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Malassezia Intra-Specific Diversity and Potentially New Species in the Skin Microbiota from Brazilian Healthy Subjects and Seborrheic Dermatitis Patients

Malassezia Intra-Specific Diversity and Potentially New Species in the Skin Microbiota from Brazilian Healthy Subjects and Seborrheic Dermatitis Patients

  • Renan Cardoso Soares, 
  • Marcelo Bergamin Zani, 
  • Ana Carolina Belini Bazán Arruda, 
  • Lucia Helena Fávaro de Arruda, 
  • Luciana Campos Paulino
PLOS
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Abstract

Malassezia yeasts are part of the resident cutaneous microbiota, and are also associated with skin diseases such as seborrheic dermatitis (SD). The role these fungi play in skin diseases and why they are pathogenic for only some individuals remain unclear. This study aimed to characterize Malassezia microbiota from different body sites in healthy and SD subjects from Brazil. Scalp and forehead samples from healthy, mild SD and severe SD subjects were collected. Non-scalp lesions from severe SD patients were also sampled. 5.8S rDNA/ITS2 amplicons from Malassezia sp. were analyzed by RFLP and sequencing. Results indicate that Malassezia microbiota did not group according to health condition or body area. Phylogenetic analysis revealed that three groups of sequences did not cluster together with any formally described species, suggesting that they might belong to potential new species. One of them was found in high proportions in scalp samples. A large variety of Malassezia subtypes were detected, indicating intra-specific diversity. Higher M. globosa proportions were found in non-scalp lesions from severe SD subjects compared with other areas, suggesting closer association of this species with SD lesions from areas other than scalp. Our results show the first panorama of Malassezia microbiota in Brazilian subjects using molecular techniques and provide new perspectives for further studies to elucidate the association between Malassezia microbiota and skin diseases.

Introduction

Fungi from Malassezia genus are lipophilic yeasts that inhabit skin from warm-blooded animals, including humans [1,2]. Molecular based studies have been showing that Malassezia yeasts are the most abundant fungi living on the human skin [3,4]. Although members of the healthy skin microbiota, these fungi are also associated with skin diseases, such as atopic dermatitis, psoriasis, pityriasis versicolor, dandruff and seborrheic dermatitis (SD) [5,6].

SD is a highly prevalent condition that affects mostly sebum rich skin sites, such as scalp, forehead, nose, chest and upper back causing skin desquamation, itching and reddish lesions [2], as well as having social and psychological negative impact [7,8]. The role Malassezia yeasts play in the development of SD is poorly understood. It also remains unclear why Malassezia organisms are pathogenic for only some individuals as they are also present in healthy skin. Among Malassezia species, M. globosa and M. restricta are the most commonly found species occurring in large proportions on human skin in both healthy individuals and subjects with skin diseases [4,913].

Malassezia genus underwent many taxonomic revisions in the last 25 years. Up until 1995, three species were accepted: M. furfur, M. pachydermatis and M. sympodialis [14]. The detection of different variants through rDNA sequencing [15] led to a taxonomic revision that resulted in 4 new species based on morphology, ultrastructure, physiology and also molecular data—M. globosa, M. restricta, M. obtusa and M. slooffiae [16]. Subsequently, other species were described, isolated from humans—M. dermatis [17], M. japonica [18] and M. yamatoensis [19]; and other animals—M. nana [20], M. caprae [21], M. equina [21] and M. cuniculi [22], isolated from cat, goat, horse and rabbit respectively. Moreover, four uncharacterized phylotypes were previously reported in healthy and psoriatic subjects [9,10] and, recently putative new Malassezia species were isolated from parrots and opossum [1], suggesting that Malassezia genus includes more species than currently known.

Despite the recent increase of studies focusing on Malassezia sp., little is still known concerning Malassezia genetic diversity. It has been proposed that specific Malassezia genotypes might be associated with skin diseases, such as atopic dermatitis [23] and also SD [24,25]. Moreover, limited information is available regarding Malassezia microbiota in Brazilian subjects based on molecular data. Therefore, this study aimed to characterize and compare Malassezia cutaneous microbiota from different body sites in Brazilian healthy subjects and patients with seborrheic dermatitis, based on Malassezia genetic diversity analysis.

Materials and Methods

Subjects and sample collection

This study was reviewed and approved by the Institutional Review Board from Pontifícia Universidade Católica de Campinas (PUC-Campinas), SP, Brazil (Protocol 604/08) and was conducted according to the principles expressed in the World Medical Association Declaration of Helsinki. Subjects were recruited in the Dermatology Department from Celso Pierro Hospital and Maternity, PUC-Campinas (Brazil). All subjects provided written informed consent prior to any study-related procedures. Exclusion criteria was comprised of antibiotic, antifungal or anti-dandruff therapy in the last 60 days prior to sampling; smoking; and the presence of cutaneous diseases that have been associated with Malassezia sp. except SD. To quantify SD severity, scalp desquamation, erythema and itching were all rated on a scale ranging from 0 to 5 points [26]. The values were totaled to indicate degrees of severity as follows: 0 points: healthy (no skin diseases related to Malassezia sp.); 1–5 points: mild SD; 6–9 points: moderate SD; 10–15 points: severe SD. Healthy (n = 5), mild SD (n = 5) and severe SD (n = 4) subjects were enrolled (S1 Table).

Samples from scalp and forehead were collected from each subject. No patient presented SD lesion on forehead. Non-scalp lesions from severe SD patients were also sampled (P02: chin, P10: shoulder; P14 and P22: interface between nape and hairline). In total, 32 samples were analyzed. Samples were collected using sterilized cotton swabs soaked in ST solution (0.15 M NaCl, 0.1% Tween 20) as previously described [10]. Two swabs were rubbed on the skin using zigzag repetitive movements, covering approximately 16 cm2 [12]. The heads of the swabs were cut, placed into microtubes containing ST solution, and centrifuged for 5 min [10]. Cotton swabs with no skin contact submitted to the same procedures were used as negative controls.

