The Influence of Tea Tree Oil (Melaleuca alternifolia) on Fluconazole Activity against Fluconazole-Resistant Candida albicans Strains

The aim of this study was to evaluate the activity of fluconazole against 32 clinical strains of fluconazole-resistant Candida albicans, and C. 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 μg/mL to an average of 38.46 μg/mL, and the fluconazole minimal fungicidal concentrations (MFC) decreased from an average of 254.67 μg/mL to an average of 66.62 μg/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.


Introduction
Essential oils are antiseptic substances produced by plants. Tea tree oil (TTO) is the essential oil obtained by steam distillation from the Australian native plant Melaleuca alternifolia and is used medicinally as a topical antiseptic. It has a broad spectrum of antimicrobial activity against a wide range of bacteria, viruses, and fungi, including yeasts and dermatophytes. TTO is a mixture of more than 100 different compounds, primarily terpenes (mainly monoterpenes and sesquiterpenes). The physical properties and chemical composition of TTO are variable, and it is, therefore, important to determine international standards. The Australian Standard for tea tree oil (AS 2782(AS -1985 includes directives relating to the levels of two components: the minimum content of terpinen-4-ol should be at least 30% and the maximum content of 1,8-cineole should be less than 15% of the oil volume [1]. The international standard for tea tree oil (ISO 4730:2004) includes maximum and minimum percentage values for the 15 most important TTO components. TTO obtained by steam distillation of the leaves and terminal branches of Melaleuca alternifolia Cheel, Melaleuca linariifolia Smith, Melaleuca dissitiflora F. Mueller, and other species of Melaleuca should conform to this standard [2].
TTO has been used for centuries in Australian folk medicine, predominantly for wound treatment [3,4]. In the 1920s, Penfold described for the first time the properties and chemical composition of TTO, and he later confirmed the antiseptic properties of TTO and its components [5][6][7][8]. In the 1930s, consecutive publications appeared which demonstrated the powerful antimicrobial activity of TTO when used in inhalation therapy, aseptic surgery, dental surgery, wound disinfection, and oral cavity rinsing [9][10][11].
Currently, TTO is used as a local agent for treating various diseases, predominantly dermatoses (e.g., recurrent herpes labialis, acne, pustules, dandruff, and rash). TTO is also used to treat Staphylococcus aureus infections of the oral cavity and the pharynx, vaginitis, and respiratory tract diseases. Numerous studies have confirmed the broad antimicrobial activity of TTO against bacteria, fungi, and viruses, as well as microorganisms that are resistant to conventional drugs [12][13][14][15][16]. This is important due to the increase in infections that are difficult to treat, as TTO can be used as an alternative to or in combination with conventional drugs (including antibiotics and chemotherapeutic agents).
Treatment of infections can be based on monotherapy (using one antimicrobial drug) or combined therapy (two or more drugs). The primary aim of combined therapy is to enhance the action of the drugs while decreasing the dosages, through synergism. When monotherapy or combined therapy based on conventional drugs is unsuccessful, then combined treatment including a natural agent may be more effective. Several recent studies have reported the increased antimicrobial activity of natural substances combined with conventional drugs as compared to conventional drug treatment alone [17][18][19][20].
The aim of this study was to evaluate the activity of fluconazole against clinical strains of fluconazole-resistant Candida albicans and reference strain C. albicans ATCC 10231, after their exposure to sublethal concentrations of TTO or its main bioactive component terpinen-4-ol.

