Journal of Food, Agriculture & Environment Vol.12 (3&4):

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1 WFL Publisher Science and Technology Meri-Rastilantie 3 B, FI Helsinki, Finland info@world-food.net Journal of Food, Agriculture & Environment Vol.12 (3&4): The in vitro antibiofilm activity of Rosmarinus officinalis L. essential oil against multiple antibiotic resistant Pseudomonas sp. and Staphylococcus sp. Ozgur Ceylan 1 *, Aysel Uğur 2, Nurdan Saraç 3, Filiz Ozcan 3 and Tuba Baygar 3 1 Apiculture Program, Ula Ali Kocman Vocational School, Mugla Sıtkı Koçman University, Ula, Mugla, Turkey. 2 Department of Basic Sciences, Section of Medical Microbiology, Faculty of Dentistry, Gazi University, Emek, Ankara, Turkey. 3 Department of Biology, Faculty of Arts and Sciences, Mugla Sıtkı Koçman University, Kotekli, Mugla, Turkey. * ozgceylan@hotmail.com Received 12 July 2014, accepted 3 September Abstract Rosmarinus officinalis (rosemary) is widely used as a flavouring agent for food and well known medicinally for its chemical composition. The aim of this study was to investigate the antibiofilm activity of the essential oil from Rosmarinus officinalis against biofilm formation of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Bacillus cereus and Bacillus subtilis. Essential oil was obtained from the aerial parts of the plant by using a Clevenger apparatus for 4 h. The antibacterial activity of the obtained essential oil from Rosmarinus officinalis was determined using disc diffusion technique, minimum inhibitory concentrations (MICs) and minimum bacterial concentration (MBC). Staphylococcus sp. and Pseudomonas sp. were evaluated as the resistant microorganisms in the antibacterial assays. The antibiofilm effect of MBC, MIC, MIC/2, MIC/4, MIC/8 and MIC/16 concentrations of Rosmarinus officinalis essential oil was assessed by the microplate biofilm assay. The essential oil exhibited significant antibacterial activity against all tested bacteria. In contrast to the antibacterial activity, MBC and subinhibitory concentrations of essential oil showed limited antibiofilm activity. MBC concentrations of essential oil attenuated the biofilm formation at 60.76% and 74.7% for Staphylococcus aureus MU 47 and Pseudomonas aeruginosa MU 187, respectively. Direct observation by scanning electron microscope (SEM) analysis further revealed an exact reduction for the bacterial biofilm formation in response to the effective concentration. This study has demonstrated that the Rosmarinus officinalis essential oil may be considered as a potent agent for the prevention of biofilm-related applications that are increasingly problematic in the food processing environments and medical industries. Key words: Rosmarinus officinalis, antimicrobial, antibiofilm, Staphylococcus, Pseudomonas. Introduction Rosmarinus officinalis L. (rosemary) is a spice and medicinal herb widely used around the world 1. The fresh and dried leaves are frequently used in traditional Mediterranean cuisine as an additive. They have a bitter, astringent taste, which complements a wide variety of foods. A tisane can also be made from them. They are extensively used in cooking, and a distinct mustard smell gives off while they are burned, therefore, they often are used to flavor foods while barbecuing 2. As medicinal plant rosemary belongs to the pool of herbs, which probably more than others, lies at the boundary between myth, superstition and traditional popular usages, but at the same time, its efficacy is largely acknowledged, being, in fact, listed in the official pharmacopoeia of several countries 3. Historically, rosemary has been used as a medicinal agent to treat renal colic and dysmenorrhea. It has also been used to relieve symptoms caused by respiratory disorders and to stimulate the growth of hair. Extracts of rosemary are used in aromatherapy to treat anxiety-related conditions and to increase alertness 4. The latest research related with rosemary essential oil has mainly focused on its antibacterial 1, 2, 5-18, antioxidant 18-27, antifungal 28, 29, anticancer 30, 31 and antibiofilm properties 12-14, 32. In many ecosystems, bacteria are growing in surfaces as a layer that is called biofilm 33. Bacterial biofilms are associated with a large number of infections. Biofilms are ubiquitous, for example in dental plaque, endocarditis, lung infections, and infections related to the use of medical devices, such as catheters and stents. Many persistent and chronic bacterial infections are now thought to be linked to biofilm formation, over 60% of all bacterial infections have been estimated to involve biofilms 34. Also, outside the medical field, biofilms cause a host of problems such as surface fouling and blocking of equipment 35. Biofilm-dwelling bacteria are particularly resistant to antibiotics, making it hard to eradicate biofilm-associated infections. Earlier investigations on plants and their active constituents have almost exclusively focused on their effects on planktonic bacteria with little emphasis on the more antimicrobial resistant pathogens and difficult to control biofilm forms 32. There are a number of studies focusing on the biological activities of R. officinalis essential oil in recent years, but to our knowledge, fewer comparative studies on antibiofilm activity including against human clinical isolates have been reported. In these studies, there are no study that reveals the antibiofilm activity 82 Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

