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An investigation of factors influencing early life gut microbiome in the piglet model, and potential functional food ingredients
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Date
2023
Authors
Skillington, Orla
Journal Title
Journal ISSN
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Publisher
University College Cork
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Abstract
Increasingly regarded as an “invisible organ”, the microbiological supersystem that is the gut microbiome, has become a central field of research in recent years. Thanks to multi-omics approaches and modern day bioinformatic techniques, characterization of the gut microbiome has advanced hugely in the last three decades, and continues to evolve. Characterization of the gut microbiome to-date, has concerned infanthood through to old-age, across a variety of species, from humans to aquatic life. Due to ethical considerations involving human-targeted therapeutics and interventions for the gut microbiome, animal models must be considered. The porcine pig model is an attractive model for putative human interventions and their associated pre-clinical trials, due to certain physiological analogies. Herein, a porcine model was developed, to mimic the early life gut microbiota and to investigate the effects of prenatal stress exposure on the seeding of this microbial consortia.
Piglets and neonatal infants share an early life gut microbiota rich in Bacteroides, Escherichia, Lactobacillus and Clostridium¸ however, whilst Lactobacillus dominate the early life porcine gut, the nascent infant gut may be characterized by high abundances of bifidobacterial species. Analysis by 16s rRNA sequencing revealed that, at two weeks of age, piglets exposed to low stress levels in utero harboured higher levels of Prevotella, Fusobacterium and Bacteroides, whilst high stress exposure influenced increased levels of Lactobacillus. The effects of stress on the gut microbiome projected into later life, following weaning, whereby piglets of the low stress group were seen to have significantly higher levels of Prevotella NK3B31 and Helicobacter and piglets of the high stress group harboured higher levels of Faecalbacterium and Megasphaera.. Taxa driving microbial clustering as the piglet aged were seen to be stress-related, with high stress piglets becoming enriched in Deferribacterota, Treponema and Pyramidobacter at the post-weaning stage.
Conversely, increased populations of Ruminococcus were observed in the low stress groups at two weeks of age, and remained higher than that of their counterpart post- weaning. Commonalities between the stress groups existed at two weeks, with phyla such as Campilobacterota, Bacteroidota, Actinobacteria and Firmicutes being enriched and later declining with age. At post-weaning, both groups were found to have clusterings of Cyanobacterota, Fibrobacterota, Spirochaetota and Patescibacteria. Alpha and beta diversity metrics were unaffected by prenatal stress exposure, however differed significantly with respect to age, at two weeks of age versus four weeks (post-weaning).
We also investigated the putative probiotic potential of a range of indigenous African fermented food that may serve to provide human health benefits. It is postulated that the succession of the development of probiotic strains from these foods, may be suited for further investigations within infant formula matrices, to help to equilibrate the microbial consortia of a dysbiotic infant gut which had been exposed to prenatal stresses. The bacterial isolates, obtained from seventeen different food sources, on a variety of growth media, were initially subjected to a preliminary subtractive process in order to reduce the 218 isolates to a smaller number. Following exopolysaccharide production screening, bile salt hydrolase production screening, and antimicrobial activity screening, seven isolates were chosen for further analyses. These were found to be dominated by lactic acid bacteria such as Enterococcus and Lactobacillus as identified by 16s rRNA Sanger Sequencing, with all isolates displaying average growth kinetics of between 8 and 10 log cfu mL-1. Analysis by whole genome sequencing revealed Enterococcus populations to be dominated by E. faecium and E. casselflavus, whilst Lactobacillus delbrueckii and Pediococcus pentosaceus were also identified in the isolated cultures. In silico mining and comparative genomic analysis revealed the presence of antimicrobial substances such as enterolysins, enterocins and helveticins in the samples, whilst predicted secondary biosynthetic metabolites revealed the presence of polyketide synthases and antimicrobial ribosomally synthesized and post-translationally modified peptides (RiPPs) such as lanthipeptides. Subjection of the seven isolates to simulated gastric environments revealed the ability of two strains, L. delbrueckii and P. pentosaceus, to survive gastric pH conditions after 4 hours. Furthermore, both of these strains were found to be capable of growing in simulated bile conditions. Haemolytic assays revealed all strains were non-hazardous to red blood cells, and produce no signs of lysis when grown on blood agar plates. African fermented foods present a largely untapped area of unidentified strains with potentially valuable technical properties, however may also serve as vehicles for antimicrobial resistant and pathogenic strains. There lies a substantial gap in knowledge surrounding these food products and their applications, which warrant investigation to benefit both the native consumers, and the market potential of these foods.
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Keywords
Gut microbiome , Probiotics , Piglet model , Early life , Metabolic health , Human health
Citation
Skillington, O. 2023. An investigation of factors influencing early life gut microbiome in the piglet model, and potential functional food ingredients. MRes Thesis, University College Cork.