The effect of thyme and tea tree oils on morphology and metabolism of C andida albicans *

Members of Candida species cause significant problems in medicine and in many industrial branches also. In order to prevent from Candida sp. development, essential oils are more and more frequently applied as natural, non-toxic, non-pollutive and biodegradable agents with a broad spectrum of antimicrobial activity. The aim of the research was to determine changes in morphology and metabolic properties of Candida albicans in the presence of thyme and tea tree oils. Changes of enzymatic activity of isolates were observed in the presence of both tested essential oils, and they were primarily associated with loss or decrease of activity of all enzymes detected for control. Furthermore, only for 3 out of 11 isolates additional activity of N-acetyl-β-glucosaminidase, α-mannosidase, α-fucosidase and trypsin was detected. Vivid changes in biochemical profiles were found after treatment with tea tree oil and they were related to loss of ability to assimilate d-xylose, d-sorbitol and d-trehalose. The main differences in morphology of isolates compared to the control strain concerned formation of pseudohyphae structures. Both examined essential oils caused changes in cell and colony morphology, as well as in the metabolism of Candida albicans. However, the extent of differences depends on the type and concentration of an essential oil. The most important finding is the broad spectrum of changes in yeast enzymatic profiles induced by thyme and tea tree oils. It can be supposed that these changes, together with loss of ability to assimilate saccharides could significantly impact Candida albicans pathogenicity.


INTRODUCTION
Candida species are currently the most common cause of fungal infections worldwide (Manolakaki et al., 2010), and the first Polish multicentre candidaemia study revealed that the most frequent fungal pathogen is Candida albicans (Nawrot et al., 2013).Many fungal species are harmless commensals or endosymbionts of hosts including humans.However, when mucosal barriers disrupted or the immune system is compromised they can invade and cause disease (Kourkoumpetis et al., 2010).In last 30 years there has been a significant increase in the incidence of fungal infections in humans (Lass-Flörl, 2009).
A number of factors have been implicated in this increased occurrence of fungal disease.Specific conditions of the organism and in particular during predisposing situations like: diabetes, pregnancy, genetic factors and the increased and widespread use of certain medical practices, such as immunosuppressive therapies, invasive surgical procedures and the use of cortisones, contraceptives, estrogen and in particular broad-spectrum antibiotics are significant (Samaranaykae et al., 2002;Hagerty et al., 2003;Kojic & Darouiche, 2004;Selvia et al., 2012).In order to prevent from Candida sp.development, essential oils are more and more frequently applied in food, cosmetic, pharmaceutical industry as well as in medicine and in the processes of washing and disinfection (Dorman & Deans, 2000;Batish et al., 2008;Kotzekidou et al., 2008).Essential oils are aromatic oily liquids plant origin, forming multicomponent mixtures of terpenes and terpenoids (Burt, 2004).Due to the broad spectrum of antimicrobial activity, low risk of side effects after their use and low risk of resistance development by microorganisms, the essential oils can provide a valuable alternative to synthetically produced substance (Budzyńska et al., 2011;Kalemba & Kunicka, 2003).Moreover, the essential oils are natural, non-toxic, non-pollutive and biodegradable compounds (Donaldson et al., 2005;Adorian & Buchbauer, 2010).
The literature data focused rather on antimicrobial activity of essential oils and their active compounds than their mechanisms of action.Therefore, the aim of this study was the evaluation of effect of thyme and tea tree oils on morphology and metabolic properties of yeast Candida albicans.

MATERIALS AND METHODS
Yeast.The study was carried out for collection strain Candida albicans ATCC 10231, which is typically used as a reference strain in the analysis of disinfectants and antifungal agents.The strain was maintained on Sabouraud dextrose agar (peptone 10 g/l, dextrose 20 g/l, agar 20 g/l) and activated through double passaging in Sabouraud liquid medium at 37°C for 24 h.
Essential oils.The effect of essential oils was estimated for thyme (Thymus vulgaris L.) and tea tree oil (Melaleuca alternifolia L.), obtained from Pollena Aroma S.A. in Warsaw (Poland).Essential oils were analyzed using Trace GC Ultra (Thermo Scientific) equipment combined with DSQ II mass spectrometer and with flame ionization detector (FID) throughout MS-FID splitter.Analysis was provided using nonpolar chromatography column Rtx-1 ms (60 m×0.25 mm, film thickness 0.25 μm, Restek).The oven temperature was programmed as followed: 50-300°C at 4°C/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 in a laboratory made MS library, com-mercial 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.
Colony morphology assay.Candida albicans cell suspension (approximately 30 cells on plate) was streaked on Sabouraud dextrose agar with addition of thyme or tea tree oil in a concentration from 0.0075 to 0.5% v/v.Plates were incubated at 37°C for up to 14 days and colonies of different morphology, in comparison with control grown only on Sabouraud dextrose agar, were used for further studies.

