The Key Role of Human Milk Microbes and Oligosaccharides in Infant Gut Microbiota Development

Academic Background

The development of infant gut microbiota is crucial in early life, and breastfeeding is one of the key drivers of this process. Human milk not only provides essential nutrients for infants but also contains a rich array of microbes and oligosaccharides, particularly Human Milk Oligosaccharides (HMOs). These components have a profound impact on the shaping of infant gut microbiota. However, despite existing studies indicating the important role of milk microbes and HMOs in infant gut microbiota development, systematic research on how they change over time during lactation remains limited. In particular, studies focusing on the Dutch population are scarce. Therefore, this study aims to explore the influence of milk microbes and HMOs on infant gut microbiota composition at 1, 3, and 9 months postpartum, filling this research gap.

Source of the Paper

This study was conducted by a research team from Wageningen University & Research, Maastricht University, and FrieslandCampina in the Netherlands. The first author of the paper is Martha F. Endika, and the corresponding author is Hauke Smidt. The study was accepted on April 26, 2024, and published in the journal Gut Microbiome under the title “Seeding and feeding milestones: the role of human milk microbes and oligosaccharides in the temporal development of infant gut microbiota.”

Research Process

1. Study Design and Sample Collection

The study included 23 mother-infant pairs, with breast milk and infant fecal samples collected at 1, 3, and 9 months postpartum. All infants were full-term, vaginally delivered, and exclusively breastfed for at least the first three months after birth. Exclusion criteria included preterm birth (gestation less than 37 weeks) and infants who received antibiotics during the first month of life.

Breast milk samples were collected by mothers in the morning before feeding, either by hand expression or using a breast pump. Fecal samples were collected from diapers using a sterile spoon. All samples were immediately refrigerated after collection and transported to the laboratory within 72 hours for further processing.

2. DNA Extraction and Microbiome Analysis

Breast milk samples were centrifuged, and DNA was extracted using the FastDNA Spin Kit for Soil. Fecal DNA was extracted using the Repeated Bead Beating Method. The V4 region of the 16S rRNA gene was sequenced using the Illumina NovaSeq 6000 platform to analyze microbial composition.

3. HMO Analysis

HMOs in breast milk were isolated and purified using Solid-Phase Extraction (SPE) and quantified using High-Performance Anion-Exchange Chromatography-Pulsed Amperometric Detection (HPAEC-PAD) and Mass Spectrometry (MS). A total of 18 HMOs were analyzed, including 8 fucosylated HMOs, 4 non-fucosylated neutral HMOs, and 6 sialylated HMOs.

4. Data Analysis

Data analysis was performed using R, with Principal Component Analysis (PCA) and PERMANOVA used to assess microbiome changes and their relationship with HMOs. Alpha diversity was calculated using the Shannon index, while beta diversity was assessed using Aitchison distance.

Key Findings

1. Temporal Dynamics of Milk and Infant Fecal Microbiota

The study found that the predominant genera in breast milk samples were Streptococcus, Staphylococcus, and unclassified Enterobacteriaceae, while the predominant genera in infant feces were Bifidobacterium, Bacteroides, and unclassified Enterobacteriaceae. Shared bacterial Amplicon Sequence Variants (ASVs) between breast milk and infant feces primarily belonged to Bifidobacterium, Streptococcus, Enterobacteriaceae, Veillonella, Bacteroides, and Haemophilus.

2. Temporal Changes in Milk Microbiota

Significant changes in milk microbiota composition were observed during lactation. At 9 months, the proportions of Neisseria, Granulicatella, Haemophilus, Actinomyces, Veillonella, and Streptococcus increased significantly in breast milk, genera typically associated with the infant oral microbiota.

3. Temporal Changes in Infant Fecal Microbiota

The alpha diversity of infant fecal microbiota was significantly higher at 9 months compared to 1 and 3 months. At 9 months, the proportions of Anaerostipes, Blautia, and Faecalibacterium increased significantly, indicating notable changes in gut microbiota after the introduction of complementary foods.

4. Temporal Changes in HMOs

The study found significant changes in HMO concentrations during lactation. The concentration of 3-Fucosyllactose (3-FL) increased significantly at 9 months, while the concentrations of other HMOs remained stable or decreased. Additionally, no significant association was observed between HMO concentrations and infant fecal microbiota composition.

Conclusions and Significance

This longitudinal study revealed the important role of milk microbes in infant gut microbiota development by analyzing changes in milk microbes and HMOs at 1, 3, and 9 months postpartum. The findings indicate significant changes in milk microbiota during lactation, and shared bacterial ASVs between breast milk and infant feces suggest that milk microbes may play a key role in the colonization of infant gut microbiota. However, the impact of HMO concentration changes on infant gut microbiota was relatively limited.

The scientific value of this study lies in its systematic exploration of the temporal changes in milk microbes and HMOs in the Dutch population and their influence on infant gut microbiota, addressing a significant research gap. Additionally, the findings provide new evidence for the importance of breastfeeding in infant health and offer a theoretical basis for the future development of infant formula based on milk components.

Research Highlights

1. Temporal Dynamics Analysis: First systematic analysis of temporal changes in milk microbes and HMOs during lactation in the Dutch population.
2. Shared Bacterial ASVs: Identification of shared bacterial ASVs between breast milk and infant feces, suggesting the potential role of milk microbes in infant gut microbiota colonization.
3. Limited Impact of HMOs: The study found that changes in HMO concentrations had a relatively limited impact on infant gut microbiota, indicating that milk microbes may play a more significant role in infant gut microbiota development.
4. Innovative Experimental Methods: The study employed efficient DNA extraction and sequencing techniques, as well as precise HMO analysis methods, providing technical references for similar research.

Additional Valuable Information

The study also found that the microbial composition of breast milk at 9 months resembled that of the infant oral microbiota, suggesting that infant oral microbiota may enter breast milk through retrograde flow. This finding offers new perspectives for further research on the sources of milk microbes and their role in infant health.