MICONAZOLE ACTIVITY AGAINST CANDIDA BIOFILMS DEVELOPED
ON ACRYLIC DISCS
Acrylic resins are the most frequently used materials in the fabrication of removable dentures. The majority of denture bases are made from heat-cured acrylic resins made of polymethylmethacrylates (PMMA). When utilized in edentulous subjects, these dentures may act as a reservoir for microorganisms, leading to infection (1-3). The adherence of microbial species to acrylic dentures and the subsequent formation of biofilms on the surfaces are contributory factors to plaque-related oral and systemic diseases. The surface roughness of heat-polymerized acrylic resin and hydrophobic interactions are regarded as the main factors that affect the adhesion of Candida and subsequent biofilm formation on acrylic surfaces.
Candida-associated denture stomatitis (CaDS) is a plaque-related oral disease, and the most common form of oral candidiasis. It is prevalent in the elderly population and affects up to 58% of acrylic denture wearers (4, 5). It is characterized by inflammation, chronic erythema and edema of the palatal mucosa, particularly in areas in contact with the acrylic surface of the denture. The infection may spread from the denture-covered oral mucosa to the angles of the mouth and the tongue (6-8). The etiology of the disease is multi-factorial, and the predisposing factors include denture trauma, continuous denture wearing, systemic diseases and cancer, drug therapy (antibiotics, corticosteroids), immunosuppression, denture cleanliness, and poor oral hygiene that results in the accumulation of denture plaque. Plaque that contains high levels of Candida typically becomes acidic as a result of microbial metabolism, which may subsequently cause the colonization and inflammation of the adjacent oral mucosal surface (4, 9, 10).
C. albicans is still considered to be the major etiologic agent of oral candidiasis. C. glabrata is the most prevalent non-albicans Candida species isolated in oral candidiasis in patients with diabetes, advanced cancer, HIV infection and patients suffering from CaDS (2, 11).
CaDS is associated strongly with Candida adherence and biofilm formation on the fitting surface of dentures made of PMMA (12, 13). Candida biofilm formation starts with the initial adherence of yeast cells to the surface of medical devices, followed by the formation of microcolonies and the development of a hyphal/pseudohyphal layer that extends outward. This is accompanied by the formation of an extracellular matrix surrounding both the hyphal and yeast layers (14). The Candida biofilms display an organized 3-dimensional structure, and consist of a dense network of yeasts and filamentous cells deeply embedded in an extracellular matrix composed of polysaccharides (15-17).
CaDS can be treated with topical antifungal therapy, however, the incidence of relapse is quite high. Miconazole, which is used commonly in the topical treatment of CaDS, inhibits the 14α-demethylation of lanosterol by interacting with cytochrome P450, a crucial enzyme in the ergosterol biosynthetic pathway. The inhibition of ergosterol biosynthesis also results in the accumulation of toxic methylated sterol intermediates, and subsequently arrests fungal cell growth (18, 19). Clinical observations also emphasize the importance of biofilm formation on the fitting surface of the acrylic denture, and the inability of current antifungal therapy to treat this condition. Candida biofilms are more resistant to antimicrobial agents than planktonic, free-living cells (12, 20-22).
In this study we have used an in vitro model of Candida biofilms formed on denture acrylic discs to examine the activity of miconazole against mature in vitro biofilms of C. albicans, C. glabrata, C. parapsilosis and C. tropicalis, including patient isolates.
MATERIALS AND METHODS
Strains and growth conditions
Six oral Candida isolates were used throughout the study. Two strains were obtained from ATCC (Manassas, VA): C. albicans UTR-14 (ATCC MYA-2732) and C. glabrata GDH1407 (ATCC MYA-275). Stock cultures were stored in liquid nitrogen, sub-cultured on Sabouraud dextrose agar (Difco, Detroit, MI), and stored at 4–6°C. Four strains, C. albicans 6122/06, C. glabrata 7531/06, C. tropicalis 8122/06, and C. parapsilosis11375/07, were isolated from patients with documented CaDS attending the Department of Prosthetic Dentistry, Poznan University of Medical Sciences, after the patient's informed consent had been obtained under a protocol approved by the Bioethics Committee. The patients were not on any antimycotic therapy. Swabs were collected from areas of palatal mucosa, inoculated onto Sabouraud's medium with chloramphenicol (bioMerieux SA, Marcy-l'Etoile, France), and incubated at 37°C for 48 hours. After obtaining pure cultures, the microorganisms were identified by the commercially available test kit ID 32 C (bioMerieux SA). Stock cultures were maintained at –20°C. A loopful of the yeast was inoculated in Yeast Nitrogen Base medium (YNB) (Difco) supplemented with 100 mM glucose (Sigma-Aldrich, St. Louis, MO), and incubated overnight at 37°C. Cells were harvested, washed with PBS, and standardized to 1×107 cells/ml YNB-glucose.
