Hydrophobic Properties of Candida Spp. under the Influence of Selected Essential Oils*

Processes of colonization of biotic and abiotic surfaces and biofilm formation depend inter alia on hydrophobic properties of Candida spp. The aim of this research was to determine the effect of tea tree, thyme and clove essential oils on hydrophobic properties of environmental and clinical Candida isolates. The relative cell surface hydrophobicity of strains tested was high, and ranged from 68.7% to 91.2%, with the highest value for a C. ru-gosa food-borne strain. The effectiveness of essential oils was diversified and depended on the type of essential oil, concentration and yeast strain. Statistically significant decrease of hydrophobicity indexes was observed after application of tea tree oil for C. krusei, clove oil for C. albicans reference strain, and all essential oils tested for C. rugosa. Only in the case of C. famata food-borne strain and C. albicans clinical isolate, solely used essential oils did not affect their hydrophobic properties. To determine the interactions of essential oils, their mixtures (1 MIC:1 MIC, 1 MIC:2 MIC and 2 MIC:1 MIC) were applied. Generally, essential oils used in combinations influenced yeast's hydrophobic properties much more than applied separately. The essential oils' mixtures reduced hydrophobicity of Candida yeasts in the range of 8.2 to 45.1%, depending on combination and strain. The interaction indexes of essential oils used in combinations predominantly indicate their additive effect. The application of tea tree, thyme and clove essential oils, especially in combinations, decreases hydrophobicity of the tested Candida isolates with implications of a probable advantageous limitation of their ability to colonize the food production industry environment. INTRODUCTION Candida spp. strains are characterized by their ability to form a biofilm structure on solid surfaces, which causes significant problems in many industrial branches, but is also threatening to human health (Kojic & Da-rouiche, 2004; Stratford, 2006). Candida biofilm is a heterogeneous , spatially well-organized structure consisting of planktonic and mycelial yeast forms which are interdependent in the quorum sensing system and surrounded by an extracellular polysaccharide substance (Chan-dra et al., 2001; Donlan, 2001). The process of biofilm formation can be divided into three stages: early, lasting up to 11 hours; intermediate, lasting between 12 and 30 hours; and maturation, lasting from 38 to 72 hours. During the first two hours, floating planktonic cells of Can-dida albicans, in blastospor forms, adhere to the materials' surfaces and the first microcolonies are noticeable after 3–4 hours after inoculation. The development of extra-cellular matrix with the …


INTRODUCTION
Candida spp.strains are characterized by their ability to form a biofilm structure on solid surfaces, which causes significant problems in many industrial branches, but is also threatening to human health (Kojic & Darouiche, 2004;Stratford, 2006).Candida biofilm is a heterogeneous, spatially well-organized structure consisting of planktonic and mycelial yeast forms which are interdependent in the quorum sensing system and surrounded by an extracellular polysaccharide substance (Chandra et al., 2001;Donlan, 2001).The process of biofilm formation can be divided into three stages: early, lasting up to 11 hours; intermediate, lasting between 12 and 30 hours; and maturation, lasting from 38 to 72 hours.Dur-ing the first two hours, floating planktonic cells of Candida albicans, in blastospor forms, adhere to the materials' surfaces and the first microcolonies are noticeable after 3-4 hours after inoculation.The development of extracellular matrix with the cell wall polysaccharides containing mannose and glycosidic bonds, as the main component, dominates in the intermediate stage (Chandra et al., 2001).A further increase of extracellular matrix until complete surface covering by the colonies of Candida occurs during maturation of the biofilm structure stage.
Biofilm-forming microorganisms are characterized by high invasiveness, the ability to cause dangerous and difficult to treat infections (Donlan, 2001).Furthermore, the cells in the biofilm, compared to planktonic forms, showed reduced sensitivity to chemical compounds with antifungal activity and increased survival under unfavorable environmental conditions (Prażyńska & Gospodarek, 2014;Douglas, 2003).Currently available biofilm controlling methods rely on the prevention of surface contamination by following the principles of hygiene, the use of antifungal agents, the exchange of material, and minimizing yeast adhesion to the abiotic surface by the application of chemicals (Simoes et al., 2010).Limitation in the use of fungicides is caused by yeasts' resistance and potential risk to the humans exposed to the products of fungicide degradation (Krisch et al., 2011).Hence, a response to the increasingly sought after alternative natural compounds may be the essential oils.Essential oils (EOs) are plant-derived volatiles that can consist of more than 50 compounds, of which 1-3 are the main components representing 85-95% of the whole volume (Burt, 2004).The chemical profiles of the oils are crucial for their antimicrobial efficacy and the mechanism of action on the target organism.EOs are well known for their broad spectrum of antifungal activity, low toxicity, good biodegradability and safety (Kalemba & Kunicka, 2003).