2 of test bacteria used in our study. The present study reports the antimicrobial and antibiofilm activity of R. officinalis essential oil against multi-antibiotic resistant Staphylococcus spp. and Pseudomonas spp. that cause clinical problems with high biofilm formation. Materials and Methods Plant material: Leaves and flowers of R. officinalis were collected from Mugla, Turkey, in May-July 2012 and a voucher specimen has been deposited in the Herbarium of Mugla Sitki Kocman Univesity. Samples were air-dried at room temperature for 2-4 days. Extraction of essential oil: One hundred gram of dried plant was submitted to hydro-distillation for 4 h using a Clevenger apparatus. Oil was recovered directly, using a micro-pipette from above the distillate without adding any solvent, and stored in dark vials at 4 C. Bacterial strains and culture conditions: The antimicrobial activity of the essential oil was individually tested against a group of bacteria including Bacillus subtilis ATCC 6633, Bacillus cereus RSKK 863, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC and Pseudomonas aeruginosa ATCC Five clinically relevant microorganisms (Staphylococcus aureus MU 38, MU 40, MU 46, MU 47 and Staphylococcus epidermidis MU 30) and three marine microorganisms (Pseudomonas aeruginosa MU 187, MU 189 and Pseudomonas fluorescens MU 180) provided from Mugla Sitki Kocman University Culture Collection (MUKK) were also studied. The above-mentioned bacteria were cultured in nutrient broth (NB) (Difco, USA) at 37±0.1 C for h except from P. aeruginosa and P. fluorescens strains which were incubated at 30±0.1 C for h. Inocula were prepared by adjusting the turbidity of the medium to match the 0.5 McFarland Standard Dilutions. The strains were maintained in their appropriate agar slants at 4 C throughout the study and used as stock cultures. Disc diffusion assay: The antibacterial activity was based on the disc diffusion method 36 using a bacterial cell suspension whose concentration was equilibrated to the 0.5 McFarland standard dilutions. Of each bacterial suspension 0.1 ml was spread on a Mueller-Hinton agar plate. Sterile 6 mm paper discs (Schleicher and Schuell) were impregnated with 10 µl of essential oil. The discs were allowed to dry and were then placed on the inoculated agar. The plates were incubated at appropriate temperature and time for the microorganisms, as mentioned above. At the end of the incubation periods, diameters of no-growth zones around the disks were measured to the nearest 0.1 mm using vernier calipers. The experiments were performed in triplicate. Determination of minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC): The inhibitory and bactericidal activities of the rosemary essential oils were determined by the tube dilution method 37. The MIC was defined as the lowest antibiotic concentration that yielded no visible growth. Mueller-Hinton Broth was used as the test medium and the density of bacteria was colony-forming units (CFU)/ml. Cell suspensions (100 µl) were inoculated into the wells of 96-well microtitre plates (Nunc F96 MİKroWell plates; NunclonTM, Denmark) in the presence of essential oil with different final concentrations ( µl/ml). The essential oil was dissolved in DMSO (Sigma, USA) and serially diluted 2-fold in MHB to give final concentrations. Negative controls (bacteria+mhb), positive controls (bacteria+mhb+essential oil), vehicle controls (bacteria+mhb+dmso) and media controls (MHB) were included. The inoculated microplates were incubated at 37ºC for Staphylococcus spp., Bacillus spp. and at 30ºC for Pseudomonas spp., for 24 h. All experiments were performed in triplicate. The MBC was obtained by subculturing 100 µl of the culture from each tube, in which the MIC assay showing no apparent growth, onto substance-free Mueller-Hinton agar plates. The plates were incubated at 37ºC or 30ºC for 24 h and the MBC was defined as the least concentration that produced subcultures growing maximum five colonies on each plate. Effect of essential oil on bacterial biofilm formation: The effect of subinhibitory concentration of R. officinalis essential oil on biofilm-forming ability of bacteria was tested with a microplate biofilm assay 38. Bacterial strains were inoculated in 2-5 ml of trypticase soy broth (TSB) and growed up to stationary phase. Cultures diluted to 1:100 in TSB, and 100 µl of each dilution was pipetted to four wells in a sterile flat bottom