The reduced fungal burden indicates that the aPDT EX 527 nmr treated cells are potentially damaged and thus the survival might be altered by the addition of another cell membrane directed bombarding compound, a structure important for the maintenance of cell wall integrity. Hence, we investigated the effects of combined treatment of aPDT with fluconazole, a compound that targets P450 and affects ergesterol synthesis, a major component of the cell membrane. This antifungal agent is used extensively because of its low host toxicity to treat fungal infections. One of the mechanisms that can be used by C. albicans to develop resistance

NVP-BGJ398 to fluconazole is related to the overexpression of cell membrane multidrug efflux systems [23, 24]. Based on the hypothesis that aPDT could damage the cell membrane of C. albicans, producing

increased membrane permeability [25] and possibly damaging efflux pumps, we used G. mellonella-C. albicans system to assess the sequential combination of PDT with fluconazole. G. mellonella were inoculated with 1.41 × 106 CFU/larva to infect the larvae with the fluconazole-resistant C. albicans strain (C. albicans Can37). Larvae treated only with PDT or only ACY-1215 chemical structure with fluconazole did not show significantly prolonged larval survival. The sequential combination with fluconazole, before or after PDT, significantly increased larvae survival in both assays (Figure  4). These results suggest that aPDT increases all the susceptibility of C. albicans Can37 to fluconazole. Figure 4 Killing of G. mellonella larvae after infection by C. albicans Can37 fluconazole resistant. The larvae received an injection of 1.4x106CFU/larva and were maintained at 37°C. a) administration of fluconazole (14 mg/kg) or PBS (Control), b) antimicrobial PDT or only MB (Control), c) administration of fluconazole

followed by aPDT in a combined therapy or PBS (Control), d) administration of aPDT followed by fluconazole in a combined therapy or PBS (Control), e) administration of aPDT or fluconazole + PDT, f) administration of aPDT or fluconazole + PDT. There was no significant difference on larvae survival when treatment was done only by injecting of fluconazole (P = 0.584) or aPDT alone (P = 0.102). The combined treatment by application of aPDT followed or before fluconazole injection resulted in significantly lower death rates when compared to a control groups (P = 0.0010 to aPDT followed by fluconazole, and P = 0.0018 when aPDT was applied after fluconazole injection). A significant difference in survival was observed for combined treatment compared to aPDT alone (P = 0.0062 for aPDT followed by fluconazole, and P = 0.0068 when aPDT was applied after fluconazole injection). Discussion and conclusion In this study we used the invertebrate model G. mellonella for the in vivo study of antifungal PDT. We verified that aPDT prolonged the survival of G. mellonella caterpillars infected by C.