Patients with atopic dermatitis (AD) are highly susceptible to viral, bacterial, and fungal skin infections because their skin is dry and this compromises the barrier function of the skin. Therefore, the skin microbiota of patients with AD is believed to be different from that of healthy individuals. In the present study, the skin fungal microbiota of nine patients with mild, moderate, or severe AD and ten healthy subjects were compared using an rRNA clone library. Fungal D1/D2 large subunit analysis of 3647 clones identified 58 species and seven unknown phylotypes in face scale samples from patients with AD and healthy subjects. Malassezia species were predominant, accounting for 63%-86% of the clones identified from each subject. Overall, the non-Malassezia yeast microbiota of the patients was more diverse than that of the healthy individuals. In the AD samples 13.0 ± 3.0 species per case were detected, as compared to 8.0 ± 1.9 species per case in the samples taken from healthy individuals. Notably, Candida albicans, Cryptococcus diffluens, and Cryptococcus liquefaciens were detected in the samples from the patients with AD. Of the filamentous fungal microbiota, Cladosporium spp. and Toxicocladosporium irritans were the predominant species in these patients. Many pathogenic fungi, including Meyerozyma guilliermondii (anamorphic name, Candida guilliermondii), and Trichosporon asahii, and allergenic microorganisms such as Alternaria alternata and Aureobasidium pullulans were found on the skin of the healthy subjects. When the fungal microbiota of the samples from patients with mild/moderate to severe AD and healthy individuals were clustered together by principal coordinates analysis they were found to be clustered according to health status.

Malassezia species colonize the skin of normal and various pathological conditions including pityriasis versicolor (PV), seborrhoeic dermatitis (SD) and atopic dermatitis (AD). To elucidate the pathogenic role of Malassezia species in SD, Malassezia microflora of 31 Japanese SD patients was analyzed using a PCR-based, culture-independent method. Nested PCR assay using the primers in the rRNA gene indicated that the major Malassezia species in SD were M. globosa and M. restricta, found in 93 and 74% of the patients, respectively. The detection rate and number of each species varied similarly in SD, PV and healthy subjects (HSs), whereas AD showed higher values. Real-time PCR assay showed that the lesional skin harbored approximately three times the population of genus Malassezia found in nonlesional skin (P<0.05), and that M. restricta is a significantly more common species than M. globosa in SD (P<0.005). Genotypic analysis of the rRNA gene showed that the M. globosa and M. restricta from SD patients fell into specific clusters, and could be distinguished from those collected from HSs, but not from those colleted from AD patients. Our results indicate that certain strains of M. restricta occur in the lesional skin of SD patients.

Malassezia species are considered to be one of the exacerbating factors in atopic dermatitis (AD). During examination of the cutaneous colonization of Malassezia species in AD patients, we found a new species on the surface of the patients' skin. Analysis of ribosomal DNA sequences suggested that the isolates belonged to the genus Malassezia. They did not grow in Sabouraud dextrose agar but utilized specific concentrations of Tween 20, 40, 60, and 80 as a lipid source. Thus, we concluded that our isolates were new members of the genus Malassezia and propose the name Malassezia dermatis sp. nov. for these isolates.Malassezia species are known causative factors in pityriasis versicolor, seborrheic dermatitis (SD), and atopic dermatitis (AD) (3). In the last decade, research has focused primarily on isolating Malassezia strains and detecting specific immunoglobulin E (IgE) antibodies from patients (9,13,14,26). A comparison of the isolation rates of Malassezia species from the skin of AD patients and healthy control subjects detected a significantly higher rate for patients than for healthy subjects (8). AD patients had specific IgE antibodies against Malassezia, while healthy subjects did not. In recent years, studies have increasingly been directed towards analyzing how the cutaneous microflora at the species level are related to each disease type (pityriasis versicolor, SD, and AD) (1,6,7,12,16). We previously compared the distribution of Malassezia species in skin lesions of AD patients and in healthy subjects using a nonculture method (nested PCR) that is not affected by the isolating medium (21). Of the seven members of the genus Malassezia, Malassezia globosa and M. restricta were the species most commonly associated with AD, while M. obtusa and M. pachydermatis were not detected in AD. In our survey of cutaneous Malassezia microflora, we isolated new Malassezia species from several patients with AD. In this paper, we propose a new species, M. dermatis, for these isolates. MATERIALS AND METHODSMalassezia isolates. Nineteen AD outpatients at Juntendo University Hospital were included in the study. To obtain samples, OpSite transparent dressings (3 by 7 cm; Smith and Nephew Medical Ltd., Hull, United Kingdom) were applied to skin lesions (erosive, erythematous, and lichenoid) on the scalp, back, and nape of the neck of AD patients. Samples were then transferred onto Leeming and Notman agar (LNA) (10.0 g of polypeptone, 5.0 g of glucose, 0.1 g of yeast extract, 8.0 g of ox gall, 1.0 mg of glycerol, 0.5 g of glycerol stearate, 0.5 mg of Tween 60, 10 ml of cow's milk [whole fat], and 12.0 g of agar per liter) plates containing 50 l of chloramphenicol (Sankyo, Tokyo, Japan) and incubated at 32°C for 2 weeks.Direct DNA sequencing of rRNA genes of the isolates. Yeast isolates recovered from LNA medium were identified by analysis of rRNA gene sequences. Nuclear DNA of the isolates was extracted by the method of Makimura et al. (15). The D1 and D2 regions of 26S ribosomal DNA (rDNA) and internal transcribed spacer (IT...

