Medical Mycology Advance Access published July 10, 2015 Medical Mycology, 2015, 00, 1–10 doi: 10.1093/mmy/myv052 Advance Access Publication Date: 0 2015 Original Article
Original Article
Effect of tyrosol on adhesion of Candida albicans and Candida glabrata to acrylic surfaces
Downloaded from http://mmy.oxfordjournals.org/ at University of Otago on July 12, 2015
Douglas Roberto Monteiro1,∗ , Leonardo Perina Feresin1 , ˜ 2, ˜ Arias1 , Valentim Adelino Ricardo Barao La´ıs Salomao Debora Barros Barbosa3 and Alberto Carlos Botazzo Delbem1 1
Department of Pediatric Dentistry and Public Health, Arac¸atuba Dental School, Univ Estadual Paulista ˜ Paulo, Brazil, 2 Department of Prosthodontics and Periodontology, (UNESP), 16015–050 Arac¸atuba/Sao ˜ Paulo, Brazil Piracicaba Dental School, University of Campinas (UNICAMP), 13414–903 Piracicaba/Sao 3 and Department of Dental Materials and Prosthodontics, Arac¸atuba Dental School, Univ Estadual Paulista ˜ Paulo, Brazil (UNESP), 16015–050 Arac¸atuba/Sao *To whom correspondence should be addressed. Douglas Roberto Monteiro, Department of Pediatric Dentistry and Public ˜ Paulo, Brazil. Health, Arac¸atuba Dental School, Univ Estadual Paulista (UNESP), 16015–050 Arac¸atuba/Sao Tel: +55 18 36363316; Fax: +55 18 36363245; E-mail: [email protected] Received 5 March 2015; Revised 1 May 2015; Accepted 1 June 2015
Abstract The prevention of adhesion of Candida cells to acrylic surfaces can be regarded as an alternative to prevent denture stomatitis. The use of quorum sensing molecules, such as tyrosol, could potentially interfere with the adhesion process. Therefore, the aim of this study was to assess the effect of tyrosol on adhesion of single and mixed cultures of Candida albicans and Candida glabrata to acrylic resin surfaces. Tyrosol was diluted in each yeast inoculum (107 cells/ml in artificial saliva) at 25, 50, 100, and 200 mM. Then, each dilution was added to wells of 24-well plates containing the acrylic specimens, and the plates were incubated at 37◦ C for 2 h. After, the effect of tyrosol was determined by total biomass quantification, metabolic activity of the cells and colony-forming unit counting. Chlorhexidine gluconate (CHG) was used as a positive control. Data were analyzed using analysis of variance (ANOVA) and the Holm–Sidak post hoc test (α = 0.05). The results of total biomass quantification and metabolic activity revealed that the tyrosol promoted significant reductions (ranging from 22.32 to 86.16%) on single C. albicans and mixed cultures. Moreover, tyrosol at 200 mM and CHG significantly reduced (p < 0.05) the number of adhered cells to the acrylic surface for single and mixed cultures of both species, with reductions ranging from 1.74 to 3.64-log10. In conclusion, tyrosol has an inhibitory effect on Candida adhesion to acrylic resin, and further investigations are warranted to clarify its potential against Candida infections. Key words: tyrosol, Candida albicans, Candida glabrata, adhesion, acrylic surface.
C The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.
All rights reserved. For permissions, please e-mail: [email protected]
1
2
Introduction
capacity [25]. Tyrosol production by Candida species has been reported by Cremer et al. [26]. These authors demonstrated that C. albicans and C. tropicalis secreted significantly higher amounts of tyrosol than C. glabrata and C. parapsilosis, implying a probable correlation with the pathogenicity of these species. Moreover, it may be produced by cells in planktonic state or forming biofilms [23]. Contrary to farnesol, tyrosol has been associated with accelerated conversion of yeasts in hyphae, contributing to the harmonized expression of virulence factors under stimulating conditions, like increased microbial density [23,24,27]. Recently, the combination of tyrosol with amphotericin B revealed a synergistic effect against C. krusei and C. tropicalis biofilms, achieving 90% reduction at 80 μM and 4 mg/l, respectively [28]. Although the involvement of tyrosol in the control of Candida morphogenesis had been elucidated, to the best of the authors’ knowledge its effect on adhesion of Candida cells to acrylic surfaces remains unknown. Therefore, the aim of this study was to assess the effect of different concentrations of tyrosol on the adhesion capacity of single and mixed cultures of C. albicans and C. glabrata to an acrylic resin surface. The hypothesis evaluated was that tyrosol displays an inhibitory effect on Candida adhesion to acrylic resin.
Materials and methods Candida strains and cultivation Two reference strains of the American Type Culture Collection (ATCC) were used in this study: C. albicans ATCC 10231 and C. glabrata ATCC 90030. All Candida strains were maintained at −70◦ C and grown aerobically for 24 h at 37◦ C on Sabouraud dextrose agar (SDA; Difco, Le Pont de Claix, France) plates. To obtain standard suspensions of each Candida cell, a loopful of the colonies from SDA plates was inoculated into Sabouraud dextrose broth (SDB; Difco) medium and incubated overnight at 37◦ C with agitation (120 rpm). Afterward, the Candida cells were collected by centrifugation (8000 rpm for 5 min), washed twice in phosphate buffered saline (PBS; pH 7, 0.1M), and resuspended in artificial saliva (AS) at 1 × 107 cells/ml using a Neubauer chamber. The composition of AS (pH 6.8) is described elsewhere [29].
Cell surface hydrophobicity The sessile drop method was used to determine the cell surface hydrophobicity. Cell suspensions of C. albicans ATCC 10231 and C. glabrata ATCC 90030 were prepared in SDB, as described above. However, for this assay, the cell
Downloaded from http://mmy.oxfordjournals.org/ at University of Otago on July 12, 2015
Denture stomatitis (DS) is an infectious process characterized by frequent episodes of recurrence, which essentially affects the palatal mucosa of complete denture wearers [1]. The most important microorganism associated with DS is the polymorphic fungus Candida albicans [2]. Nevertheless, other non-Candida albicans Candida species, such as Candida glabrata, have also taken part in the development of this condition [3,4]. DS is induced by trauma, precarious oral hygiene, lack of saliva in the oral mucosa covered by the prostheses, and microbial colonization of oral epithelium or inert surfaces [1,5,6]. The colonization of oral tissues, as well as acrylic surfaces, by Candida species depends on the adhesion capacity of these species to those surfaces. Adhesion capacity of yeasts is associated with a combination of specific and non-specific factors. The specific factors include interactions between cell wall adhesins and receptors of epithelial cells [7–9], while the hydrophobic and electrostatic interactions between microbial cells and substrate surfaces represent the non-specific factors [8,10,11]. The phenomenon of adhesion is also influenced by the surface roughness of the materials [12]. Furthermore, microbial adhesion is the preliminary stage in the processes of colonization, biofilm formation and development of infections [8]. Consequently, it is regarded as one of major virulence factors of pathogenic Candida species [8]. In this sense, it is essential to investigate novel compounds that may interfere in the yeast adhesion process to prevent subsequent biofilm formation, particularly in view of the Candida recalcitrance to conventional antifungals [13]. A promising therapy is the use of cell-cell communication molecules, known as quorum-sensing (QS) molecules [14]. QS molecules are produced naturally by microorganisms as a function of the cell density and govern a variety of behaviors [15]. The first QS molecule recognized in C. albicans was the farnesol [15], an aliphatic alcohol that may also be extracted from citrus fruits [16]. Farnesol acts by blocking the yeast-to-hyphae transformation at high microbial density [17], and several studies [18–22] have demonstrated its effect as an antibiofilm agent. Gori et al. [18] reported that farnesol at 10 μM reduced the adhesion of Debaryomyces hansenii to polystyrene. This compound was able to inhibit Staphylococcus epidermidis [19], C. albicans [20], and C. dubliniensis [21] biofilms and has shown synergistic or additive effects against C. albicans biofilms when combined with fluconazole, micafungin and amphotericin B [22]. The second well-known QS molecule in C. albicans is the aromatic alcohol tyrosol [15,23,24], which may also be found as a polyphenol of olive oil with antioxidant
Medical Mycology, 2015, Vol. 00, No. 00
Monteiro et al.
3
suspensions were resuspended in PBS to a concentration of 1 × 109 cells/ml. These cell suspensions were filtered under vacuum through cellulose acetate membranes (0.22 μm) previously soaked with 10 ml of deionized water. To standardize the humidity level, the membranes were cut into three pieces, placed on glass slides and inserted into petri dishes containing 1% agar and 10% glycerol during 3.5 h [30]. Five drops (volume of each drop: 3 μl) of deionized water were deposited on each membrane piece containing the cell lawns, and the contact angles were measured using a goniometer (Kruss DSA 100S, Kruss GMBH, Hamburg, Germany). The assays were performed on three separate occasions at 22 ± 2◦ C.
MIC was performed using the microbroth dilution method, as detailed elsewhere [29], with tyrosol (2-(4hydroxyphenyl) ethanol) purchased from Sigma-Aldrich, St Louis, USA. Briefly, C. albicans and C. glabrata cell suspensions were adjusted to turbidity 0.5 of McFarland standard in saline solution, and afterwards diluted in Roswell Park Memorial Institute (RPMI 1640; Sigma-Aldrich) medium. Then, 100 μl of each yeast suspension was added to the wells of a 96-well microtiter plate (Costar, Tewksbury, USA) containing 100 μl of each specific concentration of tyrosol (0–300 mM) diluted in RPMI 1640. The plates were incubated at 37◦ C for 48 h and the MIC was recorded as the lowest concentration of tyrosol demonstrating no yeast growth. Chlorhexidine gluconate (CHG) was used as positive control. Moreover, the minimum fungicidal concentration (MFC) was determined through plating the content of each well on SDA from the MIC point. All assays were performed in triplicate on three independent occasions.
