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The aim of this study was to investigate the early-life development of the skin microbiome in atopic derma-titis. Nineteen infants with atopic dermatitis and 19 healthy infants were evaluated 3 times, at 3 months intervals, within the first 30 months of life. Tape-strips were collected from volar forearms, cheeks, and ec-zema lesions, and the skin microbiome was assessed by 16S rRNA sequencing. Both the community struc-ture and richness of the skin microbiome of infants with atopic dermatitis differed significantly from that of healthy infants, with greater richness in healthy infants. For infants with atopic dermatitis, the community composition was not dominated by Staphylo-cocci. For healthy infants, community composition and richness correlated significantly with age, while such a pattern was not revealed in infants with atopic der-matitis. This suggests a slower maturation of the skin microbiome in atopic dermatitis, which precedes the staphylococcal predominance observed in older children and adults.

Background: Hand eczema (HE) is frequently associated with Staphylococcus aureus; however, its role in the pathogenesis of HE is poorly understood. Objective: To investigate the temporal variation in S aureus subtypes, ie, clonal complex (CC) types, on the hands and relate it to S aureus colonization in the nose and severity in a cohort of HE patients. Methods: S aureus from the hands and nose of 50 adult HE patients and 50 controls was prospectively identified at 5 visits over 3 weeks. Results: S aureus was identified on the hands of 23 (46%) patients at 2 or more visits and on the hands of 1 control once. Of the HE patients with S aureus colonization, 78% had the same S aureus CC type over time. Twenty-one patients had the same S aureus CC type on the hands and in the nose. Persistent colonization was strongly related to an increased disease severity. Limitations: A relatively small S aureus culture-positive population. Conclusion: The temporal stability of S aureus CC type and high occurrence of the identical subtypes on the hands and in the nose imply that S aureus colonization in patients with HE is of a more permanent nature. Taken together with the finding that persistent colonization and HE severity are clearly related, our results indicate that S aureus may contribute to the perpetuating course of HE.

Several factors have been shown to influence the composition of the bacterial communities inhabiting healthy skin, with variation between different individuals, differing skin depths, and body locations (spatial-temporal variation). Atopic dermatitis (AD) is a chronic skin disease also affecting the skin-associated bacterial communities. While the effects of AD have been studied on these processes individually, few have considered how AD disrupts the spatial-temporal variation of the skin bacteria as a whole (i.e., considered these processes simultaneously). Here, we characterized the skin-associated bacterial communities of healthy volunteers and lesional and nonlesional skin of AD patients by metabarcoding the universal V3-V4 16S rRNA region from tape strip skin samples. We quantified the spatial-temporal variation (interindividual variation, differing skin depths, multiple time points) of the skin-associated bacteria within healthy controls and AD patients, including the relative change induced by AD in each. Interindividual variation correlated with the bacterial community far more strongly than any other factors followed by skin depth and then AD status. There was no significant temporal variation found within either AD patients or healthy controls. The bacterial community was found to vary markedly according to AD severity, and between patients without and with filaggrin mutations. Therefore, future studies may benefit from sampling subsurface epidermal communities and considering AD severity and the host genome in understanding the role of the skin bacterial community within AD pathogenesis rather than considering AD as a presence-absence disorder. IMPORTANCE The bacteria associated with human skin may influence skin barrier function and the immune response. Previous studies have attempted to understand the factors that regulate the skin bacteria, characterizing the spatial-temporal variation of the skin bacteria within unaffected skin. Here, we quantified

Background: A growing body of evidence links various biomarkers to atopic dermatitis (AD). Still, little is known about the association of specific biomarkers to disease characteristics and severity in AD. Objective: To explore the relationship between various immunological markers in the serum and disease severity in a hospital cohort of AD patients. Methods: Outpatients with AD referred to the Department of Dermatology, Bispebjerg Hospital, Copenhagen, Denmark, were divided into groups based on disease severity (SCORAD). Serum levels of a preselected panel of immunoinflammatory biomarkers were tested for association with disease characteristics. Two machine learning models were developed to predict SCORAD from the measured biomarkers. Results: A total of 160 patients with AD were included; 53 (33.1%) with mild, 73 (45.6%) with moderate, and 34 (21.3%) with severe disease. Mean age was 29.2 years (range 6-70 years) and 84 (52.5%) were females. Numerous biomarkers showed a statistically significant correlation with SCORAD, with the strongest correlations seen for CCL17/thymus and activation-regulated chemokine (chemokine ligand-17/TARC) and CCL27/cutaneous T cell-attracting-chemokine (CTACK; Spearman R of 0.50 and 0.43, respectively, p < 0.001). Extrinsic AD patients were more likely to have higher mean SCORAD (p < 0.001), CCL17 (p < 0.001), CCL26/eotaxin-3 (p < 0.001), and eosinophil count (p < 0.001) than intrinsic AD patients. Predictive models for SCORAD identified CCL17, CCL27, serum total IgE, IL-33, and IL-5 as the most important predictors for SCORAD, but with weaker associations than single cytokines. Conclusions: Specific immunoinflammatory biomarkers in the serum, mainly of the Th2 pathway, are correlated with disease severity in patients with AD. Predictive models identified biomarkers associated with disease severity but this finding warrants further investigation.

