Evaluation of the Effects of Photodynamic Therapy Alone and Combined with Standard Antifungal Therapy on Planktonic Cells and Biofilms of Fusarium spp. and Exophiala spp

Abstract

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 (aPDT) 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 (635 ± 10 nm, 12 and 24 J/cm(2)), and evaluated the effects of photodynamic inactivation on five strains 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 strains 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 strains with high minimal inhibitory concentration (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 aPDT treatment. Biofilms of both species showed high sessile MIC50 (SMIC50) and SMIC80 of ≥16 μg/ml for all azoles tested and variable susceptibilities to amphotericin B, with SMIC ranging between 1 and 16 μg/ml. Biofilms subjected to aPDT exhibited a distinct reduction in SMIC50 and SMIC80 compared to untreated groups for both species, except SMIC80 of itraconazole 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 aPDT 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.

Keywords: Exophiala; Fusarium; antifungal susceptibility; biofilm; photodynamic inactivation; planktonic.

Figure 1

Photodynamic inactivation effects on planktonic and biofilm forms of Exophiala spp. and Exophiala spp. (A) CFU counting of Planktonic Exophiala spp. (B) CFU counting of Planktonic Fusarium spp. (C) CFU counting of Exophiala Biofilms. (D) CFU counting of Fusarium Biofilms. C1, growth in Sabouraud glucose agar without irradiation or methylene blue; C2, isolates exposed to 16 μg/ml of methylene blue without irradiation; C3 isolates exposed to irradiation without methylene blue; T1 photodynamic treatment with 8 μg/ml of methylene blue; T2 photodynamic treatment with 16 μg/ml of methylene blue; T3, photodynamic treatment with 32 μg/ml of methylene blue. Data are mean values and standard error from three replicate experiments.

Figure 2

Photodynamic effects on antifungal susceptibilities of planktonic and biofilm forms of Exophiala spp. and Exophiala spp. (A) MICs of Planktonic Exophiala spp. (B) MICs of Planktonic Fusarium spp. (C) SMIC50 of Exophiala biofilm. (D) SMIC50 of Fusarium biofilm. (E) SMIC80 of Exophiala biofilm. (F) SMIC80 of Fusarium biofilm. ITC, itraconazole; VRC, voriconazole; POS, posaconazole; AMB, amphotericin B. Data are mean values and standard error from three replicate experiments.