DNA isolation and 5.8S/ ITS2 rDNA PCR amplification

Samples were vortexed with glass beads (Sigma-Aldrich, Saint Louis, MO, USA) in order to disrupt fungal cell walls. Total genomic DNA was extracted using DNeasy Blood & Tissue kit (Qiagen, Valencia, CA, USA) following the manufacturer’s recommendations. DNA was eluted in 50 μL and stored at -20°C. Negative controls were prepared using only ST solution subjected to the same procedures.

5.8S/ITS2 rDNA region was amplified using Mal1F (5′-TCTTTGAACGCACCTTGC-3′) and Mal1R (5′-AHAGCAAATGACGTATCATG-3′; H: A, T or C) Malassezia-specific primers [10]. Amplicon length ranges from 240 to 310 bp. PCR conditions were the same as described [10].

PCR products were excised from the agarose gel and purified using the QIAquick Gel Extraction Kit (Qiagen) following the manufacturer’s recommendations. Purified PCR products were eluted in 30 μl sterile water.

Construction of PCR fragment libraries

Purified PCR products were cloned into pCR2.1 vector using TA Cloning Kit (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s recommendations. E. coli DH5α competent cells were transformed using the total volume of ligation reaction and inoculated in LB-agar media containing ampicillin (100 mg/mL). Plates were incubated for 18 h at 37°C. Colonies from each library were picked using sterilized toothpicks, and added directly to PCR reaction tubes containing 1 X Taq Buffer, 1.25 U Taq polymerase (Thermo Scientific, Waltham, MA, USA), T7 and M13 vector primers (0.1 mM of each), 1.5 mM MgCl2, 0.25 mM of each dNTP and sterile water to a total volume of 50 μl. The following amplification conditions were used: initial denaturation (94°C, 2 min); 35 cycles comprising denaturation (94°C, 45 sec), annealing (50°C, 30 sec) and extension (72°C, 1 min); final extension (72°C, 2 min).

Restriction fragment length polymorphism analyses (RFLP) and sequencing

In silico restriction analysis was performed to select endonucleases capable of identifying Malassezia species and detecting intra-specific polymorphisms (Fig. 1). Restriction enzyme digestions were performed with 5 or 2.5 μL of PCR products and were done separately at 37°C for 60 or 90 min. Endonucleases were manufactured either by Promega (Madison, WI, EUA) or Thermo Scientific. Results were analyzed by electrophoresis on 2% agarose gels.

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Fig 1. Restriction digestion steps.

Digestion step sequence for Malassezia species identification and detection of intra-specific polymorphisms.

https://doi.org/10.1371/journal.pone.0117921.g001

Representative clones from each Malassezia subtype were selected for sequencing. PCR products were purified using QIAquick PCR Purification Kit (Qiagen). Sequencing was performed using T7 primer utilizing ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA) at the Center for Study on Human Genome—University of São Paulo (Brazil).

Sequence and Phylogenetic analysis

Identical sequences were grouped using BlastClust (http://toolkit.tuebingen.mpg.de/blastclust), and compared with GenBank database using BLAST algorithm [27].

Sequences were aligned using ClustalX 2.1 software (http://www.clustal.org/). Phylogenetic trees were constructed using Maximum Likelihood method and substitution model of Tamura 3-parameter utilizing Mega 5 software (http://www.megasoftware.net/). Ten-thousand bootstrap replications were performed. 5.8S/ITS2 rDNA sequences from Malassezia type strains (12 Malassezia species available in GenBank) were included.

Statistical analysis

Clustering and non-metrical Multidimensional Scaling (nmMSD) using Bray-Curtis similarity distances [28] were performed to assess the relationships between Malassezia communities from different samples. Clustering analysis was done using SIMPROF test with 5% significance level. Individual species/subtypes contribution to microbiota groups was evaluated using SIMPER [29]. Analysis of Similarities (ANOSIM) test was applied to check differences based on health condition, body area and subjects [30]. ANOSIM global R value ranges from 1 to −1. R ≈ 1 indicates that intra-group variation is higher than inter-group variation. When R ≈ 0, there is the same level of variation within and between groups. Diversity was measured using Shannon-Weaver diversity index. These analyses were performed using Primer6 [29].

Species richness of each sample were evaluated using Chao 1 richness estimator [31]. Rarefaction plots were done to assess sampling sufficiency and community diversity analysis, using EstimateS [32].

For mean comparisons and variation analysis Two-tailed Student’s T-test and Pearson’s Variation Coefficient (CV) were performed, respectively.

Results

Malassezia species and subtypes in samples from healthy and SD subjects

Fifty to sixty clones from each skin sample were analyzed by RFLP, totaling 1763 clones. Mal1F and Mal1R primers anneal to conserved sites in 5.8S rDNA/ITS2 region, allowing the amplification of Malassezia organisms specifically. As this region is polymorphic within Malassezia genus, it is suitable for detecting intra-specific diversity, and also allows accurate species identification. Species assignment based on RFLP was confirmed by sequencing, and both methods yielded coherent results. In total, 778 sequences were obtained. Seventy-four sequences were chimeras, vector fragments, human DNA or poor quality sequences, and therefore were removed. Sequence analyses allowed species assignment and identification of subtypes that were not possible by RFLP.

