Mami Tajima

A new yeast, Malassezia yamatoensis, isolated from a patient with seborrheic dermatitis, and its distribution in patients and healthy subjects.

Over the last few years, new Malassezia species have been found regularly in Japanese subjects. We isolated another new Malassezia species from a Japanese patient with seborrheic dermatitis (SD), and named it M. yamatoensis. In its physiological characteristics and the utilization of Tween by M. yamatoensis is similar to that of M. furfur and M. dermatis. It is distinguished by its growth temperature. To examine the distribution of the microorganism in the skin of patients with SD and atopic dermatitis (AD), and healthy subjects, we applied transparent dressings to the skin, and detected M. yamatoensis DNA using a non‐culture‐based method that consisted of nested PCR with specific primers. M. yamatoensis DNA was detected from 3 of 31 SD patients (9.7%), 5 of 36 AD patients (13.9%), and 1 of 22 healthy subjects (4.6%). Therefore, M. yamatoensis is a rare member of the cutaneous microflora.

Antifungal Activities of Tacrolimus and Azole Agents against the Eleven Currently Accepted Malassezia Species

ABSTRACT The lipophilic yeast Malassezia is an exacerbating factor in atopic dermatitis (AD) and colonizes the skin surface of patients with AD. With the goal of reducing the number of Malassezia cells, we investigated the antifungal activities of a therapeutic agent for AD, tacrolimus, and the azole agents itraconazole and ketoconazole against Malassezia species in vitro. We examined 125 strains of the 11 currently accepted Malassezia species by using the agar dilution method. All strains of the 11 Malassezia species were very susceptible to both azole agents, with MICs ranging from 0.016 to 0.25 μg/ml. Tacrolimus had antifungal activities against half of the strains, with MICs ranging from 16 to 32 μg/ml. Two of the major cutaneous floras, Malassezia globosa and Malassezia restricta, have several genotypes in the intergenic spacer region of the rRNA gene; the azole agents had slightly higher MICs for specific genotype strains of both microorganisms. A combination of azole agents and tacrolimus had a synergistic effect against Malassezia isolates, based on a fractional inhibitory index of 0.245 to 0.378. Our results provide the basis for testing these agents in future clinical trials to reduce the number of Malassezia cells colonizing the skin surface in patients with AD.

Characterization of the skin fungal microbiota in patients with atopic dermatitis and in healthy subjects

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.

Quantitative analysis of cutaneous Malassezia in atopic dermatitis patients using real-time PCR

We quantified the cutaneous Malassezia in patients with atopic dermatitis using a real‐time PCR assay. Seven to 12 times more Malassezia colonized the head and neck compared to the trunk or limbs, and the species M. globosa and M. restricta accounted for approximately 80% of all Malassezia colonization at any body site.

Genotype Analysis of Malassezia restricta as the Major Cutaneous Flora in Patients with Atopic Dermatitis and Healthy Subjects

Lipophilic yeasts of the genus Malassezia colonize the skin surface of humans and are an exacerbating factor in atopic dermatitis (AD). Two species, M. restricta and M. globosa are major cutaneous microflora in both AD patients and healthy subjects. We compared the DNA sequences of the intergenic spacer (IGS) region, located between the 26S and 5S rRNA genes of M. restricta colonizing the skin surfaces of 13 AD patients and 12 healthy subjects, and of three CBS stock strains as references. The IGS 1 sequences were divided into two major groups, corresponding to AD patients and healthy subjects. These findings suggest that a specific genotype of M. restricta plays a significant role in AD, although M. restricta commonly colonizes both AD patients and healthy subjects.

Molecular analysis of Malassezia microflora in the lesional skin of psoriasis patients.

Systemic and focal infections by microorganisms have been known to induce or exacerbate psoriasis. To investigate the role of Malassezia species in the development of psoriasis, we analyzed the Malassezia microflora in psoriasis patients using a nested polymerase chain reaction (PCR) assay, and compared it with those in atopic dermatitis (AD) patients and healthy subjects. Fungal DNA was directly collected from the lesional and non‐lesional skin of the trunk of 22 psoriasis patients by applying a transparent dressing. The extracted DNA was amplified by using specific primers designed for the PCR in the intergenic spacer or internal transcribed spacer area of the ribosomal RNA. All nine of the Malassezia species were detected at different rates from the 22 psoriasis patients. The overall detection rates in lesional and non‐lesional skin of M. restricta, M. globosa and M. sympodialis were high (96%, 82% and 64%, respectively), whereas the detection rates of the other species were relatively low. However, there was no difference in the rates between lesional and non‐lesional skin areas. The average number of Malassezia species detected in overall sites of the psoriasis patients was 3.7 ± 1.6 species, although this fact showed no correlation with the severity of the symptoms. The number of Malassezia species detected was 4.1 ± 1.9 in the AD patients, and 2.8 ± 0.8 in the healthy subjects, suggesting that the skin microflora of psoriasis patients and AD patients show greater diversity than that of healthy subjects.

