Limited options of antifungals and the emergence of drug resistance in fungal pathogens has been a multifaceted clinical challenge. Combination therapy represents a valuable alternative to antifungal monotherapy.

Infections of Fusarium spp. and Exophiala spp. are often chronic, recalcitrant, resulting in significant morbidity, causing discomfort, disfigurement, social isolation. Systemic disseminations happen in compromised patients, which are often refractory to available antifungal therapies and thereby lead to death. The antimicrobial photodynamic therapy has been demonstrated to effectively inactivate multiple pathogenic fungi and is considered as a promising alternative treatment for mycoses. In the present study, we applied methylene blue (8,16 and 32 μg/ml) as a photosensitizing agent and light emitting diode (635nm ± 10nm, 12 and 24 J/cm2), and evaluated the effects of photodynamic inactivation on five stains of Fusarium spp. and five strains of Exophiala spp, as well as photodynamic effects on in vitro susceptibility to itraconazole, voriconazole, posaconazole and amphotericin B, both planktonic and biofilm forms. Photodynamic therapy was efficient in reducing the growth of all stains tested, exhibiting colony forming unit-reductions of up to 6.4 log10 and 5.6 log10 against planktonic cultures and biofilms, respectively. However, biofilms were less sensitive since the irradiation time was twice longer than that of planktonic cultures. Notably, the photodynamic effects against Fusarium stains with high MIC values of ≥16, 4-8, 4-8 and 2-4 μg/ml for itraconazole, voriconazole, posaconazole and amphotericin B, respectively, were comparable or even superior to Exophiala spp., despite Exophiala spp. showed relatively better antifungal susceptibility profile. MIC ranges against planktonic cells of both species were up to 64 times lower after PDT treatment. Biofilms of both species showed high SMIC50 and SMIC80 of ≥16 μg/ml for all azoles tested and variable susceptibilities to AMB, with SMIC ranging between 1 and 16 μg/ml. Biofilms subjected to PDT exhibited a distinct reduction in SMIC50 and SMIC80 compared to untreated groups for both species, except SMIC80 of ITC against Fusarium biofilms. In conclusion, in vitro photodynamic therapy was efficient in inactivation of Fusarium spp. and Exophiala spp., both planktonic cultures and biofilms. In addition, the combination of PDT and antifungal drugs represents an attractive alternative to the current antifungal strategies. However, further investigations are warranted for the reliable and safe application in clinical practice.

Infections of Exophiala dermatitidis are often chronic and recalcitrant. Combination therapies with novel compounds and azoles could be an effective solution. Previously, we have demonstrated that pyrvinium pamoate exerted antifungal activity alone and favorable synergy with azoles against planktonic E. dermatitidis. Herein, the underlying antifungal mode of action were investigated. Pyrvinium alone showed sessile MIC50 (SMIC50) of 8->16 μg/ml against E. dermatitidis biofilms. However, synergism of PP with itraconazole, voriconazole, and posaconazole were observed against 16 (88.9%), 9 (50%), and 13 (72.2%) strains of E. dermatitidis biofilms. In accordance with in vitro susceptibilities, pyrvinium alone at concentration of 2 μg/ml resulted in significant growth restriction of planktonic E. dermatitidis. Pyrvinium alone resulted in reduction of biofilm formation. Higher concentration of pyrvinium was associate with more progressive reduction of biofilm mass. The in vivo activity of pyrvinium alone and combined with azoles was evaluated using Galleria mellonella model. Pyrvinium alone significantly improved the survival rate of larvae (P < 0.0001). The combination of pyrvinium and voriconazole or posaconazole acted synergistically in vivo (P < 0.05). Fungal burden determination revealed significant reduction of numbers of colony forming unit (CFU) in larvae treated with pyrvinium-itraconazole and pyrvinium-posaconazole compared to itraconazole or posaconazole alone group, respectively. The effect of pyrvinium on apoptosis, expression of TOR and HSP90, and drug efflux reversal were evaluated by PI/Annexin V staining, Real-Time Quantitative PCR and Rhodamine 6G assay, respectively. Pyrvinium alone or combined with azoles significantly (P < 0.05) increased late apoptosis or necrosis of E. dermatitidis cells. Pyrvinium combined with posaconazole significantly decreased the expression of TOR and Hsp90 compared to posaconazole alone group (P < 0.05). Pyrvinium resulted in significant (P < 0.05) decrease of the efflux of Rhodamine 6G. These findings suggested pyrvinium could be a promising synergist with azoles. The underlying mechanisms could be explained by inducing apoptosis/necrosis, inhibition of drug efflux pumps, and signaling pathways related with stress response and growth control.