RESULTS AND DISCUSSION
The growth of Candida albicans strain was observed on solid medium supplemented with thyme oil in concentration below 0.25% v/v and tea tree oil (TTO) below 0.5% v/v, and these values correspond to minimal inhibitory concentrations (MICs) of examined essential oils.In analyzed oil concentration ranges colonial morphology differed from control, especially in size (Fig. 1).However, there was no correlation between the size of colonies and essential oils concentrations, and the smallest colonies were detected in the presence of thyme oil in concentration 0.125, 0.06, 0.0075 and tea tree oil -0.015% v/v.In turn, colonies of larger diameter than the control were formed after treatment with 0.03, 0.015% thyme oil and 0.25, 0.06 and 0.0075% TTO.The number of morphologically different colonies varied from 15% after treatment with 0.03% v/v TTO to 100% with 0.015% v/v thyme oil.
Colony morphology of yeast is strongly influenced by growth condition, including agar and nutrient concen- tration, pH, osmotic pressure (Vopálenská et al., 2005;Voordeckers et al., 2012).Yeasts respond to environmental conditions with a common gene-expression response, termed the environmental stress response (ESR).However, in addition to ESR response, each species responds to environmental changes with a great deal of precision in terms of the genes affected by each condition, the magnitude of their expression changes, and the kinetics of the response (Gasch, 2007).It seems that C. albicans may have evolved to respond to environmental conditions rather by triggering a change in cellular states than by ESR (Ernst, 2000).
Yeast colony morphology is controlled by a very large number of genes that are involved in different signalling cascades, including the MAPK, TORC, SNF1, RIM101 pathways (Granek & Magwene, 2010;Voordeckers et al., 2012).Other genes that affect colony morphology control protein sorting and epigenetic regulation (Voordeckers et al., 2012).Furthermore, many of the genes that are implicated in colony formation have been previously reported to control adhesion, mat formation and invasive growth (Madhani, 2000;Reynolds et al., 2006).Changes in C. albicans colony morphology are associated not only with differences in gene expression but also with the rearrangements of chromosomal DNA, and the most frequent changes involves the long chromosome VIII, which carries ribosomal DNA cistrons (Rustchenko-Bulgac, 1991).It cannot be excluded, that colonial morphology changes observed by us after treatment with essential oils may results from modifications of chromosomal DNA or gene expression, which will be checked in the further research.
Hyphae formation in C. albicans is linked to the colony morphology and associated with the processes of endocytosis and vacuolar sorting (Sudbery, 2011).This observation on dependence between colony and cell morphology is consistent with our data.
In our study differences in colony morphology were associated with different sizes of isolates' cells, expressed as morphology index, though changes did not have directional character.After treatment with 0.06, 0.03 and 0.015% v/v thyme oil in microscopic preparations, besides spherical, ovoid and elongated cells as in control, the pseudohyphae structures of morphology index ranged 3.84±0.36-3.93±0.28(Table 2, Fig. 2) occurred.Isolates Th/0.125,Th/0.0075/1,Th/0.0075/2,TTO/0.25,TTO/0.06,TTO/0.015 and TTO/0.0075 were characterized only by cells of spherical and ovoid shapes.For isolates of elongated cells, frequency distributions of these cells were usually lower compared with control and equaled from 4.0 to 13.5% (Table 2).
Cells of C. albicans are capable to develop in many diverse morphological forms, two of which are the most common i.e. ovoid, budding yeast form and the filamentous hyphal form (Odds, 1988).However, intermediate morphologies between these two forms, of morphology index in the range 2.5-4.0 occur frequently (Merson-Davies et al., 1991), which is in accordance with our results.In the presence of examined essential oils C. albicans strain showed changes in biochemical and enzymatic properties, but extensive differences were found for enzymatic profiles.Yeast ability to assimilate different sources of carbon changed for d-xylose and d-trehalose assimilation after treatment with 0.03% TTO and additionally for d-sorbitol in the presence of 0.015% TTO (Table 3).Changes in enzymatic profiles were primarily associated with loss or decrease of activity of all enzymes detected for control, i.e. alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and α-glucosidase.Furthermore, for isolate Th/0.0075/2 additional activity of N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase was detected.Interestingly, for isolate TTO/0.25 as the only one an increase of 4 enzymes activities and additional activity of N-acetyl-β-glucosaminidase was found.And isolate TTO/0.0075showed proteolytic activity of trypsin, which was not observed for control strain (Table 3).