Preparation and resin specimens
characterization
of
Germ tube formation by C. albicans The effect of tyrosol on germ tube formation by C. albicans ATCC 10231 was investigated according to Chauhan et al. [31], with some modifications. In brief, 5 ml of AS containing C. albicans ATCC 10231 at 1 × 107 cells/ml were prepared as described above and added to falcon tubes. Then, tyrosol was diluted in the inoculum to achieve concentrations of 25, 50, 100, and 200 mM. Tubes without tyrosol and with CHG at 0.37 mM were kept as negative and positive controls, respectively. After 2 h of incubation at 37◦ C, the amount of yeast and germ tube forms was determined using a Neubauer chamber. The assays were performed independently three times in quadruplicate, and the percentage of germ tubes formation compared with negative control was estimated according to the formula: =
No. germ tubes in treatment × 100. No. germ tubes in control
acrylic
The acrylic resin specimens were obtained from an aluminum matrix with internal molds which were fixed with wax (Wilson, Sao ˜ Paulo, Brazil) on a stainless steel plate, and included in a flask with a type III dental stone (Herodent, Petropolis, Brazil). Following the setting pro´ cess, the flask was opened, the wax was removed, and the matrix molds were cleansed. Afterwards, the QC20 denture acrylic resin (Dentsply Ind. e Com. Ltd., Petropolis, ´ Brazil) was manipulated, inserted into the matrix molds, and polymerized in accordance with the manufacturer’s advice. After polymerization of the acrylic resin, the specimens (10 × 10 × 3 mm) were removed from the matrix, finished using 150-, 300-, 600-, and 1200-grit sandpapers
Adhesion assays To evaluate the adhesion capacity of Candida species in the presence of tyrosol, single cultures of C. albicans ATCC 10231 and C. glabrata ATCC 90030 or mixed cultures of both yeasts were developed on acrylic specimens placed in 24-well microtiter plates (Costar, Tewksbury, USA). Tyrosol was diluted in the inoculum of each single yeast (1 × 107 cells/ml in AS) and in the mixed inoculum of two species (1 × 107 cells/ml of each strain) to obtain final concentrations of 25, 50, 100, and 200 mM. Afterward, 1 ml of each dilution was inoculated into wells containing the acrylic specimens, and the plates were incubated aerobically and statically for 2 h at 37◦ C. CHG at 0.37 mM (50 x MIC) was used as positive control. Acrylic resin
Downloaded from http://mmy.oxfordjournals.org/ at University of Otago on July 12, 2015
Determination of tyrosol minimum inhibitory concentration (MIC)
(3M/ESPE, Sumar´e, Brazil), rinsed with deionized water in an ultrasonic bath, and then sterilized by irradiation (60 Co; 25 KGy) before microbiological assays. A profilometer (Mitutoyo SJ-401; Mitutoyo Corp., Tokyo, Japan) was used to measure the average surface roughness (Ra - μm) of the acrylic specimens. Three measurements for each specimen were obtained using a cut-off of 0.25 mm and speed of 0.5 mm/s. Further, to characterize the surface hydrophobicity of specimens, the contact angle (θ ) values were determined by using an automatic goniometer (Kruss DSA 100S, Kruss GMBH, Hamburg, Germany). Three drops of deionized water were deposited at different locations on each acrylic surface and the mean was calculated. The assays were performed at 22 ± 2◦ C. Finally, these characterization experiments were carried out in order to obtain standardized acrylic surfaces.
4
specimens inoculated with yeast suspensions devoid of tyrosol were used as negative controls.
Quantification of total biomass
Metabolic activity by XTT assay Metabolic activity of the adhered cells was measured by the XTT (2,3-(2-methoxy-4-nitro-5-sulphophenyl)5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) (Sigma-Aldrich) reduction assay [32]. Following the adhesion of single and mixed cultures, the supernatants were removed and the non-adherent cells were removed by washing the specimens with PBS. Then, the specimens were transferred to new wells, and 1 ml of a solution containing 150 mg XTT/L and 10 mg of phenazine methosulphate/l (Sigma-Aldrich) was added to each well. After incubation (120 rpm) in the dark at 37◦ C for 3 h, aliquots of 200 μl were transferred to 96-well plates, and the absorbance of XTT-formazan was read at 490 nm. The values were standardized per unit area of acrylic specimens (Abs/cm2 ), and the wells containing AS without Candida cells were considered as blanks. The assays were carried out in triplicate and on three separate occasions.
surfaces. Thereafter, serial decimal dilutions (in PBS) were plated on SDA (for single cultures) and on CHROMagar Candida (Difco) (for mixed cultures), and the agar plates were incubated for 24–48 h at 37◦ C. The number of CFUs was log-transformed and standardized per unit area (Log10 CFU/cm2 ) of acrylic specimens, and the assays were also performed independently three times in triplicate.
Scanning electron microscopy (SEM) analysis Cultures of Candida species were developed during 2 h on acrylic specimens in the presence of tyrosol, as previously described. Subsequently, the adhered cells on specimens were rinsed with PBS, dehydrated in a sequence of ethanol (70% for 10 min, 95% for 10 min, and 100% for 20 min), air dried in a desiccator, and finally sputter coated with gold prior to observations (JSM 5600LV, Jeol, Tokyo, Japan).
Statistical analysis SigmaPlot 12.0 software (Systat Software Inc., San Jose, USA) was employed for the statistical analysis with a confidence level of 95%. Data passed the normality test (Shapiro–Wilk), and then parametric statistical analyses were conducted with one-way ANOVA followed by Holm– Sidak post hoc test.
Results Characterization of cell and acrylic surfaces It was possible to observe that the surfaces of all strains showed a hydrophilic behavior (θ < 65◦ ) [33], with mean values of water contact angle of 10.52◦ and 18.40◦ for C. albicans ATCC 10231 and C. glabrata ATCC 90030, respectively. Regarding acrylic specimens, the mean value of Ra was 0.11 ± 0.02 μm, and the high water contact angle (106.23◦ ± 09.41) revealed hydrophobic acrylic surfaces.
MIC and MFC Quantification of cultivable cells The number of cultivable cells from single and mixed cultures was enumerated by counting colony-forming units (CFUs). After an adhesion period (2 h), acrylic specimens were washed once with 1 ml of PBS, inserted in falcon tubes containing 1 ml of PBS, sonicated at 30 W for 30 s, and vigorously vortexed for 1.5 min. CV staining assay was used to confirm the removal of fungal cells from acrylic
C. albicans ATCC 10231 was more susceptible to tyrosol (MIC = 50 mM; MFC = 90 mM) than C. glabrata ATCC 90030 (MIC = 90 mM; MFC = 100 mM). Moreover, MIC and MFC values of CHG were 0.0074 and 0.0296 mM for C. albicans ATCC 10231, and 0.0074 and 0.0148 mM for C. glabrata ATCC 90030. Thus, for all strains assayed, MIC and MFC values of tyrosol were about 3,000–12,000 times higher than those obtained for CHG.
Downloaded from http://mmy.oxfordjournals.org/ at University of Otago on July 12, 2015
Measurement of total biomass was done by crystal violet (CV) staining assay [29]. After the adhesion period (2 h), nonadherent cells were removed by washing the acrylic specimens with 1 ml of PBS. Next, Candida adhered cells were fixed with 1 ml of 99% methanol (Sigma-Aldrich) during 15 min, dried at room temperature and stained with 1 ml of 1% CV (Sigma-Aldrich) solution for 5 min. CV bound to the adhered cells in single and mixed cultures was removed with 1 ml of 33% acetic acid (Sigma-Aldrich). The absorbance of the resulting solution was recorded at 570 nm (Eon Microplate Spectrophotometer; Bio Tek, Winooski, USA) and standardized according to the area of acrylic specimens (Abs/cm2 ). Wells containing acrylic specimens inoculated with AS devoid of Candida species were used as blanks. The assays were carried out independently three times in triplicate.
Medical Mycology, 2015, Vol. 00, No. 00
Monteiro et al.
5
Germ tube formation by C. albicans Tyrosol at 25, 50, 100, and 200 mM promoted reductions in the number of germ tubes of 67.42, 91.67, 96.97, and 97.47%, respectively. For CHG, a 92.42% reduction was observed, compared with the negative control.
Effect of tyrosol on total biomass Figure 1 displays the effect of different concentrations of tyrosol on total biomass of Candida cells adhered to acrylic surfaces. Tyrosol at 25, 50, 100, and 200 mM promoted significant reductions in the C. albicans ATCC 10231 biomass of 35.13% (P < .001), 48.64% (P < .001), 45.94% (P < .001), and 54.05% (P < .001), respectively, compared to the negative control group. The higher decrease in the total biomass (62.16%, P < .001) was noted for the group exposed to CHG. For C. glabrata ATCC 90030, only the treatment with CHG generated significant reduction in the biomass (43.75%, P = .002). In mixed culture, the treatment with tyrosol also exhibited significant decreases (P < .05) in the total biomass, ranging from 25.58% to 44.18%. When compared to the negative control, once again, CHG showed the highest decrease.
Effect of tyrosol on metabolic activity The results of XTT reduction (Fig. 2) for C. albicans ATCC 10231 showed that the highest reductions in metabolic
activity were attained with 200 mM tyrosol (86.16%, P < .001) and CHG (89.28%, P < .001), without a statistical difference between these groups. Moreover, tyrosol at 25, 50, and 100 mM promoted decreases in metabolic activity of 22.32% (P = .113), 51.33% (P = .002), and 51.78% (P = .002), respectively, compared to the negative control. The mixed Candida culture demonstrated a similar pattern, where the XTT colorimetric readings decreased with increasing concentrations of tyrosol. However, for C. glabrata ATCC 90030 the treatments did not significantly affect (P > .05) the metabolic activity of cells.
Effect of tyrosol on cultivable cells Mean values of the log10 CFU/cm2 obtained for each Candida strain in single and mixed cultures are shown in Figure 3. For all strains, after 2 h of incubation of the Candida cells, there were significant reductions (P < .05) in the number of adhered cells for the groups treated with tyrosol at 200 mM and CHG, compared to the negative controls and the groups treated with tyrosol at 25, 50, and 100 mM. The reductions in the number of CFUs ranged from 1.74 to 3.64-log10 , and there were no differences between 200 mM tyrosol and CHG. Interestingly, C. albicans ATCC 10231 and C. glabrata ATCC 90030 adhered cells in single cultures showed the same sensitivity to tyrosol and CHG than the ones in mixed cultures.
Downloaded from http://mmy.oxfordjournals.org/ at University of Otago on July 12, 2015
Figure 1. Average absorbances per cm2 obtained with crystal violet assay for Candida cells adhered (2 h) to acrylic in the presence of different concentrations of tyrosol (25, 50, 100, and 200 mM). NC = negative control (Candida culture without tyrosol). PC = positive control (chlorhexidine gluconate at 0.37 mM). Error bars display standard deviation of the means. ∗ Denotes P < .05, as compared to the negative control group, using one-way ANOVA with post hoc Holm–Sidak test.
