Establishment of a gene recombination method for a major human skin commensal fungus, Malassezia restricta, using Agrobacterium tumefaciens-mediated gene transfer system.

Malassezia restricta is the most predominant fungus in the microbiome of human skin. This microorganism can cause or exacerbate Malassezia-associated skin dermatitis, seborrheic dermatitis, atopic dermatitis, and pityriasis versicolor. The virulence factors of M. restricta have not been analyzed because a gene recombination system has not been developed. In this study, we established an Agrobacterium tumefaciens-mediated gene transfer (ATMT) system, optimized for generating gene-deficient mutants of M. restricta. A mutant of FKB1 gene, which encodes the FKBP12 protein that binds to the calcineurin inhibitor tacrolimus, was generated using the ATMT system. Subsequently, the FKB1 gene was reintroduced into the FKB1 gene-deficient mutant for obtaining a gene-complemented strain. The wild-type strain of M. restricta was sensitive to tacrolimus, whereas the FKB1 gene-deficient mutant was resistant to tacrolimus; the phenotypic drug susceptibility in the mutant was restored by reintroducing the FKB1 gene. Contrastingly, the FKB1 gene-deficient mutant was not resistant to cyclosporine A, which also inhibits calcineurin by binding to cyclophilin A. The gene recombination system for M. restricta will facilitate in elucidating the molecular mechanisms causing Malassezia-associated dermatitis.

Short sequence repeats of the intergenic spacer regions of ribosomal RNA genes in Malassezia globosa and Malassezia restricta colonizing the scalps of male individuals with and without androgenetic alopecia.

We analyzed the short sequence repeats (SSRs) of the intergenic spacer (IGS) region 1 of the ribosomal RNA genes in Malassezia globosa and Malassezia restricta, which predominantly colonize the scalp in androgenetic alopecia (AGA). No AGA-specific SSRs were found in the M. globosa IGS region, whereas a (CT)6 :(AT)8 SSR was predominantly detected in the M. restricta IGS region in the AGA group. Malassezia colonization was higher in the scalps of patients with M. restricta (CT)6 :(AT)8 SSRs than in the scalps of patients without M. restricta (CT)6 :(AT)8 SSRs. These observations suggest that this specific SSR type in M. restricta is involved in the development or exacerbation of AGA.

Development of Artificial-Sebum-Containing Leeming and Notman Agar Medium to Enhance the Growth of Malassezia.

Modified Leeming and Notman agar medium (mLNA) has been widely utilized to grow lipophilic fungi belonging to the genus Malassezia. We developed a new artificial-sebum-containing mLNA to obtain higher yields of Malassezia species. The olive oil in mLNA was replaced with an artificial sebum composed of triglyceride (triolein), diglyceride (glyceryl distearate), fatty acids (palmitic acid, myristic acid, pentadecanoic acid, and oleic acid), and squalene. Furthermore, the Tween 60 was replaced with self-emulsifying glyceryl stearate. Nine human-associated Malassezia species grew well on the artificial-sebum-containing mLNA, and the most predominant fungus on human skin, Malassezia restricta, exhibited double wet cell weight in artificial sebum-containing mLNA compared to wet cell weight in standard mLNA.

The skin mycobiome of an astronaut during a 1-year stay on the International Space Station.

Analysis of the skin mycobiome of an astronaut during a 1-year stay on the International Space Station (ISS) revealed an increased relative abundance of Malassezia restricta and level of Malassezia colonization, and the presence of Cyberlindnera jadinii and Candida boidinii, uncommon skin mycobiome taxa. Similar observations were made in astronauts during a 6-month stay on the ISS (Med Mycol. 2016; 54: 232-239). Future plans for extended space travel should consider the effect of high levels of Malassezia colonization over long periods on astronauts' skin, and the abnormal proliferation of uncommon microorganisms that may occur in closed environments such as the ISS.

Pyruvate-triggered TCA cycle regulation in Staphylococcus aureus promotes tolerance to betamethasone valerate.