Candida albicans Strains. This study included 32 clinical
Candida albicans strains, which were isolated from the following materials: swabs of the pharynx and oral cavity ( = 5), vagina ( = 15), sputum ( = 8), or faeces ( = 4). These strains were isolated from culture on Sabouraud agar (bioMèrieux, Marcy l'Etoile, France), and species identification was performed using the biochemical test ID 32C (bioMèrieux, Marcy l'Etoile, France). We also used the reference strain C. albicans ATCC 10231, which was purchased from Oxoid Ltd. (Basingstoke, Great Britain). We previously determined the sensitivity of C. albicans strains to fluconazole by the Kirby-Bauer disk diffusion susceptibility test [21] using 6 mm filter paper disks impregnated with 10 g of fluconazole obtained from DHN (Cracow, Poland) and YNB agar (Yeast Nitrogen Base-Difco 0.5%, glucose 3%, agar 1.8%, pH = 7) also obtained from DHN (Cracow, Poland). The C. albicans strains were classified as exhibiting susceptibility (diameter of growth inhibition zone ≥18 mm), intermediate susceptibility (diameter of growth inhibition zone from 14 mm to 17 mm), or resistance (diameter of growth inhibition zone <14 mm) to fluconazole (the data were described in chapter 3). The fluconazole MIC (minimal inhibitory concentration) and MFC (minimal fungicidal concentration) values were determined by the broth dilution method according to the Clinical and Laboratory Standards Institute (CLSI document M27-A3-2008) [22]. Using this standard, the C. albicans strains were classified as exhibiting susceptibility (MIC ≤ 8 g/mL), intermediate susceptibility (MIC from 9 g/mL to 63 g/mL), or resistance (MIC ≥ 64 g/mL) to fluconazole (the data were presented in chapter 3).  [2]. It was performed in the following conditions: fused-silica column (50 m × 0,20 mm i.d., film thickness 0,25 m) and flame ionisation type of detector were used, the carrier gas was hydrogen (flow rate of 1 mL/min), the oven temperature programme was from 70 ∘ C to 220 ∘ C at a rate of 2 ∘ C/min, the injector temperature was 230 ∘ C, the detector temperature was 250 ∘ C, the volume of injected TTO was 0,2 L, and the split ratio was 1 : 100. In our study, we also used terpinen-4-ol, which was obtained from Sigma-Aldrich (St. Louis, MO, USA).

Fluconazole.
In this study, we used the antifungal drug fluconazole (Polfarmex, Kutno, Poland). The structure of the fluconazole molecule is shown in Figure 1.

Preparation of the Initial Candida albicans Suspension.
C. albicans cells cultured for 24 h on Sabouraud agar were suspended in a saline solution (0.85% NaCl) and adjusted to a 0,5 McFarland density standard (1,5 × 10 8 CFU/mL). This suspension was later diluted to a density of 6 × 10 4 CFU/mL. The suspension was then used to estimate the MIC and MFC values for TTO, terpinen-4-ol, and fluconazole.

Determination of MIC and MFC Values for TTO and
Terpinen-4-ol. The TTO activity against the C. albicans strains tested was determined by broth macrodilution using the general dilution standards as described by PN-EN ISO 20776-1:2007 [24]. TTO was serially diluted in liquid Sabouraud medium with 10% Tween 80 to final TTO concentrations of 1% to 0.0075%. The Tween 80 detergent helps dissolve the TTO. The same volume of the C. albicans suspension was added to each tube to obtain a final density of 3 × 10 3 CFU/mL. After 24 h of incubation at 35 ∘ C, the cell growth was assessed visually in the tubes with TTO and the positive control tube (without TTO). The MIC was defined as the lowest concentration of TTO that led to no visible growth of the cell strains tested. The MFC value was defined as the lowest concentration of TTO that showed no growth of C. albicans colonies. The experiment was performed triply. The terpinen-4-ol MIC and MFC values were determined identically as described above. The TTO and terpinen-4-ol MICs were used to calculate the sublethal doses of TTO and terpinen-4-ol used in the following experiments.

Brief Pretreatment of Candida albicans with 1/4 MIC TTO.
For each sample, a tube was prepared containing saline solution with 10% Tween 80 and TTO to a final concentration of 1/4 MIC TTO. A control tube with no TTO was also prepared. Next, the C. albicans suspension was added to tubes to obtain a final density of 3 × 10 3 CFU/mL. The suspensions were then incubated at 35 ∘ C for 30 minutes. The samples were then rinsed twice and centrifuged between rinses (3000 ×g, 15 minutes), and the cells were resuspended to a density of 6 × 10 4 CFU/mL. The suspension was then used to determine the fluconazole MIC and the minimal fungicidal concentration (MFC) of fluconazole. The study was performed in triplicate.

