1 Program workshop Malassezia: from skin to model species July 4-6, 2018 Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584CT Utrecht, The Netherlands How to get there http://www.westerdijkinstitute.nl/DefaultInfo.aspx?Page=ContactUs Organizers Teun Boekhout & Thomas Dawson ISHAM working group Malassezia, epidemiology and pathobiology & Malassezia Research Consortium Support 2 Wednesday afternoon, July 4 Theme Ecology, diversity and microbiome 14.00-14.30 Keynote Biodiversity and ecology of Malassezia species. F.J. Cabañes Universitat Autònoma de Barcelona, BELLATERRA, BARCELONA, Spain Malassezia species form a monophyletic group of basidiomycetous yeasts unique among the fungi. They are characterized to be lipophilic microorganisms which are associated to the human and animal skin. The genus was created by Baillon in 1889 and remained limited to only two species, M. furfur and M. pachydermatis, for over a century. Nowadays, the genus includes 17 species and new species are expected to be described in the coming years. Special morphological characteristics (e.g. colony and cell morphology) have been cited for some Malassezia spp. However, separation of Malassezia species on the basis of only morphological characteristics may be considered to be subjective or unreliable. Currently, the phenotypical characterization of these yeasts is based mainly on the evaluation of their ability to use certain polyoxyethylene sorbitan esters (Tweens 20, 40, 60 and 80) and Cremophor EL. However, difficulties remain in obtaining a high level of certainty in the identification of some lipid-dependent strains by means of physiological tests without molecular characterization. Although a genetic boundary between Malassezia species has been not clearly stated, the within-species “gold standard” D1/D2 sequence similarity is above 99% (<1% of dissimilarity). Other molecular markers (e.g. ITS region, CH2 and beta-tubulin genes) may be useful to achieve discrimination among the species of this group. On the other hand, genomics has become a promising methodology to infer phylogenetic relationships among these species. Minimal standards for the use of genome data in delineating new species in this group should be discussed. As far as the ecology of these yeasts is concerned, Malassezia species have been only isolated from mammals and some birds. Recent metagenomics studies showed that it was possible to detect the presence of Malassezia-similar genes in environmental DNA samples (e.g. nematodes, marine sponges, coral colonies, marine dinoflagellates). However, the presence of Malassezia cells have been not reported in these environmental studies. Consequently, the real identity of these yeasts detected by culture-independent techniques in these environmental habitats still remain unknown. 14.30-14.45 Malassezia in the environment A. Amend1, T.L. Dawson2, T. Boekhout3 1University of Hawaii Manoa, HONOLULU, USA 2Agency for Science, Technology and Research, Skin Research Institute Singapore, SINGAPORE, Singapore 3Westerdijk Fungal Biodiversity Institute, UTRECHT, Nederland Objectives: Until recently, yeasts of the basidiomycetous genus Malassezia were known mainly from skin of warm-blooded animals, including humans. In the laboratory they could be maintained on lipid supplemented media and when grown at temperatures of ca. 30°C. Recent DNA barcoding, however, detected the occurrence of these yeasts in unexpected habitats. Further they point to a large diversity of putatively undescribed species. Methods: Here we present an overview of results from various metabarcoding and metagenomics studies on the occurrence of Malassezia in various environmental habitats. Results: Presumed Malassezia DNA was detected in a wide diversity of habitats, including terrestrial, marine, and indoor environments. Moreover, it dominated the mycobiomes of animals like sponges, corals, nematodes, snails, and insects. Sequenced transcripts and ribosomal RNA indicate that Malassezia is actively functioning in at least some of these environments. Using ITS as a marker several novel phylogenetic lineages are found indicating that the existing number of species may be higher than presently know. Future studies may focus on addressing the functional role of the yeasts in such habitats, as well as in situ detection by e.g. FISH. 3 Conclusion: The diversity of Malassezia yeasts largely extends what is known from previous clinical studies. Combining environmental microbial community data from metagenomics studies with those obtained by functional genomics, and culturing and imaging Malassezia directly from these environments will provide insight in the environmental reservoir of Malassezia yeasts as well as their ecophysiology that also may contribute to our understanding of the human/animal host interactions. 