Susceptibilities of the dermatophytes Trichophyton mentagrophytes and T. rubrum microconidia to photodynamic antimicrobial chemotherapy with novel phenothiazinium photosensitizers and red light

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Abstract

Photodynamic antimicrobial chemotherapy (PACT) is a promising alternative to conventional chemotherapy that can be used to treat localized mycosis. The development of PACT depends on identifying effective and selective PS for the different pathogenic species. The in vitro susceptibilities of Trichophyton mentagrophytes and Trichophyton rubrum microconidia to PACT with methylene blue (MB), toluidine blue O (TBO), new methylene blue N (NMBN), and the novel pentacyclic phenothiazinium photosensitizer S137 were investigated. The efficacy of each PS was determined based on its minimal inhibitory concentration (MIC). Additionally, we evaluated the effect of PACT with NMBN and S137 on the survival of the microconidia of both species. S137 showed the lowest MIC. MIC for S137 was 2.5 μM both for T. mentagrophytes and T. rubrum, when a light dose of 5 J cm−2 was used. PACT with NMBN (10 μM and 20 J cm−2) resulted in a reduction of 4 logs in the survival of the T. rubrum and no survivor of T. mentagrophytes was observed. PACT with S137 at 1 μM and 20 J cm−2 resulted in a reduction of approximately 3 logs in the survival of both species. When a S137 concentration of 10 μM was used, no survivor was observed for both species at all light doses (5, 10 and 20 J cm−2).

Highlights

Dermatophytoses are among the most common and widespread infectious diseases worldwide. ► Photodynamic antimicrobial chemotherapy is a promising alternative to conventional chemotherapy. ► Development of PACT depends on identifying effective PS for the different pathogenic species. ► PACT with the new MB derivatives S137 and NMBN was more effective than with MB and TBO. ► Complete fungal killing was achieved for both species.

Introduction

The dermatophytes are pathogenic fungi capable of invading keratinized tissue, such as the skin, hair and nails of humans and other animals to produce a group of infections named dermatophytoses. These infections include tinea capitis (scalp, eyebrows, and moustaches), tinea pedis (feet), tinea cruris (groin area), and tinea unguium (nail) [1]. Dermatophytoses are among the most common and widespread infectious diseases worldwide, and Trichophyton rubrum and Trichophyton mentagrophytes are the major etiological agents [1], [2], [3], [4], [5].

Trichophyton spp. produce different types of conidia during their asexual cycle. Conidia are specialized structures responsible for reproduction, dispersion, environmental persistence and host infection [6], [7], [8]. Under nonparasitic environments, they produce saprophytic conidia which include two morphologically different types; a small single-celled microconidium and a large multicellular macroconidium. Within infected tissues dermatophytes produce mainly arthroconidia [6]. Arthroconidium results from the fragmentation of a hypha following multiple septum formation and is responsible for the survival of the infection. The disease transmission in dermatophytoses is mediated by these different types of propagules, depending on the source of infection. From saprophytic sources, such as soil or shed animal skin scales, the disease is transmitted to humans via macroconidia or microconidia. From human sources, the disease is transmitted to other individuals mainly by arthroconidia [6].

All these conidia possess the universal properties of dormancy and resistance [6], [7], [8], [9]. Several structural and physiological properties of dermatophytic conidia, such as low metabolic inactivity, resistant layers in the cell wall, and lowered susceptibility to physical and chemical agents contribute to their resistance to harmful environmental stresses (i.e. UV radiation, chilling and freezing, lytic enzymes and antifungal agents produced by soil microorganisms or host cells) [10], [11], [12], [13]. Tolerance of conidia to currently-used fungicides is frequently pointed out as one of the causes of therapeutic failure and recurrence of the infections [13], [14], [15].

There are medications available for dermatophytic infections using primarily oral and/or topical formulations of azoles or allylamines, particularly itraconazole and terbinafine respectively [16], [17]. However, the use of systemic medications may be limited by cost, medication interaction, and possible adverse effects [16], [17]. Treatment failures and relapses occur with all current available therapeutic agents [16], [17]. Certain forms of dermatophytosis, especially those caused by T. rubrum, are particularly recalcitrant to chemotherapeutic treatments. Due to limitations of current therapeutic options, the development of new approaches to treat dermatophytoses is clearly necessary.