6
Medical Mycology, 2015, Vol. 00, No. 00
Figure 3. Mean values of the logarithm of colony forming units per cm2 (log10 CFU/cm2 ) for Candida cells adhered (2 h) to acrylic in the presence of different concentrations of tyrosol (25, 50, 100, and 200 mM). NC = negative control (Candida culture without tyrosol). PC = positive control (chlorhexidine gluconate at 0.37 mM). Error bars display standard deviation of the means. ∗ Denotes P < .05, as compared to the negative control group, using one-way ANOVA with post hoc Holm–Sidak test.
SEM analysis The images in Figure 4 represent the pattern of adhered cells found for the mixed culture, which was similar for all the others (single cultures of both species). SEM observation of the negative control group revealed agglomerates of
yeasts and filamentous forms (mainly pseudohyphae) covering most of the acrylic surface (Fig. 4a), whereas the groups exposure to tyrosol at 25 mM (Fig. 4c), 50 mM (Fig. 4d), and 100 mM (Fig. 4e) comprised sparse yeasts as well as aggregated cells mostly consisting of yeast forms. However,
Downloaded from http://mmy.oxfordjournals.org/ at University of Otago on July 12, 2015
Figure 2. Average absorbances per cm2 obtained with XTT reduction assay for Candida cells adhered (2 h) to acrylic in the presence of different concentrations of tyrosol (25, 50, 100, and 200 mM). NC = negative control (Candida culture without tyrosol). PC = positive control (chlorhexidine gluconate at 0.37 mM). Error bars display standard deviation of the means. ∗ Denotes P < .05, as compared to the negative control group, using one-way ANOVA with post hoc Holm–Sidak test.
Monteiro et al.
7
the treatment with 200 mM tyrosol and CHG resulted in few aggregated yeasts partially covering the acrylic surface (Fig. 4b and f).
Discussion Adherence of microorganisms to biotic and abiotic surfaces is a necessary condition for biofilm formation and this phenomenon depends, among other factors, on the material surface characteristics. Literature data show that the higher the roughness and the hydrophobicity degree, the greater the microbial adhesion [34,35]. The surface roughness provides pores in which the microorganisms are confined and thus protected from the oral shear forces, enabling sufficient time for irreversible adherence of cells to the surface [36]. With respect to substrate wettability, the contact angle results from the equilibrium between the surface and interfacial energy, where the greater wettability, the lower the contact angle [37]. Considering these aspects, the roughness and the hydrophobicity degree of the acrylic specimens were measured in order to standardize them prior to microbiological assays. The cell surface hydrophobicity also influences the adhesion process. A previous study demonstrated that the Candida strains with highest hydrophobicity degree adhered more to polystyrene [38]. In the present study, C. glabrata ATCC 90030 showed a more hydrophobic behavior than
C. albicans ATCC 10231. Similar results were noted by Luo & Samaranayake [39]. These authors found contact angle values of 38.48◦ e 6.00◦ , respectively, for C. glabrata and C. albicans. Li et al. [40] verified that C. glabrata did not adhere well to oral keratinocytes, but adhered better to the acrylic surface, as compared with C. albicans. It is interesting to note that the different contact angle values observed for a same species in different studies may be due to physiological differences among the strains tested. Although C. albicans is a polymorphic fungus with ability to form pseudohyphae and hyphae, MIC and MFC values for this species were lower than those found for C. glabrata, which grows only as yeast. These findings are clinically important because C. albicans is the main pathogen involved in cases of denture stomatitis [2]. On the other hand, the lower susceptibility of C. glabrata is in line with previous studies showing that this strain is resistant to conventional antifungal agents, making it difficult the combat to this species [41]. The MIC values found for tyrosol in the present study may be considered high when compared to those found for farnesol in previous studies [21,22]. Jabra-Rizk et al. [21] demonstrated that the farnesol MIC values were 200 μM and > 250 μM for C. dubliniensis and C. albicans, respectively. These differences may have occurred because farnesol blocks the formation of germ tubes at low concentrations [17]. Furthermore, the lipophilic properties of this
Downloaded from http://mmy.oxfordjournals.org/ at University of Otago on July 12, 2015
Figure 4. Scanning electron microscopy (SEM) images of Candida cells in mixed culture (C. albicans ATCC 10231 + C. glabrata ATCC 90030) adhered (2 h) to acrylic surface in the presence of tyrosol at 25 mM (c), 50 mM (d), 100 mM (e), and 200 mM (f). NC (a) = negative control (Candida culture without tyrosol). PC (b) = positive control (chlorhexidine gluconate at 0.37 mM). Magnification: x 1000. Bar, 10.0 μm.
8
compared to the negative control group. Nevertheless, there were decreases in the number of germ tubes in the groups treated with tyrosol. These findings together with the results of CFU quantification (Fig. 3) may indicate that the significant reductions in biomass for single C. albicans ATCC 10231 and mixed cultures treated with tyrosol at 25, 50, and 100 mM (Fig. 1) occurred due to decrease in the number of pseudohyphae and hyphae. In fact, hyphae are elongated multicellular structures which retain greater amounts of crystal violet than yeast cells. Moreover, tyrosol might have acted by inhibiting the incipient production of extracellular matrix. It may also be deduced that the decrease in mixed cultures biomass is due to the effect of tyrosol on C. albicans ATCC 10231, while the lack of activity on single C. glabrata ATCC 90030 (Fig. 1) is because this species grows only as yeast and has a higher hydrophobicity degree, which favors the adherence to acrylic [39,40] and hinders its removal. Literature data show that tyrosol regulates the cell density by inducing the transformation of yeasts in hyphae [23,24,27]. However, the results of this study suggest that the exogenous administration of tyrosol at high concentrations may have altered the balance of its intracellular levels and resulted in the inhibition of hyphae formation on newly generated cells. Results from an XTT assay showed that tyrosol at 50 and 100 mM was able to significantly reduce the metabolic activity of cells in single cultures of C. albicans ATCC 10231 and mixed culture of both species (Fig. 2). On the other hand, these same tyrosol concentrations did not promote significant reductions in the number of CFUs (Fig. 3). Thus, it is evident that the Candida cells exposed to 50 and 100 mM tyrosol continued with potential to grow in solid medium, even with reduced metabolic activity. Noteworthy, tyrosol at 200 mM displayed an antiadhesion effect similar to CHG in all assays. However, the effective concentration of CHG (0.37 mM) was about 540 times lower than the highest tyrosol concentration (200 mM) evaluated in this study. Tyrosol is a natural compound produced by some Candida species [26], and this feature may explain, at least in part, why its effective concentration was much higher than that found for CHG. In addition, some components of artificial saliva, such as mucin used in this study, may have bound to tyrosol, requiring higher concentrations for achieving a good antiadhesion effect. Published data with some cell types show that tyrosol has no cytotoxic effect. Anter et al. [43] demonstrated that this compound at 140 μM prevented H2 O2 induced damage, having an antigenotoxic effect. Further, tyrosol was not shown to be toxic against cells from the salivary gland [44] and kidney [45]. Nevertheless, studies are needed to assess the toxic effects of tyrosol on mammalian cells at the concentrations used in the present study.
Downloaded from http://mmy.oxfordjournals.org/ at University of Otago on July 12, 2015
compound may favor its binding to the membrane, thereby changing its fluidity [17]. However, the most essential question of this research was whether the tyrosol would be able to reduce or eradicate Candida cell adhesion to acrylic resin. Adhesion of yeasts in the presence of tyrosol was assessed by quantification of total biomass, metabolic activity, cultivable cells and SEM observations. The study hypothesis was accepted since tyrosol was shown to have an inhibitory effect on the adhesion of single and mixed cultures of Candida species to acrylic resin. The tyrosol concentrations used in adhesion assays were based on the lowest MIC value (50 mM for C. albicans ATCC 10231). Therefore, the concentrations tested were 25 (1/2 MIC), 50 (MIC), 100 (2 x MIC), and 200 mM (4 x MIC). In general, the adhesion assays revealed significant effects of tyrosol in reducing total biomass (Fig. 1) and metabolic activity (Fig. 2) against single C. albicans ATCC 10231 and mixed cultures, with the highest decreases for the groups exposed to 200 mM tyrosol. For C. glabrata ATCC 90030, tyrosol did not promote significant reduction in total biomass and metabolic activity (Figs. 1 and 2). On the other hand, for all cultures, the results of CFU quantification showed that only the treatments with tyrosol at 200 mM and CHG at 0.37 mM significantly reduced the number of adhered cells to acrylic surfaces (Fig. 3), and the SEM observations confirmed these findings (Fig. 4). The adhesion of yeasts is associated to nonspecific (cell surface hydrophobicity) and specific factors (cell wall adhesins) [42]. Considering that tyrosol reduced germ tube formation, fungal metabolism and proliferation, it is possible that this compound may have interfered with the synthesis or expression of filament-associated adhesins on the surface of the cells, thereby reducing induced-adhesion, while passive adhesion due to hydrophobic interactions might have remained unaffected. It should be noted that all quantitative methods used to evaluate the effect of tyrosol on Candida adherence have some limitation; therefore, they should always be used as complementary methods. The CV staining assay does not allow the differentiation between living and dead cells. Although the results from an XTT assay are good predictors of the effect of antimicrobial agents, they do not always ensure equivalence with cell death. Furthermore, one of the limitations of the CFU quantification is that not every viable cell is also culturable in agar medium. All these aspects may help explain why the results of CFU quantification were not similar to those found for total biomass and metabolic activity. Additionally, the SEM images (Fig. 4) clearly show that the treatments with tyrosol at 25, 50, and 100 mM did not promote significant reductions in the Candida adherence,
Medical Mycology, 2015, Vol. 00, No. 00
Monteiro et al.
Acknowledgments This study was supported by Sao ˜ Paulo Research Foundation (FAPESP; processes 2013/17767–2 and 2013/10285–2), Brazil. We thank Adaias Oliveira Matos, University of Campinas (UNICAMP), for performing SEM analysis.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.
References 1. Jeganathan S, Lin CC. Denture stomatitis: a review of the aetiology, diagnosis and management. Aust Dent J 1992; 37(2): 107–114. 2. Odds FC. Pathogenesis of Candida infections. J Am Acad Dermatol 1994; 31(3 Pt 2): S2–S5. 3. Coco BJ, Bagg J, Cross LJ et al. Mixed Candida albicans and Candida glabrata populations associated with the pathogenesis of denture stomatitis. Oral Microbiol Immunol 2008; 23(5): 377–383. 4. Vanden Abbeele A, de Meel H, Ahariz M et al. Denture contamination by yeasts in the elderly. Gerodontology 2008; 25(4): 222–228.
