Association Between Breast Milk Bacterial Communities and Establishment and Development of the Infant Gut Microbiome

Abstract

Importance: Establishment of the infant microbiome has lifelong implications on health and immunity. Gut microbiota of breastfed compared with nonbreastfed individuals differ during infancy as well as into adulthood. Breast milk contains a diverse population of bacteria, but little is known about the vertical transfer of bacteria from mother to infant by breastfeeding.

Objective: To determine the association between the maternal breast milk and areolar skin and infant gut bacterial communities.

Design, setting, and participants: In a prospective, longitudinal study, bacterial composition was identified with sequencing of the 16S ribosomal RNA gene in breast milk, areolar skin, and infant stool samples of 107 healthy mother-infant pairs. The study was conducted in Los Angeles, California, and St Petersburg, Florida, between January 1, 2010, and February 28, 2015.

Exposures: Amount and duration of daily breastfeeding and timing of solid food introduction.

Main outcomes and measures: Bacterial composition in maternal breast milk, areolar skin, and infant stool by sequencing of the 16S ribosomal RNA gene.

Results: In the 107 healthy mother and infant pairs (median age at the time of specimen collection, 40 days; range, 1-331 days), 52 (43.0%) of the infants were male. Bacterial communities were distinct in milk, areolar skin, and stool, differing in both composition and diversity. The infant gut microbial communities were more closely related to an infant's mother's milk and skin compared with a random mother (mean difference in Bray-Curtis distances, 0.012 and 0.014, respectively; P < .001 for both). Source tracking analysis was used to estimate the contribution of the breast milk and areolar skin microbiomes to the infant gut microbiome. During the first 30 days of life, infants who breastfed to obtain 75% or more of their daily milk intake received a mean (SD) of 27.7% (15.2%) of the bacteria from breast milk and 10.3% (6.0%) from areolar skin. Bacterial diversity (Faith phylogenetic diversity, P = .003) and composition changes were associated with the proportion of daily breast milk intake in a dose-dependent manner, even after the introduction of solid foods.

Conclusions and relevance: The results of this study indicate that bacteria in mother's breast milk seed the infant gut, underscoring the importance of breastfeeding in the development of the infant gut microbiome.

Figure 1.. Microbial Communities in Breast Milk, Areolar Skin, and Infant Stool

A, Principal coordinate (PC) analysis of unweighted UniFrac distances revealed that breast milk, areolar skin swabs, and stool separate into 3 distinct groups. Percentages are the percent variation explained by each PC axis. B, Faith phylogenetic diversity was used to measure α diversity. The 3 groups all significantly differed from each other in 2-way comparisons of α diversity (P < .001). Infant stool was the least diverse in terms of community membership, likely reflecting the young age of study infants at the time of stool collection and their initial states of colonization following complete or near-sterility at birth. C, Breast milk had significantly higher amounts of Moraxellaceae and Pseudomonadaceae (false discovery rate [FDR] P < .001), whereas areolar skin swabs were enriched in Streptococcaceae and Staphylococcaceae (FDR P < .001) and infant stool comprised greater amounts of Bifidobacteriaceae (FDR P < .001). Boxes indicate the 25th to 75th percentile; line within the box, median; error bars, 1.5 × interquartile range.

Figure 2.. Evaluation of Breast Milk and Infant Stool During the First Year of Life

A, Relative abundance of most bacterial taxa in 238 breast milk samples from 94 mothers remained stable with age. B, Infant stool composition changed with increasing infant age in a nonrandom pattern. A total of 259 stool samples from 112 infants are shown here. C, α and β diversity (α diversity, Faith phylogenetic diversity) increased in infant stool (P = .01) but not breast milk (P = .06) with infant age. Differences between individuals (β diversity, unweighted UniFrac) in breast milk increased during the first 6 months of life (P < .001) and then decreased. β diversity of infant stool steadily decreased (P < .001) during the first 6 months and then began to level off after 6 months of life. Nonparametric t tests were used for comparisons of α and β diversity between age groups.

Figure 3.. Source of Bacterial Community in Infant Stool by Percent Daily Breastfeeding

A, Among primarily breastfed infants of all ages, 18.5% of the microbial community found in the stool was derived from breast milk compared with only 5.7% in infants not primarily breastfed (P < .001, Wilcoxon rank sum). Similarly, 5.2% of the infant stool microbial community was derived from areolar bacteria in those who breastfed more than 75% of the time compared with 0.001% in infants who breastfed 75% or less of the time (P = .01, Wilcoxon rank sum). B, Amount of contribution from breast milk to the infant stool was highest during the first 30 days of life and decreased as the infant aged, even among those who continued to breastfeed more than 75% of the time. Error bars indicate SD.

Figure 4.. Microbiome Age Estimation

A, A subset of healthy, vaginally born, exclusively breastfed infants (n = 42) was used to train a random forest regression model that was then applied to estimate microbiota maturity. Relative microbiota maturity was calculated by microbiota age of a child relative to the microbiota age of healthy children of the same chronologic age from the spline fit line shown here. B, Differences in relative microbiota maturity based on the age at which solid food was introduced in our cohort. Early solid food introduction was associated with early microbiota maturity (<4 months vs ≥4 months, P = .02; ≥4 months vs not introduced, P = .30, Kruskal-Wallis). Boxes indicate the 25th to 75th percentile; line within the box, median; error bars, 1.5 × interquartile range.