Photodynamic antimicrobial chemotherapy (PACT) is an alternative and promising method that can be used to control localized mycosis or kill fungi in the environment [18], [19], [20], [21], [22], [23], [24], [25]. The process is based on the use of a photosensitizer (PS) that binds to and/or accumulates in the target microorganism [23]. The cell is then exposed to visible light of an appropriate wavelength and a photochemical process is initiated, producing several reactive oxygen species that may subsequently cause the death of the target organisms without significant damage to host tissues [18], [20], [25]. In comparison with systemic antifungal agents, PACT has the advantage of dual selectivity in that PS can be targeted to its destination cell and the illumination can be spatially directed to the fungal lesion thus minimizing damage to the host [26]. Additionally, the multiple targets of PACT reduce the chance of selecting tolerant microorganisms. Some previous studies reported the use of PACT with different PS to kill different dermatophytes structures in vitro, in ex vivo skin model and in animal models [18], [27], [28], [29], [30], [31], [32], [33], [34]. PACT was also successfully used to treat dermatophytoses in patients with a contraindication for systemic conventional chemotherapy or in cases in which the infection was unresponsive to previous treatment [35], [36].

Because microconidia are easily produced in vitro and enable the production of single-celled and uniform inocula, they are the fungal structure preferably used in antifungal susceptibility testing for dermatophytes [37], [38], [39]. Inocula consisting of only microconidia are recommended by Clinical and Laboratory Standards Institute standard M38-A [40]. Microconidia have also been used in experiments to evaluate the efficacies of PACT with different PS against Trichophyton spp. [31], [41].

The current work is part of an ongoing program aimed to increase the efficacy of fungal photoinactivation via novel phenothiazinium compound discovery in order to progress this approach towards clinical antifungal therapy. The objective of the present study was to evaluate the efficacies of PACT with four phenothiazinium derivatives [Methylene blue (MB), new methylene blue N zinc chloride double salt (NMBN), toluidine blue O (TBO), and the novel pentacyclic phenothiazinium photosensitizer S137] in combination with red light using T. mentagrophytes and T. rubrum microconidia as the biological model systems.

Section snippets

Trichophyton species and strains

T. mentagrophytes strain ATCC 9533 and T. rubrum strain ATCC 28188 were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, EUA).

Photosensitizers and visible light sources

Methylene blue (MB) (cat # M9140-25G), new methylene blue N zinc chloride double salt (NMBN) (cat # 202096-10G) and toluidine blue O (TBO) (cat # T3260) were purchased from Sigma–Aldrich, Inc. (St. Louis, MO, USA). The novel pentacyclic phenothiazinium photosensitizer S137 was synthesized as previously described [42]. Stock solutions of all the PS

Evaluation of PACT efficacies on T. mentagrophytes and T. rubrum microconidia based on the MIC

The efficacy of PACT with each PS (MB, NMBN, TBO and S137) combined with different doses of light (0, 5, 10, 15, 20, 25 and 30 J cm−2) was evaluated by determining the MIC for each PS at each light dose. Exposure to red light in the absence of the PS did not inhibit the growth of any species independent of the dose applied (data not shown). Treatments with the PS MB, NMBN and TBO in the absence of light, in concentrations up to 200 μM and with S137 in concentrations up to 12.5 μM (which were the

Discussion

In the present study we evaluated the efficacies of PACT with four phenothiazinium derivatives in photosensitised microconidia of the dermatophytes T. mentagrophytes and T. rubrum. The efficacies were measured initially by determining the MIC of each PS for each light dose. For the two better PS (NMBN and S137) we also evaluated microconidia survival after PACT. As the MIC experiments are less time-consuming and permit the evaluation of several treatments simultaneously they were convenient to

Acknowledgements

We are grateful to Dr. Carlos Alberto de Oliveira for supplying the LED600. This work was supported by the Grant 03/07702-9 from the State of São Paulo Research Foundation (FAPESP). We sincerely thank CAPES for a PhD fellowship to G.B.R.

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