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

Ruijun Chen,Jingjun Zhao,Xiaomo Wu,Borui Chen,Xiurong Zhong,Jinyan Zhang,Yi Sun,Libao Zheng

doi:10.3389/fmicb.2019.01228

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.

Highlights

Onychomycosis is the most prevalent onychopathy that comprises 50% of nail disorders worldwide (Gupta et al, 2017)
In an attempt to gain insight into the potential clinical implementation of antimicrobial photodynamic therapy (aPDT) tackling the dermatophytic biofilms implicated in onychomycosis, we investigated photodynamic effects on the viabilities, and the drug susceptibilities of the biofilm of dermatophytes, ranging from T. rubrum, T. mentagrophytes to M. gypseum
Biofilm formation using the strain of T. mentagrophytes 7240 islolated from clinical specimen was prepared on coverslip according to the method described by Costa-Orlandi et al (2014)

Summary

Introduction

Onychomycosis is the most prevalent onychopathy that comprises 50% of nail disorders worldwide (Gupta et al, 2017). Dermatophytic pathogens are normally susceptible to antifungal agents, it is estimated that only 25–50% of patients with onychomycosis are cured after the standard treatment (Evans and Sigurgeirsson, 1999; Sigurgeirsson et al, 2002; Gupta et al, 2004; Baran et al, 2007). Comparing to free-floating planktonic cells of the same species, biofilm pathogens can tolerate as much as 1000-fold higher levels of antimicrobial agents (Hawser and Douglas, 1995; Donlan and Costerton, 2002; Marsh, 2004) and microbial biofilms thereby account for more than 60% of all fungal and bacterial infections in humans (Cieplik et al, 2018)

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Results

Conclusion