are shown in Table 1. Streptomycin was active against L. monocytogenes at the concentration of 8 µg ml-1. This finding is similar to the MIC obtained by other authors. Zhang et al (2013) reported that the
streptomycin against L.
monocytogenes with the MIC values 8 µg ml-1. Regarding the MIC values for the four essential oils’ components (Thy, Eug, Ber, Cin), it can be seen that L. monocytogenes was more sensitive to Cin (512 µg ml-1). These results are in agreement with those obtained by Babu et al (2011) which observed a high sensitivity of L. monocytogenes to Cin essential oil. The strong microbicidal activity of Cin might be ascribed to the high electrophilic properties
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of the carbonyl group adjacent to the double bound that make this compound particularly reactive with nucleophiles, such as protein sulfhydryl and amino groups of the microorganism (Neri et al. 2006). Eug and Thy had the same antimicrobial activity against L. monocytogenes with both presenting MIC of 1024 µg ml-1. Ber was active only at very high concentration against L. monocytogenes (8192 µg ml-1), it was same as previously reported that Ber was ineffective as an antibacterial because it was readily extruded by pathogen (Hsieh et al. 1998). Synergistic effects resulting from the combination of antibiotics with different plant extracts have been studied by a number of researchers (Betoni et al. 2006; Esimone et al. 2006; Hemaiswarya et al. 2008; Klein et al. 2013), but little attention has been paid previously to the synergistic effects of an individual natural essentials’ components (Thy, Eug, Ber, Cin) and streptomycin. In this study, the effect of four specific essential oils’ components (Thy, Eug, Ber, Cin) in combination with streptomycin on the L. monocytogenes was detected by chequerboard assay. The results are presented in Table 2. Both Thy and Cin were found to present synergistic effect with streptomycin against L. monocytogenes (Fractional Inhibitory Concentration, FIC index < 0.5). While the remaining combinations between Eug or Ber and streptomycin showed additive effect in the chequerboard assay (FIC index 0.5 – 1.0). Previous studies have also reported antibiofilm activity of essential oil and their components and their synergy with antibiotics against bacterial biofilms built by Pseudomonas putida and E. coli (Kavanaugh and Ribbeck 2012; Khan and Ahmad 2012; Kerekes et al. 2013). Koraichi Saad et al (2011) observed that Thy could inhibit Pseudomonas aeruginosa adherence and biofilmformation. Amalaradjou et al (2011) reported
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that Cin effectively prevented uropathogenic E.coli biofilm on plates and catheters. However, there is very limited information about the effect of these synergistic combinations streptomycin and Cin, streptomycin and Thy on L. monocytogenes in biofilm cultures. This rendered us to ask whether these synergistic combinations were also effective in breaking down biofilms formed by L. monocytogenes. As expected, the synergistic combinations between Cin or Thy with streptomycin had stronger anti-biofilm activities (Fig 1A) than the individual components. These synergistic combinations could also cause a reduction of live bacteria, compared with that of the control (Fig 1B). Also, images from fluorescence microscopy (Fig 1C) evidenced that the architecture of L. monocytogenes biofilms exposed to the synergistic combinations displayed very few scattered cell aggregates, in which there were much less viable cells than exposed to the individual components, these results may be regarded as the effect of essential oils’ components. Previous studies have indicated that essential oil could inhibit quorum sensing (QS) system and influence the expressions of intercellular adhesion gene and biofilm-related gene in biofilm (Zhou et al. 2013). So when antibiotics treat bacteria biofilms combined with essential oil, essential oil may damage biofilms and make antibiotics easily access to biofilm bacteria.
Antimicrobial activity essential oils’ components and streptomycin against S. Typhimurium The antimicrobial activity of the tested essential oils and streptomycin against S. Typhimurium is presented in Table 1. Thy was more active against the S. Typhimurium bacterium showing a MIC value =256 µg ml-1 compared to its activity against L.