Determination of the Fluconazole MIC and MFC Values after Brief Pretreatment of Candida albicans with 1/4 MIC TTO.
The fluconazole activity against the C. albicans strains tested was determined by broth macrodilution using the general dilution standards as described by PN-EN ISO 20776-1:2007 [24]. Serial, parallel dilutions of fluconazole ranging from 256.0 g/mL to 0.125 g/mL were prepared in liquid Sabouraud medium, and a control tube without the drug was included. For each of the tubes, the same volume of C. albicans cells suspension pretreated with 1/4 MIC TTO was added, and the inoculum was adjusted to a final density of 3 × 10 3 CFU/mL. After 24 h of incubation at 35 ∘ C, the cell growth in each tube was assessed visually. The MIC value was defined as the lowest concentration of fluconazole that resulted in no visible growth of the strains tested. The cells from the tube identified as the MIC, as well as several of the surrounding dilutions, were plated to Sabouraud agar. After 24 h of incubation at 35 ∘ C, the C. albicans colonies were counted. The MFC value was defined as the lowest concentration of fluconazole that showed no growth of C. albicans colonies. The experiment was performed in triplicate. The C. albicans strains were classified as exhibiting susceptibility, intermediate susceptibility, or resistance to fluconazole according to CLSI document M27-A3-2008 [22], as described in Section 2.1.

Prolonged Pretreatment of Candida albicans with Fluconazole and Sublethal Dose of TTO or Terpinen-4-ol.
Serial, parallel dilutions of fluconazole ranging from 256.0 g/mL to 0.125 g/mL were prepared in liquid Sabouraud culture medium. Two positive controls were included. All tubes contained 10% Tween 80, and TTO was added to each dilution and one of the control tubes to achieve a final concentration of 1/4 MIC TTO. The second control tube contained only the liquid medium. Next, an equal volume of C. albicans suspension was added to each tube to a final density of 3 × 10 3 CFU/mL. All the tubes were incubated at 35 ∘ C for 24 h. After incubation, the cell growth in each tube was evaluated visually, and the fluconazole MIC and MFC values were defined, as described previously. The cells from the tube identified as the MIC, as well as several of the surrounding dilutions, were plated to Sabouraud agar. After 24 h of incubation at 35 ∘ C, the C. albicans colonies were counted, and the fluconazole MFC value was defined. The experiment was performed in triplicate. The prolonged pretreatment of C. albicans with fluconazole and terpinen-4ol was performed identically as described above.

Statistical Methods.
The results are presented as the arithmetic mean and the median. The statistical differences between the mean values were determined by Student's -test and the Mann-Whitney test, depending on how well the results correlated with a normal distribution. Values of ≤ 0.05 were considered statistically significant. The programme STATISTICA version 10 (StatSoft, Cracow, Poland) was used to perform the statistical analyses.

Results
The Candida albicans strains tested were resistant to fluconazole and susceptible to low concentrations of TTO. The clinical C. albicans strains and C. albicans ATCC 10231  Terpinen-4-ol, the main bioactive component present in TTO, strongly enhanced fluconazole activity against fluconazole-resistant C. albicans strains. The terpinen-4-ol MICs for clinical C. albicans strains ranged from 0.06% to 0.25% (average = 0.11 ± 0.09%). For C. albicans ATCC 10231 standard strain, the terpinen-4-ol MIC was 0.06%. The C. albicans strains tested did not exhibit any cross-resistance to terpinen-4-ol and fluconazole. Exposure of fluconazoleresistant clinical and standard C. albicans strains for 24 h to fluconazole and sublethal doses (1/4 MIC) of terpinen-4-ol strongly enhanced fluconazole activity against these strains, and all of C. albicans isolates were classified as susceptible (fluconazole MIC decreased to 0.125 g/mL). We summed up the results of this study, and the most important data are presented in a table form (Table 4).