5. Wilkieson C, Samaranayake LP, MacFarlane TW et al. Oral candidosis in the elderly in long term hospital care. J Oral Pathol Med 1991; 20(1): 13–16. 6. Budtz-Jorgensen E, Theilade E, Theilade J. Quantitative relation¨ ship between yeast and bacteria in denture-induced stomatitis. Scand J Dent Res 1983; 91(2): 134–142. 7. Desai C, Mavrianos J, Chauhan N. Candida glabrata Pwp7p and Aed1p are required for adherence to human endothelial cells. FEMS Yeast Res 2011; 11(7): 595–601. 8. Cotter G, Kavanagh K. Adherence mechanisms of Candida albicans. Br J Biomed Sci 2000; 57(3): 241–249. 9. Vidotto V, Mantoan B, Pugliese A et al. Adherence of Candida albicans and Candida dubliniensis to buccal and vaginal cells. Rev Iberoam Micol 2003; 20(2): 52–54. 10. Jones DS, McGovern JG, Woolfson AD et al. The effects of hexetidine (Oraldene) on the adherence of Candida albicans to human buccal epithelial cells in vitro and ex vivo and on in vitro morphogenesis. Pharm Res 1997; 14(12): 1765–1771. 11. Jabra-Risk MA, Falker WA, Merz WG et al. Cell surface hydrophobicity-associated adherence of Candida dubliniensis to human buccal epithelial cells. Rev Iberoam Micol 2001; 18(1): 17–22. 12. Quirynen M, Bollen CM. The influence of surface roughness and surface-free energy on supra- and subgingival plaque formation in man. A review of the literature. J Clin Periodontol 1995; 22(1): 1–14. 13. Chandra J, Mukherjee PK, Leidich SD et al. Antifungal resistance of candidal biofilms formed on denture acrylic in vitro. J Dent Res 2001; 80(3): 903–908. 14. Weber K, Schulz B, Ruhnke M. The quorum-sensing molecule E,E-farnesol - its variable secretion and its impact on the growth and metabolism of Candida species. Yeast 2010; 27(9): 727–739. 15. Albuquerque P, Casadevall A. Quorum sensing in fungi—a review. Med Mycol 2012; 50(4): 337–345. 16. Unnanuntana A, Bonsignore L, Shirtliff ME et al. The effects of farnesol on Staphylococcus aureus biofilms and osteoblasts: an in vitro study. J Bone Joint Surg Am 2009; 91(11): 2683–2692. 17. Hornby JM, Jensen EC, Lisec AD et al. Quorum sensing in the dimorphic fungus Candida albicans is mediated by farnesol. Appl Environ Microbiol 2001; 67(7): 2982–2992. 18. Gori K, Knudsen PB, Nielsen KF et al. Alcohol-based quorum sensing plays a role in adhesion and sliding motility of the yeast Debaryomyces hansenii. FEMS Yeast Res 2011; 11(8): 643–652. 19. Pammi M, Liang R, Hicks JM et al. Farnesol decreases biofilms of Staphylococcus epidermidis and exhibits synergy with nafcillin and vancomycin. Pediatr Res 2011; 70(6): 578–583. 20. Ramage G, Saville SP, Wickes BL et al. Inhibition of Candida albicans biofilm formation by farnesol, a quorumsensing molecule. Appl Environ Microbiol 2002; 68(11): 5459–5463. 21. Jabra-Rizk MA, Shirtliff M, James C et al. Effect of farnesol on Candida dubliniensis biofilm formation and fluconazole resistance. FEMS Yeast Res 2006; 6(7): 1063–1073. 22. Katragkou A, McCarthy M, Alexander EL et al. In vitro interactions between farnesol and fluconazole, amphotericin B or micafungin against Candida albicans biofilms. J Antimicrob Chemother 2015; 70(2): 470–478.
Downloaded from http://mmy.oxfordjournals.org/ at University of Otago on July 12, 2015
As for mechanisms of action, CHG acts on the rupture of the yeast cell membrane, resulting in alteration of the cell permeability and loss of intracellular components [46,47]. On the other hand, tyrosol’s antimicrobial mechanism of action remains unknown. For Staphylococcus aureus, Inoue et al. [48] observed a relationship between the K+ ion loss and the antimicrobial activity of terpene alcohols. Consequently, based on the idea of Pammi et al. [19], it may be hypothesized that the tyrosol acts on the integrity of yeast membrane. Finally, in view of the limitations of this in vitro study, it may be concluded that tyrosol has an inhibitory effect on the adhesion of single and mixed cultures of Candida species to acrylic resin, and this QS molecule at 200 mM behaved similarly to the positive control. Moreover, assuming that in the oral cavity forces of different magnitudes are present during mastication, phonation and deglutition [49], future studies of microbial adhesion mimetizing the oral conditions, and with a larger number of strains, should be conducted. The evaluation of tyrosol’s mechanism of action and its effect alone or in combination with conventional antifungal drugs on biofilm formation must also be investigated. All these studies will contribute significantly to improving oral health and quality of life of denture wearers through the development of formulations containing tyrosol that may be used in prevention and control of Candida-associated denture stomatitis.
9
10
37.
38.
39.
40.
41.
42.
43.
44. 45.
46. 47.
48.
49.
and its relation to adherence of Candida albicans. J Prosthodont Res 2012; 56(4): 272–280. Ellepola AN, Samaranayake LP. Antimycotic agents in oral candidosis: an overview: 1. Clinical variants. Dent Update 2000; 27(3): 111–116. Klotz SA, Drutz DJ, Zajic JE. Factors governing adherence of Candida species to plastic surfaces. Infect Immun 1985; 50(1): 97–101. Luo G, Samaranayake LP. Candida glabrata, an emerging fungal pathogen, exhibits superior relative cell surface hydrophobicity and adhesion to denture acrylic surfaces compared with Candida albicans. APMIS 2002; 110(9): 601–610. Li L, Redding S, Dongari-Bagtzoglou A. Candida glabrata: an emerging oral opportunistic pathogen. J Dent Res 2007; 86(3): 204–215. Fidel PL, Jr, Vazquez JA, Sobel JD. Candida glabrata: review of epidemiology, pathogenesis, and clinical disease with comparison to C. albicans. Clin Microbiol Rev 1999; 12(1): 80–96. Silva S, Negri M, Henriques M et al. Adherence and biofilm formation of non-Candida albicans Candida species. Trends Microbiol 2011; 19(5): 241–247. Anter J, Tasset I, Demyda-Peyras ´ S et al. Evaluation of potential antigenotoxic, cytotoxic and proapoptotic effects of the olive oil by-product “alperujo”, hydroxytyrosol, tyrosol and verbascoside. Mutat Res Genet Toxicol Environ Mutagen 2014; 772: 25–33. Babich H, Visioli F. In vitro cytotoxicity to human cells in culture of some phenolics from olive oil. Farmaco 2003; 58(5): 403–407. Loru D, Incani A, Deiana M et al. Protective effect of hydroxytyrosol and tyrosol against oxidative stress in kidney cells. Toxicol Ind Health 2009; 25(4–5): 301–310. Ellepola AN, Samaranayake LP. Oral candidal infections and antimycotics. Crit Rev Oral Biol Med 2000; 11(2): 172–198. Kuyyakanond T, Quesnel LB. The mechanism of action of chlorhexidine. FEMS Microbiol Lett 1992; 100(1–3): 211–215. Inoue Y, Shiraishi A, Hada T et al. The antibacterial effects of terpene alcohols on Staphylococcus aureus and their mode of action. FEMS Microbiol Lett 2004; 237(2): 325–331. Vissink A, Waterman HA, s-Gravenmade EJ et al. Rheological properties of saliva substitutes containing mucin, carboxymethylcellulose or polyethylenoxide. J Oral Pathol 1984; 13(1): 22–28.