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monocytogenes, the same was also evident for Ber. These observations are in agreement with previous study which show S. Typhimurium is more sensitive to Thy than L. monocytogenes (Cosentino et al. 1999). However, Eug and Cin appeared more active against L. monocytogenes. These observations are consistent with previous observations. Mith et al (2014) observed that Cin was more effective against L. monocytogenes than S. Typhimurium. Generally, Gram-positive bacteria are more sensitive to essential oils than Gram-negative bacteria (Smith-Palmer et al. 1998; Lopez et al. 2005). However, two essential oils’ components in this study did not completely follow the trend described above. This suggests that there might be some particular anti-Gram-negative substances in these two components. The results of chequerboard assay are presented in Table 2. Cin with streptomycin were found to have the similar synergistic effect against both S. Typhimurium and L. monocytogenes However, eugenol showed synergistic action with streptomycin against S. Typhimurium but not against L. monocytogenes. These observation are consistent with the descriptions in the literature. Eug showed potent synergistic activity with antibiotics against Gram negative bacteria. There was nearly a 5–1000 fold decrease in the MIC of the antibiotics tested (Gallucci et al. 2006). The remaining combinations streptomycin and Thy, streptomycin and Ber showed additive effect in the chequerboard assay. The synergistic combinations also demonstrated higher anti-biofilm activity towards S. Typhimurium (Fig 2). Obviously, the synergistic combinations streptomycin and Cin, streptomycin and Eug had a pronounced effect in the decrease of biofilm mass (Fig 2A) and live bacteria (Fig 2B) than streptomycin, Cin or Eug treatment alone. These findings were also evidenced by the visualization of biofilms by scanning electron microscopy (Fig 2C), in which the architecture
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of S. Typhimurium biofilms exposed to these synergistic combinations for 24 h displayed fewer scattered cell aggregates when compared to that of individual reagents. The tested four essential oils’ components synergisms with antibiotics have been presented in previous literatures. Palaniappan and Holley (2010) observed that treatment with low concentrations of carvacrol and Thy enhanced bacterial susceptibility to ampicillin, penicillin, tetracycline and also demonstrated the synergy between the Cin and ampicillin. The combination of Ber with oxacillin produced a large synergistic effect against methicillin-resistant Staphylococcus aureus (Yu et al. 2005). A notable study by Gallucci et al (2006) described synergistic in vitro interactions between Eug and penicillin against E. coli and methicillin-resistant Staphylococcus aureus. To our knowledge, this is the first report of synergism between streptomycin and Cin or Eug on S. Typhimurium biofilm.
Essential oils facilitated antibiotic access to biofilm bacteria There are many factors accounting for the resistance of biofilm bacteria to antibiotics (Davies 2003). One factor that is generally perceived to play a role in antibiotic resistance is the inability of the antibiotic to penetrate into biofilms, thereby reducing antibiotic available to interact with biofilm bacteria. Given that essential oils can penetrate and damage biofilms (Nuryastuti et al. 2009). It rendered us to see whether essential oils could facilitate streptomycin entry into biofilms. The results of immunofluorescence analysis are shown in Fig 3. L. monocytogenes or S. Typhimurium exposed to streptomycin alone exhibited a weak green fluorescence. In contrast, green fluorescence was observed in biofilms formed by both these bacteria after treatment
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with the combined of components of essential oils with streptomycin (Thy and streptomycin, Cin and streptomycin, Eug and streptomycin). These findings clearly show that essential oils facilitate streptomycin access into the biofilms formed by these two bacteria. These observations are in agreement with previous study. Jabra-Rizk et al (2006) tested the ability of farnesol to act in synergy with gentamicin by facilitating gentamicin entry into staphylococcal biofilms.
Materials and methods
Bacterial strains L. monocytogenes (CMCC 54004) and S. Typhimurium (SL1344) were generous gifts received from Prof. Xiaodong Xia (College of Food Science and Engineering, Northwest A&F University). The strains were cultivated in tryptic soy broth (TSB, Oxoid, Basingstoke, Hampshire, UK) at 37 °C for 12 h. Following incubation, dilution of the overnight culture with TSB matched to the 0.4 McFarland turbidity standard (approximately 108 cfu ml-1) was used for experimental procedures.
Antimicrobial compounds Four components from natural essential oils (thymol (Thy), eugenol (Eug), berberine (Ber), cinnamaldehyde (Cin)) were purchased from Ziyi reagent (Shanghai, China). Stock solutions of essential oils at concentration of 100 mg ml-1 were prepared in dimethylsulfoxide (DMSO) except Thy whose stock solution was prepared in ethanol.
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Streptomycin was USP grade, purchased from solarbio (Beijing, China), and was dissolved in sterile water to a final concentration of 10 mg ml-1 and stored at 4°C until use.
