Distribution of Malassezia species on the skin of patients with atopic dermatitis, psoriasis, and healthy volunteers assessed by conventional and molecular identification methods

The fungi of the Malassezia genus, whose first description dates back to the middle of the XIX century, have only recently gained considerable attention of the dermatological community. This is because those basidiomycetous yeasts, being components of the microbiota of human and animal skin and constituting up to 80% of the total skin fungal population [1] are now increasingly recognized as opportunistic pathogens resulting in different dermatological pathologies. Malassezia yeasts are the causative agents of pityriasis versicolor (PV), which is one of the commonest superficial mycoses in human population worldwide [2]. Malassezia species are also involved in the pathogenesis of various dermatoses with global distribution, such as seborrheic dermatitis (SD), atopic dermatitis (AD), and, more recently, psoriasis (PS) [3‐6]. A growing number of reports demonstrate the implication of Malassezia yeasts in other skin disorders, including folliculitis, onychomycosis, confluent and reticulated papillomatosis, and neonatal cephalic pustulosis [7‐10]. Finally, Malassezia yeasts have been associated with systemic infections and outbreaks in neonatal and immunocompromised adults intensive care units [11, 12].

The species content of the Malassezia genus has repeatedly been revised over the last two decades. In the early 1990s the genus Malassezia contained only three species, namely M. furfur, M. pachydermatis, and M. sympodialis. In 1996, four other species (i. e. M. globosa, M. obtusa, M. restricta, and M. slooffiae) were identified within the genus, through a comparative sequence analysis of nuclear ribosomal DNA (rDNA) operons [13]. Since 2002 seven more new species have been described, on the basis of molecular data: M. dermatis, M. equina, M. japonica, M. nana, M. yamatoensis, M. caprae, and M. cuniculi[14‐19]. Overall, the genus Malassezia now accommodates 14 species, all but one (M. pachydermatis) being lipid-dependent species.

Among the Malassezia species, one of the most frequently isolated is M. sympodialis, which has been considered to be associated with AD. One of the clearest indications to support this is the fact that almost half of the adult patients suffering from AD are sensitized to M. sympodialis, as evidenced by allergen-specific IgE and/or T-cell reactivity to the yeast, and that such reactivity is rarely observed in other allergic diseases [20, 21].

The identification of most of Malassezia species can be achieved by using a combination of morphological, biochemical, and physiological characteristics. The currently used protocol, based on the phenotypic criteria, includes the examination of colony and cell morphology, determination of urease, catalase, and β-glucosidase activities, growth at 37°C and 40°C, and the capacity to grow with different polyoxyethylene sorbate compounds (Tween 20, 40, 60, 80) and polyoxyethylene castor oil (Cremophor EL), as the sole lipid source [22].

With the advent of molecular biology tools, several new, DNA-targeted methods have been developed and tested for species identification within the Malassezia taxon. These include pulsed-field gel electrophoresis (PFGE) [23, 24], randomly amplified polymorphic DNA (RAPD) analysis [23, 25], amplified fragment length polymorphism (AFLP) analysis [25, 26], denaturing gradient gel electrophoresis (DGGE) [25], multilocus enzyme electrophoresis (MEE) [27], PCR-based single strand confirmation polymorphism (PCR-SSCP), PCR-based restriction fragment length polymorphism (PCR-RFLP) [7, 28‐34], nested PCR [32, 35‐37], real-time (RT) PCR [35, 38], and direct sequencing of various genetic loci, such as rDNA and internal transcribed spacer (ITS) 1 and 2 regions in particular, the chitin synthase (CHS2) gene, and the RNA polymerase subunit 1 (RPB1) gene [18, 26, 39‐41].

The purpose of this study was to explore the species composition of Malassezia microflora on the skin of healthy volunteers and patients with AD and PS. Species determination was performed by using both conventional, culture-based methods and molecular techniques, that is PCR-RFLP and direct sequencing targeting the rDNA cluster of the Malassezia genome.

