This article was downloaded by: [Eindhoven Technical University] On: 21 November 2014, At: 02:59 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Journal of Essential Oil Bearing Plants Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/teop20 Reversal of Resistance in MRSA Strains by Thymus kotschyanus Essential Oil Mahnaz Khanavi a , Behnaz Farahanikia a , Farideh Rafiee a , Dina Dalili b , Eliyeh Safaripour c , Yousef Ajani d & Nasrin Samadi b a Department of Pharmacognosy, Faculty of Pharmacy and Traditional Iranian Medicine and Pharmacy Research Center , Tehran University of Medical Sciences, Tehran , Iran b Department of Drug and Food Control, Faculty of Pharmacy and Pharmaceutical Quality Assurance Research Center , Tehran University of Medical Sciences, Tehran , Iran c Department of Drug and Food Control, Faculty of Pharmacy and Biotechnology Research Center , Tehran University of Medical Sciences, Tehran , Iran d Institute of Medicinal Plants (ACECR) , P.O. Box: 13145–1446, Tehran , Iran Published online: 12 Mar 2013. To cite this article: Mahnaz Khanavi , Behnaz Farahanikia , Farideh Rafiee , Dina Dalili , Eliyeh Safaripour , Yousef Ajani & Nasrin Samadi (2011) Reversal of Resistance in MRSA Strains by Thymus kotschyanus Essential Oil, Journal of Essential Oil Bearing Plants, 14:6, 684-692, DOI: 10.1080/0972060X.2011.10643990 To link to this article: http://dx.doi.org/10.1080/0972060X.2011.10643990 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. 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Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions http://www.tandfonline.com/loi/teop20 http://www.tandfonline.com/action/showCitFormats?doi=10.1080/0972060X.2011.10643990 http://dx.doi.org/10.1080/0972060X.2011.10643990 http://www.tandfonline.com/page/terms-and-conditions http://www.tandfonline.com/page/terms-and-conditions Reversal of Resistance in MRSA Strains by Thymus kotschyanus Essential Oil Mahnaz Khanavi 1, Behnaz Farahanikia 1, Farideh Rafiee 1, Dina Dalili 2, Eliyeh Safaripour 3, Yousef Ajani 4, Nasrin Samadi 2* 1 Department of Pharmacognosy, Faculty of Pharmacy and Traditional Iranian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences, Tehran, Iran 2 Department of Drug and Food Control, Faculty of Pharmacy and Pharmaceutical Quality Assurance Research Center, Tehran University of Medical Sciences, Tehran, Iran 3 Department of Drug and Food Control, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences, Tehran, Iran 4 Institute of Medicinal Plants (ACECR), P.O. Box: 13145-1446, Tehran, Iran Abstract: In this study the essential oil of Thymus kotschyanus Boiss. & Hohen, which is endemic of Iran, was investigated for its antibacterial activity and its combined effects with oxacillin or methicillin against several clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). The minimum inhibitory concentrations of oxacillin, methicillin and essential oil against MRSA strains were determined by the micro- dilution method. Synergy testing of each antibiotic and essential oil against MRSA was performed by the checkerboard technique. The results indicated that T. kotschyanus essential oil had synergistic effect with oxacillin or methicillin against MRSA strains. The best synergistic effect obtained against MRSA5 and S. aureus ATCC 33591 which resulted in 32 times decrease in MIC values of oxacillin and methicillin. The synergistic activity showed that this non-antibiotic agent manifests a broad spectrum of activity against MRSA bacteria which might open a new therapeutic approach to combat drug-resistance in bacterial infections. Keywords: Synergism, MRSA, Thymus kotschyanus, Essential oil, Oxacillin, Methicillin. Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) which is also referred to as multidrug or oxacillin resistant Staphylococcus aureus (ORSA) is a bacterium responsible for several difficult to treat infections in human. MRSA is a strain of Staphylococcus aureus that is resistant to a large group of antibiotics called the beta-lactams 1. At present, there are effective antibiotics to cure almost all major infections, but they have become under threat due to development of drug resistance among microorganisms 2,3,4. Therefore, combinations of antimicrobials are chosen because an identified resistant pathogen may be more susceptible to inhibition or killing by combination of the antimicrobials 5,6. Such compounds, having antimicrobial properties in addition to their predesignated pharmacological actions, have been called the non-antibiotics 2. Journal of Essential Oil Bearing Plants ISSN Print: 0972-060X Online: 0976-5026 www.jeobp.com *Corresponding author (Nasrin Samadi) E-mail: <
