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Elucidating how Bifidobacteria shapes the microbiota in response to infant diet.

Periodic Reporting for period 1 - INFANT MICROBIOTA (Elucidating how Bifidobacteria shapes the microbiota in response to infant diet.)

Reporting period: 2015-07-01 to 2017-06-30

After birth our body surfaces are rapidly colonised by microbial communities, collectively termed the microbiota. Within our body, our gut microbiota is the largest and most diverse bacterial community, with upwards of trillions bacteria per gram of content. These resident microbes form an intricately balanced mutualistic relationship in early life with the host, which is maintained throughout life and promotes long-term host health. The infant microbiota is established by vertical transmission through the birth canal, skin-to-skin contact and breast-feeding. Pioneer bacteria, like Bifidobacterium are one of the first bacteria to colonise and in time, reach upwards of 80% of the total population density. In contrast, Bifidobacteria represent about 5% of the gut microbiota in formula-fed infants. The predominance of Bifidobacteria in the infant microbiota is proposed to be critical for establishing a ‘healthy’ microbiota and promoting paediatric wellbeing. However, the mechanism by which this Bifidobacterium-rich ‘state’ occurs remains largely unknown. Several studies indicate that an infant’s diet (breast-milk vs. formula) plays a central role in driving intestinal microbial composition and diversity. Crucially, enhanced infant well-being, such as robust mucosal immunity and reduced inflammatory diseases, can be attributed to breast-feeding and improved microbiota development in early life. Despite the significant health benefits associated with maternal milk, the rate of infants solely breast-fed in the UK is only 45% by one-week of age, and 7% at four months; this rate is lower than that observed in neighbouring European countries such as France and Germany (Renfrew, M.J. et al., 2012, UNICEF UK). However, key questions remain to be answered as to why Bifidobacteria populations are dramatically altered in breast- and formula-fed infants. It is imperative to understand the mechanisms driving beneficial bacterial colonisation, whilst excluding potentially harmful bacteria, during infant microbiota development in response to diet. The ultimate future aim is to develop improved infant formula(s) that more closely replicate the microbial community dynamic benefits derived from breast milk.

Our research objectives were to investigate the mechanisms that enhance Bifidobacteria colonisation in the gut of breast fed infants, and not in infants fed with formula. In addition, we sought to identify how Bifidobacteria modulates the wider infant gut community; establishing and shaping these communities over time. Through our experimental set-ups we have been able to identify key factors in breast-milk that promote Bifidobacteria growth at high levels and prolonged survival in the host. This research will provide insights into the function and mechanisms of how infant diet impacts Bifidobacteria colonisation in the infant gut, with the potential to identify key components that could be incorporated in new infant formula(s) to promote ‘healthy’ infant microbiota development, thus positively impacting infant wellbeing.
The main results from this research are:

• We have isolated, fully sequenced, and annotated >24 novel Bifidobacterium strains from healthy, full-term infants. These strains have all been assessed for phenotypic ‘probiotic’ properties.
• We have also isolated 43 other bacterial strains from the stool of a healthy, breast-fed infant that is used as a ‘defined early life microbiota’ and all of these strains have also been sequenced and are currently being annotated.
• We have identified kinetic and growth differences between novel Bifidobacterium isolates when grown in breast milk compared to formula milk, and further investigated gene expression differences in the two conditions using RNASeq.
• Using a reductionist approach, we have found that bifidobacteria preferentially consumes certain breast milk components over others, which are different to those found in formula, potentially suggesting why Bifidobacteria blooms in the breast-fed infant gut.
• Metabolic labelling and identification of bifidobacterial-derived metabolites produced from growth on breast-milk (compared to formula) are currently on-going with support from collaborators.
• We have assessed how the microbial community develops, and the stability of the microbial community over the first 600 days of life. We have found that once established, maternal milk continues to promote bifidobacteria growth and predominance in the infant microbiota, even after the introduction of solid foods for a period of more than a year.
• To decipher the genetic mechanisms for bifidobacteria growth in the infant gut, and specifically in response to breast milk metabolism we are generating a massive mutant library spanning >45,000 mutants.
• In order to assess bifidobacteria interactions with other microbial species, we have generated a model colon system that is run with faeces and/or the defined early life microbiota. These experiments have provided additional data on how Bifidobacteria interacts and cross-feeds with other bacteria.
It is now believed that there is a critical window during early life development, where the composition of the infant microbiota has long-lasting effects on host health. Diet has a dominant effect in early life on shaping the gut microbiota, compared to other external factors (including ethnicity, hygiene, and geography). Differences in the microbiota of breast-fed vs. formula fed infants are indicated by a rapid drop in bifidobacteria populations upon the introduction of formula feeding, that also coincides with influx of other potentially harmful bacterial species such as Enterobacteriaceae. In summary, this research has shown key components of breast milk (that are altered or absence in formula milk) are critical for promoting key bacteria, like Bifidobacteria growth in early life. With a focus on early life-associated species, we have identified gene involvement in breast-milk metabolism and found that Bifidobacterium has an intricate relationship with other gut commensals, shaping the development of a healthy microbial community in early life. Additional experiments that have been performed have also provided insight into the role of diet on bacterial community stability and diversity in infants over time.

This research has identified key factors that drive the assembly of a healthy microbiota in early life, and provide a platform for identification of new beneficial bacteria and their substrates to benefit the growing number of formula-fed infants and improve their overall health and well-being. Additionally, we hope to provide an alternative for parents who are unable or chose not to breast-feed their infants through the enhancement of infant formulas that closely mimic the health benefits affiliated with breast-feeding and improved bacterial gut development.

Collectively, the data from these experiments have resulted in three reviews and four manuscripts that are currently being prepared for submission. Many of our findings (including strains, techniques and findings) are currently pending patent review and submission. The results obtained have been widely disseminated in presentations at national and international conferences, with public and students, and potential pharmaceutical and food partners. The research this fellowship funded has led to new and exciting avenues for ongoing future research and collaborations, which we are currently pursing.
Infant model colon system
Electron microscopy of Bifidobacteria pseudocatenulatum