Optimisation of cutaneous microbiota sampling methodology

Dario Leonardo Balacco ¹ Ajoy Bardhan ^2,3 Hadeer Ibrahim ^1,4 Sarah A. Kuehne ^5,6 Melissa M. Grant ^1,6 Josefine Hirschfeld ^1,6 Adrian H. M. Heagerty ^2,3 Iain L. Chapple ^1,6*

• ¹ Periodontal Research Group, Dentistry, School of Health Sciences, University of Birmingham and Birmingham Dental Hospital (Birmingham Community Healthcare Trust), Birmingham, United Kingdom
• ² School of Health Sciences, University of Birmingham, Birmingham, United Kingdom
• ³ Epidermolysis Bullosa Unit, Department of Dermatology, University Hospitals Birmingham, National Health Service Foundation Trust, Birmingham, United Kingdom
• ⁴ Faculty of Medicine, Suez Canal University, Ismaïlia, Ismailia, Egypt
• ⁵ School of Science and Technology, Nottingham Trent University, Nottingham, United Kingdom
• ⁶ NIHR Birmingham Biomedical Research Centre, Birmingham, United Kingdom

The cutaneous microbiome plays an essential role in guarding against invasive pathogens and maintaining healthy skin homeostasis. Several studies have demonstrated the importance of a healthy skin microbiome through its alteration in several diseases. Differing skin characteristics across the body (temperature, pH, humidity) create distinct ecological niches inhabited by diverse microbial communities. The study of cutaneous microbiota is further complicated by numerous variables at all stages of investigation, including study design, skin sampling method, sample storage, sample processing, sequencing, and data analysis. Utilisation of standardised approaches is critical for reproducibility and comparison between skin microbiome studies. However, there is a notable lack of standardisation of sampling methodologies in the literature. Studies have employed differing sampling strategies and conditions which may affect microbiota characterisation. Here we performed a comparative analysis to determine whether the type of swab (cotton/eSwab), moistening solution (saline solution/phosphate buffered saline), duration of swabbing (30 sec/1 min), and sample storage temperature (room temperature/-80°C) affect sampling and identification of skin microbial communities using 16S sequencing. We report that the conditions analysed did not influence microbiome profiling allowing consistent sampling of the microbiota.