A total of 35 Malassezia sp. cultures were obtained from clinical samples, with back being the most frequent site of isolation (16 cultures; 45.7% of all Malassezia cultures), followed by chest (10; 28.6%), face (5; 14.3%), and head (4; 11.4%). Of 24 skin samples collected from each of the three subject groups, 13 (54.2%), 12 (50%), and 10 (41.7%) gave positive culture in the control group, AD group, and PS group, respectively. The overall positive culture rate of the Malassezia yeasts from 4 different body sites of 18 patients under the study was 48.6% (35 positive samples out of 72 samples tested).

Although several Malassezia species have been associated with various dermatological diseases, the exact pathological role of individual species remains obscured. An essential and still open question is whether there exists a relationship between particular Malassezia species and various skin disorders. Other clinical questions to be resolved include whether any of the Malassezia species preferentially occupies certain sites of the body, whether there are any differences in the distribution of the yeasts between lesioned and normal-appearing skin of patients, between adult and children, or between patients and healthy individuals, and finally whether there is variation in the prevalence of Malassezia species depending on gender, age, or geographical origin of the human host. There are now a growing number of works addressing these issues. For instance, studies of the Malassezia microbiota in healthy individuals consistently indicated M. globosa and M. restricta as the predominant species, with a combined detection rate of over 50% (in most of the studies) [7, 31, 32, 34, 35, 49‐51]. Whereas the involvement of the Malassezia species in SD and PV has been quite well recognized [35, 50, 52‐58], the clinical role of these fungi in AD and PS is still controversial. The Malassezia yeasts are currently considered as contributory factors to the induction and exacerbation of both these conditions. The present study was to determine the composition of Malassezia microbiota on the skin of patients with AD and PS, and healthy volunteers from Poland. The choice of the study population was driven by two facts. First, there are much less data on the Malassezia microflora in AD and PS, than in other Malassezia-related diseases (i.e. PV, and SD). Second, AD and PS are the two most common chronic skin diseases, whose incidence has been on the rise in recent years in Poland. Among the Polish population, the prevalence of AD is around 1.4% in adults, and thrice as much (4.7%) in children aged between 3 to 16 years (but up to 32% in infants and young children, that is aged between 0–6 years) [59, 60]. Psoriasis, on the other hand, is estimated to affect up to 1 million people in Poland (ca. 2.6% of general population) [61].

