The relationship between the human milk microbiome and infant gut microbiota composition over the first six months of life

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Lyons, Katriona
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University College Cork
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Breast milk is considered the gold standard feeding regime for new-born infants. It provides complete nutrition to the infant and contains many bioactive compounds which support the growth and immune development of the new-born. Breast feeding is associated with improved infant health, less incidences of gastrointestinal disease, and lower mortality rates than formula fed infants. As well as providing fundamental nutrients to the growing infant, breast milk was found to contain its own unique microbiome, despite initially being regarded as a sterile fluid. This source of commensal and potential probiotic bacteria further enhances infant health and development by preventing pathogen adhesion and promoting gut colonisation of beneficial microbes. Our understanding of the milk microbiota and its contributions to overall infant health is still limited, however, due to increasing popularity and advances in next generation sequencing and metagenomics approaches, this will enable a more comprehensive analysis of the breast milk microbiome and its functional capacity. The ‘INFAMILK’ project was developed to (1) examine the evolution of milk microbial communities over lactation, (2) characterise the infant gut of breast fed infants and compare the findings of that with a cohort of exclusively formula fed infants and (3) isolate and characterise potential probiotic species from breast milk for their potential use in infant health and nutrition market. Firstly, we examined the impact of storage, temperature and extraction kits on the milk microbiome. As with all low biomass samples, standardisation across variables such as sample collection, storage, and extraction methods is needed to eliminate discrepancies in the microbial composition reported and allow for more comparable analyses across studies. While DNA extraction from fresh samples is regarded as the optimum approach, it may not be feasible in large-scale studies where high volumes of samples are collected. The aim of this study was to investigate how different storage methods, temperatures, preservatives and extraction kits influence the human milk microbiome, compared to fresh samples. The data from this study indicate that if DNA extraction from fresh samples is not feasible, freezing samples immediately after collection at -80°C is the next best option to preserve the integrity of the milk microbiome. Furthermore, our results demonstrated that choice of extraction kit has a profound impact on the microbiota populations detected in milk. To further evaluate the evolution of the milk microbiome over lactation from birth to six months, 16S rRNA sequencing was performed on freshly extracted milk samples from a cohort of approximately 120 women sampled at 4 different time points; week 1, week 4, week 8 and week 24. Our longitudinal study investigated the evolution of the milk microbiome throughout lactation, while also examining whether factors such as lactational stage, mode of delivery and maternal body mass index (BMI) would be reflective in the milk microbiota up to six months postpartum. Although previous studies have reported that changes in the milk microbiota are attributed to the above factors, our study concluded that lactational stage was the primary driving factor for compositional changes, resulting in significant changes over time. A decrease in alpha diversity was observed over lactation, and a core microbiome composed of 11 genera including Staphylococcus, Streptococcus, Pseudomonas, Acinetobacter, Enterobacter, Pseudaminobacter, Mesorhizobium, Brevundimonas, Flavobacterium, Microbacterium and Rhodococcus was determined. To determine the relationship between breast feeding and the infant gut microbiome over the first six months of life, we next aimed to characterise the infant gut microbiota of breast fed infants using 16S rRNA compositional sequencing and compared the data with gut microbiota composition of exclusively formula fed infants. Our results demonstrated a significant impact on the gut microbiota in response to age and feeding mode (breast fed or formula fed). While both the breast fed and formula fed infant gut were characterised by a high relative abundance of Bifidobacterium throughout the first six months of life, breast fed infants also had a higher relative abundance of Bacteroides and Lactobacillus whereas formula fed infants had increased abundances of Enterococcus and Blautia. This study reveals that the gut microbiota has distinct microbial profiles based on mode of feeding throughout the first six months of life. As formula fed infants lack the exposure to commensal and potentially probiotic bacteria found in breast milk, and therefore any health promoting benefits associated with those microbes, we isolated and characterised a number of Lactobacillus strains from breast milk for their probiotic potential. Among the 27 Lactobacillus strains isolated, 18 Lactobacillus gasseri, 4 Lacticaseibacillus paracasei (formerly Lactobacillus paracasei), 1 Lactobacillus crispatus, 3 Limosilactobacillus oris (formerly Lactobacillus oris) and 1 Limosilactobacillus fermentum (formerly Lactobacillus fermentum) were identified. Four L. gasseri isolates were regarded as lead candidates on the basis of their antimicrobial activity as well as their ability to produce EPS and tolerate bile salts. While these strains demonstrated potential for use as future probiotics, more extensive research needs to be performed to determine the full extent to which these stains may beneficially contribute to infant health. Overall, this thesis successfully characterised the breast milk microbiome over lactation, and its relationship with the infant gut microbiome. The infant gut of breast fed and exclusively formula fed infants were also compared over the first six months of life, which revealed that the gut microbiota of formula fed infants matured more rapidly than breast fed infants, and exhibited higher microbiota diversity in early life. Four L. gasseri strains were characterised for their potential probiotic use in the infant health and nutrition market. While supplementation of infant formula with beneficial health promoting bacteria isolated from breast milk is an attractive concept and may be considered as a means of promoting infant health and gut colonisation in formula fed infants, further work is necessary to understand the extent to which these strains can benefit infant health.
Breast milk , Infant gut , Microbiota , Probiotics , Human milk microbiome , Breast fed infants
Lyons, K. 2021. The relationship between the human milk microbiome and infant gut microbiota composition over the first six months of life. PhD Thesis, University College Cork.