Biofilms were developed on heat-cured PMMA discs (5 mm diameter × 1.5 mm thick) that were polished with waterproof silicon carbide paper (grit p600), obtained from Hing Lung Engineering, Inc. (Hong Kong, China). The discs were submerged in the standardized Candida cell suspension in 48-well plates (1 disc/1 µl/well). The plate was placed on a titer-plate shaker (Lab-Line Instruments, Melrose, IL) at a setting of 1, and incubated for 90 min at 37°C (adherence phase). Non-adherent cells were removed from the discs by washing with PBS. The discs were then transferred to a 96-well plate, submerged in YNB/100 mM glucose (1 disc/250 l/well) and incubated for 48 hours at 37°C on the titer-plate shaker (Lab-Line Instruments, Melrose, IL) at the same setting (biofilm formation phase).
Miconazole nitrate was obtained from Sigma-Aldrich (Switzerland) and solubilized in dimethyl sulfoxide. The stock solution (5 mg/ml) was diluted in YNB-glucose to obtain the desired final concentrations. The 48-h Candida biofilms were incubated with a dilution series of antifungals in the range 1–200 µM for 24 hours at 37°C. The viability of the Candida cells were then quantified using the XTT reduction assay.
Metabolic activity (XTT reduction assay)
Metabolic activities of the biofilms were measured by the XTT [2,3-bis(2-methoxy4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay (20), XTT (Sigma) is reduced by the mitochondrial succinoxidase and cytochrome P450 system, and by flavoprotein oxidases, into a water-soluble formazan product that is measured spectrophotometrically. Discs containing no Candida cells served as controls. After removal of YNB medium, 200 µl of the XTT solution [158 µl PBS, 40 µl XTT (1 mg/ml), 2 µl menadione (0.4 mM in acetone) was added to each well. After incubation at 37°C for 3–5 hours, 150 µl of the solution was transferred to a new well in a 96-well plate, and XTT-formazan was determined at 490 nm using a Molecular Devices (Sunnyvale, CA) Versamax microtiter plate reader.
The minimum inhibitory concentrations (MICs) of miconazole against the Candida species were determined according to the microdilution method (document M-27 A3, Clinical Laboratory Standards Institute (CLSI) (23).
The results are reported as mean values ± S.D. and were analysed using Kruskal-Wallis test. Correlation between MIC values and miconazole concentrations corresponding to 50% RMA of biofilm structure was performed by calculating the Spearman rank correlation coefficient. In all tests a p value <0.05 was considered to be significant. All analyses were carried out using StatSoft's STATISTICA v. 10 software.
In this study, all Candida strains used developed biofilm structures on the surface of PMMA after 48 hours of incubation. The concentrations of miconazole that were effective in killing biofilm-associated Candida cells were determined by measuring the metabolic activity of Candida biofilms by the XTT reduction assay. Miconazole at a concentration of 0.5 µg/ml reduced the metabolic activity of the biofilms produced by C. albicans 6122/06 (Fig. 1B), C. glabrata MYA-275 (ATCC) (Fig. 2A) and C. tropicalis 8122/06 (Fig. 3) by 14.5%, 36.1% and 34%, respectively.
|Fig. 1. Antifungal activities of miconazole against C. albicans MYA-275 (A) and C. albicans 6122/06 (B) biofilms developed on PMMA discs. The results are expressed as a percentage of the control without miconazole. The data represent the mean ± S.D. from two independent experiments performed in triplicate.|
At a concentration of 5 g/ml miconazole there was a reduction in the metabolic activity in the biofilms of all the strains studied. The effects were greatest for C. glabrata MYA-275 (ATCC) (37.1%; p<0.05; Fig. 2A) and C. tropicalis 8122/06 (33%; p<0.05; Fig. 3).
|Fig. 2. Antifungal activities of miconazole against C. glabrata MYA-2732 (A) and C. glabrata 7531/06 (B) biofilms developed on PMMA discs. The data represent the mean ± S.D. from two independent experiments performed in triplicate.|
The highest inhibition of metabolic activity was observed at 96 µg/ml miconazole. In the case of C. glabrata MYA-275 (ATCC) and C. tropicalis 8122/06, this corresponded to 83.7% and 75.4%, respectively (p<0.01; Fig. 2A and Fig. 3). The lowest reduction was observed for C. parapsilosis 11375/07-46.1% (p<0.01; Fig. 4).
