Introduction

Malassezia species are lipophilic yeasts that comprise part of the normal microflora of the skin of humans and warm-blooded animals [1]. However, this yeast can also cause lesions with distinct absence of inflammation despite the heavy fungal load (pityriasis versicolor/PV), or it can be involved with diseases that lead to characteristic inflammation, or even systemic infections under suitable environmental conditions [1, 2]. Among 14 Malassezia species, Malassezia furfur is one of the main causative agents of PV [1, 2].

PV or tinea versicolor is the most common disease caused by Malassezia yeasts and it is characterized by the development of hypo or hyperpigmented scaly spots, being more frequent in the upper trunk [1, 3]. The clinical aspect of the lesions and the residual hypochromia or achromia that the disease may cause leads to great social stigma, so the proper topical antifungal treatment is required [2].

The increasing incidence of fungal infections and antifungal resistance has prompted the search for novel and effective antifungal drugs and alternative agents [3, 4]. As one of the most promising groups of natural products, essential oils (EO) are made up of many different volatile compounds and considered as potential targets for the development of safe natural antifungals [4]. In recent years, EO obtained from different plants has been documented to have the ability to effectively inhibit the growth of phytopathogenic fungi in vitro and in vivo [4, 5]. However, there is still need for development of new effective antifungal products, especially for local fungal infections that do not require systemic antifungal therapy, such as skin diseases. In addition, use of natural antifungal products could prevent the development of resistance on antifungal drugs.

It has been documented that Myrtus communis EO has fungicidal activity against the Rhizoctonia solani, Fusarium solani, Colletotrichum lindemuthianum, Aspergillus flavus, A. ochraceus, Fusarium culmorum, C. albicans, C. glabrata, C. krusei, C. tropicalis, and C. parapsilosis [6,7,8]. Additionally, this EO has broad antimicrobial activity; there are studies in the literature that report the antimicrobial activity of M. communis EO against Escherichia coli O157:H7, Bacillus cereus, Listeria monocytogenes, and Streptococcus iniae, antiplasmodial and insecticidal activity [5, 7, 8]. The antifungal effect of M. communis EO against Malassezia species has not been investigated yet, although it could have great impact for topical treatment of skin diseases caused by Malassezia species.

Given that, the aim of this study was to explore the antifungal activity of M. communis EO against Malassezia species isolated from the skin of the patients with PV.

Materials and methods

Study population

Study included 41 patients with PV that had not received any treatment for PV 2 weeks prior their enrolment. Inclusion criterion was a confirmed presence of PV and exclusion criteria were the presence of other skin or systemic diseases. The clinical diagnosis of PV was based on the clinical picture—the presence of coppery brown, paler than surrounding skin, or pink patches on trunk, neck, and/or arms, and uncommon on other parts of the body.

Study design

This was a prospective case-series study, which was conducted at the Department of Biomedical Sciences, University of Sassari, Sardinia, and at the Faculty of Medicine, University of Belgrade. The study was approved by the Ethics Committee of the Clinical Centre of Serbia under the number 3177/2016.

Data collection

All subjects had a complete clinical history, while physical examinations and necessary procedures from the skin were done aiming to confirm the diagnosis. Trained mycologists carried out laboratory analysis and were engaged in data collection and data entry.

Sampling procedure for isolation of Malassezia yeast

For the isolation, identification and determination of density of Malassezia, samples were taken from four different locations: the surface layers of the back, thorax, arms and facial skin (area of 4 cm2), using adhesive tape (Suprasorb_F, Lohmann Rauscher, Austria) on the skin for 20 s. Four samples were taken from the skin of thorax glabella or paranasal region.

Isolation of Malassezia was made by applying the sticky side of the tape to the surface of the selective culture media containing fatty acids with a higher carbon number. Two different selective media were used mDixon agar and Leeming–Notman agar (LNA) for the isolation of lipophilic species, and standard mycological medium Sabouraud dextrose agar (SDA) was used for the isolation of M. pachydermatis. Media with samples were incubated for 10 days at 32 °C under aerobic conditions and growth on culture was observed daily. A preliminary identification of the lipophilic Malassezia species was done by macroscopic detection of colonies on mDixon and LNA, and by the lack of growth of colonies on SDA.

Identification of Malassezia yeast

Identification of isolated strains was performed by examining microscopy cell characteristics and by testing physiological and biochemical characteristics (catalase production and Tween assimilation). Malassezia colonies were suspended in 0.9% NaCl and examined under the microscope to detect shape and size of yeast cells and width of budding yeast base. Production of the enzyme catalase was tested by adding a drop of 3% H2O2 to the culture suspension. The suspension was observed microscopically and the production of gas bubbles was considered as a positive catalase reaction. Tween diffusion test was used for further identification of Malassezia. This method is based on the ability of different species to exploit a specific pattern of Tween as the only source of lipids in the SDA plate (Tween 20, 40, 60 and 80).

