A simplified bacterial community found within the epidermis than at the epidermal surface of atopic dermatitis patients and healthy controls.

There has been considerable research into the understanding of the healthy skin microbiome. Similarly, there is also a considerable body of research into whether specific microbes contribute to skin disorders, with atopic dermatitis (AD) routinely linked to increased Staphylococcus aureus (S. aureus) colonisation. In this study, the epidermal surface of participants was sampled using swabs, while serial tape-stripping (35 tapes) was performed to sample through the stratum corneum. Samples were taken from AD patients and healthy controls, and the bacterial communities were profiled by metabarcoding the universal V3-V4 16S rRNA region. Results show that the majority of bacterial richness is located within the outermost layers of the stratum corneum, however there were many taxa that were found almost exclusively at the very outermost layer of the epidermis. We therefore hypothesise that tape-stripping can be performed to investigate the 'core microbiome' of participants by removing environmental contaminants. Interestingly, significant community variation between AD patients and healthy controls was only observable at the epidermal surface, yet a number of individual taxa were found to consistently differ with AD status across the entire epidermis (i.e. both the epidermal surface and within the epidermis). Sampling strategy could therefore be tailored dependent on the hypothesis, with sampling for forensic applications best performed using surface swabs and outer tapes, while profiling sub-surface communities may better reflect host genome and immunological status.

Hand eczema and temporal variation of Staphylococcus aureus clonal complexes: A prospective observational study.

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.

Skin Microbiome in Patients with Hand Eczema and Healthy Controls: A Three-week Prospective Study.

The pathogenesis of chronic hand eczema remains unclear. Insights into the skin microbiome in hand eczema and its potential relevance to disease severity may help to elucidate the underlying mechanisms of hand eczema. The aim of this study was to characterize the microbiome in patients with hand eczema and healthy controls. A 5-visit prospective study was conducted over a period of 3 weeks. At each visit, bacterial swabs were taken from the hands of patients with hand eczema and controls. The microbiome was examined using DNA extraction and 16S rRNA amplicon sequencing (V3-V4 regions). Fifty patients with hand eczema and 50 controls were included (follow-up rate=100%). The baseline bacterial α-diversity was reduced on the hands of patients with hand eczema compared with controls (effect size=-0.31; 95% confidence interval (95% CI) -0.50; -0.11; p = 0.003). The dysbiosis on the patients' hands was stable over the study period, was associated with disease severity, and was characterized by reduced bacterial diversity and different bacterial community compositions.

Altered Maturation of the Skin Microbiome in Infants with Atopic Dermatitis.

The aim of this study was to investigate the early-life development of the skin microbiome in atopic dermatitis. 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 eczema lesions, and the skin microbiome was assessed by 16S rRNA sequencing. Both the community structure 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 Staphylococci. For healthy infants, community composition and richness correlated significantly with age, while such a pattern was not revealed in infants with atopic dermatitis. This suggests a slower maturation of the skin microbiome in atopic dermatitis, which precedes the staphylococcal predominance observed in older children and adults.

Identification of cutaneous fungi and mites in adult atopic dermatitis: analysis by targeted 18S rRNA amplicon sequencing.

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.

Immunoinflammatory Biomarkers in Serum Are Associated with Disease Severity in Atopic Dermatitis.

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.

Colonization with Staphylococcus aureus in patients with hand eczema: Prevalence and association with severity, atopic dermatitis, subtype and nasal colonization.

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.

Use of Complementary and Alternative Therapies in Outpatients with Atopic Dermatitis from a Dermatological University Department.

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 χ2 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.

Tattoos and skin barrier function: Measurements of TEWL, stratum corneum conductance and capacitance, pH, and filaggrin.

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/m2 /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.

Genomic analysis reveals different mechanisms of fusidic acid resistance in Staphylococcus aureus from Danish atopic dermatitis patients.

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.

Current knowledge on biomarkers for contact sensitization and allergic contact dermatitis.

Contact sensitization is common and affects up to 20% of the general population. The clinical manifestation of contact sensitization is allergic contact dermatitis. This is a clinical expression that is sometimes difficult to distinguish from other types of dermatitis, for example irritant and atopic dermatitis. Several studies have examined the pathogenesis and severity of allergic contact dermatitis by measuring the absence or presence of various biomarkers. In this review, we provide a non-systematic overview of biomarkers that have been studied in allergic contact dermatitis. These include genetic variations and mutations, inflammatory mediators, alarmins, proteases, immunoproteomics, lipids, natural moisturizing factors, tight junctions, and antimicrobial peptides. We conclude that, despite the enormous amount of data, convincing specific biomarkers for allergic contact dermatitis are yet to be described.

In vivo expression of antimicrobial peptides in atopic dermatitis.

The aim of this review is to present findings on expression of antimicrobial peptides (AMPs) in atopic dermatitis (AD) skin, focusing only on in vivo studies, and to discuss differences in results obtained using various skin sampling techniques and different methodology for analysis of AMPs. The review also includes a discussion of the effect of frequently used treatments on AMP expression. Many studies have shown a reduced level of AMPs in lesional AD skin when compared to psoriatic skin, explaining the high frequency of AD-related infections. Interestingly, however, non-lesional AD skin has shown the same upregulation of AMPs after barrier disruption as non-lesional psoriatic skin. Various methods have been used to analyse AMP expression in the skin, and when comparing these methods, differences are revealed in AMP expression depending on the method used for sampling and analysis. Comparisons indicate that analyses of mRNA levels of AMPs may find greater differences in expression than analyses of protein levels. Few studies evaluate the effect of topical treatments on the expression of AMPs, and these indicate an inhibition of AMP expression, particularly after use of corticosteroids. AMPs are important components of the skin as a defense against infections, and despite much research, the clinical importance of the effect of common treatments, including systemic treatments for AD and the interplay between AMPs and the skin microbiome, is still largely unknown.