|Fig. 3. Antifungal activities of miconazole against C. tropicalis 8122/06 biofilms developed on PMMA discs. The data represent the mean ± S.D. from two independent experiments performed in triplicate.|
|Fig. 4. Antifungal activities of miconazole against C. parapsilosis 11375/07 biofilms developed on PMMA discs. The data represent the mean ± S.D. from two independent experiments performed in triplicate.|
The MICs for miconazole for the different strains were as follows: C. albicans MYA-2732 (ATCC) - 1 µg/ml; C. albicans 6122/06 - 0.5 µg/ml; C. glabrata MYA-275 (ATCC) - 0.25 µg/ml, C. glabrata 7531/06 - 0.5 µg/ml, C. tropicalis 8122/06 - 0.016 µg/ml, C. parapsilosis 11375/07 - 2 µg/ml. The concentration of miconazole which resulted in a reduction of metabolic activity by 50% (50% RMA) with respect to biofilms of C. albicans 6122/06, C. glabrata MYA-275 (ATCC), C. glabrata 7531/06 and C. tropicalis 8122/06 was 50 µg/ml. In the case of C. albicans MYA-2732 (ATCC) the concentration of miconazole resulting in 50% RMA was 96 µg/ml, and >96 µg/ml for C. parapsilosis 11375/07.
For all six Candida strains there was a strong correlation between MIC values and miconazole concentrations corresponding to 50% RMA of biofilm structure, rs=0.857, p=0.029 (Fig. 5).
|Fig. 5. Correlation, using the Spearman rank correlation coefficient, between the MIC and the concentration of miconazole resulting in a 50% RMA of the biofilms on the surface of PMMA (miconazole concentration range: 0.5 – 96 µg/ml), rs = 0.857, p=0.029.|
CaDS may occur as a consequence of fungal cell adhesion to polymeric materials facilitated by London-van der Waals and electrostatic forces. When the surface free energy is increased, it activates microbial adherence and the number of adherent cells also increases (24). The surface roughness of heat-polymerized resilient liners were found to be less than those of room-temperature polymerized ones. Correspondingly, Candida adherence was reported to be significantly lower on the heat polymerized acrylic resin, used for fabrication of complete acrylic dentures. The differences in surface energies and the higher hydrophilicity could be the reason for this condition (1, 25, 26).
The presence of C. albicans and the cohabitation of different Candida species on the fitting surface of acrylic dentures have been observed frequently, and have been linked to the severity of inflammation. Clinical observations emphasize the importance of biofilm formation on the acrylic denture (12, 27, 28).
The results we have presented indicate that miconazole exhibits high antifungal activity against Candida biofilms developed on acrylic discs for all strains except C. parapsilosis 11375/07. These observations are in general agreement with previous reports indicating that miconazole has potent in vitro activity against planktonic Candida spp., including fluconazole-resistant strains (29).
Several antifungal agents (polyenes and azoles), with different modes of action are available for the treatment of oral candidiasis. The susceptibility of all Candida strains to different antifungal agents has shown that C. albicans strains were more susceptible to fluconazole and miconazole than non-albicans strains (30). However C. albicans resistance to different antifungals (triazoles and particularly fluconazole) has been recently reported in immunoicompromised patients. It has been demonstrated that C. glabrata and C. krusei are intrinsically less susceptible to triazoles and amphoptericin B. Samaranayake et al. (31) demonstrated accentuated phenotypic expression of bud formation of yeast and metallothionein production associated with fluconazole resistance in C. glabrata, which may help the fungus colonize the host.
Miconazole has been used extensively for the topical treatment of CaDS, and its effectiveness against both C. albicans and non-albicans species has been described. Kurijama et al. (32) suggest that, despite an increasingly widespread use of azole antifungal agents, resistance to these antifungals among immunocompetent outpatient populations remains rare.
