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 Mam-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 (agüitas) 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 Lactobacillus gasseri, Granulicatella elegans, Streptococcus mitis, and Streptococcus parasanguinis, 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 agüitas (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.

Human Milk Microbiota in an Indigenous Population Is Associated with Maternal Factors, Stage of Lactation, and Breastfeeding Practices.

BACKGROUND: Human milk contains a diverse community of bacteria that are modified by maternal factors, but whether these or other factors are similar in developing countries has not been explored. Our objective was to determine whether the milk microbiota was modified by maternal age, BMI, parity, lactation stage, subclinical mastitis (SCM), and breastfeeding practices in the first 6 mo of lactation in an indigenous population from Guatemala. METHODS: For this cross-sectional study, Mam-Mayan indigenous mothers nursing infants aged <6 mo were recruited. Unilateral human milk samples were collected (n = 86) and processed for 16S rRNA sequencing at the genus level. Microbial diversity and relative abundance were compared with maternal factors [age, BMI, parity, stage of lactation, SCM, and 3 breastfeeding practices (exclusive, predominant, mixed)] obtained through questionnaires. RESULTS: Streptococcus was the most abundant genus (33.8%), followed by Pseudomonas (18.7%) and Sphingobium (10.7%) but relative abundance was associated with maternal factors. First, Lactobacillus and Streptococcus were more abundant in early lactation whereas the common oral (Leptotrichia) and environmental (Comamonas) bacteria were more abundant in established lactation. Second, Streptococcus,Lactobacillus,Lactococcus,Leuconostoc, and Micrococcus had a higher abundance in multiparous mothers compared with primiparous mothers. Third, a more diverse microbiota characterized by a higher abundance of lactic acid bacteria (Lactobacillus,Leuconostoc, and Lactococcus), Leucobacter, and Micrococcus was found in mothers with a healthy BMI. Finally, distinct microbial communities differed by stage of lactation and by exclusive, predominant, or mixed breastfeeding practices. CONCLUSION: Milk bacterial communities in an indigenous community were associated with maternal factors. Higher microbial diversity was supported by having a healthy BMI, the absence of SCM, and by breastfeeding. Interestingly, breastfeeding practices when assessed by lactation stage were associated with distinct microbiota profiles.

Distinct Changes Occur in the Human Breast Milk Microbiome Between Early and Established Lactation in Breastfeeding Guatemalan Mothers.

Human breast milk contains a diverse community of bacteria, but as breast milk microbiome studies have largely focused on mothers from high income countries where few women breastfeed to 6 months, the temporal changes in the breast milk microbiome that occur during later lactation stages have not been explored. For this cross-sectional study, microbiota from breast milk samples of Mam-Mayan mothers living in eight remote rural communities in the Western Highlands of Guatemala were analyzed. All mothers delivered vaginally and breastfed their infants for 6 months. Breast milk from 76 unrelated mothers was used to compare two lactation stages, either "early" (6-46 days post-partum, n = 33) or "late" (109-184 days post-partum, n = 43). Breast milk microbial communities were assessed using 16S ribosomal RNA gene sequencing and lactation stages were compared using DESeq2 differential abundance analysis. A total of 1,505 OTUs were identified, including 287 which could be annotated as putative species. Among several maternal factors, lactation stage explained microbiome variance and inertia in ordination with the most significance (p < 0.001). Differential abundance analysis identified 137 OTUs as significantly higher in either early or late lactation. These included a general shift from Staphylococcus and Streptococcus species in early lactation to Sphingobium and Pseudomonas species in late lactation. Species enriched in early lactation included putative commensal bacteria known to colonize the infant oral and intestinal tracts whereas species enriched in late lactation had a uniform functional trait associated with aromatic compound degradation. Differentially abundant species also included several species which have not previously been reported within breast milk, such as Janthinobacterium agaricidamnosum, Novosphingobium clariflavum, Ottowia beijingensis, and Flavobacterium cucumis. These discoveries describe temporal changes to the breast milk microbiome of healthy Guatemalan mothers from early to late lactation. Collectively, these findings illustrate how studying under-represented human populations might advance our understanding of factors that modulate the human milk microbiome in low and middle income countries (LMIC).

Emerging frontiers in human milk microbiome research and suggested primers for 16S rRNA gene analysis.

Human milk is the ideal food for infants due to its unique nutritional and immune properties, and more recently human milk has also been recognized as an important source of bacteria for infants. However, a substantial amount of fundamental human milk microbiome information remains unclear, such as the origin, composition and function of the community and its members. There is emerging evidence to suggest that the diversity and composition of the milk microbiome might differ between lactation stages, due to maternal factors and diet, agrarian and urban lifestyles, and geographical location. The evolution of the methods used for studying milk microbiota, transitioning from culture dependent-approaches to include culture-independent approaches, has had an impact on findings and, in particular, primer selection within 16S rRNA gene barcoding studies have led to discrepancies in observed microbiota communities. Here, the current state-of-the-art is reviewed and emerging frontiers essential to improving our understanding of the human milk microbiome are considered.