Annals of Microbiology, 58 (3) 543-547 (2008) Comparative study of in vitro methods used to analyse the antifungal activity of propolis against Trichophyton rubrum and Trichophyton mentagrophytes Nedret Ayşe KOÇ 1 , Sibel SİLİCİ 2 * 1 Department of Microbiology, Faculty of Medicine, Erciyes University, Kayseri; 2 Department of Animal Health, S. Çıkrıkçıoğlu Vocational College, Erciyes University, 38039 Kayseri, Turkey Received 17 January 2008 / Accepted 19 May 2008 Abstract - The aim of this study was to evaluate the in vitro activity of propolis against clinical isolates of Trichophyton rubrum and Trichophyton mentagrophytes by following the NCCLS guidelines for testing filamentous fungi, and compare the two methods of evaluating the efficacy of propolis: NCCLS microdilution and agar dilution methods. A broth microdilution method following the recom- mendations established by the National Committee for Clinical Laboratory Standards (NCCLS) and agar dilution method were used to compare the in vitro activity of propolis with that of itraconazole (ITC) against 29 clinical isolates the dermatophytes belonging to two different species of Trichophyton. In terms of MICs (minimal inhibitory concentrations), ITC showed greater activity than propolis. MICs of propolis at which 50% (MIC 50 ) and 90% (MIC 90 ) of the isolates were 0.1 and 0.2 g ml -1 , respectively. Propolis merits further investiga- tions as a potentially useful agent for treatment of dermatophytosis and the broth microdilution and the agar dilution methods were good agreement for propolis against dermatophytes. Moreover, the study suggests the potential value of the broth microdilution as a convenient alternative method for testing the susceptibilities of dermatophytes to propolis. Key words: propolis, dermatophytes, Trichophyton spp., itraconazole (ITC). INTRODUCTION Cases of dermatophytosis have increased over the past few dec- ades (Weitzman and Summerbell, 1995). These infections are often recalcitrant to therapy (Korting et al., 1995). In the last few years, several antifungal agents, such as itraconazole (ITC), have become available for the treatment of these infections. In this setting, the necessary long-tem suppressive therapy with antifungal agents may lead to selection of resistant isolates (Kirkpatrick, 1984). The increasing recognition and importance of fungal infections, the difficulties encountered in their treatment and the increase in resistance to antifungals have stimulated the search for therapeutic alternatives. However, antifungal activ- ity against significant number of strains and following standard procedures has not yet been investigated. The NCCLS published a standard method, M38-P (NCCLS, 1998), for determining the MICs of several antifungal agents against conidium-forming filamentous fungi. A standardised reference method for der- matophytes is needed for use in clinical laboratories to perform susceptibility testing. Propolis is a resinous substance collected by Apis mellifera L. from various tree buds, and bees use propolis for coating hive parts and also to seal cracks and cervices in the hive. The eth- anolic extract of propolis has some activities such as antibacterial (Kujumgiev et al., 1999; Moreno et al., 1999; Bosio et al., 2000), antifungal (Cafarchia et al., 1999; Ota et al., 2001; Sawaya et al., 2002), antiviral (Amoros et al., 1994), antiinflamatory (Miyataka et al., 1997), local-anaesthetic (Paintz and Metzner, 1979), anti- oxidant (Volpert and Elstner, 1993, Orhan et al., 1999), immu- nostimulating (Dimov et al., 1991) and cytostatic (Banskota et al., 1998). Several authors have reported on the inhibitory effect of the ethanolic extract of propolis on Candida using propolis of tem- perate and tropical origin (Sawaya et al., 2002; D’Auria et al., 2003). Whether European propolis (poplar type) is active against dermatophytes is still unknown. The aim of this study has been to evaluate the in vitro activity of propolis against clinical isolates of T. rubrum and T. mentagro- phytes by following the NCCLS guidelines for testing filamentous fungi, and compare the two methods of evaluating the efficacy of propolis: NCCLS microdilution and agar dilution methods. MATERIAL AND METHODS Origin and chemical analysis of propolis. Propolis sample was collected from Kayseri (Central Anatolia) in Turkey. Hand collected propolis was kept desiccated and in the dark up to processing. Voucher specimen was deposited at Department of Microbiology, Faculty of Medicine, University of Erciyes, Kayseri, Turkey. An aliquot of crude propolis (7 g) was dissolved in 80% ethanol by shaking at 50 °C, for 3 days protected from light. The resulting aqueous-ethanol extract was filtered three times through paper filter and concentrated at 50 °C. The resin obtained was dissolved in 80% ethanol to a final concentration of 100 mg mL -1 . This final solution was employed for the antifungal assays. * Corresponding author. Phone: +05324302183; Fax: +90.352.4371383; E-mail: silicis@erciyes.edu.tr