Staphylococcus aureus is a resident skin bacterium involved in the exacerbation of atopic dermatitis. Here we report that S. aureus regulates the tricarboxylic acid (TCA) cycle via the production of pyruvate for tolerance to betamethasone valerate (BV), an anti-inflammatory drug used in the treatment of atopic dermatitis. The addition of BV or clobetasol propionate to the medium among 5 different anti-inflammatory steroids delayed the growth of S. aureus. Comprehensive gene expression analysis by RNA-seq revealed that BV increased the expression of genes related to glycolysis in S. aureus. Pyruvate, a product of glycolysis, suppressed the S. aureus growth inhibition by BV. The addition of oxaloacetate, a compound in the TCA cycle biosynthesized from pyruvate, was also suppressed the inhibitory effect of BV. Malonate, an inhibitor of succinate dehydrogenase in the TCA cycle, increased the inhibitory effect of BV on the growth of S. aureus. These findings suggest that S. aureus promotes tolerance to BV, an anti-inflammatory steroid, by regulating the TCA cycle via the production of pyruvate.

Description of four Apiotrichum and two Cutaneotrichosporon species isolated from guano samples from bat-inhabited caves in Japan.

Four new yeast species belonging to the genus Apiotrichum and two new yeast species belonging to Cutaneotrichosporon are described for strains isolated from guano samples from bat-inhabited caves in Japan. In 2005, we reported these isolates as Trichosporon species based on sequence analyses of the D1/D2 domain of large subunit (LSU) rRNA genes according to available basidiomycetous yeast classification criteria; however, to date, they have not been officially published as new species with descriptions. Their phylogenetic positions have been reanalysed based on comparison of internal transcribed spacer (ITS) region sequences (including the 5.8S rRNA gene) and the D1/D2 domain of the LSU rRNA gene with those of known species; we confirmed clear separation from previously described species. Physiological and biochemical properties of the isolates also suggest their distinctiveness. Therefore, we describe Apiotrichum akiyoshidainum (holotype JCM 12595T), Apiotrichum chiropterorum (JCM 12594T), Apiotrichum coprophilum (JCM 12596T), Apiotrichum otae (JCM 12593T), Cutaneotrichosporon cavernicola (JCM 12590T) and Cutaneotrichosporon middelhovenii (JCM 12592T) as new species. C. cavernicola showed particularly distinctive morphology including large inflated anomalous cells on the hyphae and germination from the cells, although clear clamp connections on the hyphae were not confirmed. Further study is needed to elucidate the morph of this species.

Loop-mediated isothermal amplification for the rapid detection of Gardnerella vaginalis.

The aim of this study was to develop a method for the rapid detection of Gardnerella vaginalis, which is proposed to play a key role in the pathogenesis of bacterial vaginosis. Specific loop-mediated isothermal amplification (LAMP) primers were designed and used to detect target DNA within 45 min under isothermal conditions. Comparative screening indicated that the LAMP assay is superior to PCR in terms of rapidity, and is equivalent in sensitivity and specificity. This LAMP assay can be used for rapid screening and detection of G. vaginalis in vaginal samples; the limit of detection is 10 fg DNA.

Inhibitory effects of Euphorbia supina on Propionibacterium acnes-induced skin inflammation in vitro and in vivo.

BACKGROUND: Euphorbia supina (ES) plant has been used as treatment for inflammatory conditions. The antibacterial effect and the anti-inflammatory mechanism of ES for Propionibacterium (P.) acnes-induced inflammation in THP-1 cells and acne animal model remain unclear. Therefore, the objective of the present study was to determine the antibacterial and anti-inflammatory activities of ES against P. acnes, the etiologic agent of skin inflammation. METHOD: The antibacterial activities of ES were tested with disc diffusion and broth dilution methods. Cytotoxicity of ES at different doses was evaluated by the MTT assay. THP-1 cells were stimulated by heat-killed P. acnes in the presence of ES. The pro-inflammatory cytokines and mRNA levels were measured by ELISA and real-time-PCR. MAPK expression was analyzed by Western blot. The living P. acnes was intradermally injected into the ear of BLBC/c mice. Subsequently, chemical composition of ES was analyzed by liquids chromatography-mass spectrometry (LC-MS). RESULT: ES had stronger antibacterial activity against P. acnes and inhibitory activity on lipase. ES had no significant cytotoxicity on THP-1 cells. ES suppressed the mRNA levels and production of IL-8, TNF-a, IL-1ß in vitro. ES inhibited the expression levels of pro-inflammatory cytokines and the MAPK signaling pathway. Ear thickness and inflammatory cells were markedly reduced by ES treatment. Protocatechuic acid, gallic acid, quercetin, and kaempferol were detected by LC-MS analysis in ES. CONCLUSIONS: Our results demonstrate antibacterial and anti-inflammatory activities of ES extract against P. acnes. It is suggested that ES extract might be used to treatment anti-inflammatory skin disease.

