Development of a skin microbiome diagnostic method to assess skin condition in healthy individuals: Application of research on skin microbiomes and skin condition.

OBJECTIVE: Skin microbiomes vary across individuals. They are known to play essential roles in maintaining homeostasis and preventing infectious pathogens. In recent years, cosmetic product development has begun to focus on the relationship between skin microbiomes and skin conditions. However, the statistical methods used in many studies include the standard t-test and small-scale correlation analysis, which do not take into account the internal correlation structure in data on skin microbiomes and skin features. In this study, we aimed to understand the relationship between skin microbiomes and skin features by analysing complex microbiomes and skin data. METHODS: We obtained data on 19 skin characteristics and skin microbiomes based on 16s ribosomal RNA (16S rRNA) gene analysis of 276 healthy Japanese women. We then performed the principal component analysis (PCA), a method that takes into account the internal correlation structure, on 234 panels of them that did not contain outliers or missing values. We confirmed the relationship between skin microbiomes and skin features with principal component regression analysis and hierarchical clustering analysis (HCA). RESULTS: The principal component regression analysis showed strong relationships between skin microbiomes and sebum-related skin characteristics and skin pH. In the HCA, the female panel was classified into two major groups based on the skin microbiome. Furthermore, there were significant differences in sebum-related skin characteristics and the way skin condition changes with ageing between those groups, suggesting the possibility of measuring skin condition and age-related skin risk based on microbiome data. In addition, sebum-related characteristics differed significantly among middle-aged participants, suggesting a strong relationship between skin microbiomes and sebum-related characteristics. CONCLUSION: Analysis of skin condition and skin microbiome in Japanese women, taking into account the correlation between variables, showed that skin microbiome was significantly related to the number of pores and the amount of sebum. Furthermore, it was suggested that the skin condition and the way the skin condition changes with ageing may differ depending on the type of skin microbiome. The finding of a relationship between skin condition and skin microbiome suggests the possibility of proposing a new beauty method focusing on the skin microbiome in the future.

OBJECTIF: Les microbiomes de la peau varient selon les individus. Ils sont connus pour jouer des rôles essentiels dans le maintien de l'homéostasie et la prévention des agents pathogènes infectieux. Ces dernières années, le développement de produits cosmétiques a commencé à se concentrer sur la relation entre les microbiomes cutanés et les conditions de la peau. Cependant, les méthodes statistiques utilisées dans de nombreuses études comprennent le t-test standard et l'analyse de corrélation à petite échelle, qui ne tiennent pas compte de la structure de corrélation interne dans les données sur les microbiomes cutanés et les caractéristiques de la peau. Dans cette étude, nous avons cherché à comprendre la relation entre les microbiomes cutanés et les caractéristiques de la peau en analysant des données complexes sur les microbiomes et la peau. MÉTHODES: Nous avons obtenu des données sur 19 caractéristiques de la peau et sur les microbiomes cutanés à partir de l'analyse du gène de l'ARNr 16S (16S rRNA) de 276 femmes japonaises en bonne santé. Nous avons ensuite effectué l'analyse en composantes principales (PCA: principal component analysis), une méthode qui prend en compte la structure de corrélation interne, sur 234 d'entre elles qui ne contenaient pas de valeurs aberrantes ou manquantes. Nous avons confirmé la relation entre les microbiomes cutanés et les caractéristiques de la peau à l'aide d'une analyse de régression en composantes principales et d'une analyse de regroupement hiérarchique (HCA: hierarchical clustering analysis). RÉSULTATS: L'analyse de régression en composantes principales a montré des relations fortes entre les microbiomes cutanés et les caractéristiques de la peau liées au sébum et au pH de la peau. Dans l'étude HCA, le panel de femmes a été classé en deux grands groupes sur la base du microbiome cutané. En outre, il y avait des différences significatives dans les caractéristiques de la peau liées au sébum et dans la façon dont l'état de la peau change avec l'âge entre ces groupes, ce qui suggère la possibilité de mesurer l'état de la peau et le risque cutané lié à l'âge à partir des données du microbiome. En outre, les caractéristiques liées au sébum différaient de manière significative chez les participants d'âge moyen, ce qui suggère une forte relation entre les microbiomes cutanés et les caractéristiques liées au sébum. CONCLUSION: L'analyse de l'état de la peau et du microbiome cutané chez les femmes japonaises, en tenant compte de la corrélation entre les variables, a montré que le microbiome cutané était significativement lié au nombre de pores et à la quantité de sébum. En outre, il a été suggéré que l'état de la peau et la façon dont l'état de la peau évolue avec le vieillissement peuvent différer en fonction du type de microbiome cutané. La découverte d'une relation entre l'état de la peau et le microbiome cutané suggère la possibilité de proposer à l'avenir une nouvelle méthode de beauté axée sur le microbiome cutané.