We found that miconazole at a concentration of 0.5 µg/ml reduced significantly the metabolic activity of the biofilms of three strains studied, C. albicans 6122/06, C. glabrata MYA-275 and C. tropicalis 8122/06. At a concentration of 5 µg/ml miconazole was able to reduce the metabolic activity of biofilms of all the strains studied, the greatest effects being observed for C. glabrata MYA-275 (37.1%). However, at this concentration, the metabolic activity was not reduced by 50% RMA. This extent of reduction required a concentration of 50 µg/ml miconazole for the four biofilm strains of C. glabrata MYA-275, C. tropicalis 8122/06, C. glabrata 7531/06 and C. albicans 6122/06, the highest activity being observed for C. glabrata MYA-275. The greatest inhibition of biofilm metabolic activity was observed at 96 µg/ml miconazole; however, the reduction in the activity of C. parapsilosis 11375/07 biofilms did not reach 50% RMA. Our results also showed a strong correlation between the MIC values for Candida strains and the miconazole concentrations corresponding to the 50% RMA of the biofilm of the same strain. In a recent study, Isham and Ghannoum (29) reported that miconazole was effective against 150 isolates of six species of Candida. The miconazole MIC90 against fluoconazole-resistant strains was 0.5 µg/ml. They suggested that miconazole could be used as first-line treatment for oropharyngeal candidiasis. Arias et al. (36) found that all 84 isolates of C. glabrata were sensitive to concentrations of 3.125 µg/ml of miconazole, but that was necessary to obtain 100% inhibition. About 19% of the isolates were resistant to fluconazole, and none of the isolates were resistant to miconazole.
Another factor that affects biofilm development is the presence of an acquired salivary pellicle on the PMMA surface, which is formed immediately after the denture surface is exposed to the oral environment. Salivary pellicle provides receptor sites for the adhesion of microorganisms. The presence of a salivary pellicle can also alter the surface free energy and surface roughness, which may also affect the adhesion of C. albicans (25). The role of human saliva in the Candida adhesion process, via the formation of a salivary pellicle is still unclear. Saliva produces a mechanical cleaning effect on the fitting surface of the acrylic denture. Moreover, it contains antimicrobial innate defence molecules, including lysozyme, histatin, lactoferrin and IgA, which may reduce Candida adherence and subsequent biofilm formation on the PMMA surface (34). Several investigators have reported that a saliva coating reduces the adherence of C. albicans in PMMA (25, 35). Others have found increased adhesion rates due to the saliva coating (36).
An important medical implication of Candida biofilm infections is related to the increased resistance to antifungal agents, which increases as the biofilm develops and matures (21, 37, 38). Possible reasons for this resistance are the reduced growth rate of the organisms in the biofilm, the action of efflux pumps, especially at the early stages of biofilm formation, changes in the sterol composition of the fungal membranes, and the presence of an extracellular matrix. It has been suggested that filamentation is pivotal for biofilms development; mutants of C. albicans defective in the gene EFG1 are unable to form filaments and do not form biofilms (16, 17, 22).
Clinical experience indicates that Candida infection of the mucosa of the denture bearing area sometimes responds poorly to antifungal therapy. Candida colonization is often re-established soon after treatment, and the symptoms of the disease often return shortly, pointing to the ineffectiveness of the selected antifungal agent (39, 40, 41). Disinfectants, such as sodium hypochlorite or chlorhexidine, are usually recommended for disinfecting acrylic dentures as complementary treatments to antimycotic therapy (13, 40). Additionally, effective removal of denture plaque from the fitting surface of the denture plays an important role in maintaining oral health mainly in complete acrylic denture wearers (39). It was shown that denture cleansing is essential to prevent biofilm formation and the onset of CaDS. Brushing with 2% chlorhexidine gluconate and 1% sodium hypochlorite resulted in 100% removal of the biofilm, whereas immersion in these agents reduced significantly the biofilm viability (13). In addition, a correlation between toxicity of Helicobacter pylori strains and intensity of pathological changes in the upper gastrointestinal tract has been reported (42).
A new promising method of treatment of plaque-related CaDS may be the newly developed antifungal acrylic resin. Cao et al. (43) developed in vitro rechargeable infection-responsive antifungal denture materials that might be useful in the management of CaDS. The PMMA resins bind the cationic form of miconazole through ionic interactions, which lead to a sustained drug release of miconazole for months. Moreover, the anticandidal activity of the miconazole-containing PMMA-resin discs was sustained for a prolonged period of time (weeks and months). Additionally, the drug release was much faster in acidic conditions (43).
The novel treatment modalities such as using anti-inflammatory substances may be also required for successful treatment of CaDS. One innovative approach is the use of mesenchymal stem cells which offers the possibility to simultaneously target the inflammatory response (44).
Despite the limitation of this study, in which only six Candida strains were tested and in vitro nature of this investigation, the results indicate that miconazole exhibits high antifungal activity against biofilms of various Candida species developed on heat-cured poly(methyl methacrylate) discs. The study provides support for the use of miconazole as an effective agent for the treatment of Candida-associated denture stomatitis in acrylic complete denture wearers, with a low incidence of in vitro resistance.
Conflict of interests: None declared.
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A c c e p t e d : July 7, 2014