Culture Supernatants of Lactobacillus gasseri and L. crispatus Inhibit Candida albicans Biofilm Formation and Adhesion to HeLa Cells.

PURPOSE: Vulvovaginal candidiasis (VVC) is a common superficial infection of the vaginal mucous membranes caused by the fungus Candida albicans. The aim of this study was to assess the mechanisms underlying the inhibitory effects of the culture supernatants of Lactobacillus gasseri and L. crispatus, the predominant microbiota in Asian healthy women, on C. albicans biofilm formation. The inhibition of C. albicans adhesion to HeLa cells by Lactobacillus culture supernatant was also investigated. METHODS: Candida albicans biofilm was formed on polystyrene flat-bottomed 96-well plates, and the inhibitory effects on the initial colonization and maturation phases were determined using the XTT reduction assay. The expression levels of biofilm formation-associated genes (HWP1, ECE1, ALS3, BCR1, EFG1, TEC1, and CPH1) were determined by reverse transcription quantitative polymerase chain reaction. The inhibition of C. albicans adhesion to HeLa cells by Lactobacillus culture supernatant was evaluated by enumerating viable C. albicans cells. RESULTS: The culture supernatants of both Lactobacillus species inhibited the initial colonization and maturation of C. albicans biofilm. The expression levels of all biofilm formation-related genes were downregulated in the presence of Lactobacillus culture supernatant. The culture supernatant also inhibited C. albicans adhesion to HeLa cells. CONCLUSION: The culture supernatants of L. gasseri and L. crispatus inhibited C. albicans biofilm formation by downregulating biofilm formation-related genes and C. albicans adhesion to HeLa cells. These findings support the notion that Lactobacillus metabolites may be useful alternatives to antifungal drugs for the management of VVC.

Inhibition of Propionibacterium acnes lipase activity by the antifungal agent ketoconazole.

The common skin disease acne vulgaris is caused by Propionibacterium acnes. A lipase secreted by this microorganism metabolizes sebum and the resulting metabolites evoke inflammation in human skin. The antifungal drug ketoconazole inhibits P. acnes lipase activity. We previously showed that the drug also inhibits the growth of P. acnes. Thus, ketoconazole may serve as an alternative treatment for acne vulgaris, which is important because the number of antibiotic-resistant P. acnes strains has been increasing.

Comprehensive analysis of the skin fungal microbiota of astronauts during a half-year stay at the International Space Station.

The International Space Station (ISS) is a huge manned construct located approximately 400 km above the earth and is inhabited by astronauts performing space experiments. Because the station is within a closed microgravity environment, the astronauts are subject to consistent stress. This study analyzed the temporal changes in the skin fungal microbiota of 10 astronauts using pyrosequencing and quantitative PCR assay before, during, and after their stay in the ISS. Lipophilic skin fungi, Malassezia predominated most samples regardless of the collection period, body site (cheek or chest), or subject. During their stay in the ISS, the level of Malassezia colonization changed by 7.6- ± 7.5-fold (mean ± standard deviation) and 9.5- ± 24.2-fold in cheek and chest samples, respectively. At the species level, M. restricta, M. globosa, and M. sympodialis were more abundant. In the chest samples, the ratio of M. restricta to all Malassezia species increased, whereas it did not change considerably in cheek samples. Fungal diversity was reduced, and the ratio of Malassezia to all fungal colonization increased during the astronauts' stay at the ISS. The ascomycetous yeast Cyberlindnera jadinii was detected in abundance in the in-flight sample of 5 of the 10 astronauts. The microorganism may have incidentally adhered to the skin during the preflight period and persisted on the skin thereafter. This observation suggests the ability of a specific or uncommon microorganism to proliferate in a closed environment. Our study is the first to reveal temporal changes in the skin fungal microbiota of ISS astronauts. These findings will provide information useful for maintaining the health of astronauts staying in the space environment for long periods and for preventing infection due to the human skin microbiota.

