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Antimicrobial efficacy of Mutellina purpurea essential oil and α-pinene against Staphylococcus epidermidis grown in planktonic and biofilm cultures
Industrial Crops and Products 51 (2013) 152–157
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Antimicrobial efficacy of Mutellina purpurea essential oil and ␣-pinene against Staphylococcus epidermidis grown in planktonic and biofilm cultures Elwira Sieniawska a,∗ , Renata Los b , Tomasz Baj a , Anna Malm b , Kazimierz Glowniak a a b
Department of Pharmacognosy with Medicinal Plant Unit, Medical University of Lublin, 1 Chodzki, PL-20-093 Lublin, Poland Department of Pharmaceutical Microbiology, Medical University of Lublin, 1 Chodzki, PL-20-093 Lublin, Poland
a r t i c l e
i n f o
Article history: Received 11 June 2013 Received in revised form 28 August 2013 Accepted 2 September 2013 Keywords: Staphylococcus epidermidis Mutellina purpurea Biofilm Essential oil
a b s t r a c t The staphylococcal biofilms are the most frequent causes of nosocomial infections and infections on indwelling medical devices. Staphylococcus epidermidis biofilms exhibit typically up to 1000 times greater resistance to antibiotics than the planktonic cells. Because the essential oils are described as antimicrobial agents, the aim of this study was to investigate the antimicrobial efficacy of Mutellina purpurea essential oil (EO) and ␣-pinene against planktonic and biofilm cultures of S. epidermidis ATCC 35984 and the clinical strain S. epidermidis 37IINL. The obtained values of MIC (minimal inhibitory concentration) were: 0.625 mg/mL of ␣-pinene for both strains, 0.312 and 0.625 mg/mL of EO, while 1.5 and 2.5 mg/mL of thymol as a reference substance for S. epidermidis ATCC 35984 and S. epidermidis 37IINL planktonic cells respectively. The lowest EO concentration completely inhibiting the biofilm formation (MBIC – minimal biofilm inhibitory concentration) for both strains was 0.625 mg/mL; ␣-pinene caused the same result at a slightly higher concentration with MBIC = 1.25 mg/mL. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The growth of microorganisms in the form of an organized structure of biofilm is nowadays a clinical, environmental and industrial problem. There are still attempts to search for effective substances inhibiting the growth of biofilm on biomaterials and industrial areas. Eradication of biofilm requires significantly higher concentrations of antimicrobial agents because of the physical/chemical barrier that provides the intrinsic insensitivity to antimicrobial agents comparing to free planktonic cells (Johansen et al., 1997; Saginur et al., 2006). Staphylococcal biofilms exhibit typically up to 1000 times greater resistance to antibiotics than the planktonic cells (Gilbert et al., 1997; Mah and O’Toole, 2001). This makes biofilms particularly important in clinical settings as their removal presents many difficulties, while their presence often creates serious complications with regard to patient management. The antimicrobial efficacy of essential oils has been known for several years, and many studies have demonstrated activity against bacteria, fungi and viruses (Cowan, 1999). Recently, the increased antimicrobial resistance within the clinical settings is observed. In
this light, the antimicrobial potential of essential oils for the prevention and treatment of infection has been researched in several studies (Al-Shuneigat et al., 2005; Caelli et al., 2000; Dryden et al., 2004; Messager et al., 2005; Warnke et al., 2006). The aim of this study was to investigate the antimicrobial efficacy of Mutellina purpurea essential oil and ␣-pinene against planktonic and biofilm-associated cells of S. epidermidis. 2. Materials and methods 2.1. Plant material M. purpurea herb was collected in the Medicinal Plants Garden of the Medical University of Lublin in the vegetation season of 2011. The identification was done by Michal Hajnos from the Department of Pharmacognosy with the Medicinal Plant Unit (Medical University of Lublin). The voucher specimen has been deposited at the Herbarium of the Department of Pharmacognosy (ES2011MP). 2.2. Essential oil hydrodistillation
∗ Corresponding author at: Medical University of Lublin, Department of Pharmacognosy with Medicinal Plant Unit, Chodzki 1, 20-093 Lublin, Poland. Tel.: +48 817423809; fax: +48 817423809. E-mail address: [email protected] (E. Sieniawska). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.09.001
The fresh plant material (50 g of aerial parts) was placed in the round-bottomed flask and 500 mL of distilled water was added. Hydrodistillation was performed for 3 h using the Deryng apparatus.
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2.3. GC/MS analysis of essential oil The gas chromatograph Varian 450-GC with the type triple qadrupol Varian 320-MS was used. The analytes were separated on a 30 m × 0.25 mm VF-5 ms capillary column coated with a 0.25 m film of 5% phenyl methylpolysiloxane, and they were inserted directly into the ion source of the MS. The split injection 1:100 was used for the samples. The column oven temperature was programmed at 4 ◦ C/min from an initial temperature of 50 ◦ C (kept for min) to 250 ◦ C, which was kept for 10 min. The injection temperature was 250 ◦ C and the injection volume was 1 l. Helium (99.999%) was used as carrier gas at a flow rate of 0.5 mL/min. The ionizing electron energy was 70 eV and the mass range scanned was 40–1000 m/z with 0.8 s/scan. Manifold temp. was 45 ◦ C, transfer line temp. was 289.5 ◦ C and the ion source temp. was 271.2 ◦ C.
2.4. Qualitative analysis of essential oil The qualitative analysis was carried out on the basis of MS spectra, which were compared with the spectra of the NIST library (2002) and with the data available in the literature (Adams, 2001; Joulain and König, 1998). The identity of the compounds was confirmed by their retention indices (Van Den Dool and Kratz, 1963), taken from the literature, (Adams, 2001; Joulain and König, 1998) and our own data for standards (␣-pinene, p-cymene, limonene, ␥terpinene, linalool, (E)-caryophyllene, caryophyllene oxide; (Fluka, Sigma–Aldrich Chemie GmbH, Germany).