Downloaded from http://mmy.oxfordjournals.org/ at University of Otago on July 12, 2015
23. Alem MA, Oteef MD, Flowers TH et al. Production of tyrosol by Candida albicans biofilms and its role in quorum sensing and biofilm development. Eukaryot Cell 2006; 5(10): 1770–1779. 24. Chen H, Fujita M, Feng Q et al. Tyrosol is a quorum-sensing molecule in Candida albicans. Proc Natl Acad Sci U S A 2004; 101(14): 5048–5052. 25. Moreno JJ. Effect of olive oil minor components on oxidative stress and arachidonic acid mobilization and metabolism by macrophages RAW 264.7. Free Radic Biol Med 2003; 35(9): 1073–1081. 26. Cremer J, Vatou V, Braveny I. 2,4-(hydroxyphenyl)-ethanol, an antioxidative agent produced by Candida spp., impairs neutrophilic yeast killing in vitro. FEMS Microbiol Lett 1999; 170(2): 319–325. 27. Chen H, Fink GR. Feedback control of morphogenesis in fungi by aromatic alcohols. Genes Dev. 2006; 20(9): 1150–1161. 28. Shanmughapriya S, Sornakumari H, Lency A et al. Synergistic effect of amphotericin B and tyrosol on biofilm formed by Candida krusei and Candida tropicalis from intrauterine device users. Med Mycol 2014; 52(8): 853–861. 29. Monteiro DR, Gorup LF, Silva S et al. Silver colloidal nanoparticles: antifungal effect against adhered cells and biofilms of Candida albicans and Candida glabrata. Biofouling 2011; 27(7): 711–719. 30. Silva S, Negri M, Henriques M et al. Silicone colonization by non-Candida albicans Candida species in the presence of urine. J Med Microbiol 2010; 59(Pt 7): 747–754. 31. Chauhan NM, Raut JS, Karuppayil SM. A morphogenetic regulatory role for ethyl alcohol in Candida albicans. Mycoses 2011; 54(6): e697–e703. 32. Silva S, Henriques M, Oliveira R et al. In vitro biofilm activity of non-Candida albicans Candida species. Curr Microbiol 2010; 61(6): 534–540. 33. Vogler EA. Structure and reactivity of water at biomaterial surfaces. Adv Colloid Interface Sci 1998; 74: 69–117. 34. Bulad K, Taylor RL, Verran J et al. Colonization and penetration of denture soft lining materials by Candida albicans. Dent Mater 2004; 20(2): 167–175. 35. Nikawa H, Iwanaga H, Kameda M et al. In vitro evaluation of Candida albicans adherence to soft denture-lining materials. J Prosthet Dent 1992; 68(5): 804–808. 36. Al-Dwairi ZN, Al-Quran FA, Al-Omari OY. The effect of antifungal agents on surface properties of poly(methyl methacrylate)
Medical Mycology, 2015, Vol. 00, No. 00
Original Article
Effect of tyrosol on adhesion of Candida albicans and Candida glabrata to acrylic surfaces
Downloaded from http://mmy.oxfordjournals.org/ at University of Otago on July 12, 2015
Douglas Roberto Monteiro1,∗ , Leonardo Perina Feresin1 , ˜ 2, ˜ Arias1 , Valentim Adelino Ricardo Barao La´ıs Salomao Debora Barros Barbosa3 and Alberto Carlos Botazzo Delbem1 1
Department of Pediatric Dentistry and Public Health, Arac¸atuba Dental School, Univ Estadual Paulista ˜ Paulo, Brazil, 2 Department of Prosthodontics and Periodontology, (UNESP), 16015–050 Arac¸atuba/Sao ˜ Paulo, Brazil Piracicaba Dental School, University of Campinas (UNICAMP), 13414–903 Piracicaba/Sao 3 and Department of Dental Materials and Prosthodontics, Arac¸atuba Dental School, Univ Estadual Paulista ˜ Paulo, Brazil (UNESP), 16015–050 Arac¸atuba/Sao *To whom correspondence should be addressed. Douglas Roberto Monteiro, Department of Pediatric Dentistry and Public ˜ Paulo, Brazil. Health, Arac¸atuba Dental School, Univ Estadual Paulista (UNESP), 16015–050 Arac¸atuba/Sao Tel: +55 18 36363316; Fax: +55 18 36363245; E-mail: [email protected] Received 5 March 2015; Revised 1 May 2015; Accepted 1 June 2015
Abstract The prevention of adhesion of Candida cells to acrylic surfaces can be regarded as an alternative to prevent denture stomatitis. The use of quorum sensing molecules, such as tyrosol, could potentially interfere with the adhesion process. Therefore, the aim of this study was to assess the effect of tyrosol on adhesion of single and mixed cultures of Candida albicans and Candida glabrata to acrylic resin surfaces. Tyrosol was diluted in each yeast inoculum (107 cells/ml in artificial saliva) at 25, 50, 100, and 200 mM. Then, each dilution was added to wells of 24-well plates containing the acrylic specimens, and the plates were incubated at 37◦ C for 2 h. After, the effect of tyrosol was determined by total biomass quantification, metabolic activity of the cells and colony-forming unit counting. Chlorhexidine gluconate (CHG) was used as a positive control. Data were analyzed using analysis of variance (ANOVA) and the Holm–Sidak post hoc test (α = 0.05). The results of total biomass quantification and metabolic activity revealed that the tyrosol promoted significant reductions (ranging from 22.32 to 86.16%) on single C. albicans and mixed cultures. Moreover, tyrosol at 200 mM and CHG significantly reduced (p < 0.05) the number of adhered cells to the acrylic surface for single and mixed cultures of both species, with reductions ranging from 1.74 to 3.64-log10. In conclusion, tyrosol has an inhibitory effect on Candida adhesion to acrylic resin, and further investigations are warranted to clarify its potential against Candida infections. Key words: tyrosol, Candida albicans, Candida glabrata, adhesion, acrylic surface.
C The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.
All rights reserved. For permissions, please e-mail: [email protected]
1
2
Introduction
capacity [25]. Tyrosol production by Candida species has been reported by Cremer et al. [26]. These authors demonstrated that C. albicans and C. tropicalis secreted significantly higher amounts of tyrosol than C. glabrata and C. parapsilosis, implying a probable correlation with the pathogenicity of these species. Moreover, it may be produced by cells in planktonic state or forming biofilms [23]. Contrary to farnesol, tyrosol has been associated with accelerated conversion of yeasts in hyphae, contributing to the harmonized expression of virulence factors under stimulating conditions, like increased microbial density [23,24,27]. Recently, the combination of tyrosol with amphotericin B revealed a synergistic effect against C. krusei and C. tropicalis biofilms, achieving 90% reduction at 80 μM and 4 mg/l, respectively [28]. Although the involvement of tyrosol in the control of Candida morphogenesis had been elucidated, to the best of the authors’ knowledge its effect on adhesion of Candida cells to acrylic surfaces remains unknown. Therefore, the aim of this study was to assess the effect of different concentrations of tyrosol on the adhesion capacity of single and mixed cultures of C. albicans and C. glabrata to an acrylic resin surface. The hypothesis evaluated was that tyrosol displays an inhibitory effect on Candida adhesion to acrylic resin.
Materials and methods Candida strains and cultivation Two reference strains of the American Type Culture Collection (ATCC) were used in this study: C. albicans ATCC 10231 and C. glabrata ATCC 90030. All Candida strains were maintained at −70◦ C and grown aerobically for 24 h at 37◦ C on Sabouraud dextrose agar (SDA; Difco, Le Pont de Claix, France) plates. To obtain standard suspensions of each Candida cell, a loopful of the colonies from SDA plates was inoculated into Sabouraud dextrose broth (SDB; Difco) medium and incubated overnight at 37◦ C with agitation (120 rpm). Afterward, the Candida cells were collected by centrifugation (8000 rpm for 5 min), washed twice in phosphate buffered saline (PBS; pH 7, 0.1M), and resuspended in artificial saliva (AS) at 1 × 107 cells/ml using a Neubauer chamber. The composition of AS (pH 6.8) is described elsewhere [29].
Cell surface hydrophobicity The sessile drop method was used to determine the cell surface hydrophobicity. Cell suspensions of C. albicans ATCC 10231 and C. glabrata ATCC 90030 were prepared in SDB, as described above. However, for this assay, the cell
Downloaded from http://mmy.oxfordjournals.org/ at University of Otago on July 12, 2015
Denture stomatitis (DS) is an infectious process characterized by frequent episodes of recurrence, which essentially affects the palatal mucosa of complete denture wearers [1]. The most important microorganism associated with DS is the polymorphic fungus Candida albicans [2]. Nevertheless, other non-Candida albicans Candida species, such as Candida glabrata, have also taken part in the development of this condition [3,4]. DS is induced by trauma, precarious oral hygiene, lack of saliva in the oral mucosa covered by the prostheses, and microbial colonization of oral epithelium or inert surfaces [1,5,6]. The colonization of oral tissues, as well as acrylic surfaces, by Candida species depends on the adhesion capacity of these species to those surfaces. Adhesion capacity of yeasts is associated with a combination of specific and non-specific factors. The specific factors include interactions between cell wall adhesins and receptors of epithelial cells [7–9], while the hydrophobic and electrostatic interactions between microbial cells and substrate surfaces represent the non-specific factors [8,10,11]. The phenomenon of adhesion is also influenced by the surface roughness of the materials [12]. Furthermore, microbial adhesion is the preliminary stage in the processes of colonization, biofilm formation and development of infections [8]. Consequently, it is regarded as one of major virulence factors of pathogenic Candida species [8]. In this sense, it is essential to investigate novel compounds that may interfere in the yeast adhesion process to prevent subsequent biofilm formation, particularly in view of the Candida recalcitrance to conventional antifungals [13]. A promising therapy is the use of cell-cell communication molecules, known as quorum-sensing (QS) molecules [14]. QS molecules are produced naturally by microorganisms as a function of the cell density and govern a variety of behaviors [15]. The first QS molecule recognized in C. albicans was the farnesol [15], an aliphatic alcohol that may also be extracted from citrus fruits [16]. Farnesol acts by blocking the yeast-to-hyphae transformation at high microbial density [17], and several studies [18–22] have demonstrated its effect as an antibiofilm agent. Gori et al. [18] reported that farnesol at 10 μM reduced the adhesion of Debaryomyces hansenii to polystyrene. This compound was able to inhibit Staphylococcus epidermidis [19], C. albicans [20], and C. dubliniensis [21] biofilms and has shown synergistic or additive effects against C. albicans biofilms when combined with fluconazole, micafungin and amphotericin B [22]. The second well-known QS molecule in C. albicans is the aromatic alcohol tyrosol [15,23,24], which may also be found as a polyphenol of olive oil with antioxidant
Medical Mycology, 2015, Vol. 00, No. 00
Monteiro et al.
3
suspensions were resuspended in PBS to a concentration of 1 × 109 cells/ml. These cell suspensions were filtered under vacuum through cellulose acetate membranes (0.22 μm) previously soaked with 10 ml of deionized water. To standardize the humidity level, the membranes were cut into three pieces, placed on glass slides and inserted into petri dishes containing 1% agar and 10% glycerol during 3.5 h [30]. Five drops (volume of each drop: 3 μl) of deionized water were deposited on each membrane piece containing the cell lawns, and the contact angles were measured using a goniometer (Kruss DSA 100S, Kruss GMBH, Hamburg, Germany). The assays were performed on three separate occasions at 22 ± 2◦ C.
MIC was performed using the microbroth dilution method, as detailed elsewhere [29], with tyrosol (2-(4hydroxyphenyl) ethanol) purchased from Sigma-Aldrich, St Louis, USA. Briefly, C. albicans and C. glabrata cell suspensions were adjusted to turbidity 0.5 of McFarland standard in saline solution, and afterwards diluted in Roswell Park Memorial Institute (RPMI 1640; Sigma-Aldrich) medium. Then, 100 μl of each yeast suspension was added to the wells of a 96-well microtiter plate (Costar, Tewksbury, USA) containing 100 μl of each specific concentration of tyrosol (0–300 mM) diluted in RPMI 1640. The plates were incubated at 37◦ C for 48 h and the MIC was recorded as the lowest concentration of tyrosol demonstrating no yeast growth. Chlorhexidine gluconate (CHG) was used as positive control. Moreover, the minimum fungicidal concentration (MFC) was determined through plating the content of each well on SDA from the MIC point. All assays were performed in triplicate on three independent occasions.