Comprehensive pyrosequencing analysis of the bacterial microbiota of the skin of patients with seborrheic dermatitis.

Seborrheic dermatitis (SD) is a chronic inflammatory dermatologic condition in which erythema and itching develop on areas of the body with sebaceous glands, such as the scalp, face and chest. The inflammation is evoked directly by oleic acid, which is hydrolyzed from sebum by lipases secreted by skin microorganisms. Although the skin fungal genus, Malassezia, is thought to be the causative agent of SD, analysis of the bacterial microbiota of skin samples of patients with SD is necessary to clarify any association with Malassezia because the skin microbiota comprises diverse bacterial and fungal genera. In the present study, bacterial microbiotas were analyzed at non-lesional and lesional sites of 24 patients with SD by pyrosequencing and qPCR. Principal coordinate analysis revealed clear separation between the microbiota of non-lesional and lesional sites. Acinetobacter, Corynebacterium, Staphylococcus, Streptococcus and Propionibacterium were abundant at both sites. Propionibacterium was abundant at non-lesional sites, whereas Acinetobacter, Staphylococcus and Streptococcus predominated at lesional sites; however, the extent of Propionibacterium colonization did not differ significantly between lesional and non-lesional sites according to qPCR. Given that these abundant bacteria hydrolyze sebum, they may also contribute to SD development. To the best of our knowledge, this is the first comprehensive analysis of the bacterial microbiotas of the skin of SD patients.

Low DNA Sequence Diversity of the Intergenic Spacer 1 Region in the Human Skin Commensal Fungi Malassezia sympodialis and M. dermatis Isolated from Patients with Malassezia-Associated Skin Diseases and Healthy Subjects.

As DNA sequences of the intergenic spacer (IGS) region in the rRNA gene show remarkable intraspecies diversity compared with the small subunit, large subunit, and internal transcribed spacer region, the IGS region has been used as an epidemiological tool in studies on Malassezia globosa and M. restricta, which are responsible for the exacerbation of atopic dermatitis (AD) and seborrheic dermatitis (SD). However, the IGS regions of M. sympodialis and M. dermatis obtained from the skin of patients with AD and SD, as well as healthy subjects, lacked sequence diversity. Of the 105 M. sympodialis strains and the 40 M. dermatis strains, the sequences of 103 (98.1 %) and 39 (97.5 %), respectively, were identical. Thus, given the lack of intraspecies diversity in the IGS regions of M. sympodialis and M. dermatis, studies of the diversity of these species should be performed using appropriate genes and not the IGS.

Temporal changes in the skin Malassezia microbiota of members of the Japanese Antarctic Research Expedition (JARE): A case study in Antarctica as a pseudo-space environment.

The International Space Station (ISS) is located approximately 400 km above the Earth. Astronauts staying at the ISS are under microgravity and are thus unable to bathe or shower; instead, they wash their bodies using wet tissues. For astronauts, skin hygiene management is important to maintain the quality of life during long-term stays on the ISS. In Antarctica, members of a Japanese geological investigation team negotiate their way over land using snowmobiles. During their 3-month stay, they are subject to a "pseudo-space" environment similar to that experienced by ISS astronauts, including the inability to bathe or shower. In this study, temporal changes in the colonization levels of skin lipophilic fungi, Malassezia were investigated in 16 team members. Compared to the levels before their trip to Antarctica, the fold changes in Malassezia colonization levels during the researchers' stay in Antarctica were in the range of 3.0 ± 1.9 to 5.3 ± 7.5 in cheek samples, 8.9 ± 10.6 to 22.2 ± 40.0 in anterior chest samples, 6.2 ± 5.4 to 16.9 ± 25.5 in behind-the-ear samples, and 1.7 ± 0.9 to 17.4 ± 33.4 in sole-of-the-foot samples. On the scalp, the level of Malassezia colonization increased dramatically, by 96.7 ± 113.8 to 916.9 ± 1251.5 fold. During their stay in Antarctica, the team members experienced itchy scalps and produced a large number of scales. The relative proportions of Malassezia globosa and M. restricta shifted to seborrheic dermatitis/dandruff types. These results provide useful information for the development of skin hygiene management plans for astronauts staying at the ISS.

