Composition of the human milk microbiome in the GUSTO cohort is shaped by intrapartum antibiotic prophylaxis and breastfeeding exclusivity.

Human milk contains a low biomass microbiome, which is thought to contribute to mammary and infant health. However, the determinants of the human milk microbiome across populations are poorly understood. Here, we characterized the microbiome of 266 milk samples collected from 208 mothers at 3 weeks and 3 months postpartum from the Growing Up in Singapore Toward healthy Outcomes (GUSTO) study, a multi-ethnic Asian cohort. Full-length 16S rRNA gene sequencing showed a substantial level of inter-individual variation, as well as temporal variation, highlighting the need for broad-scale longitudinal sampling in this field. Milk microbiome composition and diversity were associated with maternal, socioeconomic, and breastfeeding factors. In particular, intrapartum antibiotic prophylaxis and breastfeeding exclusivity modulated milk bacterial composition and diversity, even at 3 months postpartum. These results highlight the dynamic nature of the human milk microbiome and its relationship with clinical and breastfeeding factors, with potential implications for infant health.IMPORTANCEHuman milk exposes infants to a constant source of maternal bacteria that may influence the development of the infant immune system and gut microbiome. However, compared to other body niches, the human milk microbiome is relatively under-studied, and there is limited consensus on the factors driving variance in these bacterial communities. In this study, we performed in-depth microbiome profiling of milk samples from 208 mothers in a diverse Asian population, finding a high level of variation between individuals and over time. We found that factors such as delivery-related antibiotics, breastfeeding practices, and maternal lifestyle can influence which bacteria are present in milk. These findings suggest that the milk microbiome is not static, but dynamic and shaped by both medical and social factors. Understanding what drives variance in the milk microbiome could help inform strategies to support maternal and infant health, especially in the critical early months of life when microbial exposures can have long-term effects.