Preparation and resin specimens
characterization
of
Germ tube formation by C. albicans The effect of tyrosol on germ tube formation by C. albicans ATCC 10231 was investigated according to Chauhan et al. [31], with some modifications. In brief, 5 ml of AS containing C. albicans ATCC 10231 at 1 × 107 cells/ml were prepared as described above and added to falcon tubes. Then, tyrosol was diluted in the inoculum to achieve concentrations of 25, 50, 100, and 200 mM. Tubes without tyrosol and with CHG at 0.37 mM were kept as negative and positive controls, respectively. After 2 h of incubation at 37◦ C, the amount of yeast and germ tube forms was determined using a Neubauer chamber. The assays were performed independently three times in quadruplicate, and the percentage of germ tubes formation compared with negative control was estimated according to the formula: =
No. germ tubes in treatment × 100. No. germ tubes in control
acrylic
The acrylic resin specimens were obtained from an aluminum matrix with internal molds which were fixed with wax (Wilson, Sao ˜ Paulo, Brazil) on a stainless steel plate, and included in a flask with a type III dental stone (Herodent, Petropolis, Brazil). Following the setting pro´ cess, the flask was opened, the wax was removed, and the matrix molds were cleansed. Afterwards, the QC20 denture acrylic resin (Dentsply Ind. e Com. Ltd., Petropolis, ´ Brazil) was manipulated, inserted into the matrix molds, and polymerized in accordance with the manufacturer’s advice. After polymerization of the acrylic resin, the specimens (10 × 10 × 3 mm) were removed from the matrix, finished using 150-, 300-, 600-, and 1200-grit sandpapers
Adhesion assays To evaluate the adhesion capacity of Candida species in the presence of tyrosol, single cultures of C. albicans ATCC 10231 and C. glabrata ATCC 90030 or mixed cultures of both yeasts were developed on acrylic specimens placed in 24-well microtiter plates (Costar, Tewksbury, USA). Tyrosol was diluted in the inoculum of each single yeast (1 × 107 cells/ml in AS) and in the mixed inoculum of two species (1 × 107 cells/ml of each strain) to obtain final concentrations of 25, 50, 100, and 200 mM. Afterward, 1 ml of each dilution was inoculated into wells containing the acrylic specimens, and the plates were incubated aerobically and statically for 2 h at 37◦ C. CHG at 0.37 mM (50 x MIC) was used as positive control. Acrylic resin
Downloaded from http://mmy.oxfordjournals.org/ at University of Otago on July 12, 2015
Determination of tyrosol minimum inhibitory concentration (MIC)
(3M/ESPE, Sumar´e, Brazil), rinsed with deionized water in an ultrasonic bath, and then sterilized by irradiation (60 Co; 25 KGy) before microbiological assays. A profilometer (Mitutoyo SJ-401; Mitutoyo Corp., Tokyo, Japan) was used to measure the average surface roughness (Ra - μm) of the acrylic specimens. Three measurements for each specimen were obtained using a cut-off of 0.25 mm and speed of 0.5 mm/s. Further, to characterize the surface hydrophobicity of specimens, the contact angle (θ ) values were determined by using an automatic goniometer (Kruss DSA 100S, Kruss GMBH, Hamburg, Germany). Three drops of deionized water were deposited at different locations on each acrylic surface and the mean was calculated. The assays were performed at 22 ± 2◦ C. Finally, these characterization experiments were carried out in order to obtain standardized acrylic surfaces.
4
specimens inoculated with yeast suspensions devoid of tyrosol were used as negative controls.
Quantification of total biomass
Metabolic activity by XTT assay Metabolic activity of the adhered cells was measured by the XTT (2,3-(2-methoxy-4-nitro-5-sulphophenyl)5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) (Sigma-Aldrich) reduction assay [32]. Following the adhesion of single and mixed cultures, the supernatants were removed and the non-adherent cells were removed by washing the specimens with PBS. Then, the specimens were transferred to new wells, and 1 ml of a solution containing 150 mg XTT/L and 10 mg of phenazine methosulphate/l (Sigma-Aldrich) was added to each well. After incubation (120 rpm) in the dark at 37◦ C for 3 h, aliquots of 200 μl were transferred to 96-well plates, and the absorbance of XTT-formazan was read at 490 nm. The values were standardized per unit area of acrylic specimens (Abs/cm2 ), and the wells containing AS without Candida cells were considered as blanks. The assays were carried out in triplicate and on three separate occasions.
surfaces. Thereafter, serial decimal dilutions (in PBS) were plated on SDA (for single cultures) and on CHROMagar Candida (Difco) (for mixed cultures), and the agar plates were incubated for 24–48 h at 37◦ C. The number of CFUs was log-transformed and standardized per unit area (Log10 CFU/cm2 ) of acrylic specimens, and the assays were also performed independently three times in triplicate.
Scanning electron microscopy (SEM) analysis Cultures of Candida species were developed during 2 h on acrylic specimens in the presence of tyrosol, as previously described. Subsequently, the adhered cells on specimens were rinsed with PBS, dehydrated in a sequence of ethanol (70% for 10 min, 95% for 10 min, and 100% for 20 min), air dried in a desiccator, and finally sputter coated with gold prior to observations (JSM 5600LV, Jeol, Tokyo, Japan).
Statistical analysis SigmaPlot 12.0 software (Systat Software Inc., San Jose, USA) was employed for the statistical analysis with a confidence level of 95%. Data passed the normality test (Shapiro–Wilk), and then parametric statistical analyses were conducted with one-way ANOVA followed by Holm– Sidak post hoc test.
Results Characterization of cell and acrylic surfaces It was possible to observe that the surfaces of all strains showed a hydrophilic behavior (θ < 65◦ ) [33], with mean values of water contact angle of 10.52◦ and 18.40◦ for C. albicans ATCC 10231 and C. glabrata ATCC 90030, respectively. Regarding acrylic specimens, the mean value of Ra was 0.11 ± 0.02 μm, and the high water contact angle (106.23◦ ± 09.41) revealed hydrophobic acrylic surfaces.
MIC and MFC Quantification of cultivable cells The number of cultivable cells from single and mixed cultures was enumerated by counting colony-forming units (CFUs). After an adhesion period (2 h), acrylic specimens were washed once with 1 ml of PBS, inserted in falcon tubes containing 1 ml of PBS, sonicated at 30 W for 30 s, and vigorously vortexed for 1.5 min. CV staining assay was used to confirm the removal of fungal cells from acrylic
C. albicans ATCC 10231 was more susceptible to tyrosol (MIC = 50 mM; MFC = 90 mM) than C. glabrata ATCC 90030 (MIC = 90 mM; MFC = 100 mM). Moreover, MIC and MFC values of CHG were 0.0074 and 0.0296 mM for C. albicans ATCC 10231, and 0.0074 and 0.0148 mM for C. glabrata ATCC 90030. Thus, for all strains assayed, MIC and MFC values of tyrosol were about 3,000–12,000 times higher than those obtained for CHG.
Downloaded from http://mmy.oxfordjournals.org/ at University of Otago on July 12, 2015
Measurement of total biomass was done by crystal violet (CV) staining assay [29]. After the adhesion period (2 h), nonadherent cells were removed by washing the acrylic specimens with 1 ml of PBS. Next, Candida adhered cells were fixed with 1 ml of 99% methanol (Sigma-Aldrich) during 15 min, dried at room temperature and stained with 1 ml of 1% CV (Sigma-Aldrich) solution for 5 min. CV bound to the adhered cells in single and mixed cultures was removed with 1 ml of 33% acetic acid (Sigma-Aldrich). The absorbance of the resulting solution was recorded at 570 nm (Eon Microplate Spectrophotometer; Bio Tek, Winooski, USA) and standardized according to the area of acrylic specimens (Abs/cm2 ). Wells containing acrylic specimens inoculated with AS devoid of Candida species were used as blanks. The assays were carried out independently three times in triplicate.
Medical Mycology, 2015, Vol. 00, No. 00
Monteiro et al.
5
Germ tube formation by C. albicans Tyrosol at 25, 50, 100, and 200 mM promoted reductions in the number of germ tubes of 67.42, 91.67, 96.97, and 97.47%, respectively. For CHG, a 92.42% reduction was observed, compared with the negative control.
Effect of tyrosol on total biomass Figure 1 displays the effect of different concentrations of tyrosol on total biomass of Candida cells adhered to acrylic surfaces. Tyrosol at 25, 50, 100, and 200 mM promoted significant reductions in the C. albicans ATCC 10231 biomass of 35.13% (P < .001), 48.64% (P < .001), 45.94% (P < .001), and 54.05% (P < .001), respectively, compared to the negative control group. The higher decrease in the total biomass (62.16%, P < .001) was noted for the group exposed to CHG. For C. glabrata ATCC 90030, only the treatment with CHG generated significant reduction in the biomass (43.75%, P = .002). In mixed culture, the treatment with tyrosol also exhibited significant decreases (P < .05) in the total biomass, ranging from 25.58% to 44.18%. When compared to the negative control, once again, CHG showed the highest decrease.
Effect of tyrosol on metabolic activity The results of XTT reduction (Fig. 2) for C. albicans ATCC 10231 showed that the highest reductions in metabolic
activity were attained with 200 mM tyrosol (86.16%, P < .001) and CHG (89.28%, P < .001), without a statistical difference between these groups. Moreover, tyrosol at 25, 50, and 100 mM promoted decreases in metabolic activity of 22.32% (P = .113), 51.33% (P = .002), and 51.78% (P = .002), respectively, compared to the negative control. The mixed Candida culture demonstrated a similar pattern, where the XTT colorimetric readings decreased with increasing concentrations of tyrosol. However, for C. glabrata ATCC 90030 the treatments did not significantly affect (P > .05) the metabolic activity of cells.
Effect of tyrosol on cultivable cells Mean values of the log10 CFU/cm2 obtained for each Candida strain in single and mixed cultures are shown in Figure 3. For all strains, after 2 h of incubation of the Candida cells, there were significant reductions (P < .05) in the number of adhered cells for the groups treated with tyrosol at 200 mM and CHG, compared to the negative controls and the groups treated with tyrosol at 25, 50, and 100 mM. The reductions in the number of CFUs ranged from 1.74 to 3.64-log10 , and there were no differences between 200 mM tyrosol and CHG. Interestingly, C. albicans ATCC 10231 and C. glabrata ATCC 90030 adhered cells in single cultures showed the same sensitivity to tyrosol and CHG than the ones in mixed cultures.
Downloaded from http://mmy.oxfordjournals.org/ at University of Otago on July 12, 2015
Figure 1. Average absorbances per cm2 obtained with crystal violet assay for Candida cells adhered (2 h) to acrylic in the presence of different concentrations of tyrosol (25, 50, 100, and 200 mM). NC = negative control (Candida culture without tyrosol). PC = positive control (chlorhexidine gluconate at 0.37 mM). Error bars display standard deviation of the means. ∗ Denotes P < .05, as compared to the negative control group, using one-way ANOVA with post hoc Holm–Sidak test.
