Antifungal efficacy of propolis against fluconazole-resistant Candida glabrata isolates obtained from women with recurrent vulvovaginal candidiasis

Abstract

⁎ Corresponding author at: Mycology Research Center, Faculty of Veterinary Medicine, University of Tehran, Tehran, PO Box 14155 6453, Iran. Tel.: +98 2161117151; fax: +98 2166933222. E-mail address: Khosravi@ut.ac.ir (A.R. Khosravi). Recurrent vulvovaginal candidiasis (RVVC), which affects approximately 5% of women of reproductive age, is defined as 4 or more episodes of symptomatic Candida vaginitis within a year [1]. At present, there is no satisfactory treatment for RVVC, owing to difficulties in managing the high incidence of resistant nonalbicans Candida species such as Candida glabrata[2]. This organism is the second most common agent of candidiasis (occurring in 30% of cases) after Candida albicans and is of particular importance because of its innately increased resistance to fluconazole [3]. Natural products are currently considered to be an interesting alternative to synthetic antifungals for the control of resistant fungal agents, owing to their more effective, safe, and low-cost properties [4]. Propolis, a resinous wax-like substance produced by honey bees, is well known for its antibacterial, antifungal, antiviral, local anesthetic, anti-inflammatory, antioxidant, hepatoprotective, immunostimulating, and cytostatic activities [5]. Thepurposeof thepresent studywas to evaluate the effect of ethanol extract of propolis samples collected from the southern region of Iran (Hormozgan province) against different fluconazole-resistant C. glabrata isolated from women with RVVC. The chemical composition of thepropolis extractwasdeterminedusinggas chromatography–mass spectrometry. Female outpatients attending the Imam Khomeini Hospital, Tehran, Iran, who had experienced a symptomatic and culture-confirmed episode of RVVC and at least 4 documented episodes of Candida vaginitis within the previous 12 months were enrolled in the present study in 2010. The study was approved by the Iranian Ministry of Health and all women gave informed consent. Candida glabrata isolates were identified via urease test, differential culture medium (CHROMagar Candida; CHROMagar Microbiology, Paris, France), and sugar fermentation and assimilation tests (RapID Yeast Plus System; Remel, Lenexa, KS, USA). Minimal inhibitory concentrations (MICs) and minimal fungicidal concentrations of propolis and fluconazole (Diflucan; Pfizer, New York, NY, USA) were determined via broth microdilution [4]. Stock solutions of fluconazole were prepared at 10 times the desired concentration and diluted with different amounts of yeast nitrogen base to achieve concentrations of 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 mg/L. The propolis samples were diluted (1 g of propolis, 1 mL of ethanol, and 4 mL of sterile distilled water) to concentrations of 0.25, 0.5, 1, 2, 4, 5, 8, 10, 15, and 20 mg/mL. The density of the yeast-cell suspension in the yeast nitrogen base was adjusted via spectrophotometer to up to 1×105 cells/mL. The tests were performed in sterile, flat-bottomed, 96-well microtiter plates. The fluconazole solutions and prepared C. glabrata samples (2×100 μL of each) were dispensed into the wells. For the propolis test, 100-μL aliquots of prepared C. glabrata samples were distributed to 11 microtiter plates and propolis solutions were added at different concentrations. After 48 hours, readings from both the fluconazole and the propolis samples were obtained using a microplate reader (Digiscan; Asys Hitech, Eugendorf, Austria). Data were analyzed via t test with 95% confidence intervals (CIs). Pb0.05 was considered to be statistically significant. The propolis extract was composed mainly of phenolic acid (89.8%), sugars (3.2%), and caffeic acid (2.2%). Other elements were oleic acid (0.8%), hexadecanoic acid (0.5%), cinnamic acid (0.4%), 3quinoline carboxamide (0.4%), 12β-hydroxypicrasan-3-one (0.3%), octadecanoic acid (0.2%), butanedioic acid (0.2%), ferulic acid (0.2%), levomenol (0.2%), isoferulic acid (0.1%), and unknown components (1.5%). Under mycologic analysis, 14 C. glabrata strains (C. glabrata1– C. glabrata14) were isolated and identified as being fluconazole resistant. The ethanol extract of propolis showed efficacy against all resistant isolates of C. glabrata (Table 1). The mean MIC values of antifungal drug and propolis against the yeast strains were 109.9 μg/mL (95% CI, 0.5–128.0) and 3.8 mg/mL (95% CI, 0.5–5.0), respectively (Pb0.05). At a propolis concentration of 8 mg/mL, 57.1% of the isolates did not grow at all.