2.5. Quantitative analysis of essential oil Essential oil was diluted 100 times using n-hexane to achieve 1 mL volume, then 100 L dodecane C12 as internal standards (1 mg/mL in toluene) was added into the diluted oil. The sample prepared in this way was subjected to GC–MS determinations. The quantitative analyses of essential oil was performed on the basis of calibration curves plotted to find the dependence between the ratio of the peak area for the analyte to the area for internal standard (Aanalyte:Ai.s.) vs. the analyte concentration (Canalyte), for p- cymene, ␥-terpinene, linalool, (E)-caryophyllene, caryophyllene oxide, in appropriate concentration range (Kowalski and Wawrzykowski, 2009). The contents of the analyzed substances were read from the achieved calibration curves, the data for which originated from the peak areas for M. purpurea oil components and internal standard peak areas from GC separation. The final result took into account all dilutions during the whole analytical procedure. For comparative purposes, the percentage of components of the M. purpurea essential oil was presented, assuming that the sum of the peak areas for all identified constituents was 100%. All determinations were conducted in triplicate, and all results were calculated as mean ± standard deviation (SD).
2.6. Microorganisms The two reference strains of Staphylococcus epidermidis from the American Type Culture Collection – S. epidermidis ATCC 12228 (showing no ability to form a biofilm as a negative control) and S. epidermidis ATCC 35984 (having the ability to form a biofilm as a positive control) and the clinical strain of S. epidermidis37IINL (having the ability to form a biofilm) isolated from the nose of a patient with lung cancer were used. This strain was chosen from the collection of S. epidermidis strains described elsewhere (Juda et al., 2005).
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2.7. Determination of MICs and MBCs of M. purpurea essential oil (EO), ˛-pinene and thymol Susceptibility testing of planktonic cells of S. epidermidis strains was performed by the broth microdilution assay according to Skalicka-Wozniak et al. (2010). The stock solutions of the tested substances, i.e. EO, ␣-pinen or thymol were prepared in DMSO (20 mg/mL). Then, the two-fold dilutions of this stock solution (ranged from 0.156 to 5 mg/mL) in Tryptic soy broth (Biocorp, Poland) were made and put into the sterile 96-well polystyrene plates (200 L per well). All strains were stored at −70 ◦ C in Nutrient broth (Biocorp, Poland) containing 16% (v/v) glycerol until used. Before the experiments, each bacterial strain was subcultured on fresh Trypticase soy agar (Biocorp, Poland) at 35 ◦ C for 24 h. Inocula (0.5 McFarland standard – approximately 150 × 106 Colony Forming Units – CFU/mL) were prepared using sterile physiological saline. 2 L of the bacterial inocula were added to each well. After the 24 h incubation at 35 ◦ C the MIC (minimal inhibitory concentration) values were assessed visually as the lowest concentrations of the tested compounds showing complete bacterial growth inhibition. Appropriate DMSO growth and sterile controls were carried out. Gentamicin was used as a reference compound. After MICs reading, MBC (minimal bactericidal concentration) of the tested substances towards staphylococcal strains were determined by subculturing 10 L from each well that showed complete growth inhibition onto Trypticase soy agar. After the incubation (35 ◦ C for 24 h), the MBC values were defined as the lowest concentration of the compound at which there was no bacterial growth. The experiments were carried out in triplicates. Representative data are presented. 2.8. Effect of M. purpurea essential oil (EO) and ˛-pinene on prevention of biofilm formation by S. epidermidis – determination of MBIC The sterile 96-well polystyrene plates were filled with 200 L of Tryptic Soy Broth (TSB) with EO or ␣-pinene in the appropriate concentrations (0.156–2.5 mg/mL). The stock solutions of the EO and ␣-pinene were prepared in DMSO. Then, 2 L of the bacterial inocula of density corresponding to 0.5 McFarland standard, prepared as describes above, were added to each well. After the 24 h incubation at 37 ◦ C, the formed biofilm was rinsed twice with sterile phosphate buffer saline (PBS) to remove the non-adherent cells. The attached bacteria were stained with safranine (for 10 min). Then the biofilm was rinsed with PBS solution to remove the dye. Sodium chloride (100 L) was added to each well and the plate was sonicated to resuspend the bacterial cells. Absorbance measured at = 490 nm using a microtitre plate reader (Epoch, BioTek Instruments, USA), was expressed as optical density (OD490). The minimal biofilm inhibitory concentration (MBIC) was calculated by comparing the obtained values of OD490 with those for the negative control – S. epidermidis ATCC 12228. The experiments were carried out in triplicates. Data are presented as the mean ± SD from at least three independent experiments. 2.9. Effect of M. purpurea essential oil and ˛-pinene on eradication of S. Epidermidis biofilm – determination of MBRC Biofilms of each strain were grown in the sterile 96-well microtitre plates (24 h incubation at 37 ◦ C as described above using bacterial inoculum of density corresponding to 0.5 McFarland standard). To remove any unbound cells the biofilm was rinsed with PBS solution. Then 100 L of EO and ␣-pinene in the appropriate concentrations (2, 5–10 mg/mL) were added. The stock solutions of the EO and ␣-pinene were prepared in DMSO. After 24 h of incubation 37 ◦ C the antimicrobial agents were removed and the wells
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Fig. 1. Antibacterial activity of essential oil from Mutellina purpurea (EO) and ␣pinene on S. epidermidis ATCC 35984 planktonic cells. MIC (minimal inhibitory concentration) and MBC (minimal bactericidal concentration) [mg/mL]. Thymol was used as a reference compound. The experiments were carried out in triplicates. Representative data are presented.