Chemical composition of EO

The composition of EO M. communis was investigated by LC–MS/MS according to the previously reported method. Main compounds of M. communis EO are presented in the Table 1. Standards of the compounds were purchased from Sigma-Aldrich Chem (Steinheim, Germany), Fluka Chemie GmbH (Buchs, Switzerland) or from ChromaDex (Santa Ana, CA, USA). The Agilent 1200 series liquid chromatograph coupled with Agilent series 6410B electrospray ionization triple-quadruple mass spectrometer and controlled by MassHunter Workstation B.03.01 software was used for analysis. Components were separated using a Zorbax Eclipse XDB-C 18 4.6 mm × 50 mm × 1.8 μm (Agilent Technologies, Santa Clara, CA, USA) reversed phase column.

Table 1 Main compounds of M. communis essential oil
Full size table

Retention indexes

A hydrocarbon mixture of n-alkanes (C9–C22) was analyzed separately under the same chromatographic conditions used on the HP-5MS and the VF-Wax capillary columns to calculate the retention indexes with the generalized equation, Ix = 100[(tx − tn)/(tn + 1 − tn) + n], where t is the retention time, x is the analysis, n is the number of carbons of alkane that elutes before analysis and n + 1 is the number of carbons of alkane that elutes after analysis.

Preparation of Malassezia cell suspension

Malassezia cell suspension was prepared direct from mDixon, LNA or SDA agar using a sterile cotton swab from the surface of the colonies and re-suspended in 0.9% NaCl with 0.1% Tween 20 by mixing thoroughly using vortex mixer. After settling of the larger particles, suspensions were adjusted to 106 colony forming units per 1 ml (CFU/ml), and then diluted in RPMI 1640 (2% glucose) in a ratio of 1:10 (105/ml).

Antifungal effect assessment of EO using microdilution method

Antifungal effect of EO on Malassezia strains was tested using EUCAST broth microdilution method [9]. The final volume of the microtiter plate wells was 200 µl (100 µl/well of EO solution and 100 µl/well spore suspension). Final concentrations of EO were 0.12, 0.5 and 1%, and final inoculum size 104/well of Malassezia cells. Appropriate control including no addition of EO was included: 100 µl/well RPMI 1640 and 100 µl/well Malassezia strains were added to each well where EO was not added. Microtiter plates were incubated at 35 °C for 5 days under aerobic conditions. Cell growth was investigated by measuring absorbance at 620 nm after 5 days incubation at 35 °C. Minimum inhibitory concentration (MIC) was considered as the lowest concentration that inhibits visible growth of fungal spores in the well. Contents of all wells with no visible growth after 5 days were subcultured on mDixon, LNA and SDA plates in aim to determine minimum fungicidal concentrations (MFC). MFC was considered as the lowest concentration that kills all the fungal spores (at which no growth occurred on the SDA plates).

Data analysis

Descriptive and inferential statistical analysis were used for the evaluation of the data using the Statistical Package for Social Science (SPSS) (SPSS version 17.0; SPSS, Chicago, IL, USA). Data were expressed as mean ± SD and counts or percentages (%), where appropriate. The Mann–Whitney test was used for non-parametric data, and the Chi-square test was used for categorical variables. A p value of < 0.05 was assumed to be significant.

Results

Patient’s demographic data and isolated strains

In the PV population (n = 41), there were 22 men (~ 54%) and 19 women (~ 46%) aged 20–80 years (mean 43 ± 16). A total of 86 yeast colonies were isolated from 41 patients with PV. Seven different Malassezia species were identified as follows: Malassezia furfur (42.5%, 37/86), M. sympodialis (23.5%, 20/86), M. sllooffiae (13.9%, 12/86), M. globosa (7.5%, 7/86), M. obtusa (6%, 5/86), M. japonica (4%, 3/86) and M. restricta (2.5%, 2/86). The most and least infected sites were back (74.5, 31/41) and forehead (11.8%, 5/41), respectively.

Inhibition of fungal growth with EO

Inhibition of fungal growth with M. communis EO was effective in: 96% (36/37) of isolated M. furfur, 83% (16/20) of M. sympodialis, 78% (9/12) of M. slooffiae, 78% (5/7) of M. globosa, 75% (4/5) M. obtusa, 73% (2/3) of M. japonica and 62% (1/2) of M. restricta.

In other cases, Malassezia had shown growth despite EO presence.

MIC and MFC

MIC90 values range from 15.625 to 600 μl/ml. The less MIC and MFC of M. communis EO were 31.25 and 62.5 µl/ml, respectively. Results of inhibitory effect of Myrtus communis against different Malassezia species are presented in Table 2.