Atopic dermatitis (AD) is a common inflammatory skin disease characterized by an epidermal barrier impairment, as well as a Th2/Th22-skewed immune response, both favoring skin colonization with Staphylococcus aureus. Colonization is strongly related to severity of the disease, and a reduction of S. aureus has been found to alleviate symptoms. Lactic acid bacteria (LAB) produce antimicrobial compounds such as organic acids and bacteriocins and are widely used as probiotics. The aim of this study was to isolate LAB and screen for antibacterial effect specifically toward S. aureus clonal complex type 1. A total of 680 LAB were isolated from fermented vegetables and swab samples from healthy volunteers (vaginal, stool and skin). Screening for antibacterial activity toward S. aureus, narrowed the field of isolates down to four LAB strains with high antibacterial activity. The activity varied according to the specific LAB strain and the origin of the strain. The results suggested different modes of action, including co-aggregation, expression of bacteriocins and production of specific organic acids. However, the ability to acidify the surroundings appeared as the main effect behind inhibition of S. aureus. Broth microdilution assays showed a significant reduction of S. aureus growth when using down to 10% cell free supernatant (CFS). Our results underline the use of specific living LAB or their CFS as potential future treatment strategies to reduce S. aureus colonization of AD skin.

Background: Atopic dermatitis (AD) patients have an altered skin bacterial community, with an abundance of Staphylococcus aureus associated with flares, highlighting that microbial organisms may be important for disease exacerbation. Despite strong evidence of association between bacterial skin colonisation and AD, very limited knowledge regarding the eukaryotic microbial community, including fungi and ectoparasites, in AD exists. In this study, we compared the skin and nasal eukaryotic microbial community between adult AD patients (n = 55) and non-AD healthy controls (n = 45) using targeted 18S rRNA amplicon sequencing. Analysis was based on the presence or absence of eukaryotic microorganisms. Results: The cutaneous composition of the eukaryotic microbial community and the alpha-diversity differed significantly between AD patients and non-AD individuals, with increased species richness on AD skin. Alpha-diversity and beta-diversity were similar on lesional and non-lesional skin of patients. The ectoparasite Demodex folliculorum and the yeast Geotrichum candidum were significantly more prevalent on the skin of AD patients. The prevalence of D. folliculorum on lesional skin was greater among patients recently treated with topical corticosteroid. Malassezia was one of the most frequently detected genera at all sites, with M. globosa and M. restricta being the most prevalent. M. restricta was under represented in the anterior nares of AD patients as compared to the non-AD control population. Conclusion: Significant differences in the eukaryotic microbial communities were found between AD patients and non-AD individuals, with the most striking finding being the significantly overrepresentation of D. folliculorum on AD skin. Whether D. folliculorum can contribute to skin inflammation in AD needs further investigation.

The skin microbiota of atopic dermatitis (AD) patients is characterized by increased Staphylococcus aureus colonization, which exacerbates disease symptoms and has been linked to reduced bacterial diversity. Skin bacterial communities in AD patients have mostly been described at family and genus levels, while species‐level characterization has been limited. In this study, we investigated the role of the bacteria belonging to the Staphylococcus genus using targeted sequencing of the tuf gene with genus‐specific primers. We compared staphylococcal communities on lesional and non‐lesional skin of AD patients, as well as AD patients with healthy controls, and determined the absolute abundance of bacteria present at each site. We observed that the staphylococcal community, bacterial alpha diversity, and bacterial densities were similar on lesional and non-lesional skin, whereas AD severity was associated with significant changes in staphylococcal composition. Increased S. aureus, Staphylococcus capitis, and Staphylococcus lugdunensis abundances were correlated with increased severity. Conversely, Staphylococcus hominis abundance was negatively correlated with severity. Furthermore, S. hominis relative abundance was reduced on AD skin compared to healthy skin. In conclusion, various staphylococcal species appear to be important for skin health.