[email protected] > © 2011, Har Krishan Bhalla & Sons Jeobp 14 (6) 2011 pp 684 - 692 684 Received 07 October 2010; accepted in revised form 23 June 2011 D ow nl oa de d by [ E in dh ov en T ec hn ic al U ni ve rs ity ] at 0 2: 59 2 1 N ov em be r 20 14 Thyme is cultivated throughout the world for culinary, cosmetic and medicinal purposes. It contains fourteen species in Iran which are locally called “Avishan” 7. One of the species which is endemic and native of Iran is Thymus kotschyanus Boiss. & Hohen. It is stated to possess bactericidal, anthelmintic, astringent, carminative, antispasmodic, antitussive and expectorant properties. The aerial parts of T. kotschyanus are traditionally used in Iran for treatment of infectious diseases such as gastrointestinal disturbances, chronic gastritis and bacterial diarrhea in children 8. Phytochemical analysis of Thymus species have confirmed the occurrence of phenolic compounds such as thymol, carvacrol, thymonin, caffeic acid, rosmarinic acid, terpenoids, flavonoids and saponins in the plant 9. According to our previous investigation, the main component of T. kotchyanus essential oil was carvacrol 10 which has been shown to exhibit a range of biological effects such as antibacterial, antifungal, insecticidal, analgesic and antioxidant activities 11,12. Therefore, in a continuation of our previous study and considering the world concern on emergence of MRSA, the aim of the present study was to investigate the antibacterial activity of essential oil of Thymus kotschyanus and to determine its combined effects with oxacillin or methicillin against several clinical isolates of MRSA. Material and methods Plant material: Thymus Kotschyanus Boiss. & Hohen were collected from Yazd, province of Yazd, Iran, in June 2009, during the flowering stage. The plant was identified and authenticated by Dr. G. Amin and voucher specimen (TEH-6677) was deposited at the Herbarium of the Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. Isolation of volatile oil: The aerial parts of T. Kotschyanus were air-dried, powdered and finally subjected to hydrodistillation using a Clevenger-type apparatus according to the European Pharmacopoeia 13 for 4 h. The oil was dried over anhydrous sodium sulfate and kept at 4°C in the sealed brown vial until required. Characterization of methicillin-resistant of S. aureus (MRSA): Eight methicillin-resistant clinical isolates of S. aureus and standard microorganism ATCC 33591 as control strain were used in this study. Individual colonies were selected from Tryptic Soy agar (Merck Co.) plates, transferred to Tryptic Soy broth (Merck Co.) and incubated at 35°C for 24 h. A direct suspension of microorganisms was prepared in sterile saline. The turbidity of the suspension was adjusted to match 0.5 McFarland standard with a spectrophotometer at 600 nm, which corresponds to 1.5 ×1 08 CFU/ ml. Resistance determination was performed by disc diffusion method based on the method described by NCCLS 14. The surface of Mueller-Hinton agar (MHA, Merck Co.) plates were evenly inoculated with the bacterial suspensions. Then, filter paper discs (7 mm, Whatman No.1) containing 5 μg and 1 μg of methicillin and oxacillin respectively, were applied to the inoculated plates. After incubation (24 h at 35°C), the subsequent zones of inhibition around each disc were measured in millimeters. The interpretation made as follows: for methicillin discs, resistant (14) and for oxacillin discs, resistant (13) 15. MIC determination of oxacillin, methicillin and T. kotschyanus essential oil: The minimum inhibitory concentrations of oxacillin (Glaxo Co.), methicilin (Glaxo Co.) and essential oil against MRSA strains were determined in Mueller-Hinton broth (MHB, Merck Co.) by the micro dilution method 14. A stock concentration of 8 mg/ml from each antibiotic was prepared in MHB. Aliquot of 100 Nasrin Samadi et al. / Jeobp 14 (6) 2011 684 - 692 685 D ow nl oa de d by [ E in dh ov en T ec hn ic al U ni ve rs ity ] at 0 2: 59 2 1 N ov em be r 20 14 μl DMSO and 10 μl Tween 80 was used to dissolve 20 μl of essential oil in 1 ml MHB. Then two- fold dilution of the stock solutions of antibiotics and essential oil were prepared in MHB. Final concentration of bacteria in individual tubes was adjusted to about 5 ×1 0 5 CFU/ml. After 24 h incubation at 35°C the wells were examined for possible bacterial turbidity. The endpoint MIC is the lowest concentration of the compound at which the test bacteria does not demonstrate visible growth. The resistance of strains to oxacillin and methicillin was determined due to MICs (μg/ml). The interprations were as follows: for methicillin, susceptible (MIC < 8), resistance (MIC > 16) and for oxacillin, susceptible (MIC < 2), resistance (MIC > 4) 16. Determination of combined effects of antibiotic (oxacillin, methicillin) and T. kotschyanus essential oil: Synergy testing of each antibiotic and essential oil against MRSA was performed by the checkerboard method in microtiter trays using MHB 5. The primary concentration of antibiotics and essential oil was 2 to 3 times higher than determined MICs. Two-fold dilution of the antibiotic was prepared in the first horizontal row and two-fold dilution of the essential oil was prepared alone in the first vertical column. Aliquots of 100 μl of essential oil dilutions were added to the wells of 96-well plates in horizontal orientation and aliquots of 100 μl of antibiotic dilutions were added in vertical orientation. Dilutions from the logarithmic growth phase of bacterial cultures were prepared and dispensed into microtiter trays (10 5 CFU per microtiter well). The inoculated trays were incubated at 35°C for a period of 24 h, and then evaluated for bacterial growth. In order to evaluate the activity of combinations of agents, fractional inhibitory concentration (FIC) indices were calculated using the following formula: FIC indices=FICA + FICB in which FICA=MICA in combination/MICA alone and FICB =MICB in combination/MICB alone. The interpretation made as follows: for synergy FIC 0.5 but 1.0 3. The results of checkerboard testing were interpreted by the pattern they form on the isobologram 17,18,19. Results and discussion: The results of disc diffusion assay showed that all of the S. aureus strains except MRSA3 and MRSA7 which showed intermediate susceptibility to methicillin, were resistant to both oxacillin and methicillin (Table 1). The results of MICs were comparable with inhibition zones (Table 1). The MICs of Thymus kotschyanus essential oil against nine MRSA was in the range of 2.5-5 μl/ml. The combined effects of T. kotschyanus essential oil and oxacillin or methicillin were studied by performing the checkerboard method. As shown in Table 2, particular synergistic effects were observed between essential oil and oxacillin against MRSA4, MRSA5, MRSA6 and ATCC 33591, although no combination effect was observed against MRSA1, MRSA3, MRSA7 and MRSA8. Oxacillin and essential oil combination showed additive effect against MRSA2. In Figure 1, isobolograms that represent the results of checkerboard are shown. The best synergistic effect was observed against MRS5 at the concentration of 0.16 μl/ml for essential oil and 15.62 μg/ml for oxacillin which resulted in FIC index of 0.09 due to 32 fold reduction in MIC of oxacillin. The data showed that there were combination effects between methicillin and essential oil against most of MRSA strains except MRSA3 and MRSA7 which had shown intermediate resistance to methicillin (Table 3). In Figure 2, isobolograms that represent