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Phytomedicine 17 (2010) 142145

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Phytomedicine
journal homepage: www.elsevier.de/phymed

Short communication

Antibacterial effect of essential oils from two medicinal plants against Methicillin-resistant Staphylococcus aureus (MRSA)
A. Tohidpour a, M. Sattari a,, R. Omidbaigi b, A. Yadegar a, J. Nazemi c
a

Department of Bacteriology, School of Medical Sciences, Tarbiat Modares University, P.O. Box: 14115-158, Tehran, Iran Faculty of Horticultural Science, College of Agriculture, Tarbiat Modares University, Tehran, Iran c Faculty of Plant Protection, College of Agriculture, Kordestan University, Sanandaj, Iran
b

a r t i c l e in f o

a b s t r a c t
Antimicrobial properties of plants essential oils (EOs) have been investigated through several observations and clinical studies which purpose them as potential tools to overcome the microbial drug resistance problem. The aim of this research is to study the antibacterial effect of two traditional plants essential oils, Thymus vulgaris and Eucalyptus globulus against clinical isolates of Methicillin resistant Staphylococcus aureus (MRSA) and other standard bacterial strains through disk diffusion and agar dilution methods. Gas Chromatography (GC) and Gas Chromatography/Mass Spectrometry (GC/ MS) analysis examined the chemical composition of the oils. Results revealed both of oils to possess degrees of antibacterial activity against Gram (+) and Gram () bacteria. T. vulgaris EO showed better inhibitory effects than E. globulus essential oil. GC analysis of T. vulgaris resulted in thymol as the oil major compound whereas GC/MS assay exhibited eucalyptol as the most abundant constitute of E. globulus EO. These results support previous studies on these oils and suggest an additional option to treat MRSA infections. Clinical and further analytical trials of these data are necessary to conrm the obtained outcomes. & 2009 Published by Elsevier GmbH.

Keywords: Methicillin resistant Staphylococcus aureus (MRSA) Essential oils Thymus vulgaris Eucalyptus globulus

Introduction Staphylococcus aureus is considered as a main pathogen of causing nosocomial infections. The emergence of antibiotic resistant strains of S. aureus with infection outbreaks among hospitalized patients is a serious problem world wide (Mastoraki et al. 2008; Geha et al. 1994). Most of clinical MRSA isolates, rst reported about 40 years ago (Barber 1961), have mecA gene which encodes production of PBP 2a, an enzyme (78-kDa) in the bacteria cell wall with a low afnity for blactam antibiotics which renders cell wall formation in inactivating concentrations of drugs for other PBPs (Chambers and Sachdeva 1990; Hartman and Tomasz 1984). This has associated with hospital acquired colonizations and considerable increase in death and infection rate in nosocomial settings (Witte et al. 2005). Plant extracts and essential oils have always had choice of use for different purposes (Hammer et al. 1999). Essential oils have been searched for their antibacterial, antifungal, antiviral, insecticidal, anticancer and antioxidant properties (Burt 2004; Kordali et al. 2005; Sylvestre et al. 2006). There has been an increased concern in studying

Corresponding author. Tel.: +98 2182883563; Mobile: +98 9124544534; fax: +98 21 88013030. E-mail address: sattarim@modares.ac.ir (M. Sattari).

antimicrobial properties of plants EOs after emergence of antibiotic resistance to current antibiotics as a result of antibiotics misuse and drugs selective pressure. Hence, as an urgent need for discovering new therapeutic agents, it is reasonable to expect a selection of plant compounds in these oils with specic antibacterial activities (Ang et al. 2004; Darokar et al. 1998). It can be estimated as more than 60 individual compounds are detectable in EOs that are up to 85% constituted by Major dened compounds and only a minimal proportion is for EOs minor chemicals. The genus Eucalyptus, (family: Myrtaceae) is native to Australian region. The plant is increasingly used in traditional medicine for various medical implications such as antibacterial, anti-inammatory, and antipyretic effects. E. globulus is popular for it is cultivated in subtropical and Mediterranean regions more than other species. Eucalyptus species Essential oils are extensively consumed in cosmetics, pharmaceutical and food industries (Gomes-Carneiro et al. 1998; Takahashi et al. 2004; Gray and Flatt. 1998).The genus Thymus (family: Labiatae or Lamiaceae) consists up to 300 species. In vitro antimicrobial activity of many Thymus species EOs has been shown through several studies (Konemann 1999). T. vulgaris, also called common thyme, is native to Mediterranean region. Several studies have investigated the chemical composition of the T. vulgaris essential oils grown in different geographical regions. The plant oil has proved to have antibacterial activity which is mainly attributed to the presence of

