Relationship between skin fungal and bacterial microbiomes and skin pH.

OBJECTIVE: The present study investigated the relationship between the skin bacterial and fungal microbiomes and skin pH at sebaceous sites, namely, the forehead, cheek and upper chest. We also examined the relationship between the skin microbiome and transepidermal water loss (TEWL), which is an indicator of rough skin. METHODS: Thirty healthy Japanese subjects (15 females and 15 males) were enrolled as subjects. The forehead, cheek and upper chest were examined in August (summer) and February (winter). Skin surface samples were collected using the swab method, and the 16s rRNA and ITS regions were analysed using next-generation sequencing. Skin pH and TEWL were also assessed. RESULTS: The changes observed in the skin microbiome were associated with season-dependent changes in skin pH. An increase in skin pH at the forehead and cheek was associated with a higher relative abundance of Cutibacterium spp., a reduction in bacterial diversity, and a lower relative abundance of Staphylococcus spp. A decrease in skin pH at the upper chest was associated with a higher relative abundance of Malassezia restricta, a lower relative abundance of M. sympodialis and decrease in fungal diversity. Moreover, these changes were also associated with an increase in TEWL. CONCLUSION: The present results indicate that changes in skin pH are associated with changes in the skin bacterial and fungal microbiomes at sebaceous sites. The changes observed in the skin microbiome were also associated with rough skin.

OBJECTIF: La présente étude a examiné la relation entre les microbiomes bactériens et fongiques de la peau et le pH de la peau des sites sébacés, à savoir le front, la joue et la partie supérieure du thorax. Nous avons également examiné la relation entre le microbiome cutané et la perte d'eau transépidermique (transepidermal water loss, TEWL), qui est un indice de rugosité cutanée. MÉTHODES: Trente sujets japonais en bonne santé (15 femmes et 15 hommes) ont été inclus en tant que sujets de l'étude. Le front, la joue et la partie supérieure du thorax ont été examinés en août (été) et en février (hiver). Des échantillons de surface cutanée ont été prélevés par écouvillonnage et les régions de l'ARNr 16s et ITS ont été analysées par séquençage à haut débit. Le pH de la peau et la TEWL ont également été évalués. RÉSULTATS: Les changements observés dans le microbiome cutané étaient associés à des modifications du pH de la peau dépendant de la saison. Une augmentation du pH de la peau au niveau du front et de la joue était associée à une plus grande abondance relative de Cutibacterium spp. et à une réduction de la diversité bactérienne, ainsi qu'à une plus faible abondance relative de Staphylococcus spp. Une diminution du pH de la peau au niveau de la partie supérieure du thorax était associée à une abondance relative plus élevée de Malassezia restricta et à une abondance relative plus faible de M. sympodialis, ainsi qu'à une diversité fongique. De plus, ces changements ont également été associés à une augmentation de la TEWL. CONCLUSION: Ces résultats indiquent que les modifications du pH de la peau sont associées à des modifications des microbiomes bactérien et fongique de la peau au niveau des sites sébacés. Les changements observés dans le microbiome de la peau étaient également associés à une rugosité de la peau.

The Microbiome in Comedonal Contents of Inflammatory Acne Vulgaris is Composed of an Overgrowth of Cutibacterium Spp. and Other Cutaneous Microorganisms.

Background: Acne vulgaris (acne) and cutaneous resident microorganisms are considered to be closely related. However, the bacterial and fungal microbiota in the comedonal contents of inflammatory acne lesions have not yet been investigated in detail. Purpose: To clarify the relationship between cutaneous microorganisms and acne, we examined the microbiome in the comedonal contents of inflammatory acne and on the facial skin of patients with acne using 16s rRNA and ITS gene sequencing with a next-generation sequencer (NGS). Patients and Methods: Twenty-two untreated Japanese acne outpatients were examined. The comedonal contents of inflammatory acne lesions on the face were collected using a comedo extractor. Skin surface samples from facial skin were collected using the swab method. Results: The results obtained revealed that the predominant bacteria in the comedonal contents of inflammatory acne were Cutibacterium spp. (more prominent in areas with large amounts of sebum), while those on the skin surface were Staphylococcus spp. Malassezia spp., particularly Malassezia restricta, were the predominant fungi in both the comedonal contents of inflammatory acne and on the skin surface. The bacterial microbiome in comedonal contents exhibited stronger metabolic activity, including the production of enzymes related to acne, than that on the skin surface. Conclusion: These results indicate that acne is an inflammatory disease involving the overgrowth of Cutibacterium acnes and other cutaneous resident microorganisms, including Malassezia spp.