Five known Malassezia species were detected. M. restricta was found in all samples, being the most abundant species in 22 samples (total 32 samples). M. globosa was detected in almost all samples (28/32), although it was the most abundant species in only 4 of them. M. sympodialis, (found in 2 samples) M. dermatis (2 samples) and M. pachydermatis (1 sample) were detected in low proportions. In total, 27 Malassezia subtypes were detected, mostly from M. restricta (13 subtypes), indicating variation among species and also intra-specific polymorphisms (Fig. 2A). Some subtypes are more common than others and others are rare on the analyzed samples. Subtypes MR1, MR3, MG1, ND2 and ND1 were the most abundant in samples. MR1 proportion is higher in scalp samples than in forehead ones and the opposite is found for MR3 proportions. Malassezia subtypes abundance varied depending on the sample. Subtypes of a same species occur in different proportions depending on the sample, suggesting that each sample has its own subtypes community.

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Fig 2. Malassezia subtype communities in 32 skin samples from healthy and SD subjects.

(A) Proportion of Malassezia subtypes. (B) Clustering analysis of Malassezia microbiota. Solid black lines represent clusters significant at 95% according to Simprof test. Sample code: P: SD patient; H: healthy; S: scalp; F: forehead; L: non-scalp lesion sites.

https://doi.org/10.1371/journal.pone.0117921.g002

Three groups of sequences from uncharacterized organisms that might belong to potentially novel Malassezia species were found. Phylotypes 1 and 3 were previously detected in healthy subjects and patients with psoriasis [9,10] but were not characterized; and a new phylotype was first found on human skin in this work (Phylotype 5—nucleotide sequence deposited in the GenBank database with accession number KM205220), which did not cluster together with any formally described Malassezia species (Fig. 3). Phylotypes 1, 3 and 5 were detected in 20, 2 and 12 samples, respectively. Phylotype 1 was more abundant in forehead samples, whereas phylotype 5 predominated in scalp samples, and it was not detected in any samples from severe SD patients. Phylotype 1 was the most abundant organism in 3 samples, while in 3 other samples phylotype 5 was found in the highest proportion (Fig. 2A). Moreover, 5.8S/ITS2 rDNA fragment from phylotype 5 lengths 238 bp, being the smallest one among Malassezia genus sequences available for this region in GenBank which ranges from 240 to 310 bp. Different restriction subtypes were also detected for Phylotype 1 and Phylotype 5 (Fig. 2A).

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Fig 3. Malassezia phylogenetic tree.

Maximum Likelihood phylogenetic tree based on 5.8S/ITS2 rDNA sequences from Malassezia sp. 5.8S/ITS2 rDNA sequences are available for 12 Malassezia species. Potential new Malassezia species found in this study are highlighted. Bootstrap values > 50% are shown. Codes between parentheses correspond to Genbank accession numbers. Numbers between brackets represent the number of Malassezia species clones by body site.

https://doi.org/10.1371/journal.pone.0117921.g003

Malassezia community analysis

Malassezia communities from 32 skin samples were analyzed by Clustering, nmMDS and ANOSIM considering health status, body site and subject. Clustering analysis with SIMPROF test divided the Malassezia subtypes communities in 9 groups (Fig. 2B). Contribution of subtypes for group formation is shown in the (S2 Table).

Malassezia microbiota groups considering RLFP subtypes differ from the ones formed based on species abundance. At species level, only 6 groups are indicated by SIMPROF test (S1 Fig.). The reduction in the number of groups observed is not only due to group gathering when subtypes are counted together at species level, but also due to changes in the clustering dendogram topology. Species contribution to group formation is shown in S3 Table.

Considering health condition specifying disease severity (severe or mild SD), no grouping of samples was observed by clustering or nmMSD (Fig. 4A). This is also supported by ANOSIM test (R = 0.017). Similar results were obtained when SD severity was not specified (ANOSIM R = -0.101; Fig. 4B) Moreover, no dichotomy between Malassezia microbiota from scalp and forehead samples was detected (R = 0.03; Fig. 4C). Samples from different body areas from the same subject did not group preferentially either (global R = 0.006; Fig. 4D), indicating that each sample has its own specific microbiota.

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Fig 4. Multi Dimensional Scaling of Malassezia microbiota at subtype level in 32 skin samples from healthy subjects and SD patients.

(A) Similarity between samples highlighting health condition specifying severity; (B) health condition not specifying severity; (C) body site and (D) subjects. (E) Sample diversity. Circle diameter is proportional to the Shannon-Weaver index. Bubble size scale indicates the relative diversity, with the smallest diversity (P14-S) indexed as 1. (F) Mean diversity based on Shannon-Weaver diversity indexed by body site. Student’s T-test with bars representing Mean ± SEM. * p < 0.05. Sample code: P: SD patient; H: healthy; S: scalp; F: forehead; L: non-scalp lesion sites.

https://doi.org/10.1371/journal.pone.0117921.g004

Malassezia diversity varied among samples (Fig. 4E and S2 Fig.). For the majority of samples, rarefaction curves reached plateaus, indicating that sample diversity was sufficiently explored (S2 Fig.). Species richness estimated by Chao1 is greater than the observed richness, suggesting the presence of rare subtypes that were not covered in sampling effort (S2 Fig.). Most scalp samples are less diverse in comparison to forehead samples. Diversity measured by Shannon-Weaver index considering samples by body site shows similar results (Two-Tailed Student’s T-test p = 0.0103), even though in scalp samples are included samples from healthy subjects and from SD patients (Fig. 4F).

Analyzing each organism individually, relative data variation for M. globosa proportions was compared considering Pearson’s coefficient of variation (CV). Scalp and forehead coefficients are 5.4 (CV = 1.41) and 6.2 (CV = 1.64) times higher, respectively, than the non-scalp lesion sites coefficient (CV = 0.26), indicating that samples from these areas present more variation than the non-scalp lesions. M. globosa mean proportion in the non-scalp lesion sites is 42.28%, which is larger than proportions found in most samples from scalp and forehead. This suggests a closer association of M. globosa with lesions from areas other than scalp from severe SD patients. M. globosa also predominated in two other samples from severe SD patients (scalp from subject P10 and forehead from subject P22 (Fig. 2A).