We explored differences between lesional and non-lesional skin and anatomical variation in epidermal biomarker levels using stratum corneum (SC) tape strips in adult patients with atopic dermatitis (AD). SC tape strips from three distinct anatomical locations (upper extremity, trunk and lower extremity) and serum samples were collected from 25 adult patients with AD (mean age 34.8 years). Inflammatory cytokines in tape strips and serum, natural moisturizing factor (NMF), skin pH, and trans-epidermal water loss (TEWL) were measured. Disease severity was assessed using SCORing of Atopic Dermatitis (SCORAD) scores, respectively, global (overall) and local (lesion specific based on objective signs) SCORAD(1).

Tape stripping is a promising technique for assessment of epidermal biomarkers in inflammatory skin diseases. However, to facilitate its implementation in the clinical practice, a thorough validation regarding sampling strategy is needed. Knowledge of biomarkers variation in concentration across stratum corneum is scarce. Therefore, this study aimed to assess the variability of cytokines across stratum corneum using tape stripping technique by consecutive application of 21 adhesive tapes (D-squame) to lesional and non-lesional skin from 15 patients with atopic dermatitis (AD) and 16 healthy controls. Concentration of cytokines (IL-1α, IL-1b, IL-5, IL-18, IFN-γ, CCL17, CCL22, CCL27, CXCL8, CXCL10, TNF-α, TSLP, VEGFA) was determined in five different depths, using multiplex immunoassay. Comparing tape 4 with tape 21, no cytokine changed significantly in concentration in AD lesional skin. In AD non-lesional skin a small decrease was found for CCL17, CXCL8 and CXCL10. For healthy controls, a decrease was found for IL-1a, IL-1b, VEGFA and an increase for IL-18. Differences were found between AD skin and healthy control skin. Concentration of cytokines was stable across stratum corneum, indicating that sampling of only one tape from the stratum corneum is reliable in reflecting the overall cytokine milieu. Differences between AD and healthy skin confirm robustness of tape stripping for measuring cytokine levels.

Background: While Staphylococcus aureus (S. aureus) colonization has been thoroughly studied in atopic dermatitis (AD), where S. aureus is related to flares and considered a trigger factor, S. aureus colonization in hand eczema (HE) has only been sparsely studied. Objectives: To examine the 1-week prevalence of S. aureus colonization in HE patients, and its association with severity, HE subtype, AD, and nasal S. aureus colonization compared with healthy controls. Methods: In a case-control study of 50 adult HE patients and 50 healthy controls, bacterial swabs from lesional skin (patients only), non-lesional skin (dorsal hand), and the nasal cavity were sampled for culturing of S. aureus on days 1, 3, 5 and 8. Participants were characterized by demographics, AD, HE subtype, filaggrin gene mutation status, and HE severity. Results: Twenty-seven HE patients (54%) were colonized with S. aureus on the hand compared to one control (2%) (P <.01). Nasal S. aureus colonization was found in 72% of patients and 22% of controls (P <.01). For patients, S. aureus colonization on the hands was associated with an atopic HE subtype and HE severity (P =.01 and P <.01, respectively). Conclusions: Both hand and nasal S. aureus colonization were highly prevalent among HE-patients and may have an impact on the persistence of HE.

Tape-stripping is a minimally invasive approach for skin sampling that captures the cutaneous immune/barrier abnormalities in atopic dermatitis (AD). However, tape-strips have not been used to evaluate molecular changes with therapeutic targeting. In this study, we sought to characterize the proteomic signature of tape-strips from AD patients, before and after dupilumab therapy. Twenty-six AD patients were treated with every-other-week dupilumab 300 mg for 16 weeks. Tape-strips from lesional and non-lesional skin were collected before and after treatment, and analyzed with the Olink proteomic assay. Using criteria of fold-change>1.5 and FDR < 0.05, 136 proteins significantly decreased after dupilumab treatment, corresponding to an overall mean improvement of 66.2% in the lesional vs. non-lesional AD proteome. Significant decreases after dupilumab were observed in immune markers related to general inflammation (MMP12), Th2 (CCL13/CCL17), Th17/Th22 (IL-12B, CXCL1, S100A12), and innate immunity (IL-6, IL-8, IL-17C), while the Th1 chemokines CXCL9/CXCL10 remained elevated. Proteins related to atherosclerosis/cardiovascular risk (e.g., SELE/E-selectin, IGFBP7, CHIT1/ chitotriosidase-1, AXL) also significantly decreased after treatment. Dupilumab therapy suppressed AD-related immune biomarkers and atherosclerosis/cardiovascular risk proteins. Tape-strip proteomics may be useful for monitoring therapeutic response in real-life settings, clinical trials, and longitudinal studies for AD and beyond.