The process of candidal adhesion, a prerequisite stage of the biofilm formation, is rather complex and involves both, biological and non-biological factors.The relative cell surface hydrophobicity of Candida, which is a property of the cell wall, is widely considered as a non-biological factor of critical importance pertaining to candidal adhesion (Ellepola & Samaranayake, 2001).In the light of potential use of essential oils as natural anticandidal agents, this study was undertaken to evaluate the effect of selected essential oils on hydrophobic properties of Candida strains of various origin.
Essential oils.The effect on C. albicans cells was estimated for essential oils of Melaleuca alternifolia (Maiden & Betche) Cheel (tea tree oil), Thymus vulgaris L. (thyme oil) and Syzygium aromaticum (L.) Merr.& L.M. Perry (clove oil), commercially produced and obtained from Pollena Aroma S.A. (Warsaw, Poland).Essential oils (EOs) were analyzed using Trace GC Ultra (Thermo Scientific) equipment combined with DSQ II mass spectrometer and with flame ionization detector (FID) and MS-FID splitter.Analysis was provided using a nonpolar chro-matography column Rtx-1 ms (60 m × 0.25 mm, film thickness 0.25 μm, Restek).The oven temperature was programmed as follows: 50-300ºC at 4ºC/min; injector temp.280°C; carrier gas helium with regular pressure 200 kPa, ionization energy 70 eV, ion source temperature 200°C.Identification of components was based on the comparison of their MS spectra with those of a laboratory made MS library, commercial libraries (NIST 98.1 and Mass Finder 4) along with the retention indices associated with a series of alkanes with linear interpolation (C8-C26).A quantitative analysis (expressed as percent ages of each component) was carried out by peak area normalization measurements without correction factors.The components of essential oils are presented in Table 1.
Cell surface hydrophobicity (CSH).The hydrophobicity of Candida strains was determined by microbial adhesion with a hydrocarbon method, according to Kanatiwela et al. (2013).The tested isolates were grown overnight in Sabouraud dextrose broth at 28°C and washed with phosphate buffered saline (NaCl 8 g/l, KCl 0.2 g/l, Na 2 HPO 4 1.44 g/l, KH 2 PO 4 0.24 g/l; pH 7.4).For adhesion assay, 2.5 ml of the cell suspensions (OD 520 = 1.0) were mixed with 0.5 ml of p-xylene (Sigma-Aldrich) in an acid-washed glass tube.The samples were incubated at 37°C for 10 min and vigorously mixed for 30 sec.After 45 min of incubation at 37°C, the absorbance of aqueous phase was measured at 520 nm.The percentage of cells in the xylene layer was used to estimate the hydrophobicity index, HI, according to the formula: where: A 1 -absorbance of inoculum, A 2absorbance of the aqueous phase.
The effect of essential oils on cell surface hydrophobicity of Candida spp.Candida strains' cell surface hydrophobicity was determined by incorporating essential oils in the inoculum, using the above procedure.As reference, antifungal antibiotics caspofungin acetate ( 1 3,4,7,9,11,17,16,21,25,27,29,Poland) at the concentration of 50 µg/ ml were used.EOs were applied at their MIC concentrations, specific for a particular strain and oil (Table 2).Each essential oil was used solely and in combinations (1 MIC:1 MIC, 2 MIC:1 MIC and 1 MIC:2 MIC).
In order to determine the interactions of essential oils in mixtures, the FIH (Fractional Influence on Hydrophobicity) were calculated, analogously to FIC (Fractional Inhibitory Concentration) (Silva et al., 2011).
-The effect of essential oils on hydrophobic properties of Candida spp.
Statistical analysis of the results.Statistical calculations were carried out with the use of Statistica 6.0 software package (StatSoft).All results are expressed as the mean ± S.D. of 3 independent experiments.In order to compare the means, One-Way Anova test was performed at p=0.05 significance level using the statistical package of Origin version 6.1 (OriginLab Corporation).

RESULTS AND DISCUSSION
This study was performed for five Candida isolates, chosen conscientiously among several ones, each being a representative for their species (data unpublished).The specific Candida species were selected according to their different sensitivity to antibiotics and diverse biochemical profiles (Maroszyńska et al., 2013).In addition, among them there were both, food-borne and clinical isolates.Collating such a heterogenic biological material, we would also like to check the spectrum of the chosen EOs anticandidal activity in the light of their use against a variety of environmental strains.Four of the five strains tested showed, according to the accepted classification (Nostro et al., 2004), high hydrophobic properties and their hydrophobicity indexes ranged from 76.4 to 91.2%.On the other hand, C. albicans clinical isolate cl/MP/08 was characterized by a medium cell surface hydrophobicity (68.7 ± 6.0%).Previously, it has been demonstrated that hydrophobic yeasts are more virulent than their hydrophilic counterparts (Hazen & Hazen, 1992).However, even among clinical Candida strains their hydrophobic properties are diversified and may vary from 40% to 99% (Noumi et al., 2011).Much lower values of cell surface hydrophobicity, in the range from 2% to 41%, was obtained in a group of 50 C. albicans clinical isolates by Raut et al. (2010).Furthermore, hydrophobicity seems to be a species-dependent feature, and besides C. albicans, high values of hydrophobicity were displayed by C. tropicalis, both being the most clinically relevant Candida species (Noumi et al., 2011;Silva-Dias et al., 2015).