Table 2 Inhibition effect of Myrtus communis against different Malassezia species
Full size table

Discussion

EOs are widely used in folk medicine and cosmetic industry, but only in recent years they have been recognized as a potential antimicrobial agent that could be used in the treatment of infectious diseases [1, 2]. Up to our knowledge, this is the first study aimed to analyze inhibitory effect of M. communis EO against Malassezia sp. Our data showed that median MIC and MFC of M. communis tested against ten isolates were 31.25 and 62.5 µl/ml, respectively, while the highest inhibiting activity was shown on growth of M. furfur (96%) and M. sympodialis (83%). It can be concluded that the broad-spectrum antimicrobial activity of M. communis EO includes also potent inhibition on Malassezia growth placing this EO as a candidate for topical use in the treatment of skin diseases.

Myrtus communis, belonging to the Myrtaceae family, is a shrub that is growing in different parts of the world, including Sardinia [5, 6]. People traditionally are using the leaves and fruits of myrtle as stomachic, hypoglycaemic, anti-cough, antimicrobial, and anti-haemorrhage remedy [6,7,8, 10]. The EO obtained from myrtle leaves were found to be efficient in lung disorders and wound healing, and they showed antimicrobial, antioxidant, and anti-mutagenic properties [6,7,8]. The main constituents of Italian myrtle oil were found to be 1,8-cineole and a-pinene [10].

Ghasemi Pirbalouti et al. evaluated the antimicrobial activity of M. communis extracts and EOs against Escherichia coli O157:H7, Bacillus cereus, Listeria monocytogenes, and C. albicans, and both the extracts and EOs showed to be effective growth inhibitors of C. albicans (83.67 and 91.16%, respectively) and bacterial pathogens [5]. Moreover, these authors revealed the positive antibacterial activity of M. communis against Streptococcus iniae, as well as it’s antiplasmodial and insecticidal activities [5]. The growth inhibition of clinical isolates of Candida by M. communis EO was also studied [7], while there is no study on growth inhibition of clinical isolates of other fungi, as Malassezia. Our study revealed potent therapeutical activity of M. communis on different types of skin diseases caused by Malassezia sp. The highest inhibiting activity was shown on growth of M. furfur (96%) and M. sympodialis (83%). Our results suggest that the M. communis EO may replace antifungal drugs in the treatment of fungal infections of the skin, mucous membranes and fight against dandruff.

Antimicrobial activity of plant extracts is considered significant if MIC values are below 100 μl/ml for crude extract and moderate when MICs vary from 100 to 625 μl/ml [7, 8]. In this study, M. communis significantly inhibited the growth of all the tested Malassezia sp. with median MIC and MFC 31.25 and 62.5 µl/ml, respectively. This study indicates that the crude extract of M. communis had an in vitro antifungal activity against pathogenic fungal strains isolated from the skin. These results should be confirmed by in vivo research of efficacy of this EO on the skin redness, lesion severity, and Malassezia occurrence on the skin. There are only a very few references in the literature that refer to the efficacy of M. communis on the human skin diseases, but no one refers to PV and Malassezia sp. These studies showed that the statistically significant recurrence of hair growth in the dermatophytes infected areas in treatment models compared with control group. While clotrimazole cured T. mentagrophytes and M. canis infection on days 21 and 17, respectively; M. communis extract cured T. mentagrophytes and M. canis infection on days 9 and 13, and days 9 and 11, respectively [8,9,10].

In general, longer treatment periods (up to 4 weeks) and higher concentrations of topical regimens or doses of systemic agents result in higher cure rates, without, however, avoiding the increased relapse rate [3]. Optimal preventive regimens employing other oral antifungals or topical formulations have not been adequately evaluated to date. Our study suggests application of M. communis EO in treatment and prophylaxes of PV as safe, less expensive and non-hepatotoxic and non-nephrotoxic treatment. Various pharmacological activities such as antioxidant, anti-inflammatory, anticancer, and antimicrobial effects have been related to M. communis [5, 10]. In addition, phytochemical screening of the crude extract of M. communis has revealed the presence of tannins, alkaloids, flavonoids, and phenols in these plants. Individual activities of these compounds have also been proven [5, 6, 10]. Various studies have reported the potent antifungal effects of these compounds and their derivatives such as thymol and carvacrol against some pathogenic fungal strains [10].

The lack of this study is use of morphological and biochemical analysis for identification at species level, although, in some countries, new methods are available. Ma-assisted laser desorption–ionization time of flight mass spectrometry (MALDI-TOF MS) has been reported to be a rapid and reliable diagnostic tool to identify clinically important yeasts, but so far no data have been reported on identification of Malassezia isolates with this technique. Implementation of the MALDI-TOF MS system as a routine identification tool will contribute to correct identification of Malassezia yeasts with minimal effort and in a short turnaround time, which is especially important for the rapid identification of Malassezia in skin diseases and nosocomial outbreaks [10].

Conclusion

These results suggest that the M. communis EO may replace antifungal drugs in the treatment of fungal infections of the skin, mucous membranes and fight against dandruff. The broad-spectrum antimicrobial activity of M. communis EO should be further researched by in vivo studies with aim to considerate this EO as candidate for topical use in treatment of skin diseases.