Quantification of activated and total caspase-14 with newly developed ELISA systems in normal and atopic skin

BACKGROUND Activation of caspase-14 occurs during terminal differentiation of keratinocytes and may play a role in filaggrin degradation. Therefore, down-regulation of caspase-14 may lead to impaired barrier function. OBJECTIVE To compare the levels of active and total caspase-14 in healthy subjects in various age groups and in patients with atopic dermatitis (AD), using two enzyme-linked immunoassay (ELISA) systems. METHODS We established four clones of monoclonal antibodies to caspase-14 and used clone 3 as the immobilizing antibody. A cleavage site-directed antibody, h14D146 [4] was used for specific quantification of active caspase-14 in extracts of tape-stripped corneocytes. Total caspase-14 was measured with a commercial antibody, H-99. RESULTS The amount of caspase-14 remained constant (ca. 0.1% of extractable proteins) in healthy males from their twenties to their fifties. Caspase-14 was mostly in active form (71-94%) in these extracts. In contrast, caspase-14 level and active caspase-14 ratio were significantly decreased in females in their fifties and sixties. Contents of free amino acids were decreased in females in their sixties, and transepidermal water loss was increased in females in their forties and sixties. In patients with AD, active caspase-14 was markedly down-regulated compared to age-matched controls in both lesional (7.5%) and non-lesional skin (10.6%). Staining of active caspase-14 was considerably weaker in non-lesional skin and was hardly detectable in lesional skin with parakeratosis. CONCLUSION Our new ELISA systems are effective tools to quantify activation of caspase-14. Our results indicate a role of caspase-14 in epidermal barrier function.

Anti-Malassezia-Specific IgE Antibodies Production in Japanese Patients with Head and Neck Atopic Dermatitis: Relationship between the Level of Specific IgE Antibody and the Colonization Frequency of Cutaneous Malassezia Species and Clinical Severity

Atopic dermatitis of the head and neck (HNAD) is recognized as a separate condition. Malassezia, the predominant skin microbiota fungus, is considered to exacerbate atopic dermatitis (AD), especially HNAD. In the present study, we investigated the relationships between the levels of specific IgE antibodies, colonization frequency of eight predominant Malassezia species, and clinical severity in 61 patients with HNAD (26 mild, 24 moderate, and 11 severe cases). As clinical severity increased, the levels of specific IgE antibodies against eight Malassezia species also increased. Species diversity of the Malassezia microbiota in scale samples from patients was analyzed by nested PCR using species-specific primers. The clinical severity of HNAD was correlated with the total level of specific IgE antibodies against Malassezia species and the number of Malassezia species detected.

A New Calcineurin Inhibitor, Pimecrolimus, Inhibits the Growth of Malassezia spp.