Discussion

Malassezia genus have been widely studied in recent years due to their association with skin diseases. Different aspects have been analyzed, such as the specific fungal roles on the pathogenic process [24,25,3335] and the host immune response [3642]. The genomic sequences of M. globosa [33] and M. sympodialis [43] have been completely determined, and the partial sequence of M. restricta is also available [33]. Despite advances, the role that Malassezia yeasts play in the pathogenic processes is still poorly understood.

Studies based on molecular methods in different populations frequently show M. restricta as the most abundant Malassezia species on healthy and diseased human skin, followed by M. globosa [913,4446]. In our study with Brazilian subjects, M. restricta was detected in all samples, and was the most abundant species in the majority of them. M. globosa was detected in most samples, although in low proportions. Other formally described species such as M. sympodialis, M. pachydermatis and M. dermatis were found in few samples and in low proportions in this study, and M. furfur was not detected. M. furfur has been shown to be rare or even absent, and when detected, is found in low proportions by molecular-based studies [3,12,4750]. In contrast, it is often isolated by culture-based methods [5153], which favor faster-growing organisms like M. furfur. In culture, this species normally overtakes fastidious species such as M. restricta [54,55]. In this study, rarefaction curves were performed to verify the sampling effort of the clone libraries, and the results indicated that the observed diversity approximates or equals the estimated one. Additionally, PCR amplification tests using Mal1F/Mal1R primer set successfully amplified Malassezia type strains and clinical isolates, including M. furfur, thus indicating that the adopted methodology would allow the detection of this species.

Three groups of sequences that might belong to uncharacterized organisms were detected, two of them previously found in healthy subjects and patients with psoriasis [9,10]. Here, we report that phylotype 1 was detected in most samples, found in higher proportions in forehead samples and being the most prevalent organism in some of them. In contrast, Paulino et. al, 2006 reported that phylotype 1, although present in many studied samples, was found in low proportions. Additionally, a new phylotype (phylotype 5) was found in the Brazilian subjects. This new phylotype did not cluster with any other formally described species and has sequence identity <85% comparing to other Malassezia sp. sequences, suggesting that it might indeed be a new Malassezia species. Further studies are necessary in order to isolate, characterize and formally describe these putative new taxa, including molecular analysis of other genomic regions such as ribosomal large subunit D1/D2, as well as morphological and biochemical analyses. Phylotype 5 was found preferentially in scalp samples rather than in forehead, being the predominant organism in 3 samples. It is important to note that it was not detected in any sample from severe SD patients, suggesting that it inhabits preferentially skin from healthy and mild SD subjects. This might be related to immune response on severe SD subjects, which can potentially interfere with its growth and maintenance on skin.

Several Malassezia subtypes were identified, indicating high intra-specific diversity. Subtype proportions varied between samples, resulting in differences in clustering dendogram topologies at subtype level in comparison with species level. This suggests that Malassezia communities might vary between samples even when species proportions are similar. Therefore, studying Malassezia microbiota at intra-specific level may be useful to identify genotypes associated with skin diseases.

Samples did not group according to health condition, nor to body area and subject, and no Malassezia genotype was detected preferentially on SD patients. These findings are interesting, as they differ from other studies that reported specific Malassezia genotypes associated with SD [24,25]. DNA regions varied between studies, which might be related to the differences observed. We found that Malassezia diversity is higher in forehead than in scalp samples, regardless scalp health status, which suggests diversity patterns associated with body site and not with health condition. Although no dichotomy according to health status was observed, it is possible that specific lineages might be more closely involved with SD pathogenic process. Other possible markers might help to elucidate this association.

Considering the relative abundance of each Malassezia organism individually M. globosa occurred in high proportions in all samples from non-scalp lesions from severe SD patients. High M. globosa proportions were also found in some of the scalp and forehead samples from severe SD patients, suggesting that this species might be more closely related to SD. Previous studies suggested that M. globosa is associated with SD [24,33,45,56,57]; however, they do not differentiate disease occurrence in scalp and other body areas. We found different M. globosa proportions in non-scalp lesions as compared with scalp lesions from mild and severe SD patients, suggesting that M. globosa might be associated with SD symptoms from areas other than scalp. Perhaps this species is more efficient in evading and modulating local immune response so it can grow in these areas, despite inflammation occurrence.

Malassezia sp. has been associated with SD and it is known that only some individuals develop SD [6], suggesting that both yeasts and host plays a role in SD development. We observed no group formation according to health condition, indicating that other factors aside from Malassezia can be involved in skin diseases development, such as immune system response [5860]. In healthy individuals, probably host immune system controls Malassezia growth, allowing its maintenance without local inflammation, reaching an equilibrium on human skin [6]. Factors that turn Malassezia into a pathogen are not yet fully understood [6]. Possibly the ability of these fungi to modulate local immune response allied to host susceptibility and second metabolites production have a role in SD development [2]. High lipase gene expression and lipolytic activity of M. globosa lipases may also be associated with SD development [35,56,61]. The correlation of severe SD cases with AIDS patients suggests that host immune response indeed has an important role on SD [2,58,62]. In this context, differential gene expression of immune response genes from healthy and SD patients has been reported [60]. Patients treated with antifungal drug exhibited gene expression pattern similar to healthy subjects, supporting the relation between host gene expression and skin disease development.

In Brazil, most data regarding Malassezia organisms are provided by veterinary studies [6367]. To our knowledge, studies aiming to analyze Malassezia microbiota in Brazilian population are based on culture-dependent methods [68,69]. The same happens in other Latin American countries [52,7072]. Currently, culture-independent methods are preferentially chosen due to culture bias and the fastidious nature of many microorganisms, including Malassezia [10,45,54,7375]. Thus the available information regarding Malassezia microbiota in Brazil is still restricted and possibly influenced by limitations of culture-dependent methods.

