Gastrointestinal and Nasopharyngeal Microbiota profiles and Metabolomic changes over the course of lactation in Gambian mother/infant pairs up to 60 days of age Authors Konstantinos Karampatsas1, Sean Aller1, Isabel Garcia Perez2, Alex Shaw2, Sora Liu2, Amadou Faal3, Mustapha Jaiteh3, Adam Witney1, Kirsty Le Doare1,2 Affiliations 1 St George’s, University of London, London, United Kingdom 2 Imperial College London, London, United Kingdom 3 Medical Research Council- The Gambia Unit, Fajara, The Gambia Background: Exclusive breastfeeding for the first 6 months of life is essential for infants’ healthy development. Microbiota composition in breastmilk affects intestinal microbiota colonisation and the development of the immune system in infants. The metabolomic content of breastmilk is thought to interact with the microbiota and thus may influence the developing infant immune system. Methods: In this study we characterised the microbiota of healthy, vaginally delivered, exclusively breastfed Gambian infants. 107 mothers and their infants were included in the study. We successfully sequenced 68 breast milk samples, 51 maternal rectovaginal swabs and 35 infants’ rectal swabs at birth. We also collected 70 breast milk samples and 51 infants’ nasopharyngeal swabs 60 days post-delivery. We used 16S rRNA gene sequencing to assess the microbiota composition and compare it between mothers and infants across the different body sites. After DNA extraction, 16S amplicon libraries were generated and sequenced. Taxonomic and functional analyses were then performed. Colostrum and mature breast milk samples also underwent metabolomic profiling using a multiplatform approach combining 1H nuclear magnetic resonance spectroscopy and Gas chromatography-mass spectrometry Results: Bacterial communities were distinct in breastmilk (BM), maternal rectovaginal swabs and infant rectal swabs, differing in both composition and diversity. BM composition changed over the first 60 days of lactation. The relative abundance of α-1,4- and α-1,3-Fucosylated HMOs increased over time. We found changes in 33 metabolites between birth and day 60 of life, such as monosaccharides, sugar alcohols and fatty acids known to be important for infant immunological, neurological, and gastrointestinal development, as well as being an important source of energy. Conclusions: The results of this study indicate that infant gut microbiota at birth is a unique bacterial community in transition. Breast milk microbiota composition and metabolomic profile change throughout lactation. These changes may contribute to the development of the infant microbiome according to the changing needs of the growing infant. Further studies are required to fully understand the interplay between the metabolome, microbiome and gut immunome to fully understand their role in infant gut immune development.