Discussion
TTO is the most commonly used essential oil for its antibacterial and antifungal properties [3,25]. In this study, we evaluated the change in fluconazole activity in vitro against fluconazole-resistant clinical Candida albicans strains exposed to the sublethal concentrations of TTO or terpinen-4-ol, the main bioactive component of TTO. The earlier in vitro studies of the sensitivity of Candida spp. to TTO have shown that TTO is highly active against these microbes, as well as azole-resistant strains, for which the TTO MICs ranged from 0.25% to 0.5% [14,26]. For the C. albicans strains that were resistant to both fluconazole and itraconazole, the TTO MICs ranged from 0.25 to 1.0%, the TTO MIC 50 was 0.5%, and the TTO MIC 90 was 1% [27]. Another study showed that three fluconazole-resistant clinical C. albicans strains had very low TTO MICs (0.15% for two strains and 0.07% for the third strain) [15].
The experiments performed in this study confirm the results from previously published studies in that all of the tested fluconazole-resistant C. albicans strains were sensitive to TTO [14,15,28,29]. The determined TTO MICs were low, ranging from 0.06% to 0.5%. The TTO antimicrobial activity is attributed mainly to terpinen-4-ol, the main bioactive component present in TTO [3,14]. The determined MIC values for terpinen-4-ol were very low, ranging from 0.06% to 0.25%. Our study and other studies show that C. albicans does not exhibit cross-resistance to TTO and azole agents [14,15,26,27]. Clinical resistance to TTO has not been reported. Multicomponent nature of TTO may reduce the potential for resistance to occur spontaneously, and multiple simultaneous mutations may be required to overcome all of   the antimicrobial actions of each of the components [3]. Thus, TTO can be used as a topical antiseptic to effectively treat superficial mycoses caused by fluconazole-resistant Candida spp. and other azole-resistant yeast. Unfortunately, TTO can be potentially toxic when it is ingested in high doses, and, therefore, TTO should not be administrated orally. The acute oral toxicity of TTO is similar to the oral toxicity of other common essential oils, for example, such as eucalyptus oil [30,31]. The lipophilic nature of TTO, which enables it to penetrate the outer layers of skin, potentiates not only the antiseptic actions but also the possibility of TTO toxicity due to dermal absorption. TTO can cause skin irritation at higher concentrations and may cause allergic reactions in predisposed individuals [3,31,32]. Zhang and Robertson observed ototoxic effect of 100% TTO [33]. The toxicity of TTO is dose-dependent, and the majority of adverse events can be avoided through the use of TTO in a diluted form [31]. TTO is not mutagenic or genotoxic [34,35]. There is increasing interest not only in the activity of natural substances against resistant microbes but also in the synergistic interactions between these substances and conventional drugs [19,20,[36][37][38]. Fluconazole is one of the azole antifungal agents widely used for both prophylaxis and therapy of Candida infections [39][40][41]. In this study, we explored changes in the activity of fluconazole against fluconazole-resistant C. albicans strains after exposure to sublethal concentrations of TTO or terpinen-4-ol. We used exclusively fluconazole-resistant strains because identifying synergistic treatments for these strains would be especially important. We tested sublethal concentrations of TTO and terpinen-4-ol because we expected concentrations lower than the MIC to weaken the cell structure without killing the cells, facilitating the activity of fluconazole and consequently inhibiting C. albicans resistance to fluconazole. Our results show that brief (0.5 h) exposure of fluconazole-resistant C. albicans strains to sublethal concentration of TTO (1/4 MIC TTO) had no influence on the antifungal activity of fluconazole. However, exposing C. albicans cells to sublethal concentration of TTO and then treating them with fluconazole inhibited the resistance to fluconazole in 87.5% of the tested strains. These results suggest that there is a synergistic interaction between fluconazole and TTO against fluconazoleresistant C. albicans. TTO was used to permeabilise the yeast cell membranes, markedly increasing the susceptibility to fluconazole. The TTO becomes embedded in the lipid bilayer membrane, which disrupts its structure, resulting in increased permeability and impaired physiological function. TTO also inhibits the formation of germ tubes or mycelial conversion in C. albicans and inhibits respiration in C. albicans in dose-dependent manner [3]. Fungal cells exposed to TTO will eventually rupture. Sublethal concentrations of TTO also weaken Candida spp. cells vitality [41,42]. The mechanism of fluconazole antifungal activity is different. It was demonstrated that fluconazole interferes with the cytochrome P-450-dependent enzyme C-14 -demethylase, which is responsible for production of ergosterol. The disruption of ergosterol synthesis causes structural and functional changes in the fungal cell membrane, which predispose the fungus cells to damage. Inhibition of cytochrome oxidative and peroxidative enzymes is an additional antifungal activity of fluconazole [39]. Several mechanisms have been described for fluconazole resistance in C. albicans isolates: increased production of lanosterol 14 -demethylase encoded by ERG11 gene and decreases in the affinity of lanosterol 14demethylase for fluconazole because of mutations in ERG11 gene and a defect in Δ5-6 desaturase encoded by ERG3 gene causing loss of function in the ergosterol pathway. The other mechanism of fluconazole resistance in C. albicans is the active transport of drugs across the plasma membrane by "efflux pumps, " which requires the expression of the CDR1/2 and MDR1 genes [39,[43][44][45][46][47]. TTO-induced cell membrane damage can disrupt the function of "efflux pumps, " thus making the fungal cell more susceptible to fluconazole [48,49].
