Title: The skin microbiome is different in pediatric versus adult atopic dermatitis
Abstract: Atopic dermatitis (AD) is the most common inflammatory skin disease in the general population. The disease prevalence is associated with age. AD often starts in early childhood, affecting 15% to 30% of children.1Bieber T. Atopic dermatitis.N Engl J Med. 2008; 358: 1483-1494Crossref PubMed Scopus (1557) Google Scholar However, up to 70% of children with AD show clearing of the disease or a spontaneous remission around puberty, even in patients with filaggrin mutations. AD can persist or start in adulthood. However, the prevalence of AD in adults is only approximately 3%. Although multiple factors contribute to AD pathogenesis, skin micro-organisms are critical in driving disease development. Shifts in the skin microbiome were observed during disease progression of pediatric AD.2Kong H.H. Oh J. Deming C. Conlan S. Grice E.A. Beatson M.A. et al.Temporal shifts in the skin microbiome associated with disease flares and treatment in children with atopic dermatitis.Genome Res. 2012; 22: 850-859Crossref PubMed Scopus (1099) Google Scholar In adult AD, however, the skin microbiome is not well characterized. Comparisons of the skin microbiome among age groups of patients with AD and healthy controls will help delineate age differences in the microbial pathogenesis of this disease. In this study, we recruited 128 patients with AD: 59 young children (age, 2-12 years), 13 teenagers (age, 13-17 years), and 56 adults (age, 18-62 years). A cohort of 68 age-matched nonatopic healthy controls (age, 3-59 years) was also enrolled, which includes 13 young children, 10 teenagers, and 45 adults (see Table E1 in this article's Online Repository at www.jacionline.org). We collected 2 swab samples from the volar forearm of each patient with AD, one from lesional skin and one from adjacent normal-appearing nonlesional skin. One swab sample of the volar forearm was collected from each healthy subject. In total, 324 samples were analyzed using 16S ribosomal RNA (rRNA) gene sequencing. After data cleaning, on average 32,311 paired-end 16S rRNA sequences were obtained for each sample. Our data provided sufficient sequencing depths at the species level as indicated by the rarefaction curve (see Fig E1 in this article's Online Repository at www.jacionline.org). This large study cohort and the high sequencing coverage enabled us to robustly identify differences in the skin microbiome between age groups in healthy individuals and patients with AD. In the skin microbiome of healthy individuals, we identified 7 prevalent bacterial phyla and 20 genera (Fig 1, A). Four of the genera (Propionibacterium, Corynebacterium, Staphylococcus, and Streptococcus) were present in 90% or more of the healthy subjects, with 5% or more relative abundance in at least 1 age group. Among the 15 species identified (see Fig E2 in this article's Online Repository at www.jacionline.org), Propionibacterium acnes, Staphylococcus epidermidis, and Streptococcus mitis/oralis/pneumoniae/sanguinis were the most prevalent species in healthy subjects, accounting for 98.5% of total Propionibacterium species, 46.6% of total Staphylococcus species, and 32.5% of total Streptococcus species in relative abundance. To determine whether the stages of the human physical development have a significant effect on the skin microbiome,3Oh J. Conlan S. Polley E.C. Segre J.A. Kong H.H. Shifts in human skin and nares microbiota of healthy children and adults.Genome Med. 2012; 4: 77Crossref PubMed Scopus (225) Google Scholar we compared the microbiome among the age groups. We found that the healthy skin microbiome was significantly more diverse in young children than in adults (alpha diversity, P = .01), and was distinct between the 2 age groups as indicated by beta diversity (ANOSIM, P = .009) (Fig 2, A). At the genus level, Streptococcus, Granulicatella, Gemella, Rothia, and Haemophilus were more abundant in young children, whereas Propionibacterium, Corynebacterium, Staphylococcus, Lactobacillus, Finegoldia, and Anaerococcus were more abundant in adults (see Table E2 in this article's Online Repository at www.jacionline.org). At the species level, Streptococcus salivarius/thermophilus/vestibularis was more abundant in young children (P = .045) (see Table E3 in this