Fig. 3. Effect of essential oil from Mutellina purpurea (EO) on S. epidermidis biofilm. The experiments were carried out in triplicates. Data are presented as the mean ± SD from at least three independent experiments.
washed twice with 200 L of sterile PBS. To detect the presence of biofilm the bacteria were stained with safranine (for 10 min). Then the biofilm was rinsed with PBS solution to remove the dye. Sodium chloride (100 L) was added to each well and the plate was sonicated to resuspend the bacterial cells. Absorbance was measured OD490. The minimal biofilm reduction concentration (MBRC) was calculated by comparing the obtained values of OD490 to those for the negative control – S. epidermidis ATCC 12228 (Nuryastuti et al., 2009). The experiments were carried out in triplicates. Data are presented as the mean ± SD from at least three independent experiments. 3. Results The chemical composition of analyzed essential oil is shown in Table 1. ␣-pinene was the predominant compound (about 10%) followed by sabinene, -elemene, myrcen and -pinene. In a study concerning the antimicrobial activity of M. purpurea essential oil ␣pinene was chosen as a reference compound because it is one of the main components of the tested oil. Also a thymol was used, which is a typical antimicrobial compound derived from essential oils (Mathela et al., 2010). Among the substances tested (Figs. 1 and 2 – S. epidermidis 37IINL and S. epidermidis ATCC 35984 planktonic cells, respectively), lower MIC values for two staphylococcal strains were obtained for EO and ␣-pinene than that for thymol. MBC values for these two substances were also lower in comparison with thymol MBC value. The MBC/MIC ratio amounting to 2 or 4, depending on the substance and strain tested, shows a bactericidal activity of EO, ␣-pinene, and thymol. Based on these results, in the next step EO and ␣-pinene were used.
Fig. 2. Antibacterial activity of essential oil from Mutellina purpurea (EO) and ␣-pinene on S. epidermidis 37IINL planktonic cells. MIC (minimal inhibitory concentration) and MBC (minimal bactericidal concentration) [mg/ml]. Thymol was used as a reference compound. The experiments were carried out in triplicates. Representative data are presented.
Fig. 4. Effect of ␣-pinene on S. epidermidis biofilm. The experiments were carried out in triplicates. Data are presented as the mean ± SD from at least three independent experiments.
Figs. 3 and 4 show the results of the EO and ␣-pinene effect on biofilm formation by both strains of S. epidermidis. MBIC of EO for both strains was 0.625 mg/mL. ␣-pinene caused the same result at a slightly higher concentration with MBIC = 1.25 mg/mL. As shown in Figs. 5 and 6, none of the tested concentrations of the EO and ␣-pinene resulted in a complete eradication of staphylococcal biofilms, not allowing to calculate of MBRC. However, the tested substances in concentrations of 1.25–10 mg/mL, caused the partial removal of biofilm. The degree of eradication was proportional to the concentration. More sensitive to the action of the tested compounds was biofilm of S. epidermidis strain 37IINL whereas EO was characterized by greater efficiency comparing with a single ingredient, ␣-pinene.
Fig. 5. Eradication of the mature biofilm of S. epidermidis strains by essential oil from Mutellina purpurea (EO). The experiments were carried out in triplicates. Data are presented as the mean ± SD from at least three independent experiments.
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Table 1 Chemical composition of M. purpurea EO.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72.
Compound
RI
Percentage in the EO ± SD
Tricyclene ␣-Thujene ␣-Pinene Camphene Sabinene -Pinene Myrcen ␦-2-Carene ␣-Terpinene para-Cymene Limonene -Phellandrene (Z)--ocimene (E)--ocimene ␥-Terpinene (Z)-sabinene hydrate Terpinolene meta-Cresol Perillene Linalool (E)-sabinene hydrate Octen-3-yl acetate <1-> Octanol acetate<3-> (Z)-para-Menth-2-en-1-ol ␣-Campholenal (E)-para-Menth-2-en-1-ol (Z)-Verbenol (E)-Verbenol -Pinene oxide (Z)-para-Mentha-1,5-dien-8-ol Bornel Terpinen-4-ol Cryptone ␣-Terpineol Verbenone Pulegone (Z)-sabinene hydrate acetate Lavandulyl acetate Bornyl acetate Thujyl acetate<-3-> Terpinen-4-ol acetate ␣-Copaene Daucene -Bourbonene -Elemene Sesquithujene<7-epi-> Metyl eugenol 2.5-dimethoxy-para-Cymene (E)-Caryophyllene -Copaene (Z)--farnesene ␣-Humulene Sesquicineole <7-epi-1,2-dehydro-> ␥-Muurolene d-germacrene 1,3,6,10-Dodecatetraene, 3,7,11-trimethyl-Selinene ␣-Selinene ␣-Bulnesene A-germacrene ␥-Cadinene ␦-Amorphene (Z)-␣-bisabolene (Z)-sesquisabinene hydrate (E)-nerolidol Spathulenol Caryophyllene oxide Globulol Humulene epoxide II -Atlantol -Acorenol epi-␣-Muurolol
925 932 939 954 975 980 989 1012 1017 1024 1027 1029 1033 1043 1055 1068 1071 1093 1104 1105 1108 1110 1122 1132 1136 1150 1154 1156 1170 1172 1182 1182 1199 1201 1219 1242 1256 1287 1290 1305 1318 1381 1384 1390 1393 1405 1410 1418 1427 1437 1456 1464 1473 1482 1489 1493 1498 1504 1510 1516 1521 1531 1545 1564 1566 1589 1594 1598 1622 1624 1645 1658
0.1 0.3 9.8 1.1 8.6 1.4 7.1 1.0 0.1 1.5 3.2 1.1 0.2 0.2 0.2 0.1 0.1 0.1 0.1 0.6 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.4 0.2 0.1 0.1 1.4 0.3 0.4 0.2 0.1 0.1 0.3 1.4 0.3 0.1 0.2 0.1 0.1 9.2 0.1 0.3 0.2 3.1 0.1 1.6 0.5 1.2 0.9 2.5 0.5 1.0 1.2 0.5 1.4 0.2 0.5 0.3 0.7 0.8 5.1 1.9 0.7 0.5 0.8 0.8 3.0
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.0 0.0 0.1 0.0 0.2 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0
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Table 1 (Continued)
73. 74. 75. 76. 77. 78. 79.
Compound
RI
Percentage in the EO ± SD
␣-Muurolol Alloaromadendrene Bulnesol ␣-Bisabolol Khusinol (Z)-farnesol (Z)-␣-bisabolene epoxide
1668 1672 1691 1703 1704 1712 1726
1.4 1.2 0.6 5.5 0.3 0.4 1.8
Total
± ± ± ± ± ± ±
0.0 0.0 0.0 0.1 0.0 0.0 0.0
92.8
RI – experimental retention indices.