the results of checkerboards are shown. The highest MIC decrease was observed for S. aureus ATCC 33591 due to 32 fold reduction in MIC with the concentration of 1.25 μl/ml for essential oil and 7.81 μg/ml for methicillin which resulted in FIC index of 0.53. In addition, particular synergistic effects with FIC indices of 0.18 and 0.37 were observed against MRSA1 and MRSA4, respectively. Thyme is cultivated throughout the world for culinary, cosmetic and medicinal purposes. Its oil is manufactured commercially for use in cough drops, mouthwashes, liniments, toothpastes, detergents and perfumes. Nasrin Samadi et al. / Jeobp 14 (6) 2011 684 - 692 686 D ow nl oa de d by [ E in dh ov en T ec hn ic al U ni ve rs ity ] at 0 2: 59 2 1 N ov em be r 20 14 Fig. 1. Isobolograms for combined effects Thymus kotschyanus essential oil and oxacillin In our previous study, the analysis of T. kotschyanus oil, carried out by GC-MS, lead to the identification of different components. Major constituents in the oil isolated by hydrodistillation were reported as carvacrol (80.66 %), 1,8-cineol (2.98 %), borneol (1.49 %) and thymol (1.47 %) 10. Carvacrol is used in low concentration in human food as a flavor. Moreover, it is utilized as oral bactericidal, anti-fungal and breath freshening compound in oral cleansing products 20. Antimicrobial activity of the methanolic extract of T. kotschyanus was found against Bordetella bronchiseptica and Micrococcus luteus by disc diffusion method 21. It also showed anti-Helicobacter pylori activity Nasrin Samadi et al. / Jeobp 14 (6) 2011 684 - 692 687 D ow nl oa de d by [ E in dh ov en T ec hn ic al U ni ve rs ity ] at 0 2: 59 2 1 N ov em be r 20 14 22. The results of previous study on antimicrobial activity of carvacrol and thymol showed that both methicillin-sensitive and-resistant S. aureus were strongly inhibited by these two monoterpenoid phenols 23. The MIC values were ranged from 0.015-0.03 % v/v for carvacrol and 0.03-0.06 % v/v for thymol. Similar to our findings, they showed that MRSA susceptibility to essential oil was independent of their antibiotic susceptibility patterns. In this study all nine MRSA strains which showed different susceptibility to oxacillin and methicillin were inhibited by 2.5-5 % v/v of T. kotschyanus essential oil. Synergism, partial synergism and additivity inferences were observed against MRSA strains when oxacillin or methicillin was used in combination with T. kotschyanus essential oil. Toroglu 24 showed that Thymus eigii essential oil (0.5 μl/disc) in combination with standard vancomycin (30 μg) or erythromycin (15 μg) discs displayed antagonistic effect against different Gram-positive and Gram-negative bacteria, while synergistic, additive and antagonistic effects were observed when T. eigii essential oil was applied in combination with Pinus nigra or Cupressus sempervirens essential oil. Abascal and Yarnell 25 also indicated that combination of tea (Camellia sinensis) or epicatechin from green tea and β-lactam antibiotics had synergistic effect against MRSA and β-lactamase producing S. aureus strains. Although several investigations showed benefits of using combination of herbal extracts or vegetable oils and antibiotics in reversal of antibiotic resistance in bacteria 26,27,28, antagonistic inference which results in decrease in antibiotic effectiveness is also probable. It was obvious that synergistic activity is dependent on the bacterial strain and relative concentration of antimicrobial agents in combination. For any particular herbal extract or essential oil, the content of total phenolic compounds i.e. carvacrol and thymol is known as a main factor in producing synergistic effects 19,29,30,31. Therefore, synergistic inferences between T. kotschyanus essential oil and oxacillin or methicillin against MRSA strains could be supposed because of its high carvacrol content. Nasrin Samadi et al. / Jeobp 14 (6) 2011 684 - 692 688 D ow nl oa de d by [ E in dh ov en T ec hn ic al U ni ve rs ity ] at 0 2: 59 2 1 N ov em be r 20 14 Fig. 2. Isobolograms for combined effects of