Hair follicle microbiome

In humans, microorganisms within hair follicles differ from those on the skin surface and may contribute to autoimmune skin diseases such as alopecia areata and vitiligo. Owing to the low biomass of hair follicle microorganisms, precise sample acquisition and strategies to avoid contamination with genetic material are critical. To address this, we developed and evaluated a sampling methodology using a nonhuman wildlife proxy model – the wild roe deer (Capreolus capreolus) - to identify effective approaches for minimizing skin contamination prior to biopsy. Animal was obtained during licenced hunts and frozen at -20°C shortly postmortem. Four sets of biopsy and skin surface swab samples were collected from the right front leg: 1) untreated skin; 2) skin cleaned with isopropyl alcohol (IPA) wipes; 3) skin treated with cyanoacrylate glue (CAG); and 4) skin treated with CAG followed by IPA wipes. Swabs were collected via Copan FLOQSwabs, whereas biopsies were obtained with single-use 1 mm biopsy punches. Nucleic acids were subjected to droplet digital PCR (ddPCR) for 16S rRNA and 18S rRNA gene copy number quantification. Additionally, the swab samples were subjected to 16S rRNA V3-4 region and ITS-1 sequencing. Results: ddPCR analysis revealed greater bacterial presence on the skin surface (11,661?5,181 copies/?L) than fungal presence (87?35 copies/?L), and the microbial load was greater in the swab samples (16S: 11,661?5,181; 18S: 87?35 copies/?L) than in the biopsy samples (16S: 76?56; 18S: 8?5 copies/?L). Notably, the use of CAG reduced the microbial load in biopsy samples by 3.7-fold. 16S and ITS sequencing analysis revealed slightly greater alpha diversity in swab samples than in biopsy samples, particularly in areas treated with CAG. Beta diversity revealed distinct clustering of swab and biopsy samples, with shorter distances between CAG-treated samples and untreated samples. Fewer bacterial (n=8) and fungal (n=1) genera were detected in CAG-treated biopsies compared with untreated (n=21, n=9, resp.) and IPA-treated (n=45, n=7, resp.) biopsies. Conclusions: These results demonstrate a high probability of biopsy contamination with microbial DNA originating from the skin surface. They also confirmed that CAG effectively reduces such contamination and can serve as a practical decontamination step before biopsy. Although based on a nonhuman wildlife model, this feasibility study provides methodological insights that can inform the design of future human hair follicle microbiome investigations.