Fig. 6. Eradication of the mature biofilm of S. epidermidis strains by ␣-pinene. The experiments were carried out in triplicates. Data are presented as the mean ± SD from at least three independent experiments.
4. Discussion This is a first report concering the antimicrobial activity of M. purpurea essential oil or ␣-pinene against S. epidermidis when growing both in suspension and as biofilm. The MIC values determined for planktonic cells of S. epidermidis treated with M. purpurea essential oil – 0.625 and 0.325 mg/mL for S. epidermidis ATCC 35982 and S. epidermidis 37IINL, respectively and one of the main components of this oil: ␣-pinene – 0.625 mg/mL for both strains suggest that ␣-pinene may be responsible for the antimicrobial activity of essential oil. However, somewhat higher activity of essential oil as compared to that of ␣-pinene may be due to synergism between a-pinene and other compounds of essential oil. Similar synergistic effects between components of essential oil were also observed by other authors. Lachowicz et al. (1998) reported the results for the antimicrobial properties of crude basil oil and its two main components linalool and methyl chavicol, while Hendry et al. (2009) observed this relationship in the case of 1,8-cineole and eucalyptus oil. Essential oils and their components have been studied for anti-adhesive and anti biofilm properties against selected strains of microorganisms, which are etiologic agents of infections associated with biofilm formation. The influence of some essential oils and their components on S. epidermidis biofilms have been also deter´ mined (Budzynska et al., 2011; Hendry et al., 2009; Lachowicz et al., 1998). Karpanen et al. (2008) described the activity of the tea tree oil, eucalyptus oil and thymol against selected strains of S. epidermidis, determining the MIC and MBC values for planktonic and biofilm cells. The biofilm forming S. epidemidis RP62A, (described by Sadovskaya et al., 2005) and the clinical isolate TK1 were used. Among the substances tested, thymol was characterized as having the highest efficiency. The MIC values of thymol for planktonic cells were 4 and 0.5 mg/mL and the MBC values were 16 or 4 mg/mL (for RP62ATK1 respectively). The MIC values of biofilm cells obtained for thymol were corresponding MBIC values (0.5 mg/ml for both strains), and MBC values were corresponding MBRC values (2 or
8 mg/mL for both strains). Concentrations of ␣-pinene and EO needed to eradicate existing biofilm of S. epidermidis (>10 mg/ml) were much higher than the MIC values obtained for free planktonic cells (0.625 mg/mL) and concentrations needed for preservation of biofilm formation (0.625 mg/mL). Preventing of biofilm formation by essential oils and its components may be due to inhibition of adhesion. It is crucial, because it stops further development of the biofilm. According to Karpanen et al. (2008) thymol showed higher activity against S. epidermidis growing as a biofilm compared with planktonic cells. Thymol is a phenolic compound that both hydrophilic and hydrophobic properties, which may enhance diffusion of this compound to a biofilm and allow its access to bacterial cells where it alters the permeability of plasma membranes (Nostro et al., 2007). However, a similar phenomenon was not observed in the case of S. epidermidis strains, used in the present study, although a sensitivity of planktonic cells to thymol was similar. This phenomenon was not observed neither in the case of M. purpurea essential oil and ␣-pinene. Essential oils are valuable antimicrobial agents because of a low risk of resistance development. This is related to the complex chemical composition of the essential oils, depending on the plant chemotype. Even when the bacteria produce a mechanism of resistance, the composition of each batch of the oil may be slightly different, what limits the selection of resistant strains, and thus determines the sensitivity of bacteria to the essential oils (Reichling et al., 2009). 5. Conclusion The coagulase-negative staphylococci and, in particular, S. epidermidis, have emerged as major nosocomial pathogens associated with infections of implanted medical devices. Present study explored the promising bactericidal activity of M. purpurea essential oil against staphylococcal biofilm formation. Among samples tested, EO showed the best activity. S. epidermidis 37IINL was twofold more susceptible then S. epidermidis ATCC 35982. Concentrations of ␣-pinene and EO needed to eradicate existing biofilm were much higher than the MIC values obtained for free planktonic cells. It was also higher then concentrations needed for inhibition of biofilm formation. M. purpurea EO could be considered as effective factor preventing biofilm formation. In addition to antibiotic prophylaxis, preventive strategies to control S. epidermidis medical device-related infections are focusing on the development of improved biomaterials which can be coated or soaked with compounds preventing the adhesion, the initial stage of biofilm formation. References Adams, R.P., 2001. Identification of Essential Oil Compounds by Gas Chromatography–Quadrupole Mass Spectroscopy. Allured, Carol Stream, IL. Al-Shuneigat, J., Cox, S.D., Markham, J.L., 2005. Effects of a topical essential oilcontaining formulation on biofilm-forming coagulase-negative staphylococci. Lett. Appl. Microbiol. 41, 52–55.