micotiterplate. After incubation at 37ºC for 48 h, the wells were washed with distilled water twice to remove the planktonic bacteria. The remaining bacteria were subsequently stained with 125 µl of 0.1% crystal violet solution (Sigma Chemical Co.) at room temperature. Wells were washed once again to remove the crystal violet solution that is not specifically staining the adherent bacteria. The plates were air-dried and 200 µl of 95% ethanol and 33% glacial acetic acid (Sigma Chemical Co.) were added to each Gram-negative and Grampositive bacteria wells, respectively. Biofilm stains solubilized at room temperature. After shaking and pipetting of wells, 125 µl of the solution from each well was transferred to a sterile tube and the volume was made up to 1 ml with distilled water. Finally, optical density of each well was measured at 550 nm wavelength (Thermo Scientific Multiskan FC, Vantaa, Finland). Negative controls (cells+tsb), positive control (cells+tsb+essential oil), vehicle control (cells+tsb+dmso), and media controls (TSB) were included. Positive controls for essential oil of µl/ml were prepared via serial dilution techniques. Each strain was tested for biofilm formation in duplicate and the assay was repeated three times. Replicate absorbance readings for each concentration were averaged and the average of the media control was subtracted. This value was divided by the mean absorbance of the (cell+tsb) and multiplied by 100. Scanning electron microscopy (SEM): To observe the biofilm formation, glass coverslips were prepared for SEM observation. Coverslips at size of 3 mm x 3 mm were placed in Eppendorf tubes containing 1.5 ml TSB supplemented with 1% glucose and sterilized. Then 300 µl bacterial suspension and 200 µl essential oil were added to reach the final concentration of µl/ml. Eppendorf tubes were incubated at 37 C for Bacillus and Staphylococcus and at 30 C for Pseudomonas, for 48 h. Prior to imaging, the bacteria were fixed and dehydrated. Briefly, the coverslips were Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October

3 gently rinsed twice with 0.01 M PBS and then initially fixed by 2.5% glutaraldehyde at 4 C for 2 h. The surfaces were washed twice with 0.01 M PBS for 1 h. The coverslips were post-fixed with 0.1% osmium tetroxide for 1 h. The bacteria were dehydrated by replacing the buffer with increasing concentrations of ethanol (30%, 50%, 70%, 80%, 90%, 95% and 100%) for 10 min for each. After critical point drying and coating by gold sputter, samples were examined with a scanning electron microscope (JEOL JSM- 7600F; JEOL Ltd., Tokyo, Japan). Statistical analysis: Differences between groups were statistically analyzed using analysis of variance (ANOVA). All experiments were performed in triplicate. Results and Discussion The antimicrobial activity of R. officinalis essential oil was evaluated in vitro against 13 microorganisms which are known to cause human diseases. The measured inhibition zones and MIC/ MBC results of the rosemary essential oil against the test bacteria are given in Table 1. Based on their MIC and MBC values obtained from antimicrobial tests and the percentage inhibition of biofilm formation against the test bacteria are given in Table 2. The antibiofilm activity of different concentrations of R. officinalis essential oil for Gram-positive and Gram-negative test bacteria is shown in Figs. 1 and 2, respectively. The results indicated that the R. officinalis essential oil showed anti-bacterial activity mainly against the Gram-positive bacteria (S. aureus and S. epidermidis), similar to Jiang et al. 2, Jordan et al. 10, Okoh et al. 11, Zaouali et al. 18. The highest antibiofilm activity for the Gram-positive test bacteria was determined against S. aureus MU 47 with 1.25 µl/ml essential oil concentration (MBC) with the inhibition rate by 60.76%. S. aureus MU 47 biofilm formation was reduced to 48.14% by MIC as µl/ml. Due to the decrease in the concentration of essential oil, the biofilm formation of S. aureus MU 47 was inhibited by 33.45%, 14.11% and 8.07% in MIC/2, MIC/ 4 and MIC/8, respectively. The MBC and MIC of S. aureus ATCC were 1.25 and µl/ml, respectively. R. officinalis essential oil in MBC and MIC concentrations reduced the S. aureus ATCC biofilm formation to 58.02% and 27.6%. For S. aureus MU 38, MBC and MIC were 10 and 5 µl/ml and biofilm formation was reduced to 53.83% and 26.43% in these concentrations. Quave et al. 12 reported the effect of R. officinalis extract on planktonic growth, biofilm formation and adherence of methicillin-resistant S. aureus. In this study, MIC 50 value of 512 µg/ml and IC 50 value of 16 µg/ml were reported for R. officinalis extract. Here we provide the first report, to our knowledge, of the demonstration of the antibiofilm activity of R. officinalis essential oil against S. aureus and S. epidermidis. Table 1. Determination of MIC, MBC (µl/ml) and disc diffusion (mm) assay of R. officinalis essential oil. Microorganism