In vitro Evaluation of Photodynamic Effects Against Biofilms of Dermatophytes Involved in Onychomycosis

Abstract

Dermatophytes are the most common cause of onychomycosis, counting for 90% fungal nail infection. Although dermatophyte pathogens are normally susceptible to antifungal agents, onychomycosis often results in refractory chronic disease, and the formation of biofilms frequently underlines the inadequate responses and resistance to standard antifungal treatment. Numerous in vitro and in vivo antimicrobial photodynamic therapy (aPDT) studies have shown biofilm eradication or substantial reduction, however, such investigation has not yet been expanded to the biofilms of dermatophytes involved in onychomycosis. To shed a light on the potential application of aPDT in the clinic management of onychomycosis, in particular with the manifestation of dermatophytoma, we investigated photodynamic effects on the viabilities and the drug susceptibilities of the biofilm of dermatophytes in vitro. Here, methylene blue at the concentration of 8, 16, and 32 μg/ml applied as photosensitizing agent and LED (635 ± 10 nm, 60 J/cm²) as light source were employed against six strains of Trichophyton rubrum, ten strains of Trichophyton mentagrophytes and three strains of Microsporum gypseum isolated from clinical specimens. Our results indicated highly efficient photodynamic inhibition, exhibiting CFU (colony forming unit) reduction up to 4.6 log₁₀, 4.3 log₁₀, and 4.7 log₁₀ against the biofilms formed by T. rubrum, T. mentagrophytes, and M. gypseum, respectively. Subjected biofilms displayed considerable decreases in SMICs (sessile minimum inhibitory concentrations) to multiple antifungal agents when compared with untreated groups, indicating the biofilms of dermatophytes became more susceptible to conventional antifungal drugs after aPDT. Additionally, the obliteration of biofilm after aPDT could be observed as shattered and ruptured structures being evident in SEM (Scanning Electron Microscopy) images. These findings suggest that aPDT is an attractive alternative treatment holding great promise for combating recalcitrant onychomycosis associated with the biofilm formation.

Keywords: aPDT; biofilm; dermatophytes; dermatophytoma; onychomycosis.

FIGURE 1

SEM images of the biofilms of T. mentagrophytes SEM provided three-dimensional images for biofilm structural assessment, with low magnification of x100 displayed in (A) and high magnifications of x500 and x5000 displayed in (B) and (C), respectively. Two types of peculiar ECM architecture can be observed: (I) an extremely thin “blanket-like” layer covering the areas between hyphae (B); (II) very fine “mesh-like” layer wrapping the filaments of hyphae (C).

FIGURE 2

Photodynamic inhibition on the biofilms of T. rubrum, T. mentagrophytes, and M. gypseum. CFU counting indicated the viabilities of the biofilms of T. rubrum (A), T. mentagrophytes (B), and M. gypseum (C) under the conditions: T1, photodynamic treatment with 32 μg/ml of methylene blue; T2, photodynamic treatment with 16 μg/ml of methylene blue; T3, photodynamic treatment with 8 μg/ml of methylene blue; C1, mock condition with no irradiation and no methylene blue application; C2, exposed to 16 μg/ml of methylene blue without irradiation; C3, exposed to irradiation with no methylene blue applied. All data were represented as the mean ± SD (Two-way ANOVA, p values are in comparison to the results of C1 mock control: ^∗p ≤ 0.05; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001; ^∗∗∗∗p ≤ 0.0001; ns p > 0.05).

FIGURE 3

Photodynamic effects on the susceptibilities of dermatophytic biofilms to conventional antifungal reagents. The SMIC80 of the biofilms of T. rubrum (A), T. mentagrophytes (B), and M. gypseum (C) to TRB, ITC, CLO, and FLU were analyzed prior to or after photodynamic treatment. All data were represented as the mean ± SD [Unpaired t-test (one-tailed), ^∗p ≤ 0.05; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001; ^∗∗∗∗p ≤ 0.0001; ns p > 0.05]. (D) Comparing how aPDT altered the susceptibilities of T. rubrum, T. mentagrophytes, and M. gypseum to TRB, ITC, CLO, and FLU (Two-way ANOVA, Tukey’s test, ^∗p ≤ 0.05; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001; ^∗∗∗∗p ≤ 0.0001; ns p > 0.05).

FIGURE 4

SEM images of the bioflms of M. gypseum after photodynamic treatment. (A) M. gypseum not subjected to photodynamic treatment. (B–D) M. gypseum subjected to photodynamic treatment. (I) Formation of a relatively complete membrane-like structure. (II) Biofilms appeared to have a “hole” in their surface, with a tearing appearance. (III) Perforated ECM surrounded the macroconidia. (IV) Mycelia were fractured, sections of hyphae were broken.