Original article/Article original
Antifungal activity of a soil isolate of Pseudomonas chlororaphis against medically important dermatophytes and identification of a phenazine-like compound as its bioactive metaboliteActivité antifongique d’un isolat tellurique de Pseudomonas chlororaphis vis-à-vis des dermatophytes pathogènes et l’identification d’un composé de type phénazine comme son métabolite actif

https://doi.org/10.1016/j.mycmed.2014.01.117Get rights and content

Summary

Objective

The increasing importance of dermatophytoses and emerging resistance of dermatophytes to current synthetic antifungals have stimulated the search for safer and more effective alternative drugs from natural sources. The present study was carried out to identify antagonistic bacteria of soil origin with strong inhibitory activities on the growth of major human pathogenic dermatophytes.

Materials and methods

Antifungal activity of isolated soil bacteria was screened against the dermatophytes from three genera Microsporum (M. canis, M. gypseum), Epidermophyton (E. floccosum) and Trichophyton (T. mentagrophytes, T. rubrum, T. violaceum, T. tonsurans) by using visual plate agar assay method. A Pseudomonas chlororaphis isolate S105, identified at the species level by 16S ribosomal RNA sequence analysis, was reported as the strongest antagonistic bacterium. P. chlororaphis S105 culture supernatant (PCCS) was examined against tested dermatophytes by GY (glucose-yeast extract) broth bioassay in 6-well microplates. Antifungal compound of the bacterium was partially purified from the culture supernatant through a purification scheme of methanol extraction, Diaion HP20 ion-exchange chromatography and preparative thin layer chromatography.

Results

P. chlororaphis S105 was the most potent inhibitor of fungal growth for all tested dermatophytes with a percent inhibition ranged from 57.1% to 99.8%. The PCCS suppressed the growth of all fungi tested in the range of 18.5% to 84.8%. Partially purified antifungal compound of the bacterium was identified as a phenazine-like compound with an Rf value of 0.51. The compound inhibited fungal growth by 73.6% to 97.9% on GY broth. Fungal growth inhibition was significant for all dermatophytes tested in comparison with the controls (Anova, P < 0.05).

Conclusion

With respect to the strong inhibitory activity of P. chlororaphis against pathogenic dermatophytes reported here, it may be considered as a rich source of useful metabolites with potential application in antifungal drug discovery.

Résumé

Objectif

L’importance croissante des dermatophytes et l’émergence de la résistance de ces champignons aux antifongiques de synthèse actuels ont stimulé la recherche de médicaments plus efficaces provenant de sources naturelles. La présente étude a été réalisée pour identifier des bactéries antagonistes d’origine tellurique avec de fortes activités inhibitrices sur la croissance des principaux dermatophytes pathogènes pour l’homme.

Matériel et méthodes

L’activité antifongique de certaines bactéries d’origine tellurique a été testée contre des dermatophytes appartenant aux trois genres Microsporum (M. canis, M. gypseum), Epidermophyton (E. floccosum) et Trichophyton (T. mentagrophytes, T. rubrum, T. violaceum, T. tonsurans) en utilisant une méthode visuelle sur gélose. L’isolat S105, identifié comme Pseudomonas chlororaphis par séquençage des gènes de l’ARN ribosomial 16S, s’est révélé être le plus actif sur ces champignons. Le surnageant de culture de P. chlororaphis S105 (SCPC) a été testé contre les dermatophytes étudiés en milieu liquide glucose-extrait de levures (bouillon GY), en plaques de microtitration 6 puits. Le composé bactérien antifongique a été partiellement purifié à partir du surnageant de culture par extraction au méthanol, suivie d’une chromatographie échangeuse d’ions sur Diaion HP20 et enfin d’une chromatographie sur couche mince préparative.

Résultats

L’isolat P. chlororaphis S105 est le plus puissant inhibiteur de la croissance des dermatophytes étudiés avec un pourcentage d’inhibition allant de 57,1 % à 99,8 %. Le SCPC réduit de manière significative la croissance de tous les champignons testés de 61,5 % à 84,8 %. Le composé bactérien supportant l’activité antifongique a été identifié comme un dérivé de la phénazine avec un Rf de 0,51. Le composé partiellement purifié inhibe de 73,6 % à 97,9 % la croissance fongique en bouillon GY. Par comparaison avec les témoins, l’inhibition de la croissance fongique est significative pour tous les dermatophytes étudiés (Anova, p < 0,05).

Conclusion

Compte tenu de la forte activité inhibitrice de P. chlororaphis vis-à-vis des dermatophytes étudiés, cet isolat peut être considéré comme une source intéressante de métabolites avec des applications potentielles dans le domaine antifongique.

