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Atopic dermatitis (AD) is an inflammatory skin disease with a microbiome dysbiosis towards a high relative abundance of Staphylococcus aureus. However, information is missing on the actual bacterial load on AD skin, which may affect the cell number driven release of pathogenic factors. Here, we combined the relative abundance results obtained by next-generation sequencing (NGS, 16S V1-V3) with bacterial quantification by targeted qPCR (total bacterial load = 16S, S. aureus = nuc gene). Skin swabs were sampled cross-sectionally (n = 135 AD patients; n = 20 healthy) and longitudinally (n = 6 AD patients; n = 6 healthy). NGS and qPCR yielded highly inter-correlated S. aureus relative abundances and S. aureus cell numbers. Additionally, intra-individual differences between body sides, skin status, and consecutive timepoints were also observed. Interestingly, a significantly higher total bacterial load, in addition to higher S. aureus relative abundance and cell numbers, was observed in AD patients in both lesional and non-lesional skin, as compared to healthy controls. Moreover, in the lesional skin of AD patients, higher S. aureus cell numbers significantly correlated with the higher total bacterial load. Furthermore, significantly more severe AD patients presented with higher S. aureus cell number and total bacterial load compared to patients with mild or moderate AD. Our results indicate that severe AD patients exhibit S. aureus driven increased bacterial skin colonization. Overall, bacterial quantification gives important insights in addition to microbiome composition by sequencing.

Our skin influences our physical and mental health, and its chemical composition can reflect environmental and disease conditions. Therefore, through sampling the skin metabolome, we can provide a promising window into the mechanisms of the body. However, the broad application of skin metabolomics has recently been hampered by a lack of easy and widely applicable sampling methods. Here, we present a novel rapid, simple, and, most importantly, painless and non-invasive sampling technique suitable for clinical studies of fragile or weakened skin. The method is called WET PREP and is simply a lavage of the skin which focuses on capturing the metabolome. We systematically evaluate WET PREPs in comparison with the non-invasive method of choice in skin metabolomics, swab collection, using ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS2) on two complementary chromatographic columns (C18 reversed phase and hydrophilic interaction chromatography). We also integrate targeted analyses of key metabolites of skin relevance. Overall, WET PREP provides a strikingly more stable shared metabolome across sampled individuals, while also being able to capture unique individual metabolites with a high consistency in intra-individual reproducibility. With the exception of (phospho-)lipidomic studies, we recommend WET PREPs as the preferred skin metabolome sampling technique due to the quick preparation time, low cost, and gentleness for the patient.