And Eucalyptus Globulus Essential Oils on Oral Candida Biofilm Formation on Polymethylmethacrylate

Full Length Research Paper

In vitro activity of Melaleuca alternifolia (Tea tree) and Eucalyptus globulus essential oils on oral Candida biofilm formation on polymethylmethacrylate

Noumi Emira 1*, Snoussi Mejdi 2 and Mahjoub Aouni 1

1Laboratoire des Maladies Transmissibles et des Substances Biologiquement Actives, Faculté de Pharmacie, Université de Monastir, Tunisie. 2Laboratoire de Traitement des Eaux Usées, Centre de Recherches et des Technologies des Eaux (CERTE), Technopole de Borj-Cédria, BP 273- Soliman 8020, Tunisie.

Accepted 19 April, 2013

Melaleuca alternifolia and Eucalyptus globulus essential oils are known for their antifungal activities and efficacy in the treatment of oral candidiasis. Candida biofilm increased resistance to antifungal agents that have activity against their planktonic cells. The aim of this study was to evaluate the potential role of M. alternifolia and E. globulus essential oils in the inhibition of Candida biofilm formation on polymethylmethacrylate (PMMA). The antifungal activity of M. alternifolia and E. globulus essential oils and adhesion and biofilm on PMMA inhibition capacity were tested on two oral Candida isolates and two reference type strains. The biofilm formation by Candida strains was quantified by colorimetric method based on the reduction of the 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenyl amino) carbonyl]-2H-tetrazolium hydroxide (XTT). M. alternifolia and E. globulus essential oils were active against clinical and reference Candida albicans and Candida glabrata strains in their planktonic and adherent phases. In fact, both minimum inhibition concentration (MIC) and 1/2 MIC values of these two plants essential oils can inhibit adhesion and biofilm formation of clinical and reference strains of Candida on PMMA. Also, E. globulus essential oil was more active on Candida biofilm formation on PMMA. M. alternifolia and E. globulus essential oils can inhibit Candida biofilm formation on PMMA. This may contribute to the use of these plants as alternative products for oral Candida biofilm prevention, control and treatment.

Key words: Candida , biofilm, polymethylmethacrylate, Melaleuca alternifolia , Eucalyptus globulus .

INTRODUCTION

Medicinal plants have been used in developing countries The therapy of deep fungal infections, particularly those as alternative treatments to health problems. Many plant caused by opportunistic pathogens, such as Candida extracts and essential oils isolated from plants have been albicans , remains a difficult medical problem (Bennet, shown to exert biological activity in vitro and in vivo , 1992). which justified research on traditional medicine focused The infection of C. albicans will lead to the forming on the characterization of antimicrobial activity of these within the biofilm. Besides this, the action of antifungal plants (Snoussi et al., 2008; Noumi et al., 2010a; Hajlaoui may be limited by their penetration and chemical reaction et al., 2010). into biofilm matrix, the extracellular polymeric material

*Corresponding author. E-mail: [email protected]. Tel: +216 73 466244. Fax: +216 73 461830. 1462 J. Med. Plants Res.