The Malassezia yeasts were cultured from almost half (48.6%) of the harvested skin samples, with the back being most heavily colonized body site, accounting for ca. 46% of the Malassezia cultures. The predominant Malassezia species in two clinical groups and the healthy control group was M. sympodialis. The recovery rate for this species among AD patients, PS patients, and healthy subjects was 100%, 70%, and 76.9%, respectively, and the overall recovery rate for M. sympodialis was 82.9%. The finding of such a high prevalence of M. sympodialis was rather unexpected, since other Malassezia species have usually been much more abundant in all three aforementioned groups, as reported by other authors. In a study of Nakabayashi et al., M. sympodialis was only the third most common species in lesional skin of AD Japanese patients (7% of samples), following M. furfur (21%), and M. globosa (14%) [50]. In a study from Sweden, M. sympodialis was absent from lesional sites of AD patients, whereas other Malassezia species (i. e. M. globosa, M. obtusa, M. restricta, and M. slooffiae) occurred at low rates of 3-11% [62]. Two further studies that used culture-independent, DNA-based methods for the detection of Malassezia species, showed M. globosa and M. restricta as the predominant species in AD. They were detected at frequencies ranging from 87.5% to 100%, while M. sympodialis at 40.6% and 58.3% [35, 51]. However, in a Canadian study of Gupta et al., it was M. sympodialis that predominated in AD patients, with a detection rate of 51.5% [53]. Likewise, M. sympodialis was the dominant species among Korean AD patients, yet the isolation rate was low (16.3%) [33]. Among very few reports on the prevalence and species composition of Malassezia yeasts in PS patients, two were almost completely negative for the presence of M. sympodialis; in a study from Bosnia and Herzegovina, the predominant species in PS patients was M. globosa (55%) followed by M. slooffiae (17%) and M. restricta (10%) [63], while in a study from Iran, M. globosa, as the commonest species in PS (47%), was followed by M. furfur (39%) and M. restricta (11%) [64]. Similar data were obtained from a Japanese, culture-independent study of Takahata et al., who found M. globosa and M. restricta as the sole two Malassezia species in psoriatic scale samples, with similarly high detection frequencies of 98% and 92%, respectively [38]. However, in another study from Japan, as well as in a Canadian, culture-based study, M. sympodialis was found the third (50%), after M. restricta (91%) and M. globosa (68%) or the second (31%), after M. globosa (58%) most frequently isolated Malassezia species from psoriatic lesions [53, 65]. As for the healthy skin, none of the hitherto performed studies have shown the predominance of M. sympodialis, as that seen in the present work. This species has usually ranked third in overall abundance among Malassezia species colonizing normal human skin, with the detection rate spanned from 10% to ca. 40% [7, 31, 32, 35, 50]. Two major components of the Malassezia biota of healthy individuals, that is M. globosa and M. restricta were seriously underrepresented in the current study, with an overall isolation rate of 15.4% and 7.7%, respectively. The disparities between the studies, as discussed above, in frequencies of Malassezia species isolations from different dermatological affections and body sites may be attributable to several factors, including geographical and ethnic origin, clinical and demographic characteristics and even lifestyle habits of the subjects under the study, but also certain methodological issues, such as the use of different sampling techniques (swabbing, scraping) or culture media (modified Dixon agar, Leeming-Notman agar). However, the distribution of Malassezia species is probably most influenced by a method used for species identification. Although the traditional identification schemes, based on morphological characteristics and biochemical activities, are in many clinical laboratories the only diagnostic methods available, they suffer from apparent limitations. Phenotypic tests are time-consuming, labour-intensive and often produce variable or inconclusive results, especially for newly described species; the final result relies on subjective interpretation by a laboratory expert. These methods are thus successively being complemented or replaced by DNA-based molecular techniques, of which PCR-RFLP analysis and PCR sequencing have most extensively been used [18, 26, 29‐34, 39‐41]. Conceptually and technically PCR sequencing is the simplest. It is also the fastest and the most specific identification approach. An important advantage of DNA sequencing over PCR-RFLP is that the latter often involves a lengthy and laborious analysis of complex banding patterns, not always leading to a conclusive result. Moreover, DNA sequencing possesses a much higher discriminatory capacity, allowing intraspecies polymorphisms to be revealed. Some authors have already reported on the rDNA sequence heterogeneity within various Malassezia species, proving the existence of several individual genotypes within the species [26, 39, 40]. The intra-specific genetic diversity was also evidenced in this study. Two types of ITS sequences for M. sympodialis and two types of D1/2 sequences for M. globosa were demonstrated. Given the ability of DNA sequencing for strain typing and its potential use in phylogenetic and population genetics studies, along with the costs of sequencing rapidly plummeting, the method may soon become an integral part not only of a species identification algorithm but also of a routine epidemiological investigation.