0944-7113/$ - see front matter & 2009 Published by Elsevier GmbH. doi:10.1016/j.phymed.2009.05.007

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two plant phenolic compounds, thymol and carvacrol (Leung and Foster 1996; Dorman and Deans 2000; Chao et al. 2000). In this study, the antibacterial effect of EOs from T. vulgaris and E. globulus was studied against clinical isolates of MRSA together with analyzing the biochemical composition of EOs using GC and GC/MS analysis.

Material and methods Organisms and growth conditions 14 MRSA isolates were collected from Milad hospital infectious section, Tehran, Iran during July 2007 to June of 2008 from different sources as the following: Trachea, 8; Wound, 5; Eye, 1 and CSF, 1. These were further approved as MRSA through disk diffusion method using methicillin antibiotic disks and PCR. The following Standard strains were obtained from the culture collection of the Microbiology Department at Tarbiat Modares University (TMU): Bacillus cereus ATCC 9634, E. coli ATCC 3428, Klebsiella pneumoniae ATCC 13883, MRSA standard strain ATCC 33592 and S. aureus ATCC 25922. All the bacteria were maintained on Nutrient broth media mixed with 30% glycerol in 70 1C. To rapidly identifying the methicillin resistance, presence of mecA gene in MRSA isolates was detected using PCR method as the following. Preparation of DNA Total DNA was obtained from overnight cultured colonies suspended in 500 ml of Tris-EDTA buffer (10 mM Tris-HCL, 1 mM EDTA, pH: 8) for two times each followed with a centrifuge (8000 g, 2 min). The pellet was dissolved in 500 ml distilled water, treated with 10 ml proteinase k (10 mg/ml) and incubated for 60 min. Then the tubes were transferred to a boiling water bath for 45 min. After centrifugation (8000 g, 5 min), 1 ml of the supernatant containing the genomic DNA was used for the PCR. PCR amplication According to the method of Choi et al. (2003), the following primers were used for amplication of a 314 bp region inside the mecA gene : mecA1: 50 -CCTAGTAAAGCTCCGGAA-30 and mecA2: 50 CTAGTCCATTCGGTCCA-30 . Amplication performed in a thermocyler machine (eppendorf 5330, Germany) with the thermal conditions: 94 1C, 4 min followed by 35 cycles of amplication (94 1C, 1 min, annealing at 55 1C, 1 min and extension at 72 1C, 60 sec) with a nal extension at 72 1C, 5 min. Essential oil extraction T. vulgaris and E. globulus were prepared from the TMU College of agriculture collection and authenticated by Dr. Reza Omidbaigi, professor of botany at TMU. Air-dried aerial parts of plants (90 g, three times) were hydrodistillated for 4 h using a Clevenger-type apparatus to produce essential oils according to the method by the European pharmacopoeia (4th ed., 2002). The oils were dried over anhydrous sodium sulfate and stored in sealed vials at low temperature (4 1C). Quantication and Identication The GC/MS analysis performed for the Eucalyptus EO in a Thermoquest Finnigan Trace GC/MS instrument equipped with a DB-1 fused silica column (60 m 0.25 mm i.d., lm thickness 0.25). Oven temperature raised from 60 1C to 250 1C at