Introduction. The pathogenesis of atopic dermatitis (AD) is not yet fully understood, but the bacterial composition of AD patients' skin has been shown to have an increased abundance of Staphylococcus aureus. More recently, coagulasenegative Staphylococcus (CoNS) species were shown to be able to inhibit S. aureus, but further studies are required to determine the effects of Staphylococcus community variation in AD.Aim. Here we investigated whether analysing metabarcoding data with the more recently developed DADA2 approach improves metabarcoding analyses compared to the previously used operational taxonomic unit (OTU) clustering, and can be used to study Staphylococcus community dynamics.Methods. The bacterial 16S rRNA region from tape strip samples of the stratum corneum of AD patients (nonlesional skin) and non AD controls was metabarcoded. We processed metabarcoding data with two different bioinformatic pipelines (an OTU clustering method and DADA2), which were analysed with and without technical replication (sampling strategy).Results. We found that OTU clustering and DADA2 performed well for community level studies, as demonstrated by the identification of significant differences in the skin bacterial communities associated with AD. However, the OTU clustering approach inflated bacterial richness, which was worsened by not having technical replication. Data processed with DADA2 likely handled sequencing errors more effectively and thereby did not inflate molecular richness.Conclusion. We believe that DADA2 represents an improvement over an OTU clustering approach, and that biological replication rather than technical replication is a more effective use of resources. However, neither OTU clustering nor DADA2 gave insights into Staphylococcus community dynamics, and caution should remain in not overinterpreting the taxonomic assignments at lower taxonomic ranks.

Background: Initially after tattooing, the skin barrier function is broken. However, the long-term impact of clinically healed tattoos on this has never been studied. The aim was to investigate the long-term effect on the skin barrier function in normal tattoos and examples of tattoos with chronic inflammatory complication. Methods: Participants were recruited from the “Tattoo clinic” of the Dermatological Department on Bispebjerg Hospital in Denmark, where patients with complicated tattoo reactions are treated. Transepidermal water loss (TEWL), conductance, capacitance, and pH were measured in tattooed skin with regional control measurements in normal non-tattooed skin. Natural moisturizing factor (NMF) was measured in collected tape strips. Results: Twenty six individuals with 28 tattoos were included, that is, 23 normal tattoos without any pathologic reaction and 5 tattoos with chronic inflammatory complications. No significant differences were found in tattooed versus non-tattooed skin with respect to TEWL (median values 6.6 vs 7.2 g/m 2 /h), conductance (76 vs 78 a.u.), pH (5.94 vs 5.79), and NMF (0.58 vs 0.59 mmol/g protein). Capacitance (64 vs 57 a.u.) was higher in tattooed skin compared to non-tattooed skin (P = 0.006). Similar results were found in tattoos with inflammatory reactions. Conclusion: Overall, skin tattoos do not affect the long-term skin barrier function markedly. The skin capacitance was, however, affected in tattooed skin areas compared to non-tattooed skin areas.

Background: Treatment of atopic dermatitis (AD) may be challenging, therefore some patients seek complementary and alternative medications (CAM). We determined prevalence and predictors for CAM use in a hospital cohort of AD patients. Material and Methods: Between January 1, 2012, and December 31, 2017, AD patients referred to the dermatological outpatient clinic at Bispebjerg Hospital were included in the study. Information on CAM use, demographics and disease characteristics were obtained by questionnaire, and associations were determined by X-2 and t test separately for children (<16 years) and adults (>= 16 years). Results: In total 441 filled in the questionnaire on AD, and 433 patients responded to the questions about CAM use: 198 children and 235 adults. A total of 137 (31.6%) had used one or more CAM. CAM use in children was significantly associated with prior use of >= 2 conventional treatments (p = 0.047) and topical calcineurin inhibitors (p = 0.021), a higher number of affected eczema sites (p < 0.001) including more frequent affection of the face and extremities, a higher SCORAD score (p = 0.045), and low mean overall self-rated health (p = 0.003). CAM use in adults was significantly associated with lower age of onset of AD (p = 0.004), comorbid allergic rhinoconjunctivitis (p = 0.039), frequent use of moisturizing cream (p = 0.024), facial and neck eczema (p = 0.005) and high educational level (p = 0.043). Conclusion: CAM use is frequent in both children and adult AD patients. CAM users are characterized by long disease duration, a significant disease burden and by having a longer education. The high prevalence of CAM may indicate that patients' expectations regarding treatment of AD are not redeemed in the conventional health care system. (C) 2019 S. Karger AG, Basel