(Jain et al., 2007; Meyer, 2003; Sanglard et al., 2003). In Candida isolates this context, new agents from natural resources that can inhibit the growth of biofilm-associated microorganisms Four Candida strains including two species ( Candida albicans and Candida glabrata ) were used in this study. Two clinical Candida are greatly needed and would enhance the number of strains: C. albicans (15 B) and C. glabrata (15 T) were isolated from effective therapeutic alternatives (Alviano et al., 2005). the oral cavity of the same patient by using a swabbing method. All Biofilm-associated or sessile, C. albicans organisms isolates were incubated at 30°C for 24 to 48 h on Sabouraud demonstrate increased resistance to traditional antifungal Chloramphenicol agar (Bio-rad, France) and yeast-like colonies agents that have activity against their planktonic were isolated and identified by the ID 32 C (bio-Mérieux, Marcy- l’Étoile, France) assimilation kit. The C. albicans ATCC 90028 and counterparts (Hawser and Douglas, 1995). C. glabrata 90030 species were used as reference strains. Since adhesion is an essential prerequisite in colonisation and infection, its role in the pathogenesis of several diseases by fungus is widely acknowledged. Microdilution method of determination of the minimal Several specific interactions between the genus Candida inhibition concentration (MIC) and minimal fungicidal and other organisms, medical devices and host tissues concentration (MFC) have been described (Hawser and Douglas, 1995; Jain et The MIC and the MFC values were determined for Candida strains. al., 2007). The inoculums of the yeast strains were prepared from 12 h The essential oil of Melaleuca alternifolia contains ~100 Sabouraud dextrose broth cultures and suspensions were adjusted components, which are mostly monoterpenes, to an optical density of 0.5 at 540 nm. The 96-well plates were sesquiterpenes and related alcohols. Tea tree oil (TTO) prepared by dispensing into each well 95 µl of nutrient broth (1% has been used medicinally in Australia, with uses relating NaCl) and 5 µl of the inoculum. A 100 µl aliquot from the stock solutions of each plants extract was added into the first wells. Then, primarily to its antimicrobial (Carson and Riley, 1993; 100 µl from the serial dilutions were transferred into eleven Carson et al., 2002; Mondello et al., 2003), anti- consecutive wells. The last well containing 195 µl of nutrient broth inflammatory and antifungal especially anticandidal (1% NaCl) without plants extract and 5 µl of the inoculum on each properties (Hammer et al., 1998; Hammer et al., 2000; strip was used as the negative control. The final volume in each Noumi et al., 2010b). TTO efficiency was confirmed in the well was 200 µl. The plates were incubated at 37°C for 18 to 24 h. The plants extract tested in this study was screened two times treatment of dandruff (Satchell et al., 2002) and oral against each strain. The MIC was defined as the lowest candidiasis (Jandourek et al., 1998; Hammer et al., concentration of the compounds to inhibit the growth of the micro- 2000). In fact, oral candidiasis is the most common organisms. The MFC values were interpreted as the highest dilution mouth fungus infection in humans, especially caused by (lowest concentration) of the sample, which showed clear fluid with C. albicans (Pizzo et al., 2001, Ellepola and no development of turbidity and without visible growth. All tests Samaranayake, 1998). This yeast proliferates in resistant were performed in triplicate. biofilm (Jain et al., 2007). Also, Eucalyptus globulus essential oil shows promise as a topical antifungal agent, Biofilm formation on PMMA with recent clinical data indicating efficacy in the treatment of oral candidiasis. The essential oil extracted from Biofilm formation these plants has antimicrobial activity (Jandourek et al., 1998). The acrylic resin strips of PMMA were prepared as described by our group in a previous study (Noumi et al., 2010c). The ability of C. Our previous study showed that the essential oils of M. albicans strains to form a biofilm was tested according to the alternifolia and E. globulus possessed a good in vitro protocol described by Chandra et al. (2001). For thus, cells were antifungal activity against oral Candida isolates better grown for 24 h at 37°C in yeast nitrogen base (YNB) containing 50 than amphotericin B (Noumi et al., 2010b). Therefore, the mM of glucose. Batches of medium were inoculated with overnight aim of the this study was to evaluate the potential role of yeast cultures and incubated at 37°C in an orbital shaker operating M. alternifolia E. globulus at 150 rpm. Cells were harvested after 24 h (stationary growth and essential oils in the phase), washed once with phosphate-buffered saline (PBS, pH inhibition of Candida biofilm formation on 7.2), and standardized to a density at 1 × 10 7 cells/ml. polymethylmethacrylate (PMMA) in an attempt to A 80 µl quantity of the standardized C. albicans cells suspen- contribute to the use of these plants as alternative sions was applied to the surfaces of 1.5 cm 2 PMMA strips placed in products for oral Candida biofilm prevention, control and a 12-well tissue culture plate. The cells were allowed to adhere for treatment. 90 min at 37°C (adhesion phase). Non-adherent cells were removed from the strips by being gently washed with 5 ml PBS. Strips were then submerged in 4 ml of YNB containing 50 mM of glucose. Strips to which no cells were added served as negative MATERIALS AND METHODS controls. Control and experimental strips were incubated at 37°C for 72 h (biofilm growth phase). Plant material and essential oil