Microorganisms inhabiting follicular contents of facial acne are not only Propionibacterium but also Malassezia spp.

To clarify the relationship between major cutaneous microorganisms (Propionibacterium, Staphylococcus and Malassezia spp.) and acne vulgaris (acne), we examined the microbiota quantitatively in the follicular contents of inflammatory acne and on the facial skin of patients with acne. Fifteen Japanese untreated acne outpatients were studied. The follicular contents from inflammatory acne lesions of the face were collected using a comedo extractor. The skin surface samples were obtained by the swab method from 10 cm(2) of facial skin. The microbiota was analyzed using polymerase chain reaction. The microbiota in follicular contents was similar to that on the skin surface, namely, there were large populations of Propionibacterium spp., Staphylococcus spp. and Malassezia spp. Moreover, the number of Malassezia spp. on the skin surface was correlated with that of inflammatory acne and that in follicular contents. This study clarified that there are large populations of Propionibacterium spp., Staphylococcus spp. and Malassezia spp. in follicular contents. These results suggest the possibility that not only Propionibacterium acnes but also other cutaneous resident microorganisms are related to acne. Particularly, we considered that Malassezia spp. is closely related.

Fatty acid compositions of triglycerides and free fatty acids in sebum depend on amount of triglycerides, and do not differ in presence or absence of acne vulgaris.

To clarify the influence of the fatty acid composition of sebum in acne vulgaris, we investigated the amounts and fatty acid compositions of triglycerides (TG) and free fatty acids (FFA), and the amounts of cutaneous superficial Propionibacterium acnes in acne patients and healthy subjects. The foreheads of 18 female patients, 10 male patients, 10 healthy females and 10 healthy males were studied in a Japanese population. There were significant differences in the amounts of sebum, TG and cutaneous superficial P. acnes, as well as the fatty acid compositions of TG and FFA between acne patients and healthy subjects in females. Their fatty acid compositions were correlated with the amount of TG with or without acne. It was clarified that the fatty acid compositions of TG and FFA depended on the amount of TG, and there were no differences in the fatty acid composition in the presence and absence of acne.

Analysis of facial skin-resident microbiota in Japanese acne patients.

OBJECTIVES: We investigated the facial skin microbiota of Japanese acne patients. METHODS: Skin swab samples were obtained from 100 acne patients and 28 healthy controls to evaluate Propionibacterium and Staphylococcus spp. using a culture method. Malassezia spp. were evaluated using a nonculture method. Antibiotic resistance of Propionibacterium spp. was also examined. RESULTS: Acne patients and controls did not show significant differences in Propionibacterium and Staphylococcus spp. populations. However, the number of Malassezia globosa from patients was greater than that from controls. Moreover, the number of Propionibacterium spp. from patients carrying antibiotic-resistant strains was significantly greater than that from patients not carrying them. CONCLUSIONS: The present study characterized the facial skin microbiota of Japanese acne patients, suggesting a correlation between acne and quantitative differences in Malassezia microbiota. It was also found that the antibiotic resistance of Propionibacterium spp. may affect its abundance in the skin.

Increased hydrophobicity in Malassezia species correlates with increased proinflammatory cytokine expression in human keratinocytes.

Malassezia cells stimulate cytokine production by keratinocytes, although this ability differs among Malassezia species for unknown reasons. The aim of this study was to clarify the factors determining the ability to induce cytokine production by human keratinocytes in response to Malassezia species. M. furfur NBRC 0656, M. sympodialis CBS 7222, M. dermatis JCM 11348, M. globosa CBS 7966, M. restricta CBS 7877, and three strains each of M. globosa, M. restricta, M. dermatis, M. sympodialis, and M. furfur maintained under various culture conditions were used. Normal human epidermal keratinocytes (NHEKs) (1 × 10(5) cells) and the Malassezia species (1 × 10(6) cells) were co-cultured, and IL-1α, IL-6, and IL-8 mRNA levels were determined. Moreover, the hydrophobicity and ß-1,3-glucan expression at the surface of Malassezia cells were analyzed. The ability of Malassezia cells to trigger the mRNA expression of proinflammatory cytokines in NHEKs differed with the species and conditions and was dependent upon the hydrophobicity of Malassezia cells not ß-1,3-glucan expression.

Malassezia globosa tends to grow actively in summer conditions more than other cutaneous Malassezia species.