Inhibition zone MIC MBC B.s. ATCC B.c. RSKK S.a. ATCC S e. MU S.a. MU S.a. MU S.a. MU S.a. MU P.a. ATCC P.a. ATCC P.f. MU P.a. MU P.a. MU B.s.: B. subtilis; B.c.: B. cereus; S.a.: S. aureus; S.e.: S. epidermidis; P.a.: P. aeruginosa; P.f.: P. fluorescens; -: No inhibition. Table 2. The effect of R. officinalis essential oil on tested bacteria biofilm formation expressed as percentage inhibition. Essential oil concentration Microorganism MBC MIC MIC/2 MIC/4 MIC/8 MIC/16 Percentage (%) inhibition B.s. ATCC B.c. RSKK S.a. ATCC S.e. MU S.a. MU S.a. MU S.a. MU S.a. MU P.a. ATCC P.a. ATCC P.f. MU P.a. MU P.a. MU B.s.: B. subtilis; B.c.: B. cereus; S.a.: S. aureus; S.e.: S. epidermidis; P.a.: P. aeruginosa; P.f.: P. fluorescens; -: No inhibition. % Inhibition Essential oil concentration (µl/ml) Figure 1. The percent inhibition of R. officinalis essential oil for biofilm formation in Gram positive bacteria. % Inhibition Essential oil concentration (µl/ml) Figure 2. The percent inhibition of R. officinalis essential oil for biofilm formation in Gram negative bacteria. 84 Journal of Food, Agriculture & Environment, Vol.12 (3&4), July-October 2014

4 Essential oil of R. officinalis showed similar antimicrobial activity against B. cereus RSKK 863 and B. subtilis ATCC 6633, but the antibiofilm activity of essential oil was more effective against B. subtilis ATCC Our results were similar to those previously reported by Celiktas et al. 8, Jiang et al. 2, Okoh et al. 11, Zaouali et al. 18 for the antimicrobial activity of the R. officinalis essential oil against B. subtilis. A good to moderate antimicrobial activity of R. officinalis essential oil against B. cereus has been reported by Genena et al. 1 and Zaouali et al. 18. The R. officinalis essential oil has also exhibited an antibacterial effect against the Gram-negative bacteria (P. aeruginosa and P. fluorescens). However, this effect was less efficient than that presented against the Gram-positive bacteria, since a higher MIC value was obtained with the Gram-negative bacteria. In this study, the highest MBC and MIC were 80 and 20 µl/ ml against P. aeruginosa ATCC and P. aeruginosa MU 187, respectively. P. aeruginosa MU 187 biofilm formation has been reduced to 74.71% in MBC and 39.49% in MIC. For the same bacteria, the biofilm formation reduced by 19.91% in MIC/2, by 14.38% in MIC/4 and by 5.39% in MIC/8. MIC/16 concentrations of the rosemary essential oil did not reduce the bacterial biofilm formation of the tested bacteria. Biofilm formation of P. aeruginosa ATCC was inhibited by 58.3% in MBC, by 21.83% in MIC and by 7.73% in MIC/2. The results of the antimicrobial activity on Gram-negative bacteria are similar to Celiktas et al. 8 and Jiang et al. 2. In contrast to our results, Zaouali et al. 18 reported that the essential oil of R. officinalis has no antimicrobial activity against P. aeruginosa. The antimicrobial activity of R. officinalis extracts against P. aeruginosa have also been reported by Genena et al. 1 and Sandasi et al. 14. Sandasi et al. 14 showed that R. officinalis extract inhibited 56% of P. aeruginosa biofilm formation. Contrary to this, they also reported that R. officinalis extract was unable to inhibit the growth and development of a pre-formed biofilm of P. aeruginosa. To evaluate the relevance of biofilm formation assay, scanning electron microscopy was employed. Direct observation by scanning electron microscopy of S. aureus MU 47 showed that, in the absence of rosemary essential oil (Fig. 3), bacterial cells formed evident biofilms with matrix material. In the presence of rosemary essential oil at concentrations of 1.25 µl/ml (MBC) bacterial cells grew as looser colonies, and the amount of biofilm was reduced to 60.76% (Fig. 4). Figure 3. Control, S.aureus MU 47 biofilm without R.officinalis essential oil. Figure 4. Scanning electron micrographs showing reduction in S.aureus MU 47 biofilm with 1.25 µl/ml R.officinalis essential oil. Conclusions R. officinalis is a spice and medicinal herb widely used around the world 1. It is widely found in the lands of Aegean and Mediterranean regions of Turkey 8. In this study, the antimicrobial and antibiofilm activities of the R. officinalis essential oil was evaluated. The results obtained are confirmed by SEM observation. According to the results of antimicrobial activity, the R. officinalis essential oil is more active against Gram-positive than Gramnegative bacteria, as evidenced by the lower MIC values for the former. Data showed that R. officinalis essential oil is not only able to kill Staphylococcus spp. and Pseudomonas spp. cells efficiently but also inhibits biofilm formation, but the results also indicate that good antimicrobial activity against planktonic microorganisms does not imply good antibiofilm activity. However, the results of this study show that rosemary essential oil is capable of affecting S. aureus biofilm formation significantly. 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