APC Microbiome Ireland - Masters by Research Theses

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Neurobiological effects of food fermentation-derived metabolites for metabolic and mental health
(University College Cork, 2023) Carey, Nathan; Schellekens, Harriet; O'Mahony, Siobhain M.
Nutrition and diet are becoming increasingly popular therapeutic interventions as we discover more about the complex roles the foods we consume play in maintaining our health status. It is now clear that foods we ingest daily and their metabolites interact with systems both within and outside the gastrointestinal tract including the gut microbiome, the nervous system, immune system and hormonal system. Each of these play essential roles in the bi-directional communication pathway of the microbiota-gut-brain axis. Interactions between our food and this axis can potentially influence centrally mediated processes such as cognition, mood and even appetite. While several foods have been identified as being beneficial to our health, there is one food group that remains under investigated and holds promise as a reservoir of both beneficial bacteria and bioactive compounds – fermented foods. Fermented foods are created through the controlled enzymatic conversion of foods to simpler organics substances by microorganisms. Common examples include foods like kimchi (a fermented cabbage product), kefir (a fermented milk product) and kombucha (a fermented sweet tea beverage). While human studies on fermented foods remain sparce, one recent finding recorded lower perceived stress in human adults who underwent a dietary intervention that included fermented food intake. Recent findings in rodents suggest that fermented foods can alter social behaviour, reduce body weight and lead to reduced anxiety in animals. The mechanism by which fermented foods act is still unknown but it likely due to a number of factors such as their probiotic bacteria content, their metabolite content including short chain fatty acids (SCFA), and the ability to breakdown their starter compounds into simpler molecules and increase their bioavailability such as phenolic compounds in fruits and vegetables. The research conducted in this thesis aims to investigate the ability of food-fermentation derived metabolites, with a specific focus on SCFAs and polyphenols, to alter the neurobiological functions associated with central appetite regulation (hypothalamus) and cognition (hippocampus). Using in vitro assays, we tested the selected panel of metabolites shown to be found in fermented foods, and capable of crossing the blood brain barrier, on both immortal cell lines (hypothalamic and hippocampal) and on primary neurosphere cultures (hippocampal). A panel of SCFA were administered to hippocampal and hypothalamic cell lines and were capable of altering brain-derived neurotrophic factor (BDNF) gene expression. These metabolites were also tested on primary hippocampal cells using a neurosphere assay of proliferation. Positive trends were observed across many of the treatments, however these trends were not significant. Perhaps most interesting were our findings when submitting the same hippocampal neurosphere assay to a panel of phenolic compounds. Apigenin and kaempferol (both flavonoids) significantly increased hippocampal cell proliferation. Moreover, to enhance the efficiency of data analysis, a semi-automatic quantification pipeline was developed for high-throughput screening of primary neurosphere cultures. This pipeline offers a systematic and reliable method for evaluating neurosphere proliferation, providing a valuable tool for future studies in the field. Our results highlight the modulatory effects of SCFA and phenolic compounds on hypothalamic and hippocampal cells in vitro, emphasising the potential role of dietary metabolites and fermented foods as a whole on brain function related to metabolic and mental health. These findings also highlight the need for more in depth analysis of fermented foods and their neuromodulator effects both in vivo.
Broadening the host range of lytic phage against Methicillin-resistant Staphylococcus aureus
(University College Cork, 2018) Ní Mhaoldomhnaigh, Eimear Sinead; Ross, R. Paul; Hill, Colin; Science Foundation Ireland
Introduction In recent years the rise in antibiotic resistance in pathogenic bacteria has meant that significant research has gone into finding alternative treatments. The higher mortality and morbidity associated with multidrug resistant bacteria suggest that solutions are urgently needed. The World Health Organisation (WHO) recently published a list of 12 bacteria which are considered the greatest threat in this regard (a list which includes drug resistant Staphylococcus aureus). Unless a viable alternative to antibiotics is found global human health will suffer and we may see a return to mortality and morbidity rates similar to those seen in the pre-antibiotic era. Despite the development of vaccines, and improved hygiene and living standards, bacterial infections remain a very real treat. In particular the developing world is most at risk of unchecked antibiotic resistance disease due to insufficient access to health care, overcrowding and decreased water sanitation. Objectives Bacteriophage targeting of specific pathogens may provide an alternative to antibiotic therapy in certain clinical settings. However specific issues may limit their use. The high specificity of phage for their host makes them potentially impractical for therapeutic use, without first identifying the causative agent/strain and then having access to a phage that infects it. The current study therefore investigated the factors that affect phage specificity for S. aureus isolates which vary at the strain level. The study also investigated whether bacteriophage populations can evolve to overcome bacterial defence mechanisms and whether it is possible to use this ability to increase or shift host ranges. We hypothesize that p cocktails of phage targeting different hosts may be the solution to overcoming narrow host ranges of individual phage. In numerous previous studies phage cocktails have been shown to have success in treating infections (O'Flynn et al., 2004; Gu et al., 2012; Chan & Abedon, 2013; Örmälä & Jalasvuori, 2013; Niu et al., 2014; Chadha et al., 2016;). Methods In this study the host range of the phage was characterized by plaque assay, the anti-biotic resistance profile of the bacterial sample set examined by agar disc diffusion method and a method was used whereby MRSA phage were co-cultured with susceptible and un-susceptible strains of MRSA of clinical importance. Following the generation of mutants in Phage B1 and phage K with favourable adaptations to host range, genetic sequencing was carried out and hypothesis proposed as to what changes lead to the altered host range. Results It was found that after prolonged exposure phage evolved to be able to infect formerly non-susceptible strains. We analysed the host range of a novel staphylococcal phage, Phage B1 in comparison with reference phage commonly used in the literature. The study established key parameters such as the exposure time required and the ratio of target strains: permissive strains that provided for optimal phage adaptation. Some of the mutants had the ability to infect previously insensitive strains and others showed increased efficacy. In practical therapeutic terms, this would mean that it is possible to rapidly select adapted phage isolates to target previously non-permissive pathogenic bacterial strains. Sequencing of the original phage, phage B1 as well as the mutated/adapted phage derivatives, phage B1 0.0066 and phage B1 3488 revealed insights into genomic variation of phage during adaptation to previously non-permissive hosts. Acquisition of specific genes (including genes encoding alternative phage tail proteins) could explain altered host infection potential in the mutated/adapted phage, although further work is necessary to associate altered genes with functional properties of the phage. Conclusion The current study established the host range of a novel staphylococcal phage, phage B1 which has potential to be used in phage therapy. Whilst some S. aureus isolates were resistant to targeting by phage B1 we established conditions under which we could rapidly select for phage variants to target such strains. Genomic analysis of both phage B1 and phage variants (phage B1 0.0066 and phage B1 3488) provided molecular insights into the process of phage adaptation. Overall, the results suggest the potential for phage adaptation and rapid isolation of new phage variants in the clinical setting.