Effect of glove occlusion on the skin barrier.

Wet work tasks are the most common exposures leading to occupational irritant contact dermatitis. Use of liquid-proof gloves is recommended when performing wet work, however, gloves may also contribute to impairment of the skin barrier and development of irritant contact dermatitis. The aim of this study is to review the literature on the effects of glove occlusion on skin barrier function. The PubMed database was searched up to 1 February 2015 for articles on the association between glove occlusion and skin barrier function, including human studies only and in English. Only experimental studies including assessment of the skin barrier function were included in the data analysis. Thirteen articles were identified, 8 with focus on occlusion alone, 7 with focus on occlusion in combination with irritant exposure (some overlapping), and 2 field studies. In conclusion, data from the literature showed that the negative effect of occlusion in itself is limited, and that only extensive and long-term occlusion will cause barrier impairment. However, studies investigating combined effect of occlusion and exposure to soaps/detergents indicate that occlusion significantly enhances the skin barrier damage caused by detergents/soaps in a dose-response fashion.

Temporal and Spatial Variation of the Skin-Associated Bacteria from Healthy Participants and Atopic Dermatitis Patients.

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 the effect of AD on the skin bacteria on multiple spatial-temporal factors simultaneously. Although significant community variation between healthy controls and AD patients was observed, the effects of AD on the overall bacterial community were relatively low compared to other measured factors. Results here suggest that changes in specific taxa rather than wholesale changes in the skin bacteria are associated with mild to moderate AD. Further studies would benefit from incorporating the complexity of AD into models to better understand the condition, including AD severity and the host genome, alongside microbial composition.

Targeted Screening of Lactic Acid Bacteria With Antibacterial Activity Toward Staphylococcus aureus Clonal Complex Type 1 Associated With Atopic Dermatitis.

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.

Staphylococcal Communities on Skin Are Associated with Atopic Dermatitis and Disease Severity.

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.

Changes in Skin and Nasal Microbiome and Staphylococcal Species Following Treatment of Atopic Dermatitis with Dupilumab.

Investigation of changes in the skin microbiome following treatment of atopic dermatitis (AD) with dupilumab may provide valuable insights into the skin microbiome as a therapeutic target. The aim of this study is to assess changes in the AD skin microbiome following treatment of AD with dupilumab (n = 27). E-swabs were collected from nose, lesional, and nonlesional skin before and after 16 weeks of dupilumab therapy, and the microbiome was analyzed by 16S rRNA and tuf gene sequencing. Data for 17 patients with milder disease receiving treatment with non-targeted therapies are also presented. The results show that both groups experienced clinical improvement (p < 0.001) following dupilumab therapy and that Shannon diversity increased and bacterial community structure changed. The relative abundance of the genus Staphylococcus (S.) and S. aureus decreased, while that of S. epidermidis and S. hominis increased. No significant changes were observed for patients receiving non-targeted treatments. The increases in S. epidermidis and S. hominis and the decrease in S. aureus correlated with clinical improvement. Furthermore, changes in S. hominis and S. epidermidis correlated inversely with S. aureus. In conclusion, treatment with dupilumab significantly changed the skin microbiome and decreased S. aureus. Our results suggest a favorable role of commensal staphylococci in AD.

Transcriptomic Profiling of Tape-Strips From Moderate to Severe Atopic Dermatitis Patients Treated With Dupilumab.

BACKGROUND: Tape-strips are a minimally invasive approach to characterize skin biomarkers in atopic dermatitis (AD). However, they have not yet been used for tracking gene expression changes with systemic treatment. OBJECTIVE: The aim of the study was to evaluate gene expression changes and therapeutic response biomarkers in AD patients before and after dupilumab (interleukin 4Rα antibody) treatment using tape-strips to obtain epidermal tissue for analysis. METHODS: Lesional and nonlesional tape-stripped skin was sampled from 18 AD patients before and after dupilumab treatment and from 17 healthy subjects and analyzed by RNA-seq. RESULTS: At baseline, we detected 6745 and 4859 differentially expressed genes between lesional and nonlesional skin versus normal, respectively, whereas 841 and 977 genes were differentially expressed after treatment, respectively (fold change >1.5 and false discovery rate <0.05). Tape-strips captured significant modulation with dupilumab in key AD immune (eg, C-C motif chemokine ligand 13 [CCL13], CCL17, CCL18) and barrier (eg, periplakin, FA2H) biomarkers. Changes in biomarkers (CCL20, interleukin 34, FABP7) were also significantly correlated with clinical disease improvements (Eczema Area and Severity Index; R > 0.5 or R < -0.4, P < 0.05). CONCLUSIONS: This real-life study represents the first comprehensive RNA-seq molecular profiling of tape-strips from moderate to severe AD patients after dupilumab therapy. Analysis of tape strip specimens detected significant gene expression changes in key AD biomarkers with dupilumab treatment, suggesting that this approach may be useful to monitor therapeutic responses in inflammatory skin diseases.

Cytokine concentration across the stratum corneum in atopic dermatitis and healthy controls.

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.

Comparing DADA2 and OTU clustering approaches in studying the bacterial communities of atopic dermatitis.

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, coagulase-negative 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 (non-lesional 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.