Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 590470, 9 pages http://dx.doi.org/10.1155/2015/590470 Research Article The Influence of Tea Tree Oil (Melaleuca alternifolia)on Fluconazole Activity against Fluconazole-Resistant Candida albicans Strains Anna Mertas, Aleksandra GarbusiNska, Ewelina Szliszka, Andrzej Jureczko, Magdalena Kowalska, and Wojciech Król Department of Microbiology and Immunology, Medical University of Silesia in Katowice, Jordana 19, 41-808 Zabrze, Poland Correspondence should be addressed to Wojciech Krol;´ [email protected] Received 9 June 2014; Revised 17 September 2014; Accepted 12 October 2014 Academic Editor: Vasilis P. Valdramidis Copyright © 2015 Anna Mertas et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The aim of this study was to evaluate the activity of fluconazole against 32 clinical strains of fluconazole-resistant Candida albicans,andC. albicans ATCC 10231 reference strain, after their exposure to sublethal concentrations of tea tree oil (TTO) or its main bioactive component terpinen-4-ol. For all tested fluconazole-resistant C. albicans strains TTO and terpinen-4-ol minimal inhibitory concentrations (MICs) were low, ranging from 0.06% to 0.5%. The 24-hour exposure of fluconazole-resistant C. albicans strains to fluconazole with sublethal dose of TTO enhanced fluconazole activity against these strains. Overall, 62.5% of isolates were classified as susceptible, 25.0% exhibited intermediate susceptibility, and 12.5% were resistant. For all of the tested clinical strains the fluconazole MIC decreased from an average of 244.0 g/mL to an average of 38.46 g/mL, and the fluconazole minimal fungicidal concentrations (MFC) decreased from an average of 254.67 g/mL to an average of 66.62 g/mL. Terpinen-4-ol was found to be more active than TTO, and strongly enhanced fluconazole activity against fluconazole-resistant C. albicans strains. The results of this study demonstrate that combining natural substances such as TTO and conventional drug such as fluconazole, may help treat difficult yeast infections. 1. Introduction includes maximum and minimum percentage values for the 15 most important TTO components. TTO obtained by steam Essential oils are antiseptic substances produced by plants. distillation of the leaves and terminal branches of Melaleuca Teatreeoil(TTO)istheessentialoilobtainedbysteamdistil- alternifolia Cheel, Melaleuca linariifolia Smith, Melaleuca lation from the Australian native plant Melaleuca alternifolia dissitiflora F. Mueller, and other species of Melaleuca should and is used medicinally as a topical antiseptic. It has a conform to this standard [2]. broad spectrum of antimicrobial activity against a wide TTO has been used for centuries in Australian folk range of bacteria, viruses, and fungi, including yeasts and medicine, predominantly for wound treatment [3, 4]. In the dermatophytes. TTO is a mixture of more than 100 different 1920s, Penfold described for the first time the properties compounds, primarily terpenes (mainly monoterpenes and and chemical composition of TTO, and he later confirmed sesquiterpenes). The physical properties and chemical com- the antiseptic properties of TTO and its components [5– position of TTO are variable, and it is, therefore, important to 8]. In the 1930s, consecutive publications appeared which determine international standards. The Australian Standard demonstrated the powerful antimicrobial activity of TTO for tea tree oil (AS 2782-1985) includes directives relating when used in inhalation therapy, aseptic surgery, dental to the levels of two components: the minimum content of surgery, wound disinfection, and oral cavity rinsing [9–11]. terpinen-4-ol should be at least 30% and the maximum con- Currently,TTOisusedasalocalagentfortreatingvarious tentof1,8-cineoleshouldbelessthan15%oftheoilvolume diseases, predominantly dermatoses (e.g., recurrent herpes [1]. The international standard for tea tree oil (ISO 4730:2004) labialis,acne,pustules,dandruff,andrash).TTOisalsoused 2 BioMed Research International to treat Staphylococcus aureus infections of the oral cavity Table 1: Composition of the TTO used in this study compared to and the pharynx, vaginitis, and respiratory tract diseases. ISO standard 4730:2004 [2]. Numerous studies have confirmed the broad antimicrobial Content (%) Content (%) of TTO activity of TTO against bacteria, fungi, and viruses, as well as Components according to ISO sample microorganisms that are resistant to conventional drugs [12– standard 4730 16]. This is important due to the increase in infections that are -Pinene 1–6 2.5 difficulttotreat,asTTOcanbeusedasanalternativetoorin combination with