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Mah, T.C., O’Toole, G.A., 2001. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol. 9, 34–39. Mathela, C.S., Singh, K.K., Gupta, V.K., 2010. Synthesis and in vitro antibacterial activity of thymol and carvacrol derivatives. Acta Pol. Pharm. 67, 375–438. Messager, S., Hammer, K.A., Carson, C.F., Riley, T.V., 2005. Effectiveness of handcleansing formulations containing tea tree oil assessed ex vivo on human skin and in vivo with volunteers using European standard EN 1499. J. Hosp. Infect. 59, 220–228. Nostro, A., Roccaro, A.S., Bisignano, G., Marino, A., Cannatelli, M.A., Pizzimenti, F.C., Cioni, P.L., Procopio, F., Blanco, A.R., 2007. Effects of oregano, carvacrol and thymol on Staphylococcus aureus and Staphylococcus epidermidis biofilms. J. Med. Microbiol. 56, 519–523. Nuryastuti, T., Van der Mei, H.C., Busscher, H.J., Iravati, S., Aman, A.T., Krom, B.P., 2009. Effect of cinnamon oil on icaA expression and biofilm formation by Staphylococcus epidermidis. Appl. Environ. Microb. 75, 6850–6855. Reichling, J., Schnitzler, P., Suschke, U., Saller, R., 2009. Essential oils of aromatic plants with antibacterial, antifungal, antiviral, and cytotoxic properties, an overview. Forsch Komplementmed. 16, 79–90. Sadovskaya, I., Vinogradov, E., Flahaut, S., Kogan, G., Jabbouri, S., 2005. Extracellular carbohydrate containing polymers of a model biofilm-producing strain Staphylococcus epidermidis RP62A. Infect. Immun. 73, 3007–3017. Saginur, R., StDenis, M., Ferris, W., Aaron, S.D., Chan, F., Lee, C., Ramotar, K., 2006. Multiple combination bactericidal testing of staphylococcal biofilms from implant-associated infections. Antimicrob. Agents Chemother. 50, 55–61. Skalicka-Wozniak, K., Los, R., Glowniak, K., Malm, A., 2010. Antimicrobial activity of fatty acids from fruits of Peucedanum cervaria and P. alsaticum. Chem. Biodivers. 7, 2748–2754. Van Den Dool, H., Kratz, P.D., 1963. A generalization the retention index system including liner temperature programed gas–liquid partition chromatography. J. Chromatogr. 11, 463–471. Warnke, P.H., Sherry, E., Russo, P.A., Acil, Y., Wiltfang, J., Sivananthan, S., Sprengel, M., Roldan, J.C., Schubert, S., Bredee, J.P., Springer, I.N., 2006. Antibacterial essential oils in malodorous cancer patients: clinical observations in 30 patients. Phytomedicine 13, 463–467.
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Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Antimicrobial efficacy of Mutellina purpurea essential oil and ␣-pinene against Staphylococcus epidermidis grown in planktonic and biofilm cultures Elwira Sieniawska a,∗ , Renata Los b , Tomasz Baj a , Anna Malm b , Kazimierz Glowniak a a b
Department of Pharmacognosy with Medicinal Plant Unit, Medical University of Lublin, 1 Chodzki, PL-20-093 Lublin, Poland Department of Pharmaceutical Microbiology, Medical University of Lublin, 1 Chodzki, PL-20-093 Lublin, Poland
a r t i c l e
i n f o
Article history: Received 11 June 2013 Received in revised form 28 August 2013 Accepted 2 September 2013 Keywords: Staphylococcus epidermidis Mutellina purpurea Biofilm Essential oil
a b s t r a c t The staphylococcal biofilms are the most frequent causes of nosocomial infections and infections on indwelling medical devices. Staphylococcus epidermidis biofilms exhibit typically up to 1000 times greater resistance to antibiotics than the planktonic cells. Because the essential oils are described as antimicrobial agents, the aim of this study was to investigate the antimicrobial efficacy of Mutellina purpurea essential oil (EO) and ␣-pinene against planktonic and biofilm cultures of S. epidermidis ATCC 35984 and the clinical strain S. epidermidis 37IINL. The obtained values of MIC (minimal inhibitory concentration) were: 0.625 mg/mL of ␣-pinene for both strains, 0.312 and 0.625 mg/mL of EO, while 1.5 and 2.5 mg/mL of thymol as a reference substance for S. epidermidis ATCC 35984 and S. epidermidis 37IINL planktonic cells respectively. The lowest EO concentration completely inhibiting the biofilm formation (MBIC – minimal biofilm inhibitory concentration) for both strains was 0.625 mg/mL; ␣-pinene caused the same result at a slightly higher concentration with MBIC = 1.25 mg/mL. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The growth of microorganisms in the form of an organized structure of biofilm is nowadays a clinical, environmental and industrial problem. There are still attempts to search for effective substances inhibiting the growth of biofilm on biomaterials and industrial areas. Eradication of biofilm requires significantly higher concentrations of antimicrobial agents because of the physical/chemical barrier that provides the intrinsic insensitivity to antimicrobial agents comparing to free planktonic cells (Johansen et al., 1997; Saginur et al., 2006). Staphylococcal biofilms exhibit typically up to 1000 times greater resistance to antibiotics than the planktonic cells (Gilbert et al., 1997; Mah and O’Toole, 2001). This makes biofilms particularly important in clinical settings as their removal presents many difficulties, while their presence often creates serious complications with regard to patient management. The antimicrobial efficacy of essential oils has been known for several years, and many studies have demonstrated activity against bacteria, fungi and viruses (Cowan, 1999). Recently, the increased antimicrobial resistance within the clinical settings is observed. In
this light, the antimicrobial potential of essential oils for the prevention and treatment of infection has been researched in several studies (Al-Shuneigat et al., 2005; Caelli et al., 2000; Dryden et al., 2004; Messager et al., 2005; Warnke et al., 2006). The aim of this study was to investigate the antimicrobial efficacy of Mutellina purpurea essential oil and ␣-pinene against planktonic and biofilm-associated cells of S. epidermidis. 2. Materials and methods 2.1. Plant material M. purpurea herb was collected in the Medicinal Plants Garden of the Medical University of Lublin in the vegetation season of 2011. The identification was done by Michal Hajnos from the Department of Pharmacognosy with the Medicinal Plant Unit (Medical University of Lublin). The voucher specimen has been deposited at the Herbarium of the Department of Pharmacognosy (ES2011MP). 2.2. Essential oil hydrodistillation
∗ Corresponding author at: Medical University of Lublin, Department of Pharmacognosy with Medicinal Plant Unit, Chodzki 1, 20-093 Lublin, Poland. Tel.: +48 817423809; fax: +48 817423809. E-mail address: [email protected] (E. Sieniawska). 0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.09.001
The fresh plant material (50 g of aerial parts) was placed in the round-bottomed flask and 500 mL of distilled water was added. Hydrodistillation was performed for 3 h using the Deryng apparatus.