Cutibacterium acnes: Much ado about maybe nothing much.

Cutibacterium acnes (also known as Propionibacterium acnes) has long been implicated in the pathogenesis of acne, inspiring both therapeutic and personal care approaches aiming to control the disease by controlling the bacterium. The purported association has made people with acne feel dirty and led to the-at times excessive-use of cleansers, antiseptics and antibiotics for the condition. However, recent evidence seems to weaken the case for C. acnes' involvement. New genetics and molecular biology findings strongly suggest that abnormal differentiation of sebaceous progenitor cells causes comedones, the primary lesions in acne. Comodegenesis is initiated by androgens and is unlikely to be triggered by C. acnes, which probably doesn't affect sebaceous differentiation. Is there still a place for it in this understanding of acne? It is necessary to critically address this question because it has consequences for treatment. Antibiotic use for acne noticeably contributes to microbial drug resistance, which we can ill afford. In this Viewpoint, we explore if and how C. acnes (still) fits into the developing view on acne. We also briefly discuss the implications for therapy in the light of antibiotic resistance and the need for more targeted therapies.

Why do humans get acne? A hypothesis.

Acne is a disease unique to humans and is associated with sebaceous glands that are found at high density on the scalp, forehead and face. Despite being a near universal problem in adolescence, the reason why such troublesome sebaceous glands exist at all is not well understood. Some interesting theories have been postulated including roles for skin maintenance, immunological function and perhaps even pheromones, but pre-pubertal skin which has sebaceous glands that are largely inactive, is healthy. Dystocia, obstructed labour, is unique to humans and no other animal has as much trouble giving birth. This is thought to reflect the relatively large human foetal head and proportionally small maternal pelvis. Noting the high density of sebaceous glands on the face, chest and back; these are exactly the same structures that pose the greatest obstruction during childbirth. Sebaceous glands develop after the fourth month of gestation and are large and well-developed at birth. Sebum production is also relatively high at birth. Having extra lubrication at these sites would help make the baby more slippery for birth conferring a selective advantage to successful delivery, as does the presence of the vernix caseosa, a white creamy substance, unique to humans that coats new-born infants. It is proposed that the sebaceous glands that cause acne are present on the face and forehead as they confer a selective advantage by 'lubricating' the widest parts of the new born baby to ease the passage of childbirth. Later in life, sebaceous glands may be inappropriately and pathologically primed, driven by a combination of hormones, diet and lifestyle to create acne.

Lipolytic Activity and the Utilization of Fatty Acids Associated with Bat Sebum by Pseudogymnoascus destructans.

Pseudogymnoascus destructans is the causative agent of a fungal infection of bats known as white-nose syndrome (WNS). Since its discovery in 2006, it has been responsible for precipitous declines of several species of cave-dwelling North American bats. While numerous advancements in the understanding of the disease processes underlying WNS have been made in recent years, there are still many aspects of WNS, particularly with respect to pathogen virulence, that remain unknown. In this preliminary investigation, we sought to further elucidate the disease cycle by concentrating on the pathogen, with specific focus on its ability to utilize lipids that compose bat wing sebum and are found in wing membranes, as a substrate for energy and growth. In vitro growth experiments were conducted with the three most common fatty acids that comprise bat sebum: oleic, palmitic, and stearic acids. None of the fatty acids were observed to contribute a significant difference in mean growth from controls grown on SDA, although morphological differences were observed in several instances. Additionally, as an accompaniment to the growth experiments, bat wing explants from Perimyotis subflavus and Eptesicus fuscus were fluorescently stained to visualize the difference in distribution of 16- and 18-carbon chain fatty acids in the wing membrane. Which substrates contribute to the growth of P. destructans is important to understanding the progressive impact P. destructans has on bat health through the course of the disease cycle.