Minimum inhibitory concentration assay The minimum inhibitory concentrations (MIC) were determined for the four components (Thy, Eug, Ber, Cin) of the natural essential oils and also streptomycin by the broth dilution method, according to standards of the CLSI (Clinical Laboratory Standardization Institute) (Schwalbe et al. 2007). All assays were carried out in triplicate. The antibacterial activities were examined after incubation at 37°C for 24 h. MIC was determined as the lowest concentration of test samples that resulted in a complete inhibition of visible growth in the broth.
Chequerboard assay The synergistic antimicrobial effects of one of the following four essential oils’ components (Thy, Eug, Ber, Cin) in combination with streptomycin were assessed by the checkerboard test as previously described (Cha et al. 2007). Serial twofold dilutions of the antimicrobial compounds were prepared in TSB that the final concentrations of both drugs ranged from 1/16 to 4 times the MIC for streptomycin and from 1/256 to 4 times the MIC for four essential oils’ components. 50 µl of the streptomycin was added to the rows of a 96-well microtitre plate in diminishing concentrations, and 50 µl of each four essential oils’ components solution was added to the columns in diminishing concentrations. The wells were then inoculated with 100 µl of L. monocytogenes or S. Typhimurium suspension
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containing 105 CFU. Columns 12 served as controls containing TSB and inoculum alone. After 24 h of incubation at 37°C, the MIC was determined as the minimal concentration at which there was not visible growth. The fractional inhibitory concentration index (FICI) is the sum of the FIC of each of the drugs, which in turn is defined as the MIC of each drug when it is used in combination divided by the MIC of the drug when it is used alone.
Biofilm formation assay One hundred microlitres, approximately 108 cfu of each bacterial suspension which was prepared in TSB were added to individual wells of a sterile flat-bottomed 96-well polystyrene microtitre plates (Corning, NY). The microtitre plates were covered and incubated at 37°C for 24 h to allow cell attachment and biofilm formation. Then, the supernatant containing non-adhered cells was removed with pipette and the plate was washed (without shaking) three times using 100 µl 0.9% (w/v) NaCl. Existing biofilms were incubated at 37°C in 90 µl TSB supplemented with 10 µl compounds (Essential oils or streptomycin alone or their combination) for 24 h, and each treatment included 6 wells. Biofilms incubated with TSB were only used as control. Biofilm mass (Crystal violet staining assay) and live bacteria (MTT assay) were evaluated. All assays were performed 3 times with similar results. Error bars represent SD.
Crystal violet staining assay Biofilm formation was quantified in terms of biomass after 24 h of incubation at 37°C. At the sampling time, culture medium from each well was gently removed with pipette and the
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plates were washed three times with 0.9% NaCl to get rid of any loosely attached bacterial cells. Subsequently, attached strongly biofilm bacteria to the wells were fixed with 100 µl of absolute methanol for 15 min. The plates were air dried at room temperature for 10 min. The biofilm was then stained by adding 100 µl of crystal violet (1%, w/v) for 20 min (RT). Excess crystal violet was removed by rinsing the wells with sterile distilled water for three times. Stained material was then solubilised in 150 µl of glacial acetic acid. The level (OD) of the crystal violet present in the destaining solution was measured at 595 nm.
Methyl Tetrazolium (MTT) assay After incubation of the bacteria with antimicrobial compounds, the viable cells were determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium-bromide (MTT) assay (Mosmann 1983). The MTT salt (Sigma Aldrich, UK) was dissolved in sterile phosphate buffered saline (PBS) to get a final concentration of 5 mg ml-1. After washing the plates as in the crystal violet staining assay, 100 µl of TSB and 10 µl of MTT solution (5 mg ml-1) were pipetted into each well and incubated for 1 h at 37°C under sterile conditions. The insoluble purple formazan (obtained by enzymatic hydrolysis of MTT by the dehydrogenase found in living cells) was further dissolved in 100 µl methyl-sulfoxide (DMSO). The absorbance of each well was then measured at 570 nm using the microplate reader (Perlong, Beijing, China).