Our data show a general view of Malassezia microbiota in healthy and SD subjects through culture-independent methods in Brazilian subjects, pointing to particularities of the analyzed population. The number of subjects included in the study is limited, and is not nearly sufficient to understand the complexity of the Malassezia microbiota in human skin. Nevertheless, the data reported here establishes the basis for future studies, which should include a higher number of subjects to provide a broader understanding of Malassezia microbiota in Brazilian population and the role of these organisms as commensals and as pathogens. Three groups of sequences from uncharacterized organisms that might belong to new Malassezia species were detected in samples from both healthy and SD subjects. One of the organisms had not been previously reported on human skin, and was mainly found in scalp samples. We intend to confirm the taxonomic status and formally describe this putative new taxon. These findings suggest differences between studies, and indicate that Malassezia diversity from human skin has not been fully explored. Differences observed in samples from Brazilian subjects might be associated with genetics and cultural habits, such as hair care. Hair appearance is an important cultural feature in Brazil and different products such as gels, creams or chemical treatments for dyeing and smoothing are commonly used. Such habits can impact the microbiota.

Our results do not suggest an association between Malassezia microbiota and SD, body area or subject based on the whole Malassezia community structure, but indicate less diversity in scalp than forehead. At organism level, higher M. globosa proportions in non-scalp lesions in severe SD subjects were found, suggesting closer association of this species with SD lesions from areas other than scalp. In order to confirm whether M. globosa plays a role in SD specifically in body areas other than scalp, it would be important to increase the number of samples analyzed in further studies. It would also be important to analyze M. globosa lipase gene expression and enzyme activity, as well as the host immune response. Different Malassezia subtypes were found in different proportions in samples. This shows that Malassezia communities can differ at intra-specific level, but can be similar at species level. Other factors beyond Malassezia presence, species, subtypes and diversity patterns might have an important role in SD development, such as genetics and host immune response.

Here we showed the first panorama of Malassezia microbiota from Brazilian subjects based on molecular methods. Results revealed potential new species occurring in large proportions in most samples. Different subtypes of Malassezia species were found, demonstrating that analyses aiming to explore intra-specific diversity in depth are a significant motivation for further studies to elucidate Malassezia role in skin diseases.

Supporting Information

S1 Fig. Malassezia communities from 32 skin samples from healthy and SD subjects at species level.

(A) Malassezia species and phylotypes proportion in each sample. (B) Clustering analysis of Malassezia microbiota. Solid black lines represent clusters significant at 95% according to Simprof test. Sample code: P: SD patient; H: healthy; S: scalp; F: forehead; L: non-scalp lesions.

https://doi.org/10.1371/journal.pone.0117921.s001

(TIF)

S2 Fig. Rarefaction curves at Malassezia subtype level.

Sample code: P: SD patient; H: healthy; S: scalp; F: forehead; L: Non-scalp lesion sites. Sobs: observed subtypes. Chao1: Estimated richness. Doted lines represent Standard Deviation.

https://doi.org/10.1371/journal.pone.0117921.s002

(TIF)

S2 Table. Contribution of Malassezia subtypes.

Contribution for groups formed by clustering analysis with SIMPROF test. Higher contributing subtypes (cut-off 90%) are listed.

https://doi.org/10.1371/journal.pone.0117921.s004

(DOCX)

S3 Table. Contribution of Malassezia species or phylotypes.

Contribution for groups formed by clustering analysis with SIMPROF test. Higher contributing subtypes (cut-off 90%) are listed.

https://doi.org/10.1371/journal.pone.0117921.s005

(DOCX)

Acknowledgments

We thank Dr. Natália Pirani Ghilardi Lopes and Dr. Gustavo Muniz Dias for the support with the statistical analyses. We are also grateful to the employees and medical team from Celso Pierro Hospital and Maternity, PUC-Campinas, Brazil. We also thank all volunteers who collaborated with this work. Finally, we thank Anderson de Oliveira Ferreira for technical support.

Author Contributions

Conceived and designed the experiments: RCS MBZ ACBBA LHFA LCP. Performed the experiments: RCS MBZ. Analyzed the data: RCS MBZ LCP. Contributed reagents/materials/analysis tools: LCP. Wrote the paper: RCS LCP.