Adaptation of acneic and non acneic strains of Cutibacterium acnes to sebum-like environment.

Cutibacterium acnes, former Proprionibacterium acnes, is a heterogeneous species including acneic bacteria such as the RT4 strain, and commensal bacteria such as the RT6 strain. These strains have been characterized by metagenomic analysis but their physiology was not investigated until now. Bacteria were grown in different media, brain heart infusion medium (BHI), reinforced clostridial medium (RCM), and in sebum like medium (SLM) specifically designed to reproduce the lipid rich environment of the sebaceous gland. Whereas the RT4 acneic strain showed maximal growth in SLM and lower growth in RCM and BHI, the RT6 non acneic strain was growing preferentially in RCM and marginally in SLM. These differences were correlated with the lipophilic surface of the RT4 strain and to the more polar surface of the RT6 strain. Both strains also showed marked differences in biofilm formation activity which was maximal for the RT4 strain in BHI and for the RT6 strain in SLM. However, cytotoxicity of both strains on HaCaT keratinocytes remained identical and limited. The RT4 acneic strain showed higher inflammatory potential than the RT6 non acneic strain, but the growth medium was without significant influence. Both bacteria were also capable to stimulate ß-defensine 2 secretion by keratinocytes but no influence of the bacterial growth conditions was observed. Comparative proteomics analysis was performed by nano LC-MS/MS and revealed that whereas the RT4 strain only expressed triacylglycerol lipase, the principal C. acnes virulence factor, when it was grown in SLM, the RT6 strain expressed another virulence factor, the CAMP factor, exclusively when it was grown in BHI and RCM. This study demonstrates the key influence of growth conditions on virulence expression by C. acnesand suggest that acneic and non acneic strains are related to different environmental niches.

Minor taxa in human skin microbiome contribute to the personal identification.

The human skin microbiome can vary over time, and inter-individual variability of the microbiome is greater than the temporal variability within an individual. The skin microbiome has become a useful tool to identify individuals, and one type of personal identification using the skin microbiome has been reported in a community of less than 20 individuals. However, identification of individuals based on the skin microbiome has shown low accuracy in communities larger than 80 individuals. Here, we developed a new approach for personal identification, which considers that minor taxa are one of the important factors for distinguishing between individuals. We originally established a human skin microbiome for 66 samples from 11 individuals over two years (33 samples each year). Our method could classify individuals with 85% accuracy beyond a one-year sampling period. Moreover, we applied our method to 837 publicly available skin microbiome samples from 89 individuals and succeeded in identifying individuals with 78% accuracy. In short, our results investigate that (i) our new personal identification method worked well with two different communities (our data: 11 individuals; public data: 89 individuals) using the skin microbiome, (ii) defining the personal skin microbiome requires samples from several time points, (iii) inclusion of minor skin taxa strongly contributes to the effectiveness of personal identification.

Developing an in vitro artificial sebum model to study Propionibacterium acnes biofilms.

AIM: The aim of the present study was to develop a new model system to study Propionibacterium acnes biofilms. This model should be representative for the conditions encountered in the pilosebaceous unit. METHODS AND RESULTS: The new model, consists of an artificial sebum pellet supported by a silicone disc. Sebum pellets were inoculated with various P. acnes strains isolated from both normal and acneic skin. Growth and biofilm formation was verified by conventional plating at different time points, as well as by resazurin assays and fluorescence microscopy after LIVE/DEAD staining. The artificial sebum pellets were also used in assays to measure the production of certain virulence factors implicated in the pathogenesis of acne, including lipase, protease and the presence of CAMP factors. CONCLUSION: The artificial sebum model can sustain biofilm growth of P. acnes, as was determined by increasing CFU counts for up to 1 week after inoculation. Metabolic activity and biofilm formation were confirmed using resazurin staining and fluorescence microscopy respectively. The production of virulence factors in this model was demonstrated as well.