Thymus kotschyanus essential oil and methicillin Evidence of antibacterial activity possessed by T. kotschyanus essential oil and its synergistic activity in combination with oxacillin or methicillin shows that this non-antibiotic in suitable combinations demonstrates a broad spectrum of activity against MRSA bacteria. Therefore, such a synergistic combination might open a new therapeutic approach to combat drug-resistance in bacterial infections and further serve in the selection of an appropriate antimicrobial therapy. Acknowledgements: This research has been supported by Tehran University of Medical Sciences and Health Services grant. (Grant number: 6862-33-01-87). References 1. Lee, D., Kang, M., Hwang, H., Eom, S., Yang, J., Lee, M., Lee, W., Jeon, Y., Choi, J. and Kim, Y. (2008). Synergistic effect between dieckol from Ecklonia stolonifera and β- lactams against methicillin-resistant Staphylococcus aureus. Biotech. Bioproc. Eng. 13: 758- 764. 2. Basu, L.R., Mazumdar, K., Dautta, N.K., Karak, P. and Dastidar, S.G. (2005). Antibacterial property of the antipsychotic agent, prochlorperazine, and its synergism with methdilazine. Microbial. Res. 160: 95-100. 3. Kumar, K.A., Mazumdar, K., Dutta, N.K., Karak, P., Dastidar, S.G. and Ray, R. (2004). Evaluation of synergism between the aminoglycoside antibiotic streptomycin and the cardio- vascular agent amlodipine. Biol. Pharm. Bull. 27: 1116-1120. Nasrin Samadi et al. / Jeobp 14 (6) 2011 684 - 692 689 D ow nl oa de d by [ E in dh ov en T ec hn ic al U ni ve rs ity ] at 0 2: 59 2 1 N ov em be r 20 14 4. Raju, S., Kumar, Oli A., Patil, S.A. and Kelmani Chandrakanth, R. (2010). Prevalence of multidrug-resistant Staphylococcus aureus in diabetics clinical samples. World J. Microbiol. 5. Pillai, K., Moellering, R.C. and Eliopoulos, G.M. (2005). Antimicrobial combinations In: Lorian V. (eds.), Antibiotics in Laboratory Medicine. 5th ed. William & Wilkins, Baltimore. pp. 365-441. 6. 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. Phyto- medicine. 15: 635-638. 7. Mozaffarian, V.A. (1996). Dictionary of Iranian plant names, Farhang Moaser, Tehran, Iran, 548. 8. Zargari, A. (1989). Medicinal Plants. Tehran University, Tehran, Iran, 28-42. 9. Kosar, M., Özek, T., Göger, F., Kürkcüoglu, M. and Baser, K.H.C. (2005). Comparison of microwave-assisted hydrodistillation and hydrodistillation methods for the analysis of volatile secondary metabolites. J. Pharm. Biol. 43: 491-495. 10. Khanavi, M., Hajimehdipoor, H., Emadi, F., Fathi, M. and Hadjiakhoondi, A. (2009). Determination of the carvacrol concentration in the essential oil of Thymus kotschyanus. Planta Medica. 75: 9-19 11. Toroglu, S. (2011). Antimicrobial Activity of Essential Oil of Thymus kotschyanus subs. glabrescens. A. J. Chem. 23(3): 1072-1074. 12. WHO Monographs on Selected Medicinal Plants (1999). World Health Organization, Geneva, 263. 13. European Pharmacopoeia (1975). Maissonneue, Sainte-Ruffine, Vol. 3. p. 68. 14. NCCLS (2006). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. Approved Standard M7-A7, 7th ed, Wayne, Pennsylvania. 15. Centers for Disease Control and Prevention (CDC) (2005). Laboratory Detection of Oxacillin/Methicillin-resistant Staphylococcus aureus. 16. Salimnia, H. and Brown, W.J. (2005). Detection of oxacillin resistance in Staphylococcus aureus: comparison of phoenix oxacillin and cefoxitin MICs, microscan oxacillin MIC, oxacillin and cefoxitin disk diffusion, and mecA gene detection. ICAAC. 24: 12-20. 17. Barry, A.L. (1986). Antibiotics in Laboratory Medicine, 2nd ed. Lorian, V. (eds.), Williams & Wilkins, Baltimore, pp. 367. 18. Rosato, A.,Vitali, C., DeLaurentis, N., Armenise, D. and Milillo, M.A. (2007). Antibacterial effect of some essential oils administered alone or in combination with Norfloxacin. Phyto- medicine. 14: 727-732. 19. Wagner, H. and Ulrich-Merzenich, G. (2009). Synergy research: approaching a new genera- tion of phytopharmaceuticals. Phytomedicine. 16: 97-110. 20. Stammati, A., Bonsi, P., Zucco, F., Moezelaar, R., Alakomi, H.L. and Von Wright, A. (1999). Toxicity of selected plant volatiles in microbial and mammalian short-term assays. J. Food Chemical. Toxicol. 