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2.3. GC/MS analysis of essential oil The gas chromatograph Varian 450-GC with the type triple qadrupol Varian 320-MS was used. The analytes were separated on a 30 m × 0.25 mm VF-5 ms capillary column coated with a 0.25 m film of 5% phenyl methylpolysiloxane, and they were inserted directly into the ion source of the MS. The split injection 1:100 was used for the samples. The column oven temperature was programmed at 4 ◦ C/min from an initial temperature of 50 ◦ C (kept for min) to 250 ◦ C, which was kept for 10 min. The injection temperature was 250 ◦ C and the injection volume was 1 l. Helium (99.999%) was used as carrier gas at a flow rate of 0.5 mL/min. The ionizing electron energy was 70 eV and the mass range scanned was 40–1000 m/z with 0.8 s/scan. Manifold temp. was 45 ◦ C, transfer line temp. was 289.5 ◦ C and the ion source temp. was 271.2 ◦ C.
2.4. Qualitative analysis of essential oil The qualitative analysis was carried out on the basis of MS spectra, which were compared with the spectra of the NIST library (2002) and with the data available in the literature (Adams, 2001; Joulain and König, 1998). The identity of the compounds was confirmed by their retention indices (Van Den Dool and Kratz, 1963), taken from the literature, (Adams, 2001; Joulain and König, 1998) and our own data for standards (␣-pinene, p-cymene, limonene, ␥terpinene, linalool, (E)-caryophyllene, caryophyllene oxide; (Fluka, Sigma–Aldrich Chemie GmbH, Germany).
2.5. Quantitative analysis of essential oil Essential oil was diluted 100 times using n-hexane to achieve 1 mL volume, then 100 L dodecane C12 as internal standards (1 mg/mL in toluene) was added into the diluted oil. The sample prepared in this way was subjected to GC–MS determinations. The quantitative analyses of essential oil was performed on the basis of calibration curves plotted to find the dependence between the ratio of the peak area for the analyte to the area for internal standard (Aanalyte:Ai.s.) vs. the analyte concentration (Canalyte), for p- cymene, ␥-terpinene, linalool, (E)-caryophyllene, caryophyllene oxide, in appropriate concentration range (Kowalski and Wawrzykowski, 2009). The contents of the analyzed substances were read from the achieved calibration curves, the data for which originated from the peak areas for M. purpurea oil components and internal standard peak areas from GC separation. The final result took into account all dilutions during the whole analytical procedure. For comparative purposes, the percentage of components of the M. purpurea essential oil was presented, assuming that the sum of the peak areas for all identified constituents was 100%. All determinations were conducted in triplicate, and all results were calculated as mean ± standard deviation (SD).
2.6. Microorganisms The two reference strains of Staphylococcus epidermidis from the American Type Culture Collection – S. epidermidis ATCC 12228 (showing no ability to form a biofilm as a negative control) and S. epidermidis ATCC 35984 (having the ability to form a biofilm as a positive control) and the clinical strain of S. epidermidis37IINL (having the ability to form a biofilm) isolated from the nose of a patient with lung cancer were used. This strain was chosen from the collection of S. epidermidis strains described elsewhere (Juda et al., 2005).
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2.7. Determination of MICs and MBCs of M. purpurea essential oil (EO), ˛-pinene and thymol Susceptibility testing of planktonic cells of S. epidermidis strains was performed by the broth microdilution assay according to Skalicka-Wozniak et al. (2010). The stock solutions of the tested substances, i.e. EO, ␣-pinen or thymol were prepared in DMSO (20 mg/mL). Then, the two-fold dilutions of this stock solution (ranged from 0.156 to 5 mg/mL) in Tryptic soy broth (Biocorp, Poland) were made and put into the sterile 96-well polystyrene plates (200 L per well). All strains were stored at −70 ◦ C in Nutrient broth (Biocorp, Poland) containing 16% (v/v) glycerol until used. Before the experiments, each bacterial strain was subcultured on fresh Trypticase soy agar (Biocorp, Poland) at 35 ◦ C for 24 h. Inocula (0.5 McFarland standard – approximately 150 × 106 Colony Forming Units – CFU/mL) were prepared using sterile physiological saline. 2 L of the bacterial inocula were added to each well. After the 24 h incubation at 35 ◦ C the MIC (minimal inhibitory concentration) values were assessed visually as the lowest concentrations of the tested compounds showing complete bacterial growth inhibition. Appropriate DMSO growth and sterile controls were carried out. Gentamicin was used as a reference compound. After MICs reading, MBC (minimal bactericidal concentration) of the tested substances towards staphylococcal strains were determined by subculturing 10 L from each well that showed complete growth inhibition onto Trypticase soy agar. After the incubation (35 ◦ C for 24 h), the MBC values were defined as the lowest concentration of the compound at which there was no bacterial growth. The experiments were carried out in triplicates. Representative data are presented. 2.8. Effect of M. purpurea essential oil (EO) and ˛-pinene on prevention of biofilm formation by S. epidermidis – determination of MBIC The sterile 96-well polystyrene plates were filled with 200 L of Tryptic Soy Broth (TSB) with EO or ␣-pinene in the appropriate concentrations (0.156–2.5 mg/mL). The stock solutions of the EO and ␣-pinene were prepared in DMSO. Then, 2 L of the bacterial inocula of density corresponding to 0.5 McFarland standard, prepared as describes above, were added to each well. After the 24 h incubation at 37 ◦ C, the formed biofilm was rinsed twice with sterile phosphate buffer saline (PBS) to remove the non-adherent cells. The attached bacteria were stained with safranine (for 10 min). Then the biofilm was rinsed with PBS solution to remove the dye. Sodium chloride (100 L) was added to each well and the plate was sonicated to resuspend the bacterial cells. Absorbance measured at = 490 nm using a microtitre plate reader (Epoch, BioTek Instruments, USA), was expressed as optical density (OD490). The minimal biofilm inhibitory concentration (MBIC) was calculated by comparing the obtained values of OD490 with those for the negative control – S. epidermidis ATCC 12228. The experiments were carried out in triplicates. Data are presented as the mean ± SD from at least three independent experiments. 2.9. Effect of M. purpurea essential oil and ˛-pinene on eradication of S. Epidermidis biofilm – determination of MBRC Biofilms of each strain were grown in the sterile 96-well microtitre plates (24 h incubation at 37 ◦ C as described above using bacterial inoculum of density corresponding to 0.5 McFarland standard). To remove any unbound cells the biofilm was rinsed with PBS solution. Then 100 L of EO and ␣-pinene in the appropriate concentrations (2, 5–10 mg/mL) were added. The stock solutions of the EO and ␣-pinene were prepared in DMSO. After 24 h of incubation 37 ◦ C the antimicrobial agents were removed and the wells
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Fig. 1. Antibacterial activity of essential oil from Mutellina purpurea (EO) and ␣pinene on S. epidermidis ATCC 35984 planktonic cells. MIC (minimal inhibitory concentration) and MBC (minimal bactericidal concentration) [mg/mL]. Thymol was used as a reference compound. The experiments were carried out in triplicates. Representative data are presented.