Our data show that there is a synergistic effect in vitro of sublethal concentrations of TTO and fluconazole against fluconazole-resistant C. albicans strains. However, the fluconazole-resistant C. albicans ATCC 10231 standard strain and four clinical C. albicans strains did not increase the susceptibility to fluconazole. The differences in mechanisms of resistance of these strains to fluconazole were probable cause of this effect. In our in vitro study the TTO main component terpinen-4-ol was more active than TTO and strongly enhanced fluconazole activity against all studied fluconazoleresistant C. albicans strains. Mondello et al. [14] as well as Ninomiya et al. [50] observed that in vivo TTO and terpinen-4-ol were similarly effective against candidiasis caused by azole-resistant C. albicans. The mechanisms underlying the synergy between fluconazole and TTO did not elucidate. Yu et al. [51] confirmed the synergism between fluconazole and triclosan against clinical isolates of fluconazole-resistant C. albicans. Liu et al. [52] observed synergistic effect between fluconazole and glabridin against C. albicans related to the effect of glabridin on cell envelope. Ahmad et al. [53] described synergistic activity of thymol and carvacrol with fluconazole against Candida isolates. Both monoterpenes inhibited efflux by 70-90% showing their high potency to block drug transporter pumps.
Previous studies also have evaluated the activity of TTO against various microorganisms in combination with other antimicrobial substances. A synergistic effect was observed for itraconazole and TTO in a thermosensitive gel used to treat vaginal candidiasis [26]. Synergistic effects have also been observed between essential oils and ciprofloxacin, gentamicin, cefixime, and pristinamycin [20]. In a disc diffusion test using C. albicans, C. glabrata, C. tropicalis, C. krusei, C. guilliermondii, and C. parapsilosis, larger growth inhibition zones occurred around discs impregnated with TTO and amphotericin B than around discs containing only TTO [17]. In a study of Staphylococcus aureus, larger zones of growth inhibition occurred around discs impregnated with TTO and other essential oils compared to discs impregnated with TTO only [54].
The synergistic action of antimicrobial substances has also been shown using time-kill curves. The short pretreatment of Pseudomonas aeruginosa with a substance that disrupts the cytoplasmic membrane (carbonyl cyanide m-chlorophenylhydrazone, polymyxin B nonapeptide, or ethylenediaminetetraacetic acid) enhanced the bactericidal activity of TTO, as demonstrated by the increased speed of microbe killing in the time-kill curves [55,56]. However, in a study using the E-test method, Escherichia coli, Salmonella enteritidis, Salmonella typhimurium, Staphylococcus aureus, and coagulase-negative staphylococci (CoNS) exposed to sublethal concentrations of TTO for 72 hours exhibited increased resistance to gentamicin, streptomycin, chloramphenicol, tetracycline, erythromycin, trimethoprim, ampicillin, fusidic acid, mupirocin, linezolid, and vancomycin [57,58]. Increased antimicrobial activity was observed when essential oils were combined with their isolated components (e.g., terpinen-4-ol from Melaleuca alternifolia) [59] and when TTO was combined with silver ions [60,61].
The fractional inhibition concentration (FIC) index, also referred to as the FICI, is used to determine whether two substances are synergistic or antagonistic. FIC values can be interpreted differently, however, in general, an FIC index lower than 0.5 indicates synergism and an FIC index higher than 4 indicates antagonism [18,19,38,59]. The FIC index value for TTO and tobramycin was 0.37 for Escherichia coli and 0.62 for Staphylococcus aureus, indicating that these two substances are synergistic [19]. A minor synergistic effect was observed when treating Candida albicans with TTO and amphotericin B and Klebsiella pneumoniae with TTO and ciprofloxacin. TTO and ciprofloxacin exhibit antagonistic effects against Staphylococcus aureus [18]. There is no synergistic effect between TTO and lysostaphin, mupirocin, gentamicin, or vancomycin against methicillin-resistant Staphylococcus aureus strains. In fact, the FIC index indicated that TTO and vancomycin are antagonistic [38].
The results of this study and other previous studies demonstrate that combining natural substances such as TTO and conventional drugs such as fluconazole may help treat difficult yeast infections. However, additional in vitro studies are needed to identify the antimicrobial activity of natural medicinal substances and detect synergistic interactions with commonly used antimicrobial agents.