Malassezia globosa is a major pathogen of Malassezia folliculitis (MF) and the predominant species on human skin. The aim of this study was to clarify the differences between M. globosa and other cutaneous Malassezia species, M. restricta, M. dermatis, M. sympodialis and M. furfur. The optimum growth temperature, effects of compounds of sweat and free fatty acids on growth, and lipase activities of five cutaneous Malassezia species were determined. The growth of M. globosa was promoted strongly by the compounds of sweat and high temperature unlike that of other cutaneous Malassezia species. This result clarified that M. globosa tended to grow actively in summer conditions more than other cutaneous Malassezia species. Furthermore, M. globosa showed high lipase activity. We consider these characteristics of M. globosa to relate to the pathogenesis of MF.

Cutaneous Malassezia microbiota in atopic dermatitis patients differ by gender and body part.

BACKGROUND: Malassezia is a particularly important factor in the occurrence of atopic dermatitis (AD). AIM: The aim of this study was to quantitatively clarify the Malassezia species isolated from AD patients by gender, body part and analytical method in detail. METHODS: The subjects were 20 AD males and 47 AD females. Samples were collected from lesion and nonlesion areas on the face and upper trunk of AD patients. Malassezia DNA was analyzed using a real-time PCR system. RESULTS: The cutaneous Malassezia microbiota in AD patients differed by gender, body part and analytical method. CONCLUSIONS: The present results indicate the possibility that the influence of Malassezia antigens is different according to gender and body part.

Cutaneous Malassezia microbiota of healthy subjects differ by sex, body part and season.

Malassezia is a component of normal cutaneous resident microbiota. The aim of this study was to quantitatively clarify the differences in cutaneous Malassezia microbiota in healthy subjects by sex, body part and season. Samples were collected from the forehead, cheek, upper chest and upper back of 20 healthy men and 20 healthy women (average age 32 years) in summer and winter by the swab method. Malassezia DNA was analyzed using a real-time PCR system. As a result, in sex, body parts and season, men, the upper trunk and summer showed the highest total numbers of cutaneous Malassezia species on average. There were also differences depending on the analytical method. The predominant species were M. restricta on the face of men, M. globosa and M. dermatis on the upper trunk of men, and M. globosa and M. sympodialis on the upper trunk of women. This study clarified that the cutaneous Malassezia microbiota of healthy subjects differed by sex, body part and season.

Effects of Propionibacterium acnes on various mRNA expression levels in normal human epidermal keratinocytes in vitro.

Propionibacterium acnes is one of the most significant pathogenic factors of acne vulgaris. This bacteria relates to acne by various pathways. It has also been reported that P. acnes influences pro-inflammatory cytokine production in keratinocytes in vitro. However, the influence on the differentiation of keratinocytes by P. acnes has not been studied extensively. We analyzed the expression of keratinocyte differentiation-specific markers, keratins, and pro-inflammatory cytokines in normal human epidermal keratinocytes (NHEK) exposed to P. acnes in vitro. All P. acnes strains used in this study increased transglutaminase (TGase), keratin 17 (K17) and interleukin (IL) mRNA expression levels in NHEK, and decreased K1 and K10 expression levels. Some P. acnes strains increased involucrin and K6 mRNA expression levels in NHEK and decreased filaggrin, K6 and K16 expression levels in vitro. This experiment clarified that P. acnes influences the differentiation of NHEK in vitro. As a result, P. acnes influenced the expression of not only pro-inflammatory cytokines but also some keratinocyte differentiation-specific markers and keratins in NHEK. Our results suggest that P. acnes relates to acne pathogenesis by not only the induction of inflammation but also in the differentiation of keratinocytes. Moreover, it was considered that the reaction of NHEK to P. acnes may be different depending on the type of bacteria.

Malassezia folliculitis is caused by cutaneous resident Malassezia species.

Malassezia folliculitis [MF] is caused by the invasion of hair follicles by large numbers of Malassezia cells, but it remains unclear which Malassezia species are involved in the disease. To clarify this situation, Malassezia species isolated from lesions of MF patients were analyzed by both culture and non-culture methods. In addition, Malassezia species recovered from the non-lesion areas of the skin of MF patients and skin samples of healthy subjects were included in this study. The test population consisted of 32 MF patients and 40 healthy individuals. The lesions were obtained using a comedone extractor, while swabs were employed to obtain skin samples from non-lesion areas of the patients and healthy subjects. Malassezia DNA was analyzed using a real-time PCR technique. The detection limit of the culture method was 5 CFU/cm(2) as opposes 50 cells/cm(2) with non-culture procedures. The predominant species recovered from MF lesions were M. globosa and M. sympodialis by culture method analysis, and M. restricta, M. globosa, and M. sympodialis with non-culture methods. These results were in agreement with those found with samples from non-lesion skin areas of MF patients and healthy subjects. This study clarified that MF is caused by Malassezia species that are part of the cutaneous microflora and not by exogenous species.