Human milk microbiome is shaped by breastfeeding practices.

There is evidence that breastfeeding practices may impact the milk microbiota diversity and differential abundance at the genera level; however, the possibility that distinct feeding practices, such as exclusive (EBF) and non-exclusive breastfeeding (non-EBF), might alter the milk microbiome at the species level has not been explored. This cross-sectional study analyzed the milk microbiome of 64 Mayan indigenous mothers from San Juan Ostuncalco in Guatemala. Two breastfeeding practices [exclusive (EBF) vs non-exclusive (non-EBF)] were analyzed at two stages of lactation [early (5-46 days post-partum) vs late (109-184 days post-partum)]. EBF was defined as offering only human milk and non-EBF was defined as feeding the infant herbal teas () and/or complementary foods while continuing to breastfeed. Results identified four clusters with distinct microbial communities that segregated bacterial species by both breastfeeding practices and stage of lactation. Comparison among these clusters identified several notable patterns. First, during EBF, the microbiome differed by stage of lactation where there was a shift in differential abundance from Actinobacteria and Firmicutes in early to Bacteroidetes and Proteobacteria species in late lactation. Second, a similar comparison between non-EBF mothers by stage of lactation also identified a higher differential abundance of Actinobacteria and Firmicutes species in early lactation, but only Proteobacteria and not Bacteroidetes in late lactation, indicating a further shift in the milk microbial ecosystem with fewer oral bacteria present in late lactation. Third, comparisons between EBF and non-EBF mothers at both early and late lactation showed that mothers who exclusively breastfed had more differentially abundant species in early (11 vs 1) and late (13 vs 2) lactation. Fourth, EBF at early and late lactation had more commensal and lactic acid bacteria, including , and , compared to those who did not exclusively breastfeed. Collectively, these results show that EBF has more differentially abundant bacteria, including commensal and lactic acid bacteria, and that the addition of (herbal teas) and/or complementary foods modify the milk microbiome composition by reducing the oral bacteria and introducing more environmentally sourced bacteria to the ecosystem.