Three new basidiomycetous yeasts, Pseudozyma alboarmeniaca sp. nov., Pseudozyma crassa sp. nov. and Pseudozyma siamensis sp. nov. isolated from Thai patients.

We previously reported the first isolation of Pseudozyma species from the blood of Thai patients. In this study, three additional new Pseudozyma species were isolated from clinical specimens from Thai patients. The Pseudozyma species showed relatively low sensitivity to azole antifungal agents. The names proposed for these isolates are Pseudozyma alboarmeniaca (DMST 17135(T) = JCM 12454(T ) = CBS 9961(T)), Pseudozyma crassa (DMST 17136(T ) = JCM 12455(T) = CBS 9959(T)) and Pseudozyma siamensis (DMST 17137(T ) = JCM 12456(T) CBS 9960(T)), where DMST is Department of Medical Sciences Culture Collection, JCM is Japan Collection of Microorganisms and CBS is Centraalbureau voor Schimmelcultures.

Genotype analyses of human commensal scalp fungi, Malassezia globosa, and Malassezia restricta on the scalps of patients with dandruff and healthy subjects.

Dandruff and seborrheic dermatitis are common afflictions of the human scalp caused by commensal scalp fungi belonging to the genus Malassezia. Malassezia globosa and Malassezia restricta are the predominant species found on the scalp. The intergenic spacer regions of these species' rRNA genes contain short sequence repeats (SSR): (GT)n and (CT)n in M. globosa and (CT)n and (AT)n in M. restricta. In the present study, we compared the genotypes (SSR) of M. globosa and M. restricta colonizing the scalps of patients with dandruff and healthy individuals. For M. globosa, the genotype (GT)10:(CT)8 (40.3 %, 25/62) was predominant followed by (GT)9:(CT)8 (14.5 %, 9/62) and (GT)11:(CT)8 (14.5 %, 9/62) in patients with dandruff, whereas the genotypes in healthy subjects were diverse. For M. restricta, the genotype (CT)6:(AT)6 (59.7 %, 37/62) was predominant followed by (CT)6:(AT)8 (24.2 %, 15/62) in patients with dandruff, while four genotypes, (CT)6:(AT)6 (10.5 %, 6/57), (CT)6:(AT)7 (22.8 %, 13/57), (CT)6:(AT)8 (17.5 %, 10/57), and (CT)6:(AT)10 (21.1 %, 12/57), accounted for 71.9 % of all combinations in healthy subjects. The results of this study suggest that the M. globosa genotype (GT)10:(CT)8 and the M. restricta genotype (CT)6:(AT)6 may be involved in the development of dandruff.

Dupilumab Alters Both the Bacterial and Fungal Skin Microbiomes of Patients with Atopic Dermatitis.

The skin microbiome at lesion sites in patients with atopic dermatitis (AD) is characterized by dysbiosis. Although the administration of dupilumab, an IL-4Rα inhibitor, improves dysbiosis in the bacterial microbiome, information regarding the fungal microbiome remains limited. This study administered dupilumab to 30 patients with moderate-to-severe AD and analyzed changes in both fungal and bacterial skin microbiomes over a 12-week period. Malassezia restricta and M. globosa dominated the fungal microbiome, whereas non-Malassezia yeast species increased in abundance, leading to greater microbial diversity. A qPCR analysis revealed a decrease in Malassezia colonization following administration, with a higher reduction rate observed where the pretreatment degree of colonization was higher. A correlation was found between the group classified by the Eczema Area and Severity Index, the group categorized by the concentration of Thymus and activation-regulated chemokine, and the degree of skin colonization by Malassezia. Furthermore, an analysis of the bacterial microbiome also confirmed a decrease in the degree of skin colonization by the exacerbating factor Staphylococcus aureus and an increase in the microbial diversity of the bacterial microbiome. Our study is the first to show that dupilumab changes the community structure of the bacterial microbiome and affects the fungal microbiome in patients with AD.