Propionibacterium species and follicular keratinocyte activation in acneic and normal skin.

BACKGROUND: The pathogenesis of acne vulgaris is multifactorial with increased sebum production, alteration in the quality of sebum lipids, dysregulation of the hormone microenvironment, follicular hyperkeratinization and Propionibacterium acnes-driven inflammation as major contributory factors. Hyperproliferation of keratinocytes is believed to contribute to hypercornification and eventually leads to comedone development. While the distribution of P. acnes is relatively well documented in acneic and healthy skin, little is known about P. granulosum and P. avidum. OBJECTIVES: To visualize directly the three major Propionibacterium in 117 control and 26 acneic skin samples. In addition, keratinocyte proliferation was evaluated. METHODS: Propionibacteria were visualized by immunofluorescence microscopy, and keratinocyte proliferation was assessed by Ki67, keratin (K) 16 and p63 immunochemistry. RESULTS: P. acnes was identified in 68 samples (48%), while P. granulosum was identified in 12 (8%) samples; P. avidum was not detected at all. Unexpectedly, acne samples did not show higher keratinocyte proliferation than controls, nor was there any association between bacterial colonization and expression of Ki67/K16/p63. CONCLUSIONS: Our findings do not support earlier notions of follicular keratinocyte hyperproliferation as a cause of ductal hypercornification in acneic facial skin. Further studies on the mechanisms underlying hypercornification in acne pathogenesis are needed.

The skin microbiome: potential for novel diagnostic and therapeutic approaches to cutaneous disease.

A vast diversity of microorganisms, including bacteria, fungi, viruses, and arthropods, colonize the human skin. Culture-independent genomic approaches for identifying and characterizing microbial communities have provided glimpses into the topographical, temporal, and interpersonal complexity that defines the skin microbiome. Identification of changes associated with cutaneous disease, including acne, atopic dermatitis, rosacea, and psoriasis, are being established. In this review, our current knowledge of the skin microbiome in health and disease is discussed, with particular attention to potential opportunities to leverage the skin microbiome as a diagnostic, prognostic, and/or therapeutic tool.

A honey trap for the treatment of acne: manipulating the follicular microenvironment to control Propionibacterium acnes.

Today, as 40 years ago, we still rely on a limited number of antibiotics and benzoyl peroxide to treat inflammatory acne. An alternative way of suppressing the growth of Propionibacterium acnes is to target the environment in which it thrives. We conjecture that P. acnes colonises a relatively "extreme" habitat especially in relation to the availability of water and possibly related factors such as ionic strength and osmolarity. We hypothesise that the limiting "nutrient" within pilosebaceous follicles is water since native sebum as secreted by the sebaceous gland contains none. An aqueous component must be available within colonised follicles, and water may be a major factor determining which follicles can sustain microbial populations. One way of preventing microbial growth is to reduce the water activity (a w ) of this component with a biocompatible solute of very high water solubility. For the method to work effectively, the solute must be small, easily diffusible, and minimally soluble in sebaceous lipids. Xylose and sucrose, which fulfil these criteria, are nonfermentable by P. acnes and have been used to reduce water activity and hence bacterial colonisation of wounds. A new follicularly targeted topical treatment for acne based on this approach should be well tolerated and highly effective.

Preen gland removal increases plumage bacterial load but not that of feather-degrading bacteria.