In the present study, changes in cell surface hydrophobicity of Candida strains were evaluated under the influence of essential oils with regard to antifungal antibiotics, nystatin and caspofungin.The choice of antibiotics resulted from the following reasons: nystatin is on the World Health Organization's List of Essential Medicines (2015) as one of the most efficacious, safe and cost-effective medicines needed in a basic health care system, and caspofungin is a valuable and fairly new generation antibiotic with a broad spectrum of anticandidal activity (Wieczorek et al., 2008).The effect of essential oils and antibiotics tested was diversified and depended on the type of antimycotics, their concentration and the yeast tested.This strain-dependent susceptibility to the essential oils' action was also previously observed for the clinical isolates within C. albicans species (Rajkowska et al., 2015).The range of hydrophobicity changes observed for C. albicans isolates (Rajkowska et al., 2015) were not statistically different from those observed here by us for the Candida species employed.The hydrophobic abilities of Candida yeasts seem to be rather strain-than speciesdependent.For isolates of C. rugosa fo/BG/05, C. famata fo/LI/02, C. albicans cl/MP/08 and C. albicans ATCC 10231, statistically significant reduction in hydrophobic properties in the presence of essential oils was found (Fig. 1).Among these yeasts, the highest effect was expressed for C. rugosa fo/BG/05, and decrease in hydrophobicity index by 14.3 to 45.1% was observed under the influence of all the tested essential oils, used solely and in combinations, except for the mixture of tea tree and thyme oil (1 MIC: 2 MIC), as well as tea tree and clove oil (2 MIC: 1 MIC).
The statistically significant reduction, ranged from 17.6 to 38.2%, in HI values of C. albicans ATCC 10231 was noted for all essential oils' combinations, and additionally, for clove oil used solely (Fig. 1).Cell surface hydrophobicity of C. albicans cl/MP/08 clinical isolate was affected by six mixtures of essential oils, i.e. tea tree and thyme oil (1 MIC: 1 MIC, 1 MIC:2 MIC, 2 MIC: 1 MIC), tea tree and clove oil (1 MIC: 1 MIC), and thyme and clove oil (1 MIC:2 MIC, 2 MIC: 1 MIC), in a statistically significant way.In the case of C. famata fo/ LI/02, only two combinations of tea tree and thyme oil (1 MIC: 1 MIC and 2 MIC: 1 MIC) caused statistically significant reduction in hydrophobicity of 21.6% and 24.0%, respectively.Only for C. krusei fo/MP/02, the effect of essential oils on the hydrophobicity was ambiguous, and in the presence of tea tree oil, and combination of thyme and clove oil (1 MIC: 2 MIC), a statistically significant decrease in hydrophobic properties of almost 9% was noted.Whereas, after treatment with thyme oil and mixtures of tea tree and thyme oil (1 MIC: 2 MIC), as well as tea tree and clove oil (1 MIC: 1 MIC), hydrophobicity increased by 3.1 to 7.2% (Fig. 1).Generally, essential oils used solely, influenced the yeast hydrophobic properties less than when applied in combinations.
Among the tested antibiotics, nystatin reduced hydrophobicity of four yeasts (C.rugosa fo/BG/05, C. famata fo/LI/02, C. krusei fo/MP/02 and C. albicans cl/MP/08) by 16.1 to 25.4%, depending on the strain (Fig. 1) in a statistically significant way.The application of caspofungin resulted in decrease in hydrophobic properties of three isolates (fo/BG/05, fo/MP/02, cl/MP/08) in the range of 14.7-6.3%.Interestingly, in our previous publication we have demonstrated that C. famata fo/LI/02 and C. krusei fo/MP/02 expressed an intermediate sensitivity to nystatin (Maroszyńska et al., 2013), and the current research indicates that even in those strains nystatin may reduce their hydrophobicity.However, the primary mechanisms of action of antibiotics tested are different and involve an increase of the cell membrane permeability by nystatin, and inhibition of the synthesis of β-(1,3)d-glucan by caspofungin (Wieczorek et al., 2008).