Background: Staphylococcus aureus skin colonization is common in patients with atopic dermatitis (AD) and is associated with risk of skin infections. AD patients therefore often receive antibiotic treatments, including topical treatment with fusidic acid, which have been associated with resistance development. Objectives: To examine the prevalence of antibiotic resistance in S. aureus isolated from Danish AD patients, with a primary focus on fusidic acid resistance and the genetic mechanisms that underlie it. Methods: One hundred and thirty-eight S. aureus isolates collected from lesional skin (n=54), non-lesional skin (n=27) and anterior nares (n=57) from 71 adult AD patients were included in the study. Isolates were tested for susceptibility to 17 selected antibiotics. S. aureus whole-genome sequences were used to examine the genetic determinants of fusidic acid resistance (fusA or fusE mutations or carriage of fusB or fusC genes). Results: One hundred and nine isolates (79%) were resistant to at least one of the tested antibiotics, with the most prevalent resistances being to penicillin (55%), fusidic acid (41%) and erythromycin (11%). The primary genetic mechanisms of fusidic acid resistance were carriage of fusC (57%) or mutations in fusA (38%). The most prevalent S. aureus lineage was ST1 (23%). All ST1 isolates carried fusC. Conclusions: S. aureus fusidic acid resistance, caused by either fusA mutations or fusC gene carriage, is a major concern among AD patients. Resistant S. aureus might spread from the patients to the community, indicating the need to reduce the use of fusidic acid in the treatment of AD.

IMPORTANCE Skin microbiome correlates with disease severity for lesional and nonlesional skin, indicating a global influence of atopic dermatitis (AD). A relation between skin microbiome and filaggrin gene (FLG) mutations proposes a possible association between skin microbiome and host genetics. OBJECTIVES To assess skin and nasal microbiome diversity and composition in patients with AD and compare with healthy controls, and to investigate the microbiome in relation to disease severity and FLG mutations in patients with AD. DESIGN, SETTING, AND PARTICIPANTS An observational case-control study of 45 adult healthy controls and 56 adult patients with AD was carried out from January 2015 to June 2015 in a tertiary referral center, Department of Dermatology, Bispebjerg Hospital, Denmark. EXPOSURES Bacterial swabs were taken from patients with AD (lesional skin, nonlesional skin, and anterior nares) and from healthy controls (nonlesional skin and anterior nares). Eczema severity was assessed and FLG mutations noted. Bacterial DNA was extracted from swabs, and V3-V4 16S rDNA regions amplified with PCR. Samples were analyzed at Statens Serum Institut September 2015 to September 2016. Bioinformatics analyses of the microbiome were analyzed using R statistical software (version 3.3.1, R Foundation Inc). MAIN OUTCOMES AND MEASURES Skin microbiomeswere investigated using next-generation sequencing targeting 16S ribosomal RNA. RESULTS Microbiome alpha diversity was lower in patients with AD compared with healthy controls in nonlesional skin (effect size, 0.710; 95%CI, 0.27-1.15; P = .002), lesional skin (effect size, 0.728; 95%CI, 0.35-1.33; P = .001), and nose (effect size, 1.111; 95%CI, 0.48-0.94; P < .001). Alpha diversity was inversely correlated with disease severity for lesional (effect size, 0.530; 95%CI, 0.23-1.64; P = .02) and nonlesional skin (effect size, 0.451; 95%CI, 0.04-2.44; P = .04) in patients with AD. Microbiome composition in AD nonlesional skin was lin

Decreased levels of antimicrobial peptides (AMPs) in atopic dermatitis (AD) have previously been reported and have been linked to the increased susceptibility to skin infections found in AD patients. This study intents to identify AMPs: hBD-2, hBD-3, RNase7, psoriasin and LL-37 in AD patients and healthy controls, and determine concentrations in consecutive depths of the outer most skin layers. Tape stripping was used on lesional and non-lesional skin. From each skin site, 35 consecutive tape strips were collected and pooled in groups of 5. Commercially available ELISA kits were used to determine AMP concentration in stratum corneum samples. hBD-2, hBD-3, RNase7 and psoriasin were identified in stratum corneum samples. hBD-3-level was markedly higher in AD non-lesional skin compared to healthy controls, and a similar trend was observed for RNase7. Most AMPs were distributed evenly through 35 tape strips, implying a homogeneous distribution of antimicrobial defense in the outer most skin layers. The findings indicate that AD patients may not suffer from a general baseline deficiency in AMPs, and that the innate immune defense is present throughout the stratum corneum, both insights of importance for understanding the role of AMPs in AD.