conventional drugs (including antibiotics Sabinene Trace-3.5 0.1 and chemotherapeutic agents). -Terpinene 5–13 8.1 Treatment of infections can be based on monotherapy Limonene 0.5–1.5 1.0 (using one antimicrobial drug) or combined therapy (two or p-Cymene 0.5–8 4.4 more drugs). The primary aim of combined therapy is to enhance the action of the drugs while decreasing the dosages, 1,8-Cineole Trace-15 2.8 through synergism. When monotherapy or combined ther- -Terpinene 10–28 19.6 apy based on conventional drugs is unsuccessful, then com- Terpinolene 1.5–5 3.2 bined treatment including a natural agent may be more Terpinen-4-ol 30–48 41.0 effective. Several recent studies have reported the increased antimicrobial activity of natural substances combined with -Terpineol 1.5-8 3.0 conventional drugs as compared to conventional drug treat- Aromadendrene Trace-3 1.3 ment alone [17–20]. Ledene Trace-3 No data available The aim of this study was to evaluate the activity of (syn. viridiflorene) fluconazole against clinical strains of fluconazole-resistant -Cadinene Trace-3 No data available Candida albicans and reference strain C. albicans ATCC Globulol Trace-1 No data available 10231, after their exposure to sublethal concentrations of TTO or its main bioactive component terpinen-4-ol. Viridiflorol Trace-1 No data available 2. Materials and Methods 2.1. Candida albicans Strains. This study included 32 clinical 2.2. Tea Tree Oil (TTO). In this study, we used Australian Candida albicans strains, which were isolated from the tea tree oil (Melaleuca alternifolia)fromThursdayPlantation following materials: swabs of the pharynx and oral cavity (Integria Healthcare, Eight Mile Plains, QLD, Australia) (=5), vagina (=15), sputum (=8), or faeces series 270930 that conforms to the ISO standard 4730:2004 (=4). These strains were isolated from culture on Sabour- [2](Table 1). TTO was distilled from specially selected aud agar (bioMerieux,` Marcy l’Etoile, France), and species Melaleuca alternifolia leaves, a plant native to the coastal identification was performed using the biochemical test ID regions of northern New South Wales and south eastern 32C (bioMerieux,` Marcy l’Etoile, France). We also used Queensland in Australia. The analysis of TTO composition the reference strain C. albicans ATCC 10231, which was was carried out by gas chromatography according to the purchased from Oxoid Ltd. (Basingstoke, Great Britain). We international standard ISO 4730 [2]. It was performed in the following conditions: fused-silica column (50 m × 0,20 mm previously determined the sensitivity of C. albicans strains to fluconazole by the Kirby-Bauer disk diffusion susceptibility i.d., film thickness 0,25 m) and flame ionisation type of detector were used, the carrier gas was hydrogen (flow rate of test [21] using 6 mm filter paper disks impregnated with ∘ 1 mL/min), the oven temperature programme was from 70 C 10 g of fluconazole obtained from DHN (Cracow, Poland) ∘ ∘ to 220 Catarateof2C/min, the injector temperature was and YNB agar (Yeast Nitrogen Base-Difco 0.5%, glucose ∘ ∘ 230 C, the detector temperature was 250 C, the volume of 3%, agar 1.8%, pH = 7) also obtained from DHN (Cracow, Poland). The C. albicans strains were classified as exhibiting injected TTO was 0,2 L, and the split ratio was 1 : 100. susceptibility(diameterofgrowthinhibitionzone≥18 mm), Inourstudy,wealsousedterpinen-4-ol,whichwas intermediate susceptibility (diameter of growth inhibition obtained from Sigma-Aldrich (St. Louis, MO, USA). zone from 14 mm to 17 mm), or resistance (diameter of growth inhibition zone <14 mm) to fluconazole (the data 2.3. Fluconazole. In this study, we used the antifungal drug were described in chapter 3). The fluconazole MIC (minimal fluconazole (Polfarmex, Kutno, Poland). The structure of the inhibitory concentration) and MFC (minimal fungicidal fluconazole molecule is shown in Figure 1. concentration) values were determined by the broth dilution method according to the Clinical and Laboratory Standards 2.4. Preparation of the Initial Candida albicans Suspension. Institute (CLSI document M27-A3-2008) [22]. Using this C. albicans cells cultured for 24 h on Sabouraud agar were suspended in a saline solution (0.85% NaCl) and adjusted to standard, the C. albicans strains were classified as exhibiting 8 susceptibility (MIC ≤ 8 g/mL), intermediate susceptibility a 0,5 McFarland density standard (1,5 × 10 CFU/mL). This 4 (MIC from 9 g/mL to 63 g/mL), or resistance (MIC ≥ suspension was later diluted to a density of 6 × 10 CFU/mL. 64 g/mL) to fluconazole (the data were presented in chapter The suspension was then used to estimate the MIC and MFC 3). values for TTO,