Comparison of aseptically and non-aseptically collected human milk samples

Human milk is a low-biomass sample type with a microbiome made up largely of typical skin and oral taxa. Concerns around contamination of milk samples with maternal skin or infant oral bacteria have been raised, leading some researchers to employ aseptic sample collection techniques, including cleansing of the maternal breast/nipple skin and hands. However, to date, no study has directly compared samples collected with and without aseptic technique, leaving questions related to potential contamination within the field. In this study, we have performed metataxonomic analysis on hand-expressed human milk samples from 23 mothers immediately prior to and immediately following cleaning of the hands and breast. Taxonomic profiles were largely unaffected by sample collection technique, with only one species, Rothia mucilaginosa, found to be elevated in samples collected prior to skin cleansing (P = 0.007). This result suggests that the skin and oral taxa typically identified in the human milk microbiome are true residents of the lactating mammary gland, rather than the result of contamination from the maternal skin during sample collection. Although pre- and post-cleaning samples were taxonomically similar, there was a higher level of bacterial richness (P = 0.003) and evenness (Shannon diversity, P = 0.0002) in pre-cleaning samples, suggesting that multiple low-abundance taxa are introduced via skin contamination. Aseptic samples collection protocols are therefore necessary to more accurately characterise the diversity of human milk samples.