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Fluorescence microscopy Static biofilms were grown on glass coverslips which were submerged with 1 ml of sterile TSB in 24-well plates, After 24 h of incubation at 37°C, the medium was carefully removed and the biofilm was gently washed three times with PBS. Existing biofilms were incubated at 37°C in TSB supplemented with antimicrobial compounds for 24 h as indicated. Biofilms were fixed in 4% paraformaldehyde solution for 30 min at room temperature. After washed with 2 ml PBS, 5-(4, 6-dichlorotriazinyl) aminofluorescein (5-DTAF) was added and incubated with shaking for 2 h at room temperature. The slides were washed 3 times in PBS and inverted onto a microscopic slide. Biofilms were imaged through the following excitation and emission wavelengths: 488 nm excitation and 505 to 530 nm emission detection range for 5-DTAF.
Scanning electron microscopy (SEM) A modified SEM method was used to analyze the biofilm morphology (Kim et al. 2007). Static biofilms were fixed in 2.5% glutaraldehyde at 4°C for 24 h. Cells were rinsed with 0.1 M PBS three times with 10 min at intervals. The cultures were then dehydrated in a gradient alcohol concentration (50%, 70%, 80%, 90% and 100%) for 10 min at each concentration. The specimen was left in 100% alcohol to prevent it from drying and mounted onto an aluminum stub with carbon tape, sputter coated with gold before examination.
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Immunofluorescence The immunofluorescence analysis was used to investigate the streptomycin concentration within the bacterial biofilm. In short, biofilms on glass coverslips were fixed in 4% paraformaldehyde. After treatment with 0.25% Triton X-100 and blocking with 1% bovine serum albumin (BSA) in PBS, cover slips were incubated with a polyclonal antibody for streptomycin (rabbit anti-streptomyicn ployclone. Abcam) at 4 °C overnight, and then incubated with a second Dylight 488-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Inc) for 1 h at room temperature. Finally microscopic slides were observed under the fluorescence microscope. Immunoreactivity was quantified by using Image Pro Plus (version 5.0, Media Cybernetics, Silver Spring, MD, USA).
Statistical analysis All tests were performed at least in triplicate. All graphical evaluations were made using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA). Differences between means of biofilm mass (A595nm) and live bacteria (A570nm) were considered significant when p ≤ 0.05.
Acknowledgments This work was supported by the “Interdisciplinary Cooperation Team” Program for Science and Technology Innovation of the Chinese Academy of Sciences, Science Technology Research and Development Program of Shaanxi Province (No.2012K02-06).
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Conflict of Interest No conflict of interest declared References Amalaradjou, M.A.R., Narayanan, A. and Venkitanarayanan, K. (2011) Trans-cinnamaldehyde decreases attachment and invasion of uropathogenic Escherichia coli in urinary tract epithelial cells by modulating virulence gene expression. J Urol 185, 1526-1531. Authority, E. (2011) The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2009. EFSa J 9, 2090. Babu, A.J., Sundari, A.R., Indumathi, J., Srujan, R. and Sravanthi, M. (2011) Study on the antimicrobial activity and minimum inhibitory concentration of essential oils of spices. Vet World 4, 311-316. Betoni, J.E.C., Mantovani, R.P., Barbosa, L.N., Di Stasi, L.C. and Fernandes Junior, A. (2006) Synergism between plant extract and antimicrobial drugs used on Staphylococcus aureus diseases. Mem Ins Oswaldo Cruz 101, 387-390. Cha, J.-D., Jeong, M.-R., Jeong, S.-I. and Lee, K.-Y. (2007) Antibacterial activity of sophoraflavanone G isolated from the roots of Sophora flavescens. J Microbiol Biotechnol 17, 858-864. Chae, M.S. and Schraft, H. (2000) Comparative evaluation of adhesion and biofilm formation of different Listeria monocytogenes strains. Int J Food Microbiol 62, 103-111. Chen, X.-W., Di, Y.M., Zhang, J., Zhou, Z.-W., Li, C.G. and Zhou, S.