Decrease of enzymatic activity of C. albicans strains could significantly reduce their pathogenicity.Batura-Gabryel and Młynarczyk (2000) have found correlation between presence of oral candidosis and secretion of lipase, valine arylamidase, cystine arylamidase, naphthol-AS-BI-phosphohydrolase and β-glucosidase by C. albicans strains.Similarly, C. albicans isolates from patients with bullous and connective tissue diseases revealed high activity of acid phosphatase, and those isolated from patients with neoplasms -acid phosphatase and naphthol-AS-BI-phosphohydrolase (Kwaśniewska et al., 2001).Yeast enzymes produced and released during reproduction and cell death initiate the inflammatory process by cytopathic effect, leading to epithelial cell damage (Louie et al., 1994).It has been shown that enzymes which play a key role in the pathogenesis of Candida species are proteases and phospholipases (Abaci, 2011, Schaller et al., 2005).Phospholipases damage mucosal cells and hydrolases are responsible for degradation of macromolecules.
Both of them facilitate fungal colonization process (Zhu & Filler, 2010).Proteases cause degradation of epithelial cells, cytokines and immunoglobulins, and modification of the fungal surface antigens, thus contribute to the process of adhesion and colonization of host tissues (Abaci, 2011, Schaller et al., 2005).Furthermore, alkaline phosphatase, N-acetyl-β-glucosaminidase and α-mannosidase inhibit the migration of neutrophils to sites of infection and lipase is considered as particularly important in the early stages of infection, when lipids may be used by fungi as a carbon source necessary to their further growth and development of infection (Nowicki & Korting, 1995).On the other hand, it has been shown that lipolytic activity of Candida spp.depends on carbon sources available in medium (Wróblewska et al., 2011).
In our study, in the presence of thyme and tea tree oils changes in C. albicans colony and cell morphology as well as, in most cases, reduction of enzymatic activity and loss of ability to assimilate saccharides were observed.These modifications may be caused by principal active constituents of examined essential oils, i.e. pinene, terpinen-4-ol, γ-terpinene, thymol, p-cymene.The antimicrobial action of essential oils and their monoterpenoid components is generally explained by toxic effects on membrane structure and function (Cox et al., 2000;Uribe et al., 1985).In yeast cells α-pinene and β-pinene, being compounds inter alia tea tree oil, destroy cellular integrity, inhibit respiration and ion transport processes and increase membrane permeability (Uribe et al., 1985).Apart of the cytotoxic effects, the interactions with the cell membrane, the antimicrobial action of monoterpenes such as thymol, carvacrol, p-cymene, and γ-terpinene may also result from a loss of ATP synthesis capacity required to support the regulation of several cell functions (Custódio et al., 2011).Moreover, essential oils show a concentration dependent post-antifungal effect (PAFE) and significantly reduce tolerance to oxidative stress (Budzyńska et al., 2013).
In recent years, there has been an increase in the number of scientific publications concerning the thera-  (Tullio et al., 2012).The therapeutic effectiveness of essential oils for mucosal candidiasis has been evaluated in a murine oral candidiasis model.The treatment with a tea tree oil and its main component terpinen-4-ol showed a decrease in the symptom score of tongues and in the viable Candida cell number (Ninomiya et al., 2012).It has been demonstrated, that the reduction of antifungal agents concentrations required to achieve lethal effect is possible by the application of essential oils into liposome-encapsulated combined preparations with silver ions (Low et al., 2013).These data may encourage the development of essential oils products for a practical and safe approach to topical treatment.The explanation of the mechanism of Candida albicans response to the essential oils requires multiplatform research, including rarely reported in the literature data phenotypic changes in yeast morphology and particularly changes within the metabolic profile.Changes that we observed for C. albicans in the presence of essential oils, especially in yeast enzymatic activity and carbon sources assimilation, may be associated with yeast response to environmental stress conditions, and may contribute to the reduction of C. albicans pathogenicity.

Figure 1 .
Figure 1.Candida albicans ATCC 10231 colony morphology in the presence of (A) thyme oil (Th) and (B) tea tree oil (TTO) in concentration from 0.0075 to 0.25% v/v; (C) control.Isolate symbol is composed as follows: essential oil type/ the oil concentration.

Table 2 . Morhology index (Mi) values for C. albicans after treatment with thyme (Th) and tea tree oil (TTO). Isolate symbol is composed as follows: essential oil type/ the oil concen- tration.
Changes in Candida morphology and metabolism caused by essential oils