References

  1. 1. Castella G, Coutinho SD, Cabanes FJ (2013) Phylogenetic relationships of Malassezia species based on multilocus sequence analysis. Med Mycol 52: 99–105.
  2. 2. Gaitanis G, Magiatis P, Hantschke M, Bassukas ID, Velegraki A (2012) The Malassezia genus in skin and systemic diseases. Clin Microbiol Rev 25: 106–141. pmid:22232373
  3. 3. Findley K, Oh J, Yang J, Conlan S, Deming C, et al. (2013) Topographic diversity of fungal and bacterial communities in human skin. Nature 498: 367–370. pmid:23698366
  4. 4. Grice EA, Segre JA (2011) The skin microbiome. Nat Rev Microbiol 9: 244–253. pmid:21407241
  5. 5. Gupta AK, Batra R, Bluhm R, Boekhout T, Dawson TL Jr. (2004) Skin diseases associated with Malassezia species. J Am Acad Dermatol 51: 785–798. pmid:15523360
  6. 6. Gaitanis G, Velegraki A, Mayser P, Bassukas ID (2013) Skin diseases associated with Malassezia yeasts: facts and controversies. Clin Dermatol 31: 455–463. pmid:23806162
  7. 7. Grimalt R (2007) A practical guide to scalp disorders. J Investig Dermatol Symp Proc 12: 10–14. pmid:18004290
  8. 8. Dessinioti C, Katsambas A (2013) Seborrheic dermatitis: etiology, risk factors, and treatments: facts and controversies. Clin Dermatol 31: 343–351. pmid:23806151
  9. 9. Paulino LC, Tseng CH, Blaser MJ (2008) Analysis of Malassezia microbiota in healthy superficial human skin and in psoriatic lesions by multiplex real-time PCR. FEMS Yeast Res 8: 460–471. pmid:18294199
  10. 10. Paulino LC, Tseng CH, Strober BE, Blaser MJ (2006) Molecular analysis of fungal microbiota in samples from healthy human skin and psoriatic lesions. J Clin Microbiol 44: 2933–2941. pmid:16891514
  11. 11. Nagata R, Nagano H, Ogishima D, Nakamura Y, Hiruma M, et al. (2012) Transmission of the major skin microbiota, Malassezia, from mother to neonate. Pediatr Int 54: 350–355. pmid:22300401
  12. 12. Clavaud C, Jourdain R, Bar-Hen A, Tichit M, Bouchier C, et al. (2013) Dandruff is associated with disequilibrium in the proportion of the major bacterial and fungal populations colonizing the scalp. PLoS One 8: e58203. pmid:23483996
  13. 13. Sugita T, Tajima M, Tsubuku H, Tsuboi R, Nishikawa A (2006) Quantitative analysis of cutaneous Malassezia in atopic dermatitis patients using real-time PCR. Microbiol Immunol 50: 549–552. pmid:16858146
  14. 14. Ashbee HR (2007) Update on the genus Malassezia. Med Mycol 45: 287–303. pmid:17510854
  15. 15. Guillot J, Gueho E (1995) The diversity of Malassezia yeasts confirmed by rRNA sequence and nuclear DNA comparisons. Antonie Van Leeuwenhoek 67: 297–314. pmid:7778898
  16. 16. Guého E, Midgley G, Guillot J (1996) The genus Malassezia with description of four new species. Antonie Van Leeuwenhoek 69: 337–355. pmid:8836432
  17. 17. Sugita T, Takashima M, Shinoda T, Suto H, Unno T, et al. (2002) New yeast species, Malassezia dermatis, isolated from patients with atopic dermatitis. J Clin Microbiol 40: 1363–1367. pmid:11923357
  18. 18. Sugita T, Takashima M, Kodama M, Tsuboi R, Nishikawa A (2003) Description of a new yeast species, Malassezia japonica, and its detection in patients with atopic dermatitis and healthy subjects. J Clin Microbiol 41: 4695–4699. pmid:14532205
  19. 19. Sugita T, Tajima M, Takashima M, Amaya M, Saito M, et al. (2004) A new yeast, Malassezia yamatoensis, isolated from a patient with seborrheic dermatitis, and its distribution in patients and healthy subjects. Microbiol Immunol 48: 579–583. pmid:15322337
  20. 20. Hirai A, Kano R, Makimura K, Duarte ER, Hamdan JS, et al. (2004) Malassezia nana sp. nov., a novel lipid-dependent yeast species isolated from animals. Int J Syst Evol Microbiol 54: 623–627. pmid:15023986
  21. 21. Cabanes FJ, Theelen B, Castella G, Boekhout T (2007) Two new lipid-dependent Malassezia species from domestic animals. FEMS Yeast Res 7: 1064–1076. pmid:17367513
  22. 22. Cabanes FJ, Vega S, Castella G (2011) Malassezia cuniculi sp. nov., a novel yeast species isolated from rabbit skin. Med Mycol 49: 40–48. pmid:20560865
  23. 23. Sugita T, Kodama M, Saito M, Ito T, Kato Y, et al. (2003) Sequence diversity of the intergenic spacer region of the rRNA gene of Malassezia globosa colonizing the skin of patients with atopic dermatitis and healthy individuals. J Clin Microbiol 41: 3022–3027. pmid:12843037
  24. 24. Tajima M, Sugita T, Nishikawa A, Tsuboi R (2008) Molecular analysis of Malassezia microflora in seborrheic dermatitis patients: comparison with other diseases and healthy subjects. J Invest Dermatol 128: 345–351. pmid:17671514
  25. 25. Hiruma M, Cho O, Hiruma M, Kurakado S, Sugita T, et al. (2014) Genotype Analyses of Human Commensal Scalp Fungi, Malassezia globosa, and Malassezia restricta on the Scalps of Patients with Dandruff and Healthy Subjects. Mycopathologia 177: 263–269. pmid:24792417
  26. 26. Emtestam L, Svensson A, Rensfeldt K (2011) Treatment of seborrhoeic dermatitis of the scalp with a topical solution of urea, lactic acid, and propylene glycol (K301): results of two double-blind, randomised, placebo-controlled studies. Mycoses 55: 393–403. pmid:21966947
  27. 27. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403–410. pmid:2231712