6
Medical Mycology, 2015, Vol. 00, No. 00
Figure 3. Mean values of the logarithm of colony forming units per cm2 (log10 CFU/cm2 ) for Candida cells adhered (2 h) to acrylic in the presence of different concentrations of tyrosol (25, 50, 100, and 200 mM). NC = negative control (Candida culture without tyrosol). PC = positive control (chlorhexidine gluconate at 0.37 mM). Error bars display standard deviation of the means. ∗ Denotes P < .05, as compared to the negative control group, using one-way ANOVA with post hoc Holm–Sidak test.
SEM analysis The images in Figure 4 represent the pattern of adhered cells found for the mixed culture, which was similar for all the others (single cultures of both species). SEM observation of the negative control group revealed agglomerates of
yeasts and filamentous forms (mainly pseudohyphae) covering most of the acrylic surface (Fig. 4a), whereas the groups exposure to tyrosol at 25 mM (Fig. 4c), 50 mM (Fig. 4d), and 100 mM (Fig. 4e) comprised sparse yeasts as well as aggregated cells mostly consisting of yeast forms. However,
Downloaded from http://mmy.oxfordjournals.org/ at University of Otago on July 12, 2015
Figure 2. Average absorbances per cm2 obtained with XTT reduction assay for Candida cells adhered (2 h) to acrylic in the presence of different concentrations of tyrosol (25, 50, 100, and 200 mM). NC = negative control (Candida culture without tyrosol). PC = positive control (chlorhexidine gluconate at 0.37 mM). Error bars display standard deviation of the means. ∗ Denotes P < .05, as compared to the negative control group, using one-way ANOVA with post hoc Holm–Sidak test.
Monteiro et al.
7
the treatment with 200 mM tyrosol and CHG resulted in few aggregated yeasts partially covering the acrylic surface (Fig. 4b and f).
Discussion Adherence of microorganisms to biotic and abiotic surfaces is a necessary condition for biofilm formation and this phenomenon depends, among other factors, on the material surface characteristics. Literature data show that the higher the roughness and the hydrophobicity degree, the greater the microbial adhesion [34,35]. The surface roughness provides pores in which the microorganisms are confined and thus protected from the oral shear forces, enabling sufficient time for irreversible adherence of cells to the surface [36]. With respect to substrate wettability, the contact angle results from the equilibrium between the surface and interfacial energy, where the greater wettability, the lower the contact angle [37]. Considering these aspects, the roughness and the hydrophobicity degree of the acrylic specimens were measured in order to standardize them prior to microbiological assays. The cell surface hydrophobicity also influences the adhesion process. A previous study demonstrated that the Candida strains with highest hydrophobicity degree adhered more to polystyrene [38]. In the present study, C. glabrata ATCC 90030 showed a more hydrophobic behavior than
C. albicans ATCC 10231. Similar results were noted by Luo & Samaranayake [39]. These authors found contact angle values of 38.48◦ e 6.00◦ , respectively, for C. glabrata and C. albicans. Li et al. [40] verified that C. glabrata did not adhere well to oral keratinocytes, but adhered better to the acrylic surface, as compared with C. albicans. It is interesting to note that the different contact angle values observed for a same species in different studies may be due to physiological differences among the strains tested. Although C. albicans is a polymorphic fungus with ability to form pseudohyphae and hyphae, MIC and MFC values for this species were lower than those found for C. glabrata, which grows only as yeast. These findings are clinically important because C. albicans is the main pathogen involved in cases of denture stomatitis [2]. On the other hand, the lower susceptibility of C. glabrata is in line with previous studies showing that this strain is resistant to conventional antifungal agents, making it difficult the combat to this species [41]. The MIC values found for tyrosol in the present study may be considered high when compared to those found for farnesol in previous studies [21,22]. Jabra-Rizk et al. [21] demonstrated that the farnesol MIC values were 200 μM and > 250 μM for C. dubliniensis and C. albicans, respectively. These differences may have occurred because farnesol blocks the formation of germ tubes at low concentrations [17]. Furthermore, the lipophilic properties of this
Downloaded from http://mmy.oxfordjournals.org/ at University of Otago on July 12, 2015
Figure 4. Scanning electron microscopy (SEM) images of Candida cells in mixed culture (C. albicans ATCC 10231 + C. glabrata ATCC 90030) adhered (2 h) to acrylic surface in the presence of tyrosol at 25 mM (c), 50 mM (d), 100 mM (e), and 200 mM (f). NC (a) = negative control (Candida culture without tyrosol). PC (b) = positive control (chlorhexidine gluconate at 0.37 mM). Magnification: x 1000. Bar, 10.0 μm.
8
compared to the negative control group. Nevertheless, there were decreases in the number of germ tubes in the groups treated with tyrosol. These findings together with the results of CFU quantification (Fig. 3) may indicate that the significant reductions in biomass for single C. albicans ATCC 10231 and mixed cultures treated with tyrosol at 25, 50, and 100 mM (Fig. 1) occurred due to decrease in the number of pseudohyphae and hyphae. In fact, hyphae are elongated multicellular structures which retain greater amounts of crystal violet than yeast cells. Moreover, tyrosol might have acted by inhibiting the incipient production of extracellular matrix. It may also be deduced that the decrease in mixed cultures biomass is due to the effect of tyrosol on C. albicans ATCC 10231, while the lack of activity on single C. glabrata ATCC 90030 (Fig. 1) is because this species grows only as yeast and has a higher hydrophobicity degree, which favors the adherence to acrylic [39,40] and hinders its removal. Literature data show that tyrosol regulates the cell density by inducing the transformation of yeasts in hyphae [23,24,27]. However, the results of this study suggest that the exogenous administration of tyrosol at high concentrations may have altered the balance of its intracellular levels and resulted in the inhibition of hyphae formation on newly generated cells. Results from an XTT assay showed that tyrosol at 50 and 100 mM was able to significantly reduce the metabolic activity of cells in single cultures of C. albicans ATCC 10231 and mixed culture of both species (Fig. 2). On the other hand, these same tyrosol concentrations did not promote significant reductions in the number of CFUs (Fig. 3). Thus, it is evident that the Candida cells exposed to 50 and 100 mM tyrosol continued with potential to grow in solid medium, even with reduced metabolic activity. Noteworthy, tyrosol at 200 mM displayed an antiadhesion effect similar to CHG in all assays. However, the effective concentration of CHG (0.37 mM) was about 540 times lower than the highest tyrosol concentration (200 mM) evaluated in this study. Tyrosol is a natural compound produced by some Candida species [26], and this feature may explain, at least in part, why its effective concentration was much higher than that found for CHG. In addition, some components of artificial saliva, such as mucin used in this study, may have bound to tyrosol, requiring higher concentrations for achieving a good antiadhesion effect. Published data with some cell types show that tyrosol has no cytotoxic effect. Anter et al. [43] demonstrated that this compound at 140 μM prevented H2 O2 induced damage, having an antigenotoxic effect. Further, tyrosol was not shown to be toxic against cells from the salivary gland [44] and kidney [45]. Nevertheless, studies are needed to assess the toxic effects of tyrosol on mammalian cells at the concentrations used in the present study.
Downloaded from http://mmy.oxfordjournals.org/ at University of Otago on July 12, 2015
compound may favor its binding to the membrane, thereby changing its fluidity [17]. However, the most essential question of this research was whether the tyrosol would be able to reduce or eradicate Candida cell adhesion to acrylic resin. Adhesion of yeasts in the presence of tyrosol was assessed by quantification of total biomass, metabolic activity, cultivable cells and SEM observations. The study hypothesis was accepted since tyrosol was shown to have an inhibitory effect on the adhesion of single and mixed cultures of Candida species to acrylic resin. The tyrosol concentrations used in adhesion assays were based on the lowest MIC value (50 mM for C. albicans ATCC 10231). Therefore, the concentrations tested were 25 (1/2 MIC), 50 (MIC), 100 (2 x MIC), and 200 mM (4 x MIC). In general, the adhesion assays revealed significant effects of tyrosol in reducing total biomass (Fig. 1) and metabolic activity (Fig. 2) against single C. albicans ATCC 10231 and mixed cultures, with the highest decreases for the groups exposed to 200 mM tyrosol. For C. glabrata ATCC 90030, tyrosol did not promote significant reduction in total biomass and metabolic activity (Figs. 1 and 2). On the other hand, for all cultures, the results of CFU quantification showed that only the treatments with tyrosol at 200 mM and CHG at 0.37 mM significantly reduced the number of adhered cells to acrylic surfaces (Fig. 3), and the SEM observations confirmed these findings (Fig. 4). The adhesion of yeasts is associated to nonspecific (cell surface hydrophobicity) and specific factors (cell wall adhesins) [42]. Considering that tyrosol reduced germ tube formation, fungal metabolism and proliferation, it is possible that this compound may have interfered with the synthesis or expression of filament-associated adhesins on the surface of the cells, thereby reducing induced-adhesion, while passive adhesion due to hydrophobic interactions might have remained unaffected. It should be noted that all quantitative methods used to evaluate the effect of tyrosol on Candida adherence have some limitation; therefore, they should always be used as complementary methods. The CV staining assay does not allow the differentiation between living and dead cells. Although the results from an XTT assay are good predictors of the effect of antimicrobial agents, they do not always ensure equivalence with cell death. Furthermore, one of the limitations of the CFU quantification is that not every viable cell is also culturable in agar medium. All these aspects may help explain why the results of CFU quantification were not similar to those found for total biomass and metabolic activity. Additionally, the SEM images (Fig. 4) clearly show that the treatments with tyrosol at 25, 50, and 100 mM did not promote significant reductions in the Candida adherence,
Medical Mycology, 2015, Vol. 00, No. 00
Monteiro et al.
Acknowledgments This study was supported by Sao ˜ Paulo Research Foundation (FAPESP; processes 2013/17767–2 and 2013/10285–2), Brazil. We thank Adaias Oliveira Matos, University of Campinas (UNICAMP), for performing SEM analysis.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.
References 1. Jeganathan S, Lin CC. Denture stomatitis: a review of the aetiology, diagnosis and management. Aust Dent J 1992; 37(2): 107–114. 2. Odds FC. Pathogenesis of Candida infections. J Am Acad Dermatol 1994; 31(3 Pt 2): S2–S5. 3. Coco BJ, Bagg J, Cross LJ et al. Mixed Candida albicans and Candida glabrata populations associated with the pathogenesis of denture stomatitis. Oral Microbiol Immunol 2008; 23(5): 377–383. 4. Vanden Abbeele A, de Meel H, Ahariz M et al. Denture contamination by yeasts in the elderly. Gerodontology 2008; 25(4): 222–228.
