Optimisation of the sampling method for skin microbiome studies in healthy children: a pilot cohort study.

Introduction: Recent interest in the diverse ecosystem of bacteria, fungi and viruses that make up the skin microbiome has led to numerous studies investigating the skin microbiome in healthy skin and in dermatological conditions. However, skin microbiome analysis is challenging due to relatively low numbers of skin microorganisms compared to mucosal sites, such as the respiratory or gastrointestinal tracts. Microbiome results are heavily influenced by sampling methods. Previous sampling methods include that of cotton swabs, tape stripping, patch sampling and punch biopsies. It is essential to have a standardised sampling method for microbiome studies to have comparable results between studies. Two non-invasive methods of sampling the skin microbiome; a skin scraping versus a flocked swab were chosen as methodologies likely to be efficient, effective, and easy to access for future skin microbiome studies in children. Here we compare the two sampling methods to describe the composition of the skin microbiome in healthy children. Method: Samples were collected from six healthy children aged three to nine years from the skin overlying the cubital fossa, cheek and axilla using (i) flocked swabs and (ii) skin scrapings with a glass slide. Samples were collected from the left and right sides of the body at two separate time points, one week apart. Quantitative PCR of the gene encoding 16S ribosomal ribonucleic acid (rRNA) was performed to compare the bacterial load collected by each sampling method. Full-length 16S rRNA gene amplicon sequencing was performed to compare the relationship of sampling method and time with the diversity and ecology of bacteria between different body sites. Results: From six children, 78 flocked swabs and 78 skin scraping samples were collected, along with details of their overall health and skin care practices. qPCR results indicated higher total bacterial load from flocked swabs compared with skin scrapings. Flocked swabs and skin scraping methods had very similar bacterial compositional profiles. The skin microbiome was diverse between individuals and remained relatively stable within individuals over time. Discussion: Overall, results were similar between sample types, however bacterial DNA yield was higher for flocked swab samples (compared to skin scraping methods) and with a simpler protocol is the preferred sampling method for future studies.