M. alternifolia (Tea tree) essential oil (leaves) was purchased from Quantitative measurement of biofilm Arkomédika (Laboratories Pharmaceutiques, BP 28-06511 Carros, France). E. globulus commercialized essential oil was kindly The biofilm formation by Candida strains was quantified by a colori- provided by the Laboratory of Pharmacognosy from the Faculty of metric method which determines the mitochondrial dehydrogenase Pharmacy (Monastir, Tunisia). activity of the metabolic fungal cells. This colorimetric assay Emira et al. 1463

Table 1. Antifungal activity of Melaleuca alternifolia and Eucalyptus globulus essential oils against Candida strains.

Inhibition zone in diameter (mm ± SD) Strain M. alternifolia (mg/ml) E. globules (mg/ml) AmB (10 mg/ml) IZ MIC MFC IZ MIC MFC IZ MIC MFC C. albicans ATCC 90028 19.33 ± 0.577 0.312 >10 17.66 ± 0.577 0.156 10 11 ± 0 0.097 0.781

15 B 15.33 ± 0.577 0.625 10 19.33 ± 0.577 0.156 10 11 ± 1 0.012 0.781

C. glabrata ATCC 90030 14.33 ± 0.57 0.625 10 22.33 ± 0.57 0.078 10 14.33 ± 0.57 0.195 1.562

15 T 12 ± 0 0.625 10 12.66 ± 1.15 0.078 10 10.66 ± 0.57 0.39 0.39

M. alternifolia : Melaleuca alternifolia , E. globulus: Eucalyptus globulus ; MIC: minimal inhibitory concentration; MFC: minimal fungicidal concentration; AmB: amphotericin B; IZ: inhibition zone; SD: standard deviation.

investigates metabolic reduction of 2, 3-bis (2-methoxy-4-nitro-5- described by Chandra et al. (2001) to quantify the biofilm formation sulfophenyl)-5-[(phenyl amino) carbonyl]-2H-tetrazolium hydroxide by C. albicans strains by determining mitochondrial dehydrogenase (XTT) to a water-soluble brown formazan product. activity of the metabolic fungal cells. Strips with biofilms were transferred to 12-well tissue culture plates containing 4 ml PBS/well. Fifty microliters of XTT (1 mg/ml in PBS) and 4 µl of menadione solution (1 mM in acetone) were RESULTS added to each well. Plates were incubated at 37°C for 5 h. The entire contents of the well were transferred into a tube and centrifuged (5 min, 6000 g). XTT formazan in the supernatant was In this study, the anticandidal activities of M. alternifolia determined spectrophotometrically at 492 nm. Experiments were and E. globulus essential oils were investigated. performed in triplicate. Antifungal effects were reported as inhibition zones using

the disc diffusion method and in vitro activity as MIC and Effect of plants oils on adhesion and biofilm formation on MFC values (Table 1). PMMA The two plant essential oils showed significant antifungal activity against the four tested Candida strains. Effect on adhesion Overall, the best antifungal activity was against C. The ability of Candida strains to form a biofilm was tested according albicans ATCC 90028 for M. alternifolia (19.33 mm) and to the protocol described by Chandra et al. (2001). Thus, cells were against C. glabrata ATCC 90030 for E. globulus oil (22.33 grown for 24 h at 37°C in yeast nitrogen base (YNB) containing 50 mm). Essential oil of E. globulus was more efficient and mM of glucose. Batches of medium were inoculated with overnight had the best antifungal effect for oral C. albicans strain yeast cultures and incubated at 37°C in an orbital shaker operating at 150 rpm. Cells were harvested after 24 h (stationary growth (15 B) (IZ = 19.33 mm) as compared to the results phase), washed once with phosphate-buffered saline (PBS, pH obtained with amphotericin B (IZ = 11 mm) and also for 7.2), and standardized to a density at 1 × 10 7 cells/ml. C. glabrata ATCC 90030 strain (IZ = 22.33 mm) as A 80 µl quantity of the standardized Candida cells suspensions compared to amphotericin B results (IZ = 14.33 mm). and 40 µl of the MIC or 1/2 of MIC was applied to the surfaces of Table 1 summarizes the MIC and MFC of the two 1.5 cm2 PMMA strips placed in a 12-well tissue culture plate. The cells were allowed to adhere for 90 min at 37°C (adhesion phase). plants essential oils. The lowest values of MIC were seen Non-adherent cells were removed from the strips by being gently against two C. glabrata isolates with E. globulus essential washed with 5 ml PBS. oil (strains 15 T and ATCC 90030; MIC: 0.078 mg/ml), followed by 0.156 mg/ml for C. albicans isolates (strains