14.45-15.00 Identification of Malassezia species in seborrheic dermatitis and fungal microbiota in androgenetic alopecia Y.P. Ran1, H. Zhang2, J.H. Huang2, X. Ran1 1West China Hospital, CHENGDU, China Objectives: Seborrheic dermatitis (SD) is a sub-acute or chronic superficial inflammatory skin condition, characterized by pruritic, erythematous plaques with greasy, yellow-gray scales,scales, which appear on areas rich in sebaceous glands such as the face, scalp, upper chest, and back. Androgenetic alopecia (AGA) is the most common cause of hair loss and affects up to 70% of men and 40% of women. Although genetic and environmental factors play a role, the causes of SD and AGA are complex and incompletely understood and the pathogenesis remains unclear. We explored Malassezia spp. composition in SD skin and fungal microbiota in AGA by non-culture based molecular identification. Methods: We analyzed Malassezia microflora in SD patients by applying a transparent dressing to the lesional skin and using direct detection of fungal DNA using nested PCR. We collected samples from the lesional skin of 146 SD patients in China and extracted fungal DNA directly from the lesional samples without culture. Specific primers for each Malassezia species were designed to amplify existing yeasts in each sample. Some samples were randomly selected to culture and identified by morphological and physiologic criteria. We focused on fungal microbiota of AGA: (1) pulling out the loose hair from the calvaria region (vulnerable to baldness) and occipital region (non-baldness) of AGA patients, to observe the fungus with microscope, SEM and TEM; (2) high- throughput sequencing and fluorescence quantification real-time PCR (QRT-PCR) combining the specific primers and probe of the Malassezia species to analyze the fungal micro-ecology. Results: M. globosa and M. restricta were found in 87.0 and 81.5 % of SD patients, respectively, which together accounted for more than 50 % of Malassezia spp. recovered in these Chinese patients. The majority of SD patients (82.9 %) showed co-colonization of two or more Malassezia species. The positive rate of Malassezia in 10 AGA patients (60%) was higher than 10 control (40%). SEM observed many Malassezia yeasts adhering and inserted to the surface of hair root, TEM proved the yeasts invaded into the fair follicle dermal root sheath. High-throughput sequencing the hair roots from AGA revealed Basidiomycota (61.03%), Ascomycota (35.58%), Zygomycota (0.40%) in calvaria region, and Basidiomycota (54.21%), Ascomycota (41.18%), Zygomycota (1.87%) in occipital region. In the control group, Basidiomycota (24.94%), Ascomycota (73.16%), Zygomycota (0.49%) revealed in calvaria region, and Basidiomycota (34.37%), Ascomycota (63.78%), Zygomycota (0.62%) in occipital region. The scalp scales by OpSiteTM sterile transparent sticking were detected with QRT-PCR. In AGA group, the constitute of M. globose (37%) and M. restricta (53%) in calvaria is significantly higher than in occipital region of M. globose (22%) and M. restricta (31%). In the control group no statistical difference existed. Conclusion: Our results indicated M. globosa and M. restricta predominated in Malassezia colonization in Chinese SD patients. Non-culture-based methods may more accurately reflect Malassezia microflora constitution. The pathogenicity of Malassezia to AGA is worth further investigation. 15.00-15.15 Socio-demographic characteristics and spectrum of Malassezia species in seborrhoeic dermatitis in urban and rural population of India S.M. Rudramurthy1, P. Honnavar2, A. Chakrabarti2, S. Dogra2, S. Handa2, P.V. M Lakshmi2 1Postgraduate institute of medical Education and Research, Chandigarh, CHANDIGARH, India 4 2PGIMER, CHANDIGARH, India Objectives: Seborrheic dermatitis/dandruff (SD/D) is a common, persistent, relapsing inflammatory condition affecting the areas rich in sebaceous glands. Though cases of SD/D are widely prevalent in India, Malassezia species implicated are not well studied. The aim of our study was to estimate the prevalence and spectrum of Malassezia species in patients with SD/D in north India. Methods: Two hundred SD/D patients and 100 healthy controls from both rural and urban background of north India were enrolled. SD/D severity was clinically graded as mild, moderate, severe and very severe. The isolates were identified by phenotypic characters and confirmed by ITS2 PCR-RFLP band pattern and sequencing of ITS region of rDNA. Results: Severe (59%) and very severe (71%) form of SD/D was higher in the rural population compared to urban population (p=0.004). Isolation rate of Malassezia was significantly higher in overall SD/D patients scalp (82%) compared to healthy controls