Essential oils are complex mixtures of a wide diversity of components and their antimicrobial activity and mode  of action is therefore related to their composition, concentration and their possible interactions (Kalemba & Kunicka-Styczyńska 2003;Burt, 2004).In essential oil mixtures, four effects of their interaction can be highlighted: additive, antagonist, indifferent and synergistic.In our study, in the group of essential oil combinations, the additive effect was dominant (Table 3), which occurs when the combined effect of the components is equal to the sum of the individual effects (Lis-Balchin et al., 1998).Much less frequently, the use of oils in mixtures resulted in an indifferent effect, thus in these cases the activity of the combined substances was equal to the individual activities.
The literature data are focused on antimicrobial activity of essential oils and their components rather than on their influence on hydrophobic properties.It has been reported that EOs containing aldehydes or phenols, such as eugenol or thymol, as major compounds, showed the highest antimicrobial activity, followed by EOs containing terpene alcohols (Burt, 2004).Some studies have demonstrated that whole EOs usually have higher bioactivity than the mixtures of their major components, although interactions between components may lead not only to synergistic activity, but also to additive and antagonistic effects (Lambert et al., 2001;Burt, 2004;Bassolé & Juliani, 2012).Generally, compounds with similar structures exhibit additive rather than synergistic effects, and the occurrence of additive interactions of some EOs has been related to their major phenolic compounds (Lambert et al., 2001;Bassolé & Juliani, 2012).These findings may explain the predominant additive effect of essential oils tested in our study.
The relative cell surface hydrophobicity, CSH, of Candida spp. is considered to be a factor closely related to the adhesion properties of yeast (Ellepola & Samaranayake, 2001).The relationship between the hydrophobicity and enhanced adhesion of Candida species to plastic surfaces has been reported (Klotz et al., 1985).Some previous studies described a statistically significant positive correlation between CSH and candidal adhesion to buccal epithelial cells and denture acrylic surfaces (Samaranayake et al., 1995;Panagoda et al., 2000).It has been also reported, that biofilm biomass and biofilm formation has been associated with CSH (Blanco et al., 2010;Silva et al., 2011).On the other hand, Candida albicans is known to regulate cell surface hydrophobicity according to growth phase, environmental and nutritional conditions (Hazen et al., 1986).
Recently, a positive effect on Candida yeast cell surface hydrophobicity, adhesion abilities and biofilm formation has been demonstrated for variety of naturally derived substances or their constituents: eugenol (De Paula et al., 2014), human serum (Ding et al., 2014), Lactobacillus-derived biosurfactant (Ceresa et al., 2015), tyrosol (Monteiro et al., 2015), magnolol and honokiol (Sun et al., 2015), extracts of Brucea javanica and Piper betle (Nordin et al., 2013).Moreover, a positive correlation between the reduction in the CSH values and reduction of adhesion abilities and biofilm formation has been previously reported for Candida spp.(Borecká-Melkusová & Bujdáková, 2008;Nordin et al., 2013;De Paula et al., 2014).A significant decrease in the hydrophobicity (> 40%), resulted in reduction in the number of adhered Candida cells to mammalian HEp-2 cells of up to 68.9% (De Paula et al., 2014), and to salivary pellicle of up to 86.0% (Nordin et al., 2013).Effectively reduced Candida CSH may also be related to limitation of biofilm formation by up to 66.7%.However, meaningfully reduced ability to form biofilm has also been noted for yeasts, which did not show changes in their hydrophobic properties (Borecká-Melkusová & Bujdáková, 2008).
An important issue is also the safety of essential oils and their components that are not orally administered.In vivo studies have demonstrated the safety of the topical use of eugenol and carvacrol, components of clove and thyme oils, for the treatment of vaginal (Chami et al., 2004) and oral (Chami et al., 2005) candidosis in rats.Toxicity of essential oils depends distinctly on concentration and the form of application (Hammer et al., 2006).It has been reported that the irritant capacity of tea tree oil, tested at concentration of 5% and 25% in cream, ointment and gel formulations, and in patients, who were patch tested with 10% TTO, was very low (Aspres & Freeman, 2003;Veien et al., 2004).Irritant and contact allergy reactions may be usually avoided through the use of lower concentrations of the irritant and therefore the use of 100% essential oils is not recommended (Hammer et al., 2006).
Due to the fact, that surface hydrophobicity of Candida spp.cells may affect cellular behavior and adhesion, the reduction of yeasts' hydrophobic properties may limit their colonization abilities.In this context, the application of tea tree, thyme and clove essential oils, especially in combinations, may advantageously modulate the invasiveness of Candida yeasts by lowering their hydrophobicity.The mixtures of tea tree and thyme oils seem to be the promising agents with the spectrum of activity against all the Candida representatives tested.The presented results, demonstrating a decrease in surface hydrophobicity of food-borne strains, indicate a possibility of application of essential oils as antifungal agents under production conditions, notably in the food industry.
FIH of A oil = HI of mixture of oils/HI of A oil FIH of B oil = HI of mixture of oils/HI of B oil FIHi = FIH A + FIH B