We compared cutaneous colonization levels of Malassezia species in patients with AD and healthy subjects using nested PCR. Malassezia-specific DNA was detected in all 32 of the patients with AD. M. globosa and M. restricta were detected in approximately 90 % of these patients, with M. furfur and M. sympodialis being detected in approximately 40% of the cases. In healthy subjects, Malassezia DNA was detected in 78% of the samples, M, globosa, M. restricta and M. sympodialis were detected at frequencies ranging from 44 to 61%, and M. furfur was found in 11% of healthy subjects.Our results suggest that M. furfur, M. globosa, M. restricta and M. sympodialis are common inhabitants of the skin of both AD patients and healthy subjects, while the skin microflora of patients with AD shows more diversity than that of healthy subjects.

Lipophilic yeasts of the genus Malassezia are part of the normal cutaneous microflora and are considered one of the factors that trigger atopic dermatitis (AD). We isolated two strains of Malassezia from a healthy Japanese female. Analysis of the D1/D2 26S ribosomal DNA and internal transcribed spacer region sequences of the isolates suggested that they are new members of the genus Malassezia. We propose the name Malassezia japonica sp. nov. for the isolates. M. japonica is easily distinguished from the seven known lipophilic species by its ability to assimilate Tween 40 and Tween 60 and its inability to assimilate Tween 20 and Tween 80 and to grow at 40°C. Furthermore, by applying transparent dressings to the skin lesions of 36 patients with AD and the skin of 22 healthy subjects, M. japonica DNA was detected by a non-culture-based method consisting of nested PCR with M. japonica species-specific primers. M. japonica DNA was detected from 12 of the 36 patients (33.3%) and 3 of the 22 healthy subjects (13.6%). Although it is not known whether M. japonica plays a role in AD, this species was part of the microflora in both patients with AD and healthy subjects.Lipophilic yeasts of the genus Malassezia are members of the normal human cutaneous microflora and are also associated with several skin diseases. It is strongly suspected that Malassezia species are responsible for pityriasis versicolor, seborrheic dermatitis, Malassezia folliculitis, and atopic dermatitis (AD) (2,4,5,19). The genus Malassezia includes eight species: Malassezia globosa, M. restricta, M. obtusa, M. slooffiae, M. furfur, M. sympodialis, M. dermatis, and M. pachydermatis (7, 22). M. pachydermatis is not a lipophilic species and is associated several animal skin diseases (1,8). Recently, our research group found M. dermatis on Japanese patients with AD (22). Much research has examined the relationships between these eight species and their roles as causative agents of disease or factors that trigger disease. Most studies indicate that pityriasis versicolor and seborrheic dermatitis are likely affected by M. globosa and M. sympodialis (3,9,10,15). The distribution of Malassezia species in the skin lesions of AD patients and healthy subjects was previously compared by a non-culturebased method (nested PCR) that is not affected by the isolation medium (21). Of the members of the genus Malassezia, M. globosa and M. restricta were associated with disease in more than 90% of AD patients, while the other species were detected in less than 50% of the patients. In our survey of the cutaneous Malassezia microflora, we isolated a new Malassezia species from a healthy subject. In the present study, we propose a new species, M. japonica, for the isolates from this subject and investigated the skin surfaces of patients with AD and healthy subjects for the presence of this species. MATERIALS AND METHODSYeast isolate. Malassezia strains were isolated from the left forearm of a healthy 22-year-old Japanese female. OpSite transparent dressings (3 by 7 cm; Smit...