Malassezia spp. are a component of the cutaneous microflora that colonizes lipid-rich areas, especially the head and neck, because the microorganisms require a lipid for its growth. An anti-Malassezia-specific immunoglobulin E antibody is produced in patients with atopic dermatitis who have disrupted skin barrier function, while healthy subjects do not produce the immunoglobulin E antibody. In addition, antifungal agents can improve the symptoms of atopic dermatitis. Based on this evidence, Malassezia spp. are considered one of the factors involved in exacerbating atopic dermatitis (1, 4). The genus Malassezia consists of 11 species (M. dermatis, M. globosa, M. furfur, M. japonica, M. obtusa, M. restricta, M. slooffiae, M. sympodialis, M. yamatoensis, M. nana, and M. pachydermatis), although the last two species show affinities to nonhuman animals. Of the 11 species, M. globosa and M. restricta have been isolated from almost all patients with atopic dermatitis, while the other species are found in fewer than 60% of patients, suggesting that M. globosa and M. restricta play a major role in atopic dermatitis (5). Pimecrolimus, an ascomycin macrolactam derivative, is a new calcineurin inhibitor that binds to the cytosolic receptor macrophilin-12 with high affinity, inhibiting the calcium-dependent phosphatase calcineurin, an enzyme required for the dephosphorylation of the cytosolic form of the nuclear factor of the activated T cell. Therefore, it targets T-cell activation and proliferation by blocking the release of both TH1 and TH2 cytokines (7). Previously, we demonstrated that the calcineurin inhibitor tacrolimus, which has a similar chemical structure, inhibits the growth of Malassezia in vitro (6). Therefore, we postulated that pimecrolimus might have an antifungal effect. In this study, we examined the in vitro drug susceptibility to pimecrolimus of 109 strains of the nine human-related Malassezia species. The strains were isolated from patients with atopic dermatitis or from healthy subjects. Pimecrolimus was kindly supplied by Novartis (Basel, Switzerland). In vitro drug susceptibility was determined according to the method of Gupta et al. (3), with slight modification (6). Drug susceptibility testing was conducted at least three times. The MICs of pimecrolimus are shown in Table ​Table1.1. Pimecrolimus had an antifungal effect against the 109 Malassezia strains, with MICs ranging from 16 to 64 μg/ml. It inhibited the growth of approximately 90% of the strains at a concentration of 16 or 32 μg/ml. No differences in MICs were seen across the Malassezia species. The calcineurin inhibitor tacrolimus, which has a similar chemical structure, also inhibited the growth of Malassezia in vitro, with MICs of 16 to 32 μg/ml, which were the same as the MICs of pimecrolimus (6). Interestingly, fungal cells also contain a calcineurin homologue, although its function is unknown. Recent studies indicate that cyclosporine and tacrolimus are toxic to the pathogenic fungi Candida albicans and Cryptococcus neoformans (2). TABLE 1. Antifungal susceptibilities of Malassezia strains to pimecrolimus Malassezia spp. are one of the factors that exacerbate atopic dermatitis. The growth-inhibitory effect of pimecrolimus might contribute to improving the symptoms of atopic dermatitis in addition to the inhibitory effect of calcineurin as its main action. A 1% concentration of pimecrolimus is used for clinical purposes, which sufficiently exceeds the growth-inhibitory concentration for Malassezia.

Atopic Dermatitis and Skin Fungal Microorganisms

A wide variety of bacteria and fungi are found on the human skin. Although some skin microorganisms produce antibacterial peptides that inhibit invasion by pathogens or promote the integrity of cutaneous defenses by eliciting host immune responses, the normal microbiome can also cause several skin diseases. Atopic dermatitis (AD) is a chronic disease that causes pruritus and involves cycles of remission and deterioration. AD is the result of dry hypersensitive skin. When the skin is dry, the protective barrier function of the cutaneous surface horny layer is compromised, and the skin readily develops dermatitis in response to various external stimuli, including skin microorganisms. Serum from almost all AD patients contains IgE antibodies against some skin microorganisms. For example, staphylococcal superantigen-specific IgE is present in the serum of AD patients, but not in the serum of healthy individuals. Normally, the weakly acidic condition of healthy skin prevents colonization by Staphylococcus aureus. However, in patients with AD, the skin pH is shifted toward neutrality, allowing S. aureus to grow and exacerbate AD. In the cutaneous fungal microbiome, lipophilic yeasts of the genus Malassezia are the predominant species on human skin. As Malassezia species require lipids for growth, they preferentially colonize sebum-rich areas such as the head, face, and neck, as opposed to the limbs or trunk. Specific IgE antibody against Malassezia species is found in the serum of AD patients. Antifungal therapy improves the symptoms of AD by decreasing the level of Malassezia colonization, suggesting that these microorganisms also exacerbate AD. Malassezia species, unlike S. aureus, colonize both AD patients and healthy subjects. Currently, the genus Malassezia consists of 14 species. Of these, M. globosa and M. restricta have been detected in almost all AD patients, suggesting that these two Malassezia species play a significant role in AD. The level of specific IgE antibody against both species is greater than that against other Malassezia species. This chapter discusses cutaneous fungi as an exacerbating factor in AD, focusing on: the fungal microbiome in patients with AD. immunological aspects of fungal colonization, and treatment with antifungal agents.