The two PSs used are hypericin (HYP) and 1,9-dimethyl methylene blue (DMMB). LY2606368 cost HYP is a natural naphthodianthrone endowed with fungicidal activity on yeast, especially on C. albicans.[9] DMMB is a hydrophobic derivative of the well-known

phenothiazinium PS methylene blue (MB).[16] The activity of different phenothiazinium salts against C. albicans has been studied only very recently.[11] Considering that phenothiazines are the most widely used PSs in clinical aPDT, especially in oral infections where C. albicans is an important pathogen, we studied whether HYP could provide any advantages over phenothiazinium salts for clinical candidiasis. DMMB was chosen as it is substantially more hydrophobic than MB or toluidine blue, the phenothiazinium salts currently in use, and it has not been studied for C. albicans. In addition, we investigate VX-770 in vitro the reactive oxygen species (ROS) involved in the phototoxic effect to ascertain mechanistic differences between both PS. Culture Media: Sabouraud Dextrose

Agar CM0041 (Oxoid Ltd., Hampshire, England). Chloramphenicol (Sigma-Aldrich®, St Louis, MO, USA). ROS quenchers: catalase (CAT) and superoxide dismutase (SOD), both from Sigma-Aldrich®. Sodium azide (SA) and mannitol (MAN) were purchased from Panreac® (Barcelona, Spain). Solvents and chemicals: ethanol (Alcohocel®; Barcelona, Spain), PBS buffer (Bio-Rad® Laboratories, Redmond, WA, USA), distiled water and physiological serum (Fresenius Kabi España®, Barcelona, Spain) and dimethyl sulphoxide (DMSO) (Panreac®). Hypericin was purchased from Sigma-Aldrich® and HWI-Analytik® Gmbh (Ruelzheim, Germany). A stock solution was prepared in DMSO and diluted immediately prior to use with distiled water or PBS to the desired concentration. 1,9-dimethyl methylene blue was purchased from Sigma-Aldrich® (Gillingham, UK). A stock solution was prepared in distiled water. Working solutions were prepared in distiled water or

PBS immediately before using with the desired concentrations. Yeast were irradiated Thymidine kinase using light-emitting diode (LED)-based lamps. For DMMB, the lamp emitted at 639.8 ± 10 nm with and irradiance 19.0 mW cm−2, whereas for HYP the wavelength was 602 ± 10 nm and the irradiance 10.3 mW cm−2. Two fluences were used namely 18 and 37 J cm−2. Azole-resistant C. albicans strains namely AZN9635, 456325H and AMO7/0267, were obtained from Canisius Wilhemina Hospital (Nijmegen, The Netherlands). The susceptible C. albicans ATCC 10231 strain was acquired from the American Type Culture Collection (ATCC, Rockville, MD, USA) and C. albicans CETC 1001 from the Spanish Type Culture Collection (CECT, Valencia, Spain). The yeast were grown aerobically overnight in Sabouraud dextrose agar added with chloramphenicol (0.5 mg l−1) plates (SB) at 35 °C.