a rate 8 1C/min and held for 20 min; transfer line temperature was 250 1C. Helium was as the carrier gas at a ow rate of 1.1 ml/min with a split ratio equal to 1/50. The quadruple mass spectrometer was scanned over the 35-465 amu with an ionizing voltage of 70 eV and an ionizing current of 150 mA. Identication of individual compounds was made by comparison of mass spectra with those of internal reference library or authentic compounds. The Thymus EO was analyzed using the GC method with a shimadzu GC-9A gas chromatograph equipped with a DB-5 fused silica column (30 m 0.25 mm, lm thickness 0.25 mm). Oven temperature held at 40 1C for 5 min and programmed to 250 1C at a rate of 3 1C/min. Injector and detector (FID) temperature was 260 1C; helium was used as carrier gas with a line velocity of 32 cm/s. Percentages were calculated by electronic integration of FID peak areas without the use of response factor correction. Antimicrobial assay Disk diffusion assay A 100 ml suspension of any tested bacteria containing about 108 cells/ml spreaded on Muller Hinton Agar (MHA) mediums using sterile swabs. 12 ml from each one of the EOs was dropped on 6 mm lter papers and placed on the agar surface. Standard antibiotic disks were also used as reference. Plates were incubated at 37 1C for 24 hours and then the inhibition zones were measured in diameters. Agar dilution method The agar dilution method followed as approved by NCCLS with minor modications: a series of twofold dilutions of each oil, (ranging: 0.03% (v/v) to 2% (v/v)), was prepared in MHA. To enhance the EO solubility, 0.5% (v/v) concentration of Tween-20% was added into medium. Inoculated plates with 2 ml of inoculums
1 2 3 4 5 6 7 M

mecA (314 bp)

10

11

12

13

14 15

mecA (314bp)

Figs. 1 and 2. Gel electrophoresis of the amplied fragment of mecA gene. Lanes: M, 100 bp DNA ladder (Fermentas Laboratories); 1, MRSA standard strain ATCC 33592 M2-M15.; MRSA clinical isolates.

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Table 1 Antimicrobial activity of Thymus vulgaris and Eucalyptus globulus essential oils against tested bacteria. Test microorganisms Thymus vulgaris Inhibition zone (mm) Minimum Inhibitory concentration (MIC) (mg/ml) 37 9.25 55.50 18.50 18.50 37 37 18.50 18.50 18.50 18.50 37 37 37 37 37 37 37 18.50 Eucalyptus globulus Inhibition zone (mm) Minimum Inhibitory concentration (MIC) (mg/ml) 85.6 51.36 68.48 85.6 51.36 34.24 34.24 34.24 8.56 34.24 51.36 85.6 34.24 85.6 34.24 85.6 34.24 51.36 85.6 Antibioticsa Inhibition zone (mm)

Bacillus cereus ATCC 9634 Escherchia coli ATCC 25922 Klebsiella pneumoniae ATCC 13883 MRSA ATCC 33592 Staphylococcus aureus ATCC 25922 M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14

16 15 12 12 19 22 20 28 28 35 24 25 21 24 23 31 29 21 27

11 7 8 8 17 13 16 20 19 30 18 15 17 22 10 23 21 15 19

38(IMI), 25(Cip), 25(T) 23(TS), 22(GM), 28(NOR) 19(GM), 28(Cip)19(SXT) 15(VA) 0(GM)0(TS) 40(IMI), 16(VA) 21(VA), 0(ME), 0(GM), 22(C) 18(VA), 7(ME) 0(GM), 23(C) 16(VA), 7(ME) 0(GM), 23(C) 19(VA) 7(ME) 0(GM) 19(C) 17(VA) 7(ME) 0(GM) 22(C) 16(VA), 7(ME) 0(GM) 19(C) 19(VA), 7(ME)0(GM), 17(C) 18(VA), 8(ME) 0(GM), 23(C) 19(VA), 0(ME) 0(GM), 24(C) 19(VA), 7(ME) 0(GM), 23(C) 19(VA) 9(ME) 0(GM), 24(C) 21(VA) 0(ME) 7(GM), 21 (C) 16(VA) 0(ME) 0(GM), 24(C) 22(VA), 7(ME) 0(GM), 25(C)

a Abbreviations; Standard antibiotic disks: imipenem, IMI; tetracycline, T; ciprooxacin, Cip; cotrimoxazole,TS; gentamicin, GM; noroxacin, NOR, trimethoprimesulfamethoxazol, SXT; Ticarcillin, TN; carbenicillin, CB; vancomycine, VA; methicillin, ME; chloramphenicol, C.

