Susceptibility of Microsporum canis arthrospores to a mixture of chemically defined essential oils: a perspective for environmental decontamination

1 Introduction

Dermatophytosis due to Microsporum canis represents the most common fungal skin infection in cats [1]. This zoophilic dermatophyte is able to infect a wide range of hosts, including humans, where different clinical features have been reported. The incidence of skin disease due to M. canis has increased in the recent years, in particular in Mediterranean countries, being the most prevalent agent of tinea capitis in children [2]. Tinea faciei and tinea corporis have also been reported recently, mostly as result of contact with pets [3]. Infected cats have been proven to contaminate the environment by shedding arthroconidia (arthrospores) which are asexual spores derived from fragmentation of fungal hyphae that are able to invade hairs and scales [4]. These fungal elements, embedded in hairs and skin debris are highly resistant at room temperature, may be viable up to 18 months and are thus responsible for infection and/or reinfection [5]. For these reasons, a reliable control program should include an antifungal treatment of both animals and their living environment. Aggressive removal of contaminated material followed by thorough application of commercial ready-to-use disinfectants is strongly suggested, and several commercially available sporocidal products have been successfully checked [6]. Users are advised to read labels to avoid toxicity risk for pets and children and to follow the manufacturer’s recommendations regarding target surfaces [7]. Furthermore, these products frequently present a variety of environmental concerns [8]. Hence, there is a growing interest of consumers in ingredients from natural sources; phytochemicals would be considered as alternatives to synthetic disinfectants/antimycotics also because many of them are relatively non-toxic to mammals and the environment. Essential oils (EOs) are gaining increasing interest because of their relatively safe status, their wide acceptance by consumers and their exploitation for potential multipurpose functional use. EOs are complex mixtures comprising many single compounds, chemically mostly of terpenoid origin, and have been used for many thousands of years in food preservation, pharmaceuticals, alternative medicine and natural therapies [9]. Antifungal activity of EOs has been demonstrated both against pathogenic fungi causing mycoses [10–12] and against fungi causing food spoilage and producing mycotoxins [13].

Essential oils from a number of plants exhibited good anti- M. canis activity both in vitro and in topical treatment of naturally infected cats [11], while no information was available on their environmental use. For these reasons, the aims of this article were to formulate an herbal mixture and to assess its antifungal activity against M. canis arthrospores with special reference to an environmental application.

2 Materials and methods

3 Results

4 Discussion

In this article, the antifungal activity of a mixture composed of 1% L.cubeba, 0.5% I. verum, 0.5% F. vulgare and 0.5% P. graveolens, in 95% ethanol against the parasitic form of M. canis (arthrospores) in infected cat hairs was investigated.

Considering that arthrospores, the infective element, are present on the broken hairs, and contamination of the environment from these sources is common, this work represents the first attempt to assess the antifungal role of EOs in environmental decontamination from M. canis. Our results can be only partially compared with those of Mugnaini et al. [11], who topically administered a mixture composed by 5% O. vulgare, 5% R. officinalis and 2% T. serpillum essential oil, respectively, in sweet almond oil to seven M. canis infected cats with satisfactory results. In fact, in this article EOs were dissolved in 95% ethanol, which is a solvent appropriate for environmental usage and not suitable for in vivo administration.

Litsea cubeba showed the lowest MIC and MFC values, probably due to its high content of geranial (36.8%) and neral (31.9%) which exhibited a strong antifungal activity as single compounds. P. graveolens also gave good results, probably due to the presence of geraniol and citronellol accounting for 13.7% and 44.4% of the total oils. The MIC and MFC shown by I. verum were 2% and 2.5%, respectively. These values were slightly higher in F. vulgare. The difference could be due to the lower amount of anethole present in the fennel EO.

Essential oils selected for the mixture are known to have antiseptic properties. All EOs showed antifungal effects and have previously been tested mostly against plant pathogens and postharvest pathogenic fungi, except for P. graveolens [24] and I. verum [11] that were assayed also against several Trichophyton species and M. canis, respectively, with antifungal activities agreeing to those found in the present study. L. cubeba was effective against Aspergillus niger, Alternaria alternata, Fusarium moniliforme and Fusarium solani [25], while F. vulgare inhibited several fungi responsible for food spoilage [26]. Furthermore P. graveolens and I. verum were active against plant pathogens [17, 27].

Most of the represented components, except for limonene, provided good antimycotic activity in agreement with literature data [28–30], in particular the isomers of citral (neral and geranial) are the main components of L. cubeba EO. Citral has a strong influence on fungi [31], being able to injure the wall and the membrane of Aspergillus flavus spores. This action results in a decrease of elasticity, and in a changed aggregation of protein-like macromolecules.

Tests of the sensitivity of dermatophytes to EOs described in the literature have been performed with fungal mycelia cultured from clinical isolates, which is not the form of spreading in the environment as represented by arthrospores [7]. In the present work, M. canis arthrospores embedded in infected hairs were employed to reproduce an in-field situation to set up an effective mixture for environmental usage.

The EO mixture showed MIC and MFC values lower than that obtained for L. cubeba, the most effective ingredient. These findings could be ascribed to synergism of the different active components.

Conventional treatment of infected environments has been exhaustively investigated by Moriello et al. [7], who reported complete inhibition of M. canis by commercial disinfectants at concentrations ranging from 3.2% for disinfectants containing lactic acid, to 0.22% for disinfectants based on quaternary ammonium derivatives. Thus, the mixture prepared by us, being effective at a concentration of 0.25%, showed antimycotic activity comparable to that of the most effective commercial product.

The direct application of the mixture on infected hairs inhibited fungal growth at day 4, with a total amount of 800 μL sprayed on the specimens. This volume was lower than that required to obtain the same results with some commercial disinfectants [7]. The mixture sprayed on soft furnishing did not stain (data not shown), unlike some conventional disinfectants such as sodium hypochlorite and lime sulphur. Furthermore this last product is not available worldwide.

P. graveolens and L. cubeba at concentrations used in the present work do not act as potential contact allergens [32] and the other selected oils are not reported to induce cutaneous adverse reactions in contrast with common household cleaning products [33]. These preliminary results seem to be promising for a further application of the EO’s mixture as a suitable tool to inactivate the environmental form of M. canis.

Conflict of interest statement: None declared.