37: 813-823. 21. Bonjar, S.G.H. (2004). Inhibition of clotrimazole-resistant Candida albicans by plants used in Iranian folkloric medicine. Fitoterapia. 75: 74-76. 22. Nariman, F., Eftekhar, F., Habibi, Z. and Falsafi, T. (2004). Anti-Helicobacter pylori activities of six Iranian plants. Helicobacter. 9: 146-151. 23. Nostro, A., Blanco A.R. Canatelli, M.A., Enea, V., Flamini, G., Morelli, I., Roccaro, A.S. and Alonzo, V. (2006). Susceptibility of methicillin-resistant staphylococci to oregano essential oil, carvacrol and thymol. FEMS Microbiol. Lett. 230: 191-195. 24. Toroglu, S. (2007). In vitro antimicrobial activity and antagonistic effect of essential oils from plant species. J. Environ. Biol. 28: 551-559. 25. Abascal, K. and Yarnell, E. (2002). Potential of botanical in drug-resistant microbes. Herbs Nasrin Samadi et al. / Jeobp 14 (6) 2011 684 - 692 690 D ow nl oa de d by [ E in dh ov en T ec hn ic al U ni ve rs ity ] at 0 2: 59 2 1 N ov em be r 20 14 and drug resistance. Part-1. American Botanical Council. Herbalgram Org. Alternative and Complementary Therapies. pp. 237-241. 26. Adeleke, O.E. and Olaitan, J.O. (2003). The effect of a fixed non-mineral oil on the anti- bacterial activity of ampicillin trihydrate against resistant clinical strains of Staphylococcus aureus. Afr. J. Biomed. Res. 6: 91-94. 27. Nascrimento, G.G.F., Locatelli, J., Freitas, P.C. and Silva, G.L. (2000). Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Braz. J. Microbiol. 31: 247-256. 28. Toroglu, S. (2011). In-vitro antimicrobial activity and synergistic/antagonistic effect of interactions between antibiotics and some spice essential oils. J. Envirion. Biol. 32: 23-29. 29. Komali, A.S., Zheng, Z. and Shetty, K.A. (1999). Mathematical model for the growth kinetics and synthesis of phenolics in oregano (Origanum vulgare) shoot cultures inoculated with Pseudomonas speices. Process Biochem. 35: 227-235. 30. Moller, J.K.S., Madsen, H.L., Altonen, T. and Skibsted, L.H. (1999). Dittany (Origanum dictamnus) as a source of water-extractable antioxidants. Food Chem. 64: 215-219. 31. Tepe, B., Daferera, D., Sokmen, M., Polissiou, M. and Sokmen, A. (2004). In-vitro antimicrobial and antioxidant activities of the essential oils and various extracts of Thymus eigii M. Zohary et. P.H. Davis. J. Agric. Food Chem. 52: 1132-1137. Table 1. Inhibition zones and MICs of Thymus kotschyanus essential oil and antibiotics against clinical isolates of S. aureus MRSA Essential oil Oxacillin Methicillin strain MIC (μμμμμl/ml) zone (mm) MIC (μμμμμg/ml) zone (mm) MIC (μμμμμg/ml) ATCC 33591 2.5 NZ1 31.25 NZ 250 MRSA1 2.5 NZ >2000 NZ 125 MRSA2 2.5 NZ 1000 NZ 125 MRSA3 2.5 9 31.25 12 15.62 MRSA4 2.5 NZ 1000 NZ 125 MRSA5 2.5 NZ 500 NZ 125 MRSA6 5.0 NZ 1000 NZ 500 MRSA7 2.5 NZ 31.25 13 15.62 MRSA8 2.5 NZ 15.62 NZ 62.5 1NZ: No Zone; Inhibition zones were determined by using oxacillin (1 μg) and methicillin (5 μg) discs Table 2. Combined effects of Thymus kotschyanus essential oil and oxacillin against MRSA strains MRSA strain Essential oil (μμμμμl/ml) Oxacillin (μμμμμg/ml) FIC Indices Inference ATCC 33591 0.31 3.90 0.24 Synergism MRSA2 1.25 500 1 Additivity MRSA4 0.31 62.5 0.18 Synergism MRSA5 0.16 15.62 0.09 Synergism MRSA6 1.25 125 0.37 Synergism Nasrin Samadi et al. / Jeobp 14 (6) 2011 684 - 692 691 D ow nl oa de d by [ E in dh ov en T ec hn ic al U ni ve rs ity ] at 0 2: 59 2 1 N ov em be r 20 14 Table 3. Combined effect of Thymus kotschyanus essential oil and methicillin against MRSA strains MRSA strain Essential oil Methicillin FIC Indices Inference (μμμμμl/ml) (μμμμμg/ml) ATCC 33591 1.25 7.81 0.53 Partial synergism MRSA2 0.16 62.5 0.56 Partial synergism MRSA4 0.31 31.25 0.37 Synergism MRSA5 0.16 62.5 0.56 Partial synergism MRSA6 0.16 250 0.53 Partial synergism MRSA1 0.16 15.62 0.18 Synergism MRSA8 0.16 31.25 0.56 Partial synergism Nasrin Samadi et al. / Jeobp 14 (6) 2011 684 - 692 692 D ow nl oa de d by [ E in dh ov en T ec hn ic al U ni ve rs ity ] at 0 2: 59 2 1 N ov em be r 20 14