The preen gland is a holocrine sebaceous gland of the avian integument which produces an oily secretion that is spread on the plumage during preening. It has been suggested that birds may defend themselves against feather-degrading bacteria (FDB) and other potential pathogens using preen gland secretions. However, besides some in vitro studies, the in vivo bacterial inhibitory effects of the preen oil on the abundance of feather-associated bacterial species has not yet been studied in passerines. Here we tested the effect of gland removal on the abundance of FDB and other-cultivable bacterial loads (OCB) of male house sparrows (Passer domesticus). Our results did not support earlier results on in vitro antibacterial activity of preen oil against FDB since the absence of the preen gland did not significantly affect their loads related to the control birds. In contrast, we found that preen gland removal led to higher loads of OCB. This result suggests that the antimicrobial spectrum of the preen oil is broader than previously thought and that, by reducing the overall feather bacterial loads, the preen gland could help birds to protect themselves against a variety of potentially harmful bacteria.

Epigallocatechin-3-gallate improves acne in humans by modulating intracellular molecular targets and inhibiting P. acnes.

Acne vulgaris is a highly prevalent skin disorder characterized by hyperseborrhea, inflammation, and Propionibacterium acnes overgrowth. Only isotretinoin and hormonal therapy reduce sebum production. To identify a new drug candidate that modulates sebum, we examined the effects of EGCG, the major polyphenol in green tea, on human SEB-1 sebocytes and in patients with acne. In SEB-1 sebocytes, we found that EGCG reduced sebum by modulating the AMPK-SREBP-1 signaling pathway. EGCG also reduces inflammation by suppressing the NF-κB and AP-1 pathways. EGCG also induces cytotoxicity of SEB-1 sebocytes via apoptosis and decreases the viability of P. acnes, thus targeting almost all the pathogenic features of acne. Finally, and most importantly, EGCG significantly improved acne in an 8-week randomized, split-face, clinical trial, and was well tolerated. Our data provide a therapeutic rationale for the use of EGCG in acne.

The Staphylococcus aureus surface protein IsdA mediates resistance to innate defenses of human skin.

Resistance to human skin innate defenses is crucial for survival and carriage of Staphylococcus aureus, a common cutaneous pathogen and nasal colonizer. Free fatty acids extracted from human skin sebum possess potent antimicrobial activity against S. aureus. The mechanisms by which S. aureus overcomes this host defense during colonization remain unknown. Here, we show that S. aureus IsdA, a surface protein produced in response to the host, decreases bacterial cellular hydrophobicity rendering them resistant to bactericidal human skin fatty acids and peptides. IsdA is required for survival of S. aureus on live human skin. Reciprocally, skin fatty acids prevent the production of virulence determinants and the induction of antibiotic resistance in S. aureus and other Gram-positive pathogens. A purified human skin fatty acid was effective in treating systemic and topical infections of S. aureus suggesting that our natural defense mechanisms can be exploited to combat drug-resistant pathogens.

Matrix metalloproteinases of epithelial origin in facial sebum of patients with acne and their regulation by isotretinoin.

Acne vulgaris is a skin disorder of the sebaceous follicles, involving hyperkeratinization and perifollicular inflammation. Matrix metalloproteinases (MMP) have a predominant role in inflammatory matrix remodeling and hyperproliferative skin disorders. We investigated the expression of MMP and tissue inhibitors of MMP (TIMP) in facial sebum specimens from acne patients, before and after treatment with isotretinoin. Gelatin zymography and Western-blot analysis revealed that sebum contains proMMP-9, which was decreased following per os or topical treatment with isotretinoin and in parallel to the clinical improvement of acne. Sebum also contains MMP-1, MMP-13, TIMP-1, and TIMP-2, as assessed by ELISA and western blot, but only MMP-13 was decreased following treatment with isotretinoin. The origin of MMP and TIMP in sebum is attributed to keratinocytes and sebocytes, since we found that HaCaT keratinocytes in culture secrete proMMP-2, proMMP-9, MMP-1, MMP-13, TIMP-1, and TIMP-2. SZ95 sebocytes in culture secreted proMMP-2 and proMMP-9, which was also confirmed by microarray analysis. Isotretinoin inhibited the arachidonic acid-induced secretion and mRNA expression of proMMP-2 and -9 in both cell types and of MMP-13 in HaCaT keratinocytes. These data indicate that MMP and TIMP of epithelial origin may be involved in acne pathogenesis, and that isotretinoin-induced reduction in MMP-9 and -13 may contribute to the therapeutic effects of the agent in acne.