article's Online Repository at www.jacionline.org), whereas P acnes and S epidermidis were more abundant in adults (P = .01 and P < 1 × 10−5, respectively) (Fig 1, B). Staphylococcus aureus was detected in 20.6% of the healthy subjects, but had very low relative abundance (<1%) in all age groups. Increased host sebum production and changes in the skin structure at and after puberty may facilitate the colonization and growth of lipophilic bacteria Propionibacterium and Corynebacterium,4Hannigan G.D. Grice E.A. Microbial ecology of the skin in the era of metagenomics and molecular microbiology.Cold Spring Harb Perspect Med. 2013; 3: a015362Crossref Scopus (70) Google Scholar which replace Streptococcus and become dominant in adulthood. Teenagers are in transition from young children to adults in physical development, and their skin microbiome is in transition as well, with a higher similarity to adults than to young children,3Oh J. Conlan S. Polley E.C. Segre J.A. Kong H.H. Shifts in human skin and nares microbiota of healthy children and adults.Genome Med. 2012; 4: 77Crossref PubMed Scopus (225) Google Scholar reflected in the representative micro-organisms (Fig 1, B) and the overall microbial community structure (Fig 2, A). In subsequent analyses, we combined teenagers and adults into one group and compared with young children. Similar to the healthy skin microbiome, in the AD skin microbiome we identified significant differences between young children and adults-teenagers (beta diversity, ANOSIM, P < .001) (Fig 2, A). In AD nonlesional skin, the microbiome diversity was significantly higher in young children than in adults-teenagers (alpha diversity, P = .036). The 20 prevalent genera identified in healthy controls were also detected in most of the patients with AD (Fig 1, C). Eight of the genera were significantly different in relative abundance between young children and adults-teenagers in both lesional and nonlesional skin (see Table E4 in this article's Online Repository at www.jacionline.org). The age differences were consistent with those observed in healthy controls. The microbiome differences in lesional and nonlesional skin were identified previously in patients with AD5Flores G.E. Seite S. Henley J.B. Martin R. Zelenkova H. Aguilar L. et al.Microbiome of affected and unaffected skin of patients with atopic dermatitis before and after emollient treatment.J Drugs Dermatol. 2014; 13: 1365-1372PubMed Google Scholar; however, it was unclear whether the differences were associated with age. In this study, we found that the microbiome diversity was significantly decreased in lesional skin compared with nonlesional skin in both young children (P < .001) and adults-teenagers (P = .013). In both age groups, Staphylococcus was significantly more abundant in lesional skin (P ≤ .012) and was also more abundant in nonlesional skin compared with healthy skin (P < .003), suggesting that nonlesional skin is susceptible to pathogen colonization and is at risk to progress toward diseased state. In contrast, skin commensals Streptococcus and Propionibacterium were observed in lower relative abundance in lesional skin compared with nonlesional skin and in nonlesional skin compared with healthy skin, but their changes were specific to young children and adults-teenagers, respectively. To better understand the bacterial associations with age in the skin microbiome, we calculated correlations among the 20 prevalent genera on the basis of their relative abundances in patients with AD and healthy controls. Three distinct bacterial clusters were identified: adult-associated, childhood-associated, and AD-associated (Fig 2, B). Most of the bacterial organisms were clustered in either adult-associated group or in childhood-associated group (see Tables E2-E5 in this article's Online Repository at www.jacionline.org), suggesting that age differences in the skin microbiome may be attributed to shifts in skin micro-organisms that are coordinated with each other in abundance during host maturation. AD-associated cluster consisted of only Staphylococcus. We identified multiple species within the major genera. Except for S aureus, species within each genus were positively correlated in relative abundance (see Fig E3 in this article's Online Repository at www.jacionline.org). S aureus was inversely correlated