containing 104 of each tested bacteria were incubated at 37 1C for 24 hours. The MICs were determined as the oils lowest concentrations in which no visible growth was detected. The presence of one or two colonies was ignored.

Results PCR detection of mecA gene. The PCR procedure was applied to 14 MRSA isolates and a standard MRSA strain ATCC 33592, and yielded in amplication of a 314 bp DNA fragment, compatible to the predicted size designation with no nonspecic background (Figs. 1 and 2). There was a complete agreement between PCR results with those of standard disk diffusion method in which no inhibition zone was visible for any of the tested bacteria. Antibacterial activity. Table 1 shows the antibacterial activity of EOs and also the inhibition zones resulted from standard antibiotic disks. The disk diffusion and agar dilution methods resulted in a semi-quantitative antimicrobial activity between two EOs. Thyme oil had the largest inhibition zones for all tested bacteria followed by the Eucalyptus EO. Results were conrmed by the MIC values (ranging: 0.14 v/v %), in which thyme oil revealed the lower MICs than eucalyptus essential oil. All the bacteria were sensitive to EOs but in variable degrees. There was no growth inhibition due to the effect of the solubelizer (Tween20%) control. Composition of the essential oils. The GC/MS (Thymus) and GC (Eucalyptus) analysis identied major compounds in EOs. Thymol was the most abundant individual compound in thyme oil (48.1%) followed by r-Cymene (15.6%) and g-Terpinene (15.4%). Eucalyptol (47.2%), (+) Spathulenol (18.1%) and a-Pinene (9.6%) were the major compounds of the eucalyptus EO.

et al. 1994). Infection control Management requires a suitable treatment strategy and a rapid resistance identication procedure. PCR was chosen due to its high sensitivity for quick identication of resistant organisms which in case of mecA gene in S. aureus was a valuable add-on to standard susceptibility testing methods. The overuse of antibiotic drugs has led to the extensive antibiotic resistance in human pathogenic bacteria, which highlights the research need on new antimicrobial agents. In this study, T. vulgaris and E. globulus EOs were tested for their putative antibacterial activity against 14 clinical MRSA isolates and 5 standard (Gram (+) and Gram ()) bacteria. Both of EOs showed antibacterial activity at different degrees. The antibacterial activities of these EOs could be associated to the presence of Thymol (48.1%) in T. vulgaris and to Eucalyptol (47.2%) in E. globulus as the most characteristic components of the EOs. In vitro and in vivo examinations should be designed to understand the synergistic effect of the secondary metabolites of the tested plants extracted through their EOs in order to understand the therapeutical effects of herbal drug combinations and overcome the problem of antibacterial drugs resistance along with reducing their disadvantageous side effects (Wagner and Ulrich-Merzenich 2009; Hemaiswarya et al. 2008). Due to variations in plants EOs composition which is affected by their chemotypes, and/or extraction procedure, and also differences in antimicrobial analysis methods and results among publications, it is hard to make a complete comparison between these results with previously issued data. As a conclusion, the present study together with previous analysis supports the antibacterial properties of T. vulgaris and E. globulus EOs and suggests them as antibacterial additives. Additional clinical trials of these oils have to be performed if they are to be used for medicinal purposes.

Conclusion and discussion The methicillin resistance rate in S. aureus isolates varies extensively amongst different sources between 5% to 80% (Geha

Acknowledgment We are thankful of Dr. Pyrmoradi (Tarbiat Modares University, College of Agriculture), and Dr. Jalil fallah Mehrabadi (MARS

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