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Intervention studies in young children to assess the ability of foods to enhance levels of health associated microbes in the gut
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Date
2022-02
Authors
Ahern, Grace Juliette
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Publisher
University College Cork
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Abstract
Complex microbial communities composed of bacteria, archaea, eukarya, viruses, bacteriophages, and their products, are collectively referred to as the microbiome. These dynamic entities are generally favoured in maintaining symbiotic relationships with humans and other microorganisms, and are capable of directly or indirectly altering host physiology, both in health and disease across the lifespan. In the context of early life, infancy not only represents a period of nutritional vulnerability but also instability, in terms of the intestinal microbiota, which continues to evolve and mature throughout childhood and preadolescence. Indeed, there is supporting evidence from epidemiological studies that prenatal, perinatal and postnatal factors mediate disproportional colonisation patterns of gut microbes in infants. Herein, this thesis involved a number of research foci with a view to gain a better understanding of (1) the developments in the area of infant nutrition, (2) the influence of early life nutrition and environmental factors on the infant gut microbiota, (3) potential probiotic species (or pharmabiotics) isolated from various infant-associated ecosystems with particular emphasis on characterising their mechanisms of action, and (4) the genetic determinants that relate to certain microbiota having a unique ability to convert essential fatty acids into health-promoting/beneficial forms. In Chapter 1, advancements to infant feeding research, particularly in the area of infant milk formula, are discussed in relation to the addition of new ingredients and bioactives to formulations. Such functional components that are inherently present in human milk, and investigation of their use in vivo by means of preclinical and randomised controlled trials, provides new and emerging translational research and opportunities for future development in the area of infant nutrition and gut health. Chapter 2 explores the translational period when infants are exposed to non-milk foods for the first time and when accompanied by breast milk and/or formula. Such infant feeding types and their impact on the gut microbiota were examined as part of the Cork Nutrition and Microbiome Maternal-Infant Cohort Study (COMBINE), based in Cork, Ireland. Noticeable alterations in the gut microbiota composition and predicted functionality among infants who were breastfed (n = 18), formula-fed (n = 26) and mixed-fed (n = 6) before and during complementary feeding were identified utilising 16S rRNA amplicon sequencing and PICRUSt. Results showed that infants were characterised as having high relative abundances of Actinobacteria and Firmicutes at 4-6 months and complementary feeding had a significant impact on community composition and microbial richness over time. The median age for initiating solids was 22 (19, 23.75) weeks; the median duration of breastfeeding was 16.5 (6, 43.25) weeks, and a longer duration of breastfeeding was associated with reduced microbial diversity. Furthermore, short-chain fatty acids (SCFAs) were significantly higher following complementary foods. Taken together, this study demonstrates that breastfeeding, formula feeding and mixed feeding result in different microbial profiles and alterations, which are particularly evident during the initial complementary feeding period. In Chapter 3, a randomised, double-blind, parallel, placebo-controlled trial was undertaken to investigate the safety and effectiveness of Lactobacillus paracasei N1115 (LP N1115) as a probiotic to enhance gut development in young children born by caesarean section. A total of 60 infants and young children between the ages of 6 months and 3 years were randomised to receive LP N1115 or placebo over an 8 week intervention period. After treatment, stool consistency was measured as the primary efficacy outcome and secondary outcomes including gastrointestinal symptoms (flatulence, bloating and abdominal pain), faecal microbiota, salivary cortisol and SCFA levels were also assessed. Overall, the placebo functioned similarly to the probiotic in relation to stool consistency, gastrointestinal symptoms, salivary cortisol and SCFAs. Enrichment of potentially pathogenic microbes such as Klebsiella and Enterococcus was evident in both groups. Differential abundance analysis indicated an increase of Lactobacillus in the probiotic group at week 4 (p < 0.05) and week 8 (p < 0.05) in comparison to the placebo group. Chapter 4 examines 250 bacterial isolates which emanated from the two paediatric cohorts developed in the studies outlined in Chapters 2 and 3. Phenotypic and genotypic screening enabled the identification of probiotic attributes amongst several robust, infant-derived strains including a novel, narrow-spectrum lantibiotic isolated from saliva. Chapter 5.1 explores the significance of bioactive fatty acids, namely conjugated linoleic acid (CLA) that is generally recognised as safe (GRAS) and known to be isomerised from polyunsaturated fatty acids. Additionally, quantitative and qualitative methods used to identify and quantify CLA isomers produced by bifidobacteria and lactic acid bacteria (LAB) were examined. Finally, in Chapter 5.2, a site-direct mutagenesis approach was employed in Bifidobacterium breve CCFM683, a CLA-producing strain isolated from the faeces of a breastfed infant with a high bioconversion efficiency. The creation of two similar insertion mutants by homologous recombination were verified by whole genome sequencing and the function of a putative linoleate isomerase target gene was successfully disrupted. As a result, B. breve CCFM683 mutant strains showed poor growth in the presence of linoleic acid in comparison to the wild-type strain. Moreover, using a rapid, UV spectrophotometric method, the mutants were shown to be incapable of producing CLA. Atomic Force Microscopy (AFM) also revealed the impact of linoleic acid concentration on the morphology and structure of the CLA-producing bacterium. This study provides key insights into the molecular mechanisms involved in the biotransformation process of linoleic acid to CLA using an alternative mutagenesis strategy. The results outlined in this thesis converge with multiple areas of infant research and expand upon current knowledge in the first few years of life as a means to elucidate a healthy growth and development trajectory.
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Keywords
Infant gut microbiota , Probiotics , Nutrition
Citation
Ahern, G. J. 2022. Intervention studies in young children to assess the ability of foods to enhance levels of health associated microbes in the gut. PhD Thesis, University College Cork.