Tacrolimus (FK506) Exhibits Fungicidal Effects against Candida parapsilosis Sensu Stricto via Inducing Apoptosis.

Tacrolimus (FK506), an immunosuppressant and calcineurin inhibitor, has fungicidal effects. However, its fungicidal effect is thought to be limited to basidiomycetes, such as Cryptococcus and Malassezia, and not to ascomycetes. FK506 had no fungicidal effect on Candida albicans, C. auris, C. glabrata, C. guilliermondii, C. kefyr, C. krusei, and C. tropicalis (>8 µg/mL); however, C. parapsilosis was susceptible to it at low concentrations of 0.125-0.5 µg/mL. C. metapsilosis and C. orthopsils, previously classified as C. parapsilosis, are molecularly and phylogenetically closely related to C. parapsilosis, but neither species was sensitive to FK506. FK506 increased the mitochondrial reactive oxygen species production and cytoplasmic and mitochondrial calcium concentration and activated metacaspases, nuclear condensation, and DNA fragmentation, suggesting that it induced mitochondria-mediated apoptosis in C. parapsilosis. Elucidating why FK506 exhibits fungicidal activity only against C. parapsilosis will provide new information for developing novel antifungal drugs.

[Malassezia Display a Hyphae-like "Spaghetti-and-Meatballs" Configuration in Keratotic Plugs].

Malassezia are lipophilic yeasts in the skin microbiome that abundantly colonize all parts of human skin except for the soles of the feet. Fungal microbiome analysis of keratotic plugs from the noses of 10 healthy individuals identified Malassezia restricta as the predominant species, followed by Malassezia globosa. Malassezia hyphae were observed in 5 of the 10 individuals. The hyphae were curved and thick-walled with spherical thick-walled and grouped blastoconidia, described as a "spaghetti-and-meatballs" configuration. In this study, we observed Malassezia hyphae in keratotic plugs of healthy subjects, although abundant Malassezia hyphae have previously only been observed in lesional sites of patients with pityriasis versicolor.

Delftia acidovorans secretes substances that inhibit the growth of Staphylococcus epidermidis through TCA cycle-triggered ROS production.

The proportion of Staphylococcus aureus in the skin microbiome is associated with the severity of inflammation in the skin disease atopic dermatitis. Staphylococcus epidermidis, a commensal skin bacterium, inhibits the growth of S. aureus in the skin. Therefore, the balance between S. epidermidis and S. aureus in the skin microbiome is important for maintaining healthy skin. In the present study, we demonstrated that the heat-treated culture supernatant of Delftia acidovorans, a member of the skin microbiome, inhibits the growth of S. epidermidis, but not that of S. aureus. Comprehensive gene expression analysis by RNA sequencing revealed that culture supernatant of D. acidovorans increased the expression of genes related to glycolysis and the tricarboxylic acid cycle (TCA) cycle in S. epidermidis. Malonate, an inhibitor of succinate dehydrogenase in the TCA cycle, suppressed the inhibitory effect of the heat-treated culture supernatant of D. acidovorans on the growth of S. epidermidis. Reactive oxygen species production in S. epidermidis was induced by the heat-treated culture supernatant of D. acidovorans and suppressed by malonate. Further, the inhibitory effect of the heat-treated culture supernatant of D. acidovorans on the growth of S. epidermidis was suppressed by N-acetyl-L-cysteine, a free radical scavenger. These findings suggest that heat-resistant substances secreted by D. acidovorans inhibit the growth of S. epidermidis by inducing the production of reactive oxygen species via the TCA cycle.