Fig. 3. Effect of essential oil from Mutellina purpurea (EO) on S. epidermidis biofilm. The experiments were carried out in triplicates. Data are presented as the mean ± SD from at least three independent experiments.
washed twice with 200 L of sterile PBS. To detect the presence of biofilm the bacteria were stained with safranine (for 10 min). Then the biofilm was rinsed with PBS solution to remove the dye. Sodium chloride (100 L) was added to each well and the plate was sonicated to resuspend the bacterial cells. Absorbance was measured OD490. The minimal biofilm reduction concentration (MBRC) was calculated by comparing the obtained values of OD490 to those for the negative control – S. epidermidis ATCC 12228 (Nuryastuti et al., 2009). The experiments were carried out in triplicates. Data are presented as the mean ± SD from at least three independent experiments. 3. Results The chemical composition of analyzed essential oil is shown in Table 1. ␣-pinene was the predominant compound (about 10%) followed by sabinene, -elemene, myrcen and -pinene. In a study concerning the antimicrobial activity of M. purpurea essential oil ␣pinene was chosen as a reference compound because it is one of the main components of the tested oil. Also a thymol was used, which is a typical antimicrobial compound derived from essential oils (Mathela et al., 2010). Among the substances tested (Figs. 1 and 2 – S. epidermidis 37IINL and S. epidermidis ATCC 35984 planktonic cells, respectively), lower MIC values for two staphylococcal strains were obtained for EO and ␣-pinene than that for thymol. MBC values for these two substances were also lower in comparison with thymol MBC value. The MBC/MIC ratio amounting to 2 or 4, depending on the substance and strain tested, shows a bactericidal activity of EO, ␣-pinene, and thymol. Based on these results, in the next step EO and ␣-pinene were used.
Fig. 2. Antibacterial activity of essential oil from Mutellina purpurea (EO) and ␣-pinene on S. epidermidis 37IINL planktonic cells. MIC (minimal inhibitory concentration) and MBC (minimal bactericidal concentration) [mg/ml]. Thymol was used as a reference compound. The experiments were carried out in triplicates. Representative data are presented.
Fig. 4. Effect of ␣-pinene on S. epidermidis biofilm. The experiments were carried out in triplicates. Data are presented as the mean ± SD from at least three independent experiments.
Figs. 3 and 4 show the results of the EO and ␣-pinene effect on biofilm formation by both strains of S. epidermidis. MBIC of EO for both strains was 0.625 mg/mL. ␣-pinene caused the same result at a slightly higher concentration with MBIC = 1.25 mg/mL. As shown in Figs. 5 and 6, none of the tested concentrations of the EO and ␣-pinene resulted in a complete eradication of staphylococcal biofilms, not allowing to calculate of MBRC. However, the tested substances in concentrations of 1.25–10 mg/mL, caused the partial removal of biofilm. The degree of eradication was proportional to the concentration. More sensitive to the action of the tested compounds was biofilm of S. epidermidis strain 37IINL whereas EO was characterized by greater efficiency comparing with a single ingredient, ␣-pinene.
Fig. 5. Eradication of the mature biofilm of S. epidermidis strains by essential oil from Mutellina purpurea (EO). The experiments were carried out in triplicates. Data are presented as the mean ± SD from at least three independent experiments.
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Table 1 Chemical composition of M. purpurea EO.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72.
Compound
RI
Percentage in the EO ± SD
Tricyclene ␣-Thujene ␣-Pinene Camphene Sabinene -Pinene Myrcen ␦-2-Carene ␣-Terpinene para-Cymene Limonene -Phellandrene (Z)--ocimene (E)--ocimene ␥-Terpinene (Z)-sabinene hydrate Terpinolene meta-Cresol Perillene Linalool (E)-sabinene hydrate Octen-3-yl acetate <1-> Octanol acetate<3-> (Z)-para-Menth-2-en-1-ol ␣-Campholenal (E)-para-Menth-2-en-1-ol (Z)-Verbenol (E)-Verbenol -Pinene oxide (Z)-para-Mentha-1,5-dien-8-ol Bornel Terpinen-4-ol Cryptone ␣-Terpineol Verbenone Pulegone (Z)-sabinene hydrate acetate Lavandulyl acetate Bornyl acetate Thujyl acetate<-3-> Terpinen-4-ol acetate ␣-Copaene Daucene -Bourbonene -Elemene Sesquithujene<7-epi-> Metyl eugenol 2.5-dimethoxy-para-Cymene (E)-Caryophyllene -Copaene (Z)--farnesene ␣-Humulene Sesquicineole <7-epi-1,2-dehydro-> ␥-Muurolene d-germacrene 1,3,6,10-Dodecatetraene, 3,7,11-trimethyl-Selinene ␣-Selinene ␣-Bulnesene A-germacrene ␥-Cadinene ␦-Amorphene (Z)-␣-bisabolene (Z)-sesquisabinene hydrate (E)-nerolidol Spathulenol Caryophyllene oxide Globulol Humulene epoxide II -Atlantol -Acorenol epi-␣-Muurolol
925 932 939 954 975 980 989 1012 1017 1024 1027 1029 1033 1043 1055 1068 1071 1093 1104 1105 1108 1110 1122 1132 1136 1150 1154 1156 1170 1172 1182 1182 1199 1201 1219 1242 1256 1287 1290 1305 1318 1381 1384 1390 1393 1405 1410 1418 1427 1437 1456 1464 1473 1482 1489 1493 1498 1504 1510 1516 1521 1531 1545 1564 1566 1589 1594 1598 1622 1624 1645 1658
0.1 0.3 9.8 1.1 8.6 1.4 7.1 1.0 0.1 1.5 3.2 1.1 0.2 0.2 0.2 0.1 0.1 0.1 0.1 0.6 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.4 0.2 0.1 0.1 1.4 0.3 0.4 0.2 0.1 0.1 0.3 1.4 0.3 0.1 0.2 0.1 0.1 9.2 0.1 0.3 0.2 3.1 0.1 1.6 0.5 1.2 0.9 2.5 0.5 1.0 1.2 0.5 1.4 0.2 0.5 0.3 0.7 0.8 5.1 1.9 0.7 0.5 0.8 0.8 3.0
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.0 0.0 0.1 0.0 0.2 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0
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Table 1 (Continued)
73. 74. 75. 76. 77. 78. 79.