with other species, including those from the same genus, suggesting an antagonistic relationship between S aureus and skin commensals. We further investigated differences in the gene functions encoded in the genomes of age-specific and AD-associated skin bacteria. We analyzed 46 genomes of 13 major species found in our cohort (see this article's Methods section in the Online Repository at www.jacionline.org). A total of 1910 KEGG orthologous groups (KO genes) were identified, 833 of which were unique to 1 of the 3 skin bacterial clusters (see Fig E4 and Table E6 in this article's Online Repository at www.jacionline.org). Among the 316 KO genes unique to AD-associated cluster, 63 KO genes were S aureus–specific, involved in disease-associated pathways including S aureus infection and bacterial invasion of epithelial cells. Among the 517 KO genes specific to skin commensals, 113 were unique to childhood-associated Streptococcus spp and 404 were unique to adult-associated P acnes and Corynebacterium spp. It has been suggested that Streptococcus can inhibit S aureus growth by producing hydrogen peroxide,6Regev-Yochay G. Trzcinski K. Thompson C.M. Malley R. Lipsitch M. Interference between Streptococcus pneumoniae and Staphylococcus aureus: in vitro hydrogen peroxide-mediated killing by Streptococcus pneumoniae.J Bacteriol. 2006; 188: 4996-5001Crossref PubMed Scopus (150) Google Scholar while adult-associated commensals Propionibacterium and Corynebacterium harbor genes involved in porphyrin metabolism and can reduce S aureus infection.7Orenstein A. Klein D. Kopolovic J. Winkler E. Malik Z. Keller N. et al.The use of porphyrins for eradication of Staphylococcus aureus in burn wound infections.FEMS Immunol Med Microbiol. 1997; 19: 307-314Crossref PubMed Scopus (81) Google Scholar In addition, metabolites of adult-associated skin commensals can decrease skin pH and enhance antimicrobial activities, thus suppressing adherence and growth of S aureus in human keratinocytes.4Hannigan G.D. Grice E.A. Microbial ecology of the skin in the era of metagenomics and molecular microbiology.Cold Spring Harb Perspect Med. 2013; 3: a015362Crossref Scopus (70) Google Scholar, 8Shu M. Wang Y. Yu J. Kuo S. Coda A. Jiang Y. et al.Fermentation of Propionibacterium acnes, a commensal bacterium in the human skin microbiome, as skin probiotics against methicillin-resistant Staphylococcus aureus.PLoS One. 2013; 8: e55380Crossref PubMed Scopus (173) Google Scholar, 9Wang Y. Dai A. Huang S. Kuo S. Shu M. Tapia C.P. et al.Propionic acid and its esterified derivative suppress the growth of methicillin-resistant Staphylococcus aureus USA300.Benef Microbes. 2014; 5: 161-168Crossref PubMed Scopus (40) Google Scholar In summary, we identified significant differences in the AD skin microbiome between young children and adults-teenagers. Among many other factors that we examined, including host factors, clinical parameters, disease history, and history of concomitant medications (see Table E7 in this article's Online Repository at www.jacionline.org), we found that the microbiome was also correlated with disease severity in AD lesional skin. This is consistent with previous observations that the skin microbiome changed with disease progression.2Kong H.H. Oh J. Deming C. Conlan S. Grice E.A. Beatson M.A. et al.Temporal shifts in the skin microbiome associated with disease flares and treatment in children with atopic dermatitis.Genome Res. 2012; 22: 850-859Crossref PubMed Scopus (1099) Google Scholar Although AD pathogenic factors drive the disease development, age-specific skin commensals possess various potentials in defending against pathogens and maintaining skin health at different development stages. Our findings, from a new perspective of the skin microbiome, may partly explain the age differences in AD. The sequence data from this study have been deposited to NCBI BioProject accession number 268694. We thank Joanne Streib, Gayle Spears, and Caroline Bronchick for their invaluable assistance in the processing and collection of skin swabs as well as recruitment of study subjects into this Atopic Dermatitis Research Network protocol. We also thank Keli Artis, Denise Babineau, and Alice Lail from Rho Federal Systems Division, Inc, for their help on this study. 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