5. Wilkieson C, Samaranayake LP, MacFarlane TW et al. Oral candidosis in the elderly in long term hospital care. J Oral Pathol Med 1991; 20(1): 13–16. 6. Budtz-Jorgensen E, Theilade E, Theilade J. Quantitative relation¨ ship between yeast and bacteria in denture-induced stomatitis. Scand J Dent Res 1983; 91(2): 134–142. 7. Desai C, Mavrianos J, Chauhan N. Candida glabrata Pwp7p and Aed1p are required for adherence to human endothelial cells. FEMS Yeast Res 2011; 11(7): 595–601. 8. Cotter G, Kavanagh K. Adherence mechanisms of Candida albicans. Br J Biomed Sci 2000; 57(3): 241–249. 9. Vidotto V, Mantoan B, Pugliese A et al. Adherence of Candida albicans and Candida dubliniensis to buccal and vaginal cells. Rev Iberoam Micol 2003; 20(2): 52–54. 10. Jones DS, McGovern JG, Woolfson AD et al. The effects of hexetidine (Oraldene) on the adherence of Candida albicans to human buccal epithelial cells in vitro and ex vivo and on in vitro morphogenesis. Pharm Res 1997; 14(12): 1765–1771. 11. Jabra-Risk MA, Falker WA, Merz WG et al. Cell surface hydrophobicity-associated adherence of Candida dubliniensis to human buccal epithelial cells. Rev Iberoam Micol 2001; 18(1): 17–22. 12. Quirynen M, Bollen CM. The influence of surface roughness and surface-free energy on supra- and subgingival plaque formation in man. A review of the literature. J Clin Periodontol 1995; 22(1): 1–14. 13. Chandra J, Mukherjee PK, Leidich SD et al. Antifungal resistance of candidal biofilms formed on denture acrylic in vitro. J Dent Res 2001; 80(3): 903–908. 14. Weber K, Schulz B, Ruhnke M. The quorum-sensing molecule E,E-farnesol - its variable secretion and its impact on the growth and metabolism of Candida species. Yeast 2010; 27(9): 727–739. 15. Albuquerque P, Casadevall A. Quorum sensing in fungi—a review. Med Mycol 2012; 50(4): 337–345. 16. Unnanuntana A, Bonsignore L, Shirtliff ME et al. The effects of farnesol on Staphylococcus aureus biofilms and osteoblasts: an in vitro study. J Bone Joint Surg Am 2009; 91(11): 2683–2692. 17. Hornby JM, Jensen EC, Lisec AD et al. Quorum sensing in the dimorphic fungus Candida albicans is mediated by farnesol. Appl Environ Microbiol 2001; 67(7): 2982–2992. 18. Gori K, Knudsen PB, Nielsen KF et al. Alcohol-based quorum sensing plays a role in adhesion and sliding motility of the yeast Debaryomyces hansenii. FEMS Yeast Res 2011; 11(8): 643–652. 19. Pammi M, Liang R, Hicks JM et al. Farnesol decreases biofilms of Staphylococcus epidermidis and exhibits synergy with nafcillin and vancomycin. Pediatr Res 2011; 70(6): 578–583. 20. Ramage G, Saville SP, Wickes BL et al. Inhibition of Candida albicans biofilm formation by farnesol, a quorumsensing molecule. Appl Environ Microbiol 2002; 68(11): 5459–5463. 21. Jabra-Rizk MA, Shirtliff M, James C et al. Effect of farnesol on Candida dubliniensis biofilm formation and fluconazole resistance. FEMS Yeast Res 2006; 6(7): 1063–1073. 22. Katragkou A, McCarthy M, Alexander EL et al. In vitro interactions between farnesol and fluconazole, amphotericin B or micafungin against Candida albicans biofilms. J Antimicrob Chemother 2015; 70(2): 470–478.
Downloaded from http://mmy.oxfordjournals.org/ at University of Otago on July 12, 2015
As for mechanisms of action, CHG acts on the rupture of the yeast cell membrane, resulting in alteration of the cell permeability and loss of intracellular components [46,47]. On the other hand, tyrosol’s antimicrobial mechanism of action remains unknown. For Staphylococcus aureus, Inoue et al. [48] observed a relationship between the K+ ion loss and the antimicrobial activity of terpene alcohols. Consequently, based on the idea of Pammi et al. [19], it may be hypothesized that the tyrosol acts on the integrity of yeast membrane. Finally, in view of the limitations of this in vitro study, it may be concluded that tyrosol has an inhibitory effect on the adhesion of single and mixed cultures of Candida species to acrylic resin, and this QS molecule at 200 mM behaved similarly to the positive control. Moreover, assuming that in the oral cavity forces of different magnitudes are present during mastication, phonation and deglutition [49], future studies of microbial adhesion mimetizing the oral conditions, and with a larger number of strains, should be conducted. The evaluation of tyrosol’s mechanism of action and its effect alone or in combination with conventional antifungal drugs on biofilm formation must also be investigated. All these studies will contribute significantly to improving oral health and quality of life of denture wearers through the development of formulations containing tyrosol that may be used in prevention and control of Candida-associated denture stomatitis.
9
10
37.
38.
39.
40.
41.
42.
43.
44. 45.
46. 47.
48.
49.
and its relation to adherence of Candida albicans. J Prosthodont Res 2012; 56(4): 272–280. Ellepola AN, Samaranayake LP. Antimycotic agents in oral candidosis: an overview: 1. Clinical variants. Dent Update 2000; 27(3): 111–116. Klotz SA, Drutz DJ, Zajic JE. Factors governing adherence of Candida species to plastic surfaces. Infect Immun 1985; 50(1): 97–101. Luo G, Samaranayake LP. Candida glabrata, an emerging fungal pathogen, exhibits superior relative cell surface hydrophobicity and adhesion to denture acrylic surfaces compared with Candida albicans. APMIS 2002; 110(9): 601–610. Li L, Redding S, Dongari-Bagtzoglou A. Candida glabrata: an emerging oral opportunistic pathogen. J Dent Res 2007; 86(3): 204–215. Fidel PL, Jr, Vazquez JA, Sobel JD. Candida glabrata: review of epidemiology, pathogenesis, and clinical disease with comparison to C. albicans. Clin Microbiol Rev 1999; 12(1): 80–96. Silva S, Negri M, Henriques M et al. Adherence and biofilm formation of non-Candida albicans Candida species. Trends Microbiol 2011; 19(5): 241–247. Anter J, Tasset I, Demyda-Peyras ´ S et al. Evaluation of potential antigenotoxic, cytotoxic and proapoptotic effects of the olive oil by-product “alperujo”, hydroxytyrosol, tyrosol and verbascoside. Mutat Res Genet Toxicol Environ Mutagen 2014; 772: 25–33. Babich H, Visioli F. In vitro cytotoxicity to human cells in culture of some phenolics from olive oil. Farmaco 2003; 58(5): 403–407. Loru D, Incani A, Deiana M et al. Protective effect of hydroxytyrosol and tyrosol against oxidative stress in kidney cells. Toxicol Ind Health 2009; 25(4–5): 301–310. Ellepola AN, Samaranayake LP. Oral candidal infections and antimycotics. Crit Rev Oral Biol Med 2000; 11(2): 172–198. Kuyyakanond T, Quesnel LB. The mechanism of action of chlorhexidine. FEMS Microbiol Lett 1992; 100(1–3): 211–215. Inoue Y, Shiraishi A, Hada T et al. The antibacterial effects of terpene alcohols on Staphylococcus aureus and their mode of action. FEMS Microbiol Lett 2004; 237(2): 325–331. Vissink A, Waterman HA, s-Gravenmade EJ et al. Rheological properties of saliva substitutes containing mucin, carboxymethylcellulose or polyethylenoxide. J Oral Pathol 1984; 13(1): 22–28.
Downloaded from http://mmy.oxfordjournals.org/ at University of Otago on July 12, 2015
23. Alem MA, Oteef MD, Flowers TH et al. Production of tyrosol by Candida albicans biofilms and its role in quorum sensing and biofilm development. Eukaryot Cell 2006; 5(10): 1770–1779. 24. Chen H, Fujita M, Feng Q et al. Tyrosol is a quorum-sensing molecule in Candida albicans. Proc Natl Acad Sci U S A 2004; 101(14): 5048–5052. 25. Moreno JJ. Effect of olive oil minor components on oxidative stress and arachidonic acid mobilization and metabolism by macrophages RAW 264.7. Free Radic Biol Med 2003; 35(9): 1073–1081. 26. Cremer J, Vatou V, Braveny I. 2,4-(hydroxyphenyl)-ethanol, an antioxidative agent produced by Candida spp., impairs neutrophilic yeast killing in vitro. FEMS Microbiol Lett 1999; 170(2): 319–325. 27. Chen H, Fink GR. Feedback control of morphogenesis in fungi by aromatic alcohols. Genes Dev. 2006; 20(9): 1150–1161. 28. Shanmughapriya S, Sornakumari H, Lency A et al. Synergistic effect of amphotericin B and tyrosol on biofilm formed by Candida krusei and Candida tropicalis from intrauterine device users. Med Mycol 2014; 52(8): 853–861. 29. Monteiro DR, Gorup LF, Silva S et al. Silver colloidal nanoparticles: antifungal effect against adhered cells and biofilms of Candida albicans and Candida glabrata. Biofouling 2011; 27(7): 711–719. 30. Silva S, Negri M, Henriques M et al. Silicone colonization by non-Candida albicans Candida species in the presence of urine. J Med Microbiol 2010; 59(Pt 7): 747–754. 31. Chauhan NM, Raut JS, Karuppayil SM. A morphogenetic regulatory role for ethyl alcohol in Candida albicans. Mycoses 2011; 54(6): e697–e703. 32. Silva S, Henriques M, Oliveira R et al. In vitro biofilm activity of non-Candida albicans Candida species. Curr Microbiol 2010; 61(6): 534–540. 33. Vogler EA. Structure and reactivity of water at biomaterial surfaces. Adv Colloid Interface Sci 1998; 74: 69–117. 34. Bulad K, Taylor RL, Verran J et al. Colonization and penetration of denture soft lining materials by Candida albicans. Dent Mater 2004; 20(2): 167–175. 35. Nikawa H, Iwanaga H, Kameda M et al. In vitro evaluation of Candida albicans adherence to soft denture-lining materials. J Prosthet Dent 1992; 68(5): 804–808. 36. Al-Dwairi ZN, Al-Quran FA, Al-Omari OY. The effect of antifungal agents on surface properties of poly(methyl methacrylate)
Medical Mycology, 2015, Vol. 00, No. 00