Compound
RI
Percentage in the EO ± SD
␣-Muurolol Alloaromadendrene
1668 1672 1691 1703 1704 1712 1726
1.4 1.2 0.6 5.5 0.3 0.4 1.8
Total
± ± ± ± ± ± ±
0.0 0.0 0.0 0.1 0.0 0.0 0.0
92.8
RI – experimental retention indices.
Fig. 6. Eradication of the mature biofilm of S. epidermidis strains by ␣-pinene. The experiments were carried out in triplicates. Data are presented as the mean ± SD from at least three independent experiments.
4. Discussion This is a first report concering the antimicrobial activity of M. purpurea essential oil or ␣-pinene against S. epidermidis when growing both in suspension and as biofilm. The MIC values determined for planktonic cells of S. epidermidis treated with M. purpurea essential oil – 0.625 and 0.325 mg/mL for S. epidermidis ATCC 35982 and S. epidermidis 37IINL, respectively and one of the main components of this oil: ␣-pinene – 0.625 mg/mL for both strains suggest that ␣-pinene may be responsible for the antimicrobial activity of essential oil. However, somewhat higher activity of essential oil as compared to that of ␣-pinene may be due to synergism between a-pinene and other compounds of essential oil. Similar synergistic effects between components of essential oil were also observed by other authors. Lachowicz et al. (1998) reported the results for the antimicrobial properties of crude basil oil and its two main components linalool and methyl chavicol, while Hendry et al. (2009) observed this relationship in the case of 1,8-cineole and eucalyptus oil. Essential oils and their components have been studied for anti-adhesive and anti biofilm properties against selected strains of microorganisms, which are etiologic agents of infections associated with biofilm formation. The influence of some essential oils and their components on S. epidermidis biofilms have been also deter´ mined (Budzynska et al., 2011; Hendry et al., 2009; Lachowicz et al., 1998). Karpanen et al. (2008) described the activity of the tea tree oil, eucalyptus oil and thymol against selected strains of S. epidermidis, determining the MIC and MBC values for planktonic and biofilm cells. The biofilm forming S. epidemidis RP62A, (described by Sadovskaya et al., 2005) and the clinical isolate TK1 were used. Among the substances tested, thymol was characterized as having the highest efficiency. The MIC values of thymol for planktonic cells were 4 and 0.5 mg/mL and the MBC values were 16 or 4 mg/mL (for RP62ATK1 respectively). The MIC values of biofilm cells obtained for thymol were corresponding MBIC values (0.5 mg/ml for both strains), and MBC values were corresponding MBRC values (2 or
8 mg/mL for both strains). Concentrations of ␣-pinene and EO needed to eradicate existing biofilm of S. epidermidis (>10 mg/ml) were much higher than the MIC values obtained for free planktonic cells (0.625 mg/mL) and concentrations needed for preservation of biofilm formation (0.625 mg/mL). Preventing of biofilm formation by essential oils and its components may be due to inhibition of adhesion. It is crucial, because it stops further development of the biofilm. According to Karpanen et al. (2008) thymol showed higher activity against S. epidermidis growing as a biofilm compared with planktonic cells. Thymol is a phenolic compound that both hydrophilic and hydrophobic properties, which may enhance diffusion of this compound to a biofilm and allow its access to bacterial cells where it alters the permeability of plasma membranes (Nostro et al., 2007). However, a similar phenomenon was not observed in the case of S. epidermidis strains, used in the present study, although a sensitivity of planktonic cells to thymol was similar. This phenomenon was not observed neither in the case of M. purpurea essential oil and ␣-pinene. Essential oils are valuable antimicrobial agents because of a low risk of resistance development. This is related to the complex chemical composition of the essential oils, depending on the plant chemotype. Even when the bacteria produce a mechanism of resistance, the composition of each batch of the oil may be slightly different, what limits the selection of resistant strains, and thus determines the sensitivity of bacteria to the essential oils (Reichling et al., 2009). 5. Conclusion The coagulase-negative staphylococci and, in particular, S. epidermidis, have emerged as major nosocomial pathogens associated with infections of implanted medical devices. Present study explored the promising bactericidal activity of M. purpurea essential oil against staphylococcal biofilm formation. Among samples tested, EO showed the best activity. S. epidermidis 37IINL was twofold more susceptible then S. epidermidis ATCC 35982. Concentrations of ␣-pinene and EO needed to eradicate existing biofilm were much higher than the MIC values obtained for free planktonic cells. It was also higher then concentrations needed for inhibition of biofilm formation. M. purpurea EO could be considered as effective factor preventing biofilm formation. In addition to antibiotic prophylaxis, preventive strategies to control S. epidermidis medical device-related infections are focusing on the development of improved biomaterials which can be coated or soaked with compounds preventing the adhesion, the initial stage of biofilm formation. References Adams, R.P., 2001. Identification of Essential Oil Compounds by Gas Chromatography–Quadrupole Mass Spectroscopy. Allured, Carol Stream, IL. Al-Shuneigat, J., Cox, S.D., Markham, J.L., 2005. Effects of a topical essential oilcontaining formulation on biofilm-forming coagulase-negative staphylococci. Lett. Appl. Microbiol. 41, 52–55.
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