Development of a synergistic synbiotic containing arabinoxylan and Bifidobacterium longum using in vivo selection
(University College Cork, 2024) Jones, Evan; Walter, Jens; van Sinderen, Douwe; University College Cork; Synbiotic Health
Colonisation and metabolic activity of orally ingested bacteria in the colon rely on competitive ecological and niche-based factors that often limit the functionality of commonly used probiotics. Synergistic synbiotics, which involve the parallel administration of a microorganism with its cognate substrate, have the potential to improve persistence and ecological performance of putative probiotic microbes. However, real synergism has not yet been established for synbiotics in human trials, and most synbiotic combinations have not been designed using an approach that accounts for the ecological constraints of the GI tract. Here we use in vivo selection (IVS) to identify strains of Bifidobacterium longum that are adapted toward the utilization of arabinoxylan (AX) in the human gut. To achieve this, bifidobacteria were quantitatively cultured from fecal samples collected during a human trial which showed that a high dose of corn bran AX leads to a significant but highly individualised increase of B. longum. Isolates were randomly picked and genotyped by a rapid, high throughput gyrB sequencing method that was developed for this project. Bacterial counts and strain composition were compared between baseline and week 6, and B. longum strains enriched in vivo were then tested through in vitro fermentations to investigate their growth on AX and its constituents. These monoculture experiments confirmed the ability of representative isolates to use free arabinose, xylo-oligosaccharides (XOS) and the complete corn AX fibre, which suggests that these B. longum strains are primary AX degraders. Viable cell counts revealed a high level of consistency in growth patterns among the fecal isolates compared to reference strains on AX. Whole genome sequencing (WGS) of selected strains followed by comparative genomic analysis revealed an enrichment of relevant glycoside hydrolase family 43 (GH43) genes and the presence of three specific carbohydrate utilisation clusters associated with xylan and AX metabolism in a number of in vivo-selected isolates which was not observed in reference strains. Finally, gas production experiments helped to further characterise the fermentation profiles of the AX-degrading isolates and highlighted their capacity to facilitate cross-feeding with other members of the microbiota. This study demonstrates the value of an ecologically relevant process for selecting improved synbiotic combinations, with the B. longum strains identified here representing promising candidates based on their predicted ecological performance in vivo.
Microbial regulation of barrier function in the gut-brain axis
(University College Cork, 2023) Sánchez Díaz, Paula; Clarke, Gerard; Cryan, John; Leigh, Sarah-Jane; Advancement in Neurosciences (Geneva, Switzerland)
The gut microbiome plays a critical role in host health through modulation of gut and blood-brain barrier integrity, responding to factors such as diet, stress, and medication. A key pathway by which the gut microbiota affects gut and bloodbrain barrier integrity is through the production of bioactive metabolites. This thesis explores the role of barriers in the microbiota-gut-brain axis, which are essential for the proper functioning of body systems and homeostasis. Chapter 2 investigates how fermenting infant nutrient formulations with different bifidobacteria strains isolated from infant gut microbiome can influence the integrity of gut and blood-brain barriers in vitro. The study found that the presence of bifidobacteria strains, in some cases, had protective effects on the barriers, and these effects sometimes differed depending on the barrier studied. Chapter 3 explores the effects of indole and two of its derivates, indole-3-acetate and indole- 3-propionate, on gut barrier function in vitro. The results indicate that indole has a protective effect on barrier function, particularly at higher concentrations, and indole-3-acetate has a protective effect at the lowest concentrations tested. Surprisingly, indole-3-propionate was not protective and at higher concentrations exacerbated the effects of LPS-induced disruption. Finally, Chapter 4 focuses on the effect of cancer therapy, specifically cisplatin, on gut and blood-brain barrier structure in mice, to further explore the role of gut microbiome in cancer-related cognitive impairment. The study used mice treated with cisplatin to investigate the expression of genes involved in the structural function of barriers and inflammation, as well as gene expression of receptors activated by microbial ligands in the ileum, colon, and hippocampus. The results showed that cisplatin affected gene expression in a region- and dose-dependent manner, leading to changes in anxiety-like and fatigue behaviours in mice. Overall, this research highlights the critical role of the gut microbiome in gut barrier and blood-brain barrier function. Microbial metabolite supplementation may present a useful therapeutic option for disease processes involving disruption of the gut and/or blood-brain barriers.
An investigation of factors influencing early life gut microbiome in the piglet model, and potential functional food ingredients
(University College Cork, 2023) Skillington, Orla; Stanton, Catherine; Ross, R. Paul; Vistamilk; Science Foundation Ireland
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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