-F. (2012) Interaction of herbal compounds with biological targets: a case study with berberine. The Sci World J 2012. Cosentino, S., Tuberoso, C., Pisano, B., Satta, M., Mascia, V., Arzedi, E. and Palmas, F. (1999) In‐vitro antimicrobial activity and chemical composition of Sardinian thymus essential oils. Lett Appl Microbiol 29, 130-135. Costerton, J., Stewart, P.S. and Greenberg, E. (1999) Bacterial biofilms: a common cause of persistent infections. Science 284, 1318-1322. Costerton, J.W. (1999) Introduction to biofilm. International journal of antimicrobial agents 11, 217-221; discussion 237-219. Davies, D. (2003) Understanding biofilm resistance to antibacterial agents. Nat Rev Drug Discov 2, 114-122. de Andrade, F.B., Midena, R.Z., Koga-Ito, C.Y. and Duarte, M.A.H. (2013) Conventional and natural products against oral infections. Dorman, H. and Deans, S. (2000) Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J Appl Microbiol 88, 308-316. Elgayyar, M., Draughon, F., Golden, D. and Mount, J. (2001) Antimicrobial activity of essential oils from plants against selected pathogenic and saprophytic microorganisms. J Food Prot 64, 1019-1024. Esimone, C., Iroha, I., Ibezim, E., Okeh, C. and Okpana, E. (2006) In vitro evaluation of the interaction between tea extracts and penicillin G against Staphylococcus aureus. Afr J Biotechnol 5. Flemming, H.-C. and Wingender, J. (2010) The biofilm matrix. Nat Rev Microbiol 8, 623-633. Gallucci, N., Casero, C., Oliva, M., Zygadlo, J. and Demo, M. (2006) Interaction between terpenes and penicillin on bacterial strains resistant to beta-lactam antibiotics. Mol Med Chem 10, 30-32. Grycová, L., Dostál, J. and Marek, R. (2007) Quaternary protoberberine alkaloids. Phytochemistry 68, 150-175. Hammer, K.A., Carson, C. and Riley, T. (1999) Antimicrobial activity of essential oils and other plant extracts. J Appl Microbiol 86, 985-990.
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Figure legends: Fig 1 Biofilms formed by L. monocytogenes (Lis) were exposed to 32 µg ml-1 Cin, 128 µg ml-1 Thy or 2 µg ml-1 streptomycin (Str) alone or their combination (Cin+Str and Thy+Str ). Biofilms incubated in TSB containing phosphate-buffered saline were used as control. Biofilm mass (A) and live bacteria (B) were quantified. Lis biofilm architectures after 24 h This article is protected by copyright. All rights reserved.
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treatment were further examined by fluorescence microscopy (C). These experiments were performed three times with similar results each time. Error bars represent SD. Fig 2 S. Typhimurium (Sal) biofilms were exposed to 256 µg ml-1 Eug, 128 µg ml-1 Cin or 1 µg ml-1 Str alone or their combination (Eug+Str and Cin+Str). Biofilms incubated in TSB containing phosphate-buffered saline were used as control. Biofilm mass (A) and live bacteria (B) were quantified and biofilm architectures after 24 h treatment were examined by scanning electron microscopy (C). These experiments were performed three times with similar results each time. Error bars represent SD. Fig 3 Essential oils’ components facilitated antibiotic access to certain biofilm bacteria. Lis biofilms were exposed to Str (2 µg ml-1), Str+Thy (128 µg ml-1) and Str+Cin (32 µg ml-1) for 1 h. Sal biofilms were exposed to Str (1 µg ml-1), Str+Eug (256 µg ml-1) and Str+Cin (128 µg ml-1) for 1 h. Biofilms incubated in TSB were used as control. Str residing in biofilms is examined by immunofluorescence. Immunoreactivity was quantified by using Image Pro Plus. These experiments were performed twice with similar results each time. Fig S1 Schematic diagram showing that the synergistic effect of essential oil’s components and streptomycin against biofilm-associated foodborne pathogens
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Table 1. MIC of four essential oils’ components and streptomycin against food-borne bacteria
MIC(µg ml-1) Bacterial species L. monocytogenes S. Typhimurium
Thy
Cin
Eug
Ber
Str
1024 256
512 1024
1024 2048
8192 2048
8 4
Table 2. Synergistic effect of the four essential oils’ components with streptomycin against food-borne bacteria
Combination Str+Thy Str+Cin Str+Eug Str+Ber
Bacteria strains
FIC
FIC index
Remark
L. monocytogenes
0.25/0.125
0.375
Synergy
S. Typhimurium
0.25/0.50
0.75
Additive
L. monocytogenes
0.25/0.0625
0.3125
Synergy
S. Typhimurium
0.25/0.125
0.375
Synergy
L. monocytogenes
0.25/0.28125
0.53125
Additive
S. Typhimurium
0.25/0.125
0.375
Synergy
L. monocytogenes
0.25/0.50
0.75
Additive
S. Typhimurium
0.25/0.75
1
Additive
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