The phenotypic and molecular identification results were discordant in ca. 35% of cases (12/35). The reason for this might either be the misidentification or the co-occurrence of different Malassezia species in one culture. The latter explanation is even more likely, as in the third of the discrepant cases, molecular methods did not invalidate the presence of a species identified by conventional phenotypic approach but only uncovered another, co-occurring Malassezia species. This in turn relates to the fact that the establishment of an axenic culture of a Malassezia species, uncontaminated by other Malassezia and non-Malassezia yeasts is rather challenging. A mixture of Malassezia species may not only be present in a clinical sample but even in a seemingly pure single colony on a culture medium. The co-isolation of two Malassezia species from the same specimen was recorded in 4 (11.4%) cases evaluated in this study. A higher prevalence of mixed Malassezia cultures was reported by other authors. For instance, in a Korean study of Lee et al., 15% of SD patients and 21% of healthy volunteers showed co-colonization of two or more Malassezia species [34]. In two European studies, M. globosa, the most commonly observed species, was associated in culture with other Malassezia species in 18% of PV patients from Greece [52] and 40% of PV patients from Spain [55]. Interestingly, in two cases in this study, M. sympodialis was co-cultured with a non-Malassezia species, namely Cryptococcus diffluens in an AD patient and Aureobasidium pullulans in a PS patient. Finding of C. diffluens on AD-affected skin is perfectly in line with previous research demonstrating this species to be a frequent colonizer of the skin surface of AD patients [66]. As for A. pullulans, it is a ubiquitous dematiaceous fungus that has emerged as an opportunistic human pathogen, especially among immunocompromised patients; it is a frequently isolated skin contaminant but rarely a causative agent of fungemia, systemic infections and abscesses in different viscera [67]. The discordance between phenotypic and molecular identification may also relate to the growth rate of different Malassezia species. It means that in a mixed culture, the fast-growing species, such as M. sympodialis may conceal the presence of the slow growers, such as M. globosa, M. restrica, or M. obtusa. This is also a possible explanation for the overall high frequency of M. sympodialis isolations in this study. Against this possibility is the fact that always a selected single separated colony from a primary culture served as an inoculum for the culture used for species identification tests. The six mixed-species cultures represent the only cases in which a primary culture colony already contained a mixture of distinct yeast species. Nevertheless, it seems that a molecular-based but culture-independent method would serve as a more accurate and reliable approach for the assessment of the diversity of Malassezia microbiota [28, 35, 38, 51]. The use of culture-based approach for characterizing Malassezia communities from skin samples posed a limitation to the present study. Due to the fastidious nature of Malassezia fungi and difficulties in culture arising therefrom, the obtained results may understate the size and complexity (species structure) of the Malassezia microbiota.

Culture-based methods essentially select for those species that readily grow under the typical nutritional and physiological conditions supported by commonly used artificial media. These species may not represent the most abundant or influential organisms within a given locality [68].

There is a paucity of reports on drug resistance in Malassezia spp., and this mainly stems from the lack of a standardized protocol for Malassezia susceptibility testing. The observed variability of the results from laboratory to laboratory precludes any association of in vitro and in vivo responses of Malassezia yeasts to antifungals. In this study, the Neo-Sensitabs tablet diffusion assay was employed to test the susceptibilities of selected Malassezia strains to six drugs most widely used in the treatment of Malassezia infections, that is the azoles (FLZ, ECZ, ITZ, KTZ, and MNZ) and cyclopiroxolamine (CPO). All the analysed isolates were susceptible to those compounds, albeit the triazoles (FLZ and ITZ) and CPO were found to be more active than the azole derivatives (ECZ, MNZ, and KTZ). Noteworthy, strains of M. furfur generally appeared less susceptible than strains of M. slooffiae or M. sympodialis, indicating that certain Malassezia species develop mechanisms of drug tolerance more easily than other species do. Some variations between different Malassezia species in the susceptibilities to antifungal agents were also recorded by other authors [69, 70]. Although the molecular bases of drug resistance in Malassezia fungi are largely unknown, there is an optimism that this will change now with an increasing body of data from the whole genome sequencing projects [71, 72]. Recently, Kim et al. have demonstrated that genetic alterations in the amino acid sequence of a putative lanosterol 14α-demethylase (CYP51) from M. globosa may be responsible for resistance to azoles by blocking substrate access channels of the enzyme [73].

To conclude, this study provides an important insight into the species composition of Malassezia microbiota in human skin. The most important findings can be summarized in three points. First, M. sympodialis was the predominant species in both normal and pathologic (AD- and PS-affected) skin. Furthermore, AD patients yielded exclusively M. sympodialis isolates, whereas isolates of M. furfur were observed only in PS patients. Whether this mirrors any predilection of particular Malassezia species for certain clinical conditions, and whether the overall dominance of M. sympodialis reflects geographical specificity needs to be evaluated on a much larger scale. Second, although the overall concordance between phenotypic and molecular methods was quite high (65%), with the discordant results being rather due to the presence of multiple species in a single culture (co-colonization) than true misidentification, for the identification of Malassezia species, molecular typing approach is preferred, as its results are more reliable and straightforward. Third, all Malassezia isolates were susceptible to cyclopiroxolamine and azole drugs, with M. furfur isolates being somewhat more drug tolerant than other Malassezia species.