Phlyctenular keratitis associated with meibomitis in young patients.

PURPOSE: To describe the unique clinical features of phlyctenular keratitis, including the association with meibomitis, in young patients. DESIGN: Observational case series. METHODS: The study population consisted of 23 Japanese patients aged under 35 years with phlyctenular keratitis. We examined their clinical history, signs and symptoms, human leukocyte antigen (HLA), bacterial cultures of meibum, and the efficacy of antibiotics. The minimal diagnostic criteria included corneal nodules consisting of cellular infiltrates and superficial neovascularization. RESULTS: Of the 23 patients, 20 (87%) were women, and 13 (56.5%) had a history of chalazia. In all cases, the lesions and the severity of corneal nodules and neovascularization corresponded well with the location and the severity of meibomitis. The frequency of HLA-A26 and HLA-B35 was significantly increased in our patients (P = .003 and .016, respectively). Propionibacterium acnes in bacterial cultures of pure meibum in 12 of the 20 patients (60%) was statistically more highly positive than those in four of the 17 age-matched normal control subjects (23.5%; P = .028). CONCLUSION: The characteristics of phlyctenular keratitis in our cases include significantly higher prevalence in female patients, severity variation of ocular surface manifestation corresponding to meibomitis, specific HLA association, and possible P. acnes involvement.

[Normal and pathologic sebaceous function. Research in a shallow milieu?]. / Fonction sébacée normale et pathologique. Des recherches au milieu du gué?

Modern therapeutic approaches allow to control sebaceous secretion but knowledge about the sebaceous gland and its precise function within the pilosebaceous unit is still insufficient. Steroid hormones are the principal albeit not exclusive regulators of the sebaceous glands. Three phases may be distinguished in sebaceous physiology: secretion-production, stocking in the follicular reservoir, and excretion. Human "native" intracellular sebum, before secretion, is composed of squalene, waxes, and triglycerides. Once secreted, the sebum is colonised by various xenobiots whose development is controlled by several defensive humoral mechanisms and by the contact with ambient oxygen. Oxygen and micro-organisms transform "native" sebum, lysis of triglycerides to fatty acids being the most pronounced activity. Certain components of this complex mixture of molecules present in the sebum are clearly cytotoxic or irritant, provoking reactive follicular hyperkeratosis and comedone formation--the first step to acne. Some lipophilic organisms like Malassezia yeast may be highly antigenic and induce chronic inflammatory reactions like in seborrhoeic dermatitis. Demodex is an inrafollicular parasite feeding on sebum that frequently causes blepharitis. Sebum is also a vehicle transporting and transmitting several endogenous and exogenous molecules, including potential regulatory factors of hair follicles. Recent development of in vitro cultures of functional sebocytes should help to better understand several aspects of the sebaceous gland's biology.

The permissive effect of sebum in seborrhoeic dermatitis: an explanation of the rash in neurological disorders.

The hypothesis that sebum permits the growth of Pityrosporum ovale, and hence the development of seborrhoeic dermatitis, was tested by observing whether a reduction of sebum production by isotretinoin would improve the disorder. In 10 male patients with seborrhoeic dermatitis, treatment with isotretinoin for 6 weeks reduced the mean sebum excretion rate by 70% and improved the severity of the rash, but with a site difference in magnitude of response. It is concluded that the residual pool of sebum is important for the growth of P. ovale and that, within the physiological range, sebum has a permissive effect on the growth of this yeast. Variation in the pools of residual sebum explains a number of features of the disease such as site of involvement and greater prevalence in males than females. The pathological increase in the residual pool of sebum due to immobility explains the frequent occurrence of seborrhoeic dermatitis in patients with a variety of neurological disorders.