  28. 28. Bray JR, Curtis JT (1957) An Ordination of the Upland Forest Communities of Southern Wisconsin. Ecological Monographs 27: 325–349.
  29. 29. Clarke KR, Gorley RN (2006) PRIMER v6: User Manual/Tutorial. 6 ed. Plymouth.
  30. 30. Clarke KR (1993) Non-parametric multivariate analyses of changes in community structure. Australian Journal of Ecology 18: 117–143.
  31. 31. Chao A (1987) Estimating the population size for capture-recapture data with unequal catchability. Biometrics 43: 783–791. pmid:3427163
  32. 32. Colwell RK (2005) EstimateS: Statistical estimation of species richness and shared species from samples. User’s Guide and application. Version 9.1.0. ed. Available: http://viceroy.eeb.uconn.edu/EstimateS/EstimateSPages/EstSUsersGuide/EstimateSUsersGuide.htm. Accessed 2014 September.
  33. 33. Xu J, Saunders CW, Hu P, Grant RA, Boekhout T, et al. (2007) Dandruff-associated Malassezia genomes reveal convergent and divergent virulence traits shared with plant and human fungal pathogens. Proc Natl Acad Sci U S A 104: 18730–18735. pmid:18000048
  34. 34. DeAngelis YM, Gemmer CM, Kaczvinsky JR, Kenneally DC, Schwartz JR, et al. (2005) Three etiologic facets of dandruff and seborrheic dermatitis: Malassezia fungi, sebaceous lipids, and individual sensitivity. J Investig Dermatol Symp Proc 10: 295–297. pmid:16382685
  35. 35. Juntachai W, Oura T, Murayama SY, Kajiwara S (2009) The lipolytic enzymes activities of Malassezia species. Med Mycol 47: 477–484. pmid:18798119
  36. 36. Faergemann J, Bergbrant IM, Dohse M, Scott A, Westgate G (2001) Seborrhoeic dermatitis and Pityrosporum (Malassezia) folliculitis: characterization of inflammatory cells and mediators in the skin by immunohistochemistry. Br J Dermatol 144: 549–556. pmid:11260013
  37. 37. Kerr K, Darcy T, Henry J, Mizoguchi H, Schwartz JR, et al. (2011) Epidermal changes associated with symptomatic resolution of dandruff: biomarkers of scalp health. Int J Dermatol 50: 102–113. pmid:21182510
  38. 38. Kerr K, Schwartz JR, Filloon T, Fielo A, Wehmeyer K, et al. (2011) Scalp Stratum Corneum Histamine Levels: Novel Sampling Method Reveals Association with Itch Resolution in Dandruff/Seborrhoeic Dermatitis Treatment. Acta Derm Venereol 91: 404–408. pmid:21336476
  39. 39. Baroni A, Paoletti I, Ruocco E, Agozzino M, Tufano MA, et al. (2004) Possible role of Malassezia furfur in psoriasis: modulation of TGF-beta1, integrin, and HSP70 expression in human keratinocytes and in the skin of psoriasis-affected patients. J Cutan Pathol 31: 35–42. pmid:14675283
  40. 40. Baroni A, Orlando M, Donnarumma G, Farro P, Iovene MR, et al. (2006) Toll-like receptor 2 (TLR2) mediates intracellular signalling in human keratinocytes in response to Malassezia furfur. Arch Dermatol Res 297: 280–288. pmid:16283346
  41. 41. Perkins MA, Cardin CW, Osterhues MA, Robinson MK (2002) A non-invasive tape absorption method for recovery of inflammatory mediators to differentiate normal from compromised scalp conditions. Skin Res Technol 8: 187–193. pmid:12236889
  42. 42. Trznadel-Grodzka E, Blaszkowski M, Rotsztejn H (2012) Investigations of seborrheic dermatitis. Part I. The role of selected cytokines in the pathogenesis of seborrheic dermatitis. Postepy Hig Med Dosw (Online) 66: 843–847. pmid:23175340
  43. 43. Gioti A, Nystedt B, Li W, Xu J, Andersson A, et al. (2013) Genomic insights into the atopic eczema-associated skin commensal yeast Malassezia sympodialis. MBio 4: e00572–00512. pmid:23341551
  44. 44. Sugita T, Suto H, Unno T, Tsuboi R, Ogawa H, et al. (2001) Molecular analysis of Malassezia microflora on the skin of atopic dermatitis patients and healthy subjects. J Clin Microbiol 39: 3486–3490. pmid:11574560
  45. 45. Zhang H, Ran Y, Xie Z, Zhang R (2013) Identification of Malassezia species in patients with seborrheic dermatitis in China. Mycopathologia 175: 83–89. pmid:23247810
  46. 46. Takahata Y, Sugita T, Hiruma M, Muto M (2007) Quantitative analysis of Malassezia in the scale of patients with psoriasis using a real-time polymerase chain reaction assay. Br J Dermatol 157: 670–673. pmid:17634085
  47. 47. Lee YW, Byun HJ, Kim BJ, Kim DH, Lim YY, et al. (2011) Distribution of Malassezia species on the scalp in korean seborrheic dermatitis patients. Ann Dermatol 23: 156–161. pmid:21747613
  48. 48. Kaga M, Sugita T, Nishikawa A, Wada Y, Hiruma M, et al. (2009) Molecular analysis of the cutaneous Malassezia microbiota from the skin of patients with atopic dermatitis of different severities. Mycoses 54: e24–e28. pmid:20002882
  49. 49. Takahata Y, Sugita T, Kato H, Nishikawa A, Hiruma M, et al. (2007) Cutaneous Malassezia flora in atopic dermatitis differs between adults and children. Br J Dermatol 157: 1178–1182. pmid:17916215
  50. 50. Lyakhovitsky A, Shemer A, Amichai B (2013) Molecular analysis of Malassezia species isolated from Israeli patients with pityriasis versicolor. Int J Dermatol 52: 231–233. pmid:23347312
  51. 51. Lian CH, Shen LL, Gao QY, Jiang M, Zhao ZJ, et al. (2014) Identification of Malassezia species in the facial lesions of Chinese seborrhoeic dermatitis patients based on DNA sequencing. Mycoses: [Epub ahead of print]. . pmid:25530423