Introduction

Dermatophytes are a group of specialized filamentous fungi classified in three genera including Trichophyton, Epidermophyton and Microsporum that invade the keratinized tissues of skin, hair and nails. They are considered as an important group of zoonotic fungi with worldwide distribution due to causing different types of superficial infections named “dermatophytoses” [34]. Increasing frequency of dermatophytosis, rapid emergence of resistant pathogenic dermatophytes to currently available antibiotics and life-threatening side effects of chemical antifungal drugs especially in long-term treatments of chronic dermatophytoses provoke the researchers to find alternative management strategies focusing on natural biodiversity [8]. Primary resistance of T. rubrum, the main agent of chronic dermatophytosis to terbinafine as one of the most used anti-dermatophytic drugs, shows the possibility of cross-resistance to various other inhibitors of squalene epoxidase, including naftifine, butenafine, tolnaftate and tolciclate, among human pathogenic dermatophytes [11], [20]. These aspects are becoming a health problem prompting testing of novel therapeutic drugs from natural sources possessing less toxicity and high bioactivity with new modes of action.

The primary current means for the identification of new antifungal agents are represented by screening of the vast biodiversity prevalent in natural resources such as soil samples, marine waters, insects, and tropical plants [10], [13], [19], [25], [26], [27], [28], [29]. In recent years, a large number of studies focused on the treatment of fungal infections by using bioactive metabolites identified in various microorganisms, especially bacteria existing as an important part of biodiversity in natural habitats. Antagonistic bacteria are in the first line of investigation not only for their extreme population diversity, but also for capability to producing a wide array of bioactive metabolites with antimicrobial properties [2], [10], [17], [23], [24], [25], [33].

Among antagonistic bacteria, members of the genus Pseudomonas have been recognized as one of the most important groups producing structurally different natural antibiotics like bactericins and phenazines capable of inhibiting the growth of a diverse range of fungi either plant or human pathogenic [14], [18], [22], [32]. As another important role, some species of Pseudomonas have been reported to present antagonistic activity against some medically important dermatophytes belonging to the genera Trichophyton and Microsporum [3], [4]. It has been suggested that P. aeruginosa may exert its growth inhibitory effect on dermatophytes and non-dermatophyte molds within the infected nail [9].

Despite a large data that has now been published about antifungal activity of various bacteria, little has been documented about the effect of bacterial antagonists and their bioactive metabolites on the growth of human pathogenic fungi especially dermatophytes as major public health hazards. In the present study, antifungal activity of a novel soil isolate of P. chlororaphis strain S105 was evaluated against major human pathogenic dermatophytes from three genera Microsporum, Trichophyton and Epidermophyton. A phenazine-like compound was purified from the bacterium culture supernatant as its bioactive metabolite.

Section snippets

Fungal strains and preparation of inoculums

Fungal strains used in the present work including Trichophyton rubrum PFCC51431, T. mentagrophytes PFCC50541, T. tonsurans PFCC88-1352, T. violaceum PFCC80-137, Microsporum canis PFCC50691, M. gypseum PFCC50701, Epidermophyton floccosum PFCC88-1437 were provided from the Pathogenic Fungi Culture Collection of the Pasteur Institute of Iran (http://en.pasteur.ac.ir/pages.aspx?id=586). For preparing of fungal spores, all the fungi were cultured on Potato Dextrose Agar (PDA; E. Merck, Darmshdt,

Identification of antagonistic bacteria

Antagonistic bacteria with anti-dermatophytic activity selected by agar plate bioassay on GY medium were identified by 16S ribosomal RNA (rRNA ∼1 kb) sequencing using ABI prism Big Dye Terminator Cycle Sequencing Apparatus. Bacterial sequences were compared with the sequences of databases in NCBI using BLAST. They were identified as B. amyloliquefaciens (6 isolates), B. subtilis (5 isolates), B. valismortis (2 isolates) and one isolate of each Acinetobacter baumannii, Streptomyces sp. and

Discussion

In the present study, a total of 16 antagonistic bacteria with strong inhibitory activity against the major human pathogenic dermatophytes were isolated from 148 soil samples and identified in the genera Bacillus, Pseudomonas, Acinetobacter and Streptomyces using 16S rRNA sequence analysis. P. chlororaphis was reported as the most potent inhibitory bacterium affecting the growth of all fungi tested. A phenazine-like compound was purified from the bacterium as its inhibitory component. There are

Disclosure of interest

The authors declare they have no conflicts of interest concerning this article.

Acknowledgments

This work was supported financially by a grant from Deputy of Research of the Tarbiat Modares University, Tehran-Iran.

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