Effect on biofilm 15B B and ATCC 90028). The MFC values were similar for all Candida tested strains (10 mg/ml). The standard Eighty microliters of the standardized Candida cells suspensions antifungal drug, amphotericin B, was more active against and 2 ml of the MIC or 1/2 of MIC was applied to the surfaces of 1.5 all oral and reference Candida strains (MIC range: 0.012 cm 2 PMMA strips placed in a 12-well tissue culture plate. The cells were allowed to adhere for 90 min at 37°C (adhesion phase). Non- to 0.39 mg/ml; MFC range: 0.195 to 1.562 mg/ml) adherent cells were removed from the strips by being gently comparing the two essential oils. washed with 5 ml PBS. Strips were then submerged in 4 ml of YNB The metabolic activity and total biomass of C. albicans containing 50 mM of glucose. Strips to which no cells were added and C. glabrata biofilms was determined by reduction of served as negative controls. Control and experimental strips were XTT dye. It was found out that the metabolic activity of incubated at 37°C for 72 h (biofilm growth phase). Candida biofilm formed on PMMA did not differ between tested strains (Table 2). Quantitative measurement of biofilm The optical densities were 0.356 for C. albicans ATCC Same colorimetric method protocol has been adopted as previously 90028 and 0.353 for the oral strain (15 B) of C. albicans . 1464 J. Med. Plants Res.

Table 2. Quantitative estimation of Candida biofilm formation on polymethylmethacrylate biomaterial.

Strain XTT reduction (mean OD 492nm ± SD) C. albicans ATCC 90028 0.356 ± 0.006

15 B 0.353 ± 0.004

C. glabrata ATCC 90030 0.325 ± 0.019

15 T 0.352 ± 0.01

Table 3. Effect of ½ MIC and MIC of E. globulus and M. alternifolia essential oils comparatively to amphotericin B on biofilm formation potency by Candida strains.

XTT reduction (OD λ=492 nm ± SD) Essential oil of Strain Amphotericin B E. globulus M. alternifolia ½ MIC MIC ½ MIC MIC ½ MIC MIC C. albicans ATCC 90028 0.242 ± 0.006 0.181 ± 0.006 0.211 ± 0.002 0.160 ± 0.006 0.207 ± 0.006 0.141 ± 0.004

15 B 0.218 ± 0.003 0.146 ± 0.005 0.210 ± 0.002 0.131 ± 0.002 0.150 ± 0.007 0.107 ± 0.009

C. glabrata ATCC 90030 0.240 ± 0.004 0.144 ± 0.004 0.223 ± 0.006 0.126 ± 0.008 0.207 ± 0.006 0.111 ± 0.007

15 T 0.210 ± 0.008 0.158 ± 0.003 0.223 ± 0.006 0.142 ± 0.006 0.175 ± 0.004 0.123 ± 0.006

MIC: Minimal inhibitory concentration.