  52. 52. Rincon S, Celis A, Sopo L, Motta A, Cepero de Garcia MC (2005) Malassezia yeast species isolated from patients with dermatologic lesions. Biomedica 25: 189–195. pmid:16022373
  53. 53. Arsic-Arsenijevic VS, Milobratovic D, Barac AM, Vekic B, Marinkovic J, et al. (2014) A laboratory-based study on patients with Parkinson’s disease and seborrheic dermatitis: the presence and density of Malassezia yeasts, their different species and enzymes production. BMC Dermatol 14: 5. pmid:24628775
  54. 54. Gemmer CM, DeAngelis YM, Theelen B, Boekhout T, Dawson TL Jr (2002) Fast, noninvasive method for molecular detection and differentiation of Malassezia yeast species on human skin and application of the method to dandruff microbiology. J Clin Microbiol 40: 3350–3357. pmid:12202578
  55. 55. Gueho E, Boekhout T, Begerow D (2010) Biodiversity, Phylogeny and Ultrastructure. In: Boekhout T, Kellermann-Guého E, Mayser P, Velegraki A, editors. Malassezia and the Skin. Berlin, Germany: Springer. pp. 17–63.
  56. 56. DeAngelis YM, Saunders CW, Johnstone KR, Reeder NL, Coleman CG, et al. (2007) Isolation and expression of a Malassezia globosa lipase gene, LIP1. J Invest Dermatol 127: 2138–2146. pmid:17460728
  57. 57. Midgley G (2000) The lipophilic yeasts: state of the art and prospects. Med Mycol 38 Suppl 1: 9–16. pmid:11204169
  58. 58. Bukvic Mokos Z, Kralj M, Basta-Juzbasic A, Lakos Jukic I (2012) Seborrheic dermatitis: an update. Acta Dermatovenerol Croat 20: 98–104. pmid:22726283
  59. 59. Ashbee HR, Bond R (2010) Malassezia species and immunity: host-pathogen interactions. In: Boekhout T, Kellermann-Guého E, Mayser P, Velegraki A, editors. Malassezia and the Skin. Berlin, Germany: Springer. pp. 139–173.
  60. 60. Mills KJ, Hu P, Henry J, Tamura M, Tiesman JP, et al. (2012) Dandruff/seborrhoeic dermatitis is characterized by an inflammatory genomic signature and possible immune dysfunction: transcriptional analysis of the condition and treatment effects of zinc pyrithione. Br J Dermatol 166 Suppl 2: 33–40. pmid:22670617
  61. 61. Juntachai W, Oura T, Kajiwara S (2011) Purification and characterization of a secretory lipolytic enzyme, MgLIP2, from Malassezia globosa. Microbiology 157: 3492–3499. pmid:22016565
  62. 62. Munoz-Perez MA, Rodriguez-Pichardo A, Camacho F, Colmenero MA (1998) Dermatological findings correlated with CD4 lymphocyte counts in a prospective 3 year study of 1161 patients with human immunodeficiency virus disease predominantly acquired through intravenous drug abuse. Br J Dermatol 139: 33–39. pmid:9764146
  63. 63. Machado ML, Cafarchia C, Otranto D, Ferreira RR, Bianchi SP, et al. (2010) Genetic variability and phospholipase production of Malassezia pachydermatis isolated from dogs with diverse grades of skin lesions. Med Mycol 48: 889–892. pmid:20105099
  64. 64. Machado ML, Ferreiro L, Ferreira RR, Corbellini LG, Deville M, et al. (2011) Malassezia dermatitis in dogs in Brazil: diagnosis, evaluation of clinical signs and molecular identification. Vet Dermatol 22: 46–52. pmid:20609207
  65. 65. Dizotti CE, Coutinho SD (2007) Isolation of Malassezia pachydermatis and M. sympodialis from the external ear canal of cats with and without otitis externa. Acta Vet Hung 55: 471–477. pmid:18277706
  66. 66. Gandra RF, Gambale W, de Cassia Garcia Simao R, da Silva Ruiz L, Durigon EL, et al. (2008) Malassezia spp. in acoustic meatus of bats (Molossus molossus) of the Amazon Region, Brazil. Mycopathologia 165: 21–26. pmid:18046623
  67. 67. Duarte EP, Melo MM, Hahn RC, Hamdan JS (1999) Prevalence of Malassezia spp. in the ears of asymptomatic cattle and cattle with otitis in Brazil. Med Mycol 37: 159–162. pmid:10498444
  68. 68. Petry V, Tanhausen F, Weiss L, Milan T, Mezzari A, et al. (2011) Identification of Malassezia yeast species isolated from patients with pityriasis versicolor. An Bras Dermatol 86: 803–806. pmid:21987156
  69. 69. Duarte ER, Hamdan JS (2010) RAPD differentiation of Malassezia spp. from cattle, dogs and humans. Mycoses 53: 48–56. pmid:19298360
  70. 70. Giusiano G, Sosa Mde L, Rojas F, Vanacore ST, Mangiaterra M (2010) Prevalence of Malassezia species in pityriasis versicolor lesions in northeast Argentina. Rev Iberoam Micol 27: 71–74. pmid:20346299
  71. 71. Hernández F, Méndez LJ, Bazán E, Arévalo A, Valera A, et al. (2003) Especies de Malassezia asociadas a diversas dermatosis y a piel sana en población mexicana. Revista iberoamericana de micología 20: 141–144.
  72. 72. Rendic E, Diaz C, Fich F (2003) [Characterization of species of the gender Malassezia in patients with seborrheic dermatitis and subjects without skin lesions]. Rev Med Chil 131: 1295–1300. pmid:14743691
  73. 73. Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59: 143–169. pmid:7535888
  74. 74. Gupta AK, Kohli Y, Summerbell RC (2000) Molecular differentiation of seven Malassezia species. J Clin Microbiol 38: 1869–1875. pmid:10790115
  75. 75. Saunders CW, Scheynius A, Heitman J (2012) Malassezia fungi are specialized to live on skin and associated with dandruff, eczema, and other skin diseases. PLoS Pathog 8: e1002701. pmid:22737067