The oral strain (15 T) of C. glabrata was more adhesive with the two plants essential oils and with amphotericin B (OD = 0.352) than the strain C. glabrata ATCC 90030 (Table 4). (OD = 0.325). Table 4 summarizes the variation in XTT reduction The results of this study showed that M. alternifolia and (biofilm formation) in function of MIC values of M. E. globulus essential oils were active against clinical and alternifolia and E. globulus essential oils. A MIC value of reference C. albicans and C. glabrata strains in their 0.625 mg/ml of E. globulus essential oils reduced the planktonic and adherent phases. In fact, for the effect on biofilm formation from (OD = 0.353) to (OD= 0.146) for adhesion, the MIC value (0.156 mg/ml) of E. globulus the oral isolate of C. albicans and from (OD = 0.352) to essential oil can inhibit adhesion of C. albicans ATCC (OD= 0.158) for the oral isolate of C. glabrata . The same 90028 strain on PMMA (OD = 0.119); a similar result has concentration of M. alternifolia essential oil inhibited the been observed with amphotericin B. Also, 0.625 mg/ml of biofilm formed by C. albicans (OD 1 = 0.353, OD 2 = 0.131) tea tree essential oil was active against adhesive oral and also by C. glabrata strain (OD 1 = 0.352, OD 2 = strains of C. albicans (15 B, OD = 0.133) and C. glabrata 0.142). These results are approximatively similar to (15 T, OD = 0.168) on PMMA. All these results are those obtained for inhibition of adhesion potency. This illustrated in Table 3. study demonstrated that E. globulus essential oil was Concerning the study of the effect of these two plants more active on Candida biofilm formation on PMMA each essential oils on biofilm formation by type and oral strains times that MIC and 1/2 MIC values of this essential oil of Candida on PMMA, our results demonstrated that 1/2 were lower than those of tea tree essential oil. MIC E. globulus (0.039 mg/ml) can inhibit biofilm formation by C. glabrata (15 T) on PMMA (OD= 0.21) more than to amphotericin B (1/2 MIC = 0.195 mg/ml, OD DISCUSSION = 0.175). The reduction of the number of active Candida cells Antifungal agents may enter the oral cavity in implicated on biofilm formation on PMMA is proportional concentrations, which are equal to or over MIC (Pizzo, to the reduction of the absorbance values of XTT. This 2001). TTO is employed for its antimicrobial property and result has been observed for the four Candida strains and is incorporated as the active ingredient in many tropical Emira et al. 1465

formulations used to treat cutaneous infections. In fact, bioactive compound(s) in tea tree and E. globulus our results agree with previous works dealing about the essential oils have distinct influence on Candida cell high susceptibility of a wide range of yeasts, growth, function and biofilm formation by interfering with dermatophytes, and other filamentous fungi (Carson et any of the steps involved in biofilm development. al., 2006). In this context, TTO has been clinically eva- Braga et al. (2007) demonstrated that thymol interfered luated for the treatment of several fungal infections with the starting phases of biofilm formation by reducing including oral candidiasis (Jandourek et al., 1998). the amount of metabolically active yeast as well as the Hammer et al. (1998 ) tested in vitro the antifungal dimorphic switching from yeasts to filamentous forms. activity of 24 essential oils against 14 Candida species Also, the effects of fresh garlic extract against planktonic isolates. They founded that E. globulus essential oil inhi- Candida spp. have been demonstrated in vitro and have bit the growth of C. albicans ATCC 10231 at MIC = 1% been attributed to the action of allicin (Ghannoum, 1988). (v, v) and from 0.12 to 0.5% for all Candida spp. tested. The results of this study suggest that M. alternifolia and MIC assays showed that M. alternifolia and E. globulus E. globulus essential oils have a potential anti-adhesive essential oils have a fungicidal effect on Candida tested effect of Candida strains on PMMA. Generally, the strains. This result is similar to that obtained by our group beneficial effects attributed to the plants may be due to researchers who verified the decrease in the growth of one or more photochemicals, including antioxidants, Candida strains cultured in medium supplemented with flavins, and other substances present in the extract different concentrations of each plant essential oil. Also, (Balasenthil et al., 1999). Therefore, the results of this TTO and E. globulus essential oils inhibit germ tube study reinforce the possible effect of the plants essential formation and mycelia conversion in C. albicans . In fact, oils in the prevention of diseases of the buccal cavity. only 1/2 MIC (0.312 mg/ml) of M. alternifolia was able to inhibit totally mycelium in C. albicans isolate while 2 MIC (0.312 mg/ml) of the second essential oil was necessary Conclusion to inhibit germ tube formation in the same strain (Noumi et al., 2010b). Conclusively, the preliminary evidence for the antifungal Tampieri et al. (2005) found that 1, 8-cineole (81,4%) activity of M. alternifolia and E. globulus essential oils and limonene (7,01%) were the main components of E. against oral Candida were presented. 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