APC Microbiome Ireland - Doctoral Theses

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Microbiota-gut-immune-brain communication across the lifespan
(University College Cork, 2022-12-21) Cruz-Pereira, Joana S.; Cryan, John; Clarke, Gerard; Science Foundation Ireland
While the exploration of the gut microbiome in health and disease evolves, the implications of the microorganisms that inhabit the gut for host brain health also multiply. As we grow up and grow old, the gut microbiota along with the host physiological systems, undergoes significant remodelling. The influences of the gut microbiome on host physiology are relevant across the lifespan, with continuous communication between the gut microbiome and the central nervous system (CNS) representing an important aspect of this host-microbe dialogue. A growing body of research into dissecting the involvement of the gut microbiota in the behaviour and functioning of the CNS raises the need to understand how these integrated systems communicate throughout the lifespan of the host, and how it is phenotypically reflected. Understanding the gut-brain axis across the lifespan is imperative, as these insights can be used to further support healthy brain aging, along with the development of better biomarkers towards the development of personalized therapeutic strategies. In this thesis, we aimed to investigate the influence of the gut microbiome in immune and behavioural features throughout the lifespan of the host: in early-life and aging. To this end, we assessed the effect of microbiota depletion in aged mice and demonstrated for the first time that the gut microbiome is associated with social behaviour and restricts the accumulation of T-helper cells in the choroid plexus in aged mice. This was accompanied by modulation of caecal metabolite levels, and in particular, some metabolites previously associated with age-dependent processes, namely argininosuccinic acid and N-formylmethionine. To further examine the involvement of the gut microbiome in aging, we explored whether supplementation with the prebiotic FOS-Inulin could alter behavioural and physiological aspects along the gut-brain axis in stressed aged mice. We demonstrated that FOS-Inulin supplementation can ameliorate the disrupted social behavioural responses that arise following a stress exposure, including the alterations in social interaction with a non-aggressive mouse and social novelty, while promoting the remodelling of caecal and prefrontal cortex metabolite levels. More specifically, dietary supplementation with FOS-Inulin promotes the amelioration of the levels of 4-Hydroxybenzaldehyde and spermine in the prefrontal cortex of stressed aged mice. Additionally, we evaluated if FOS-Inulin supplementation could alter adult social, depressive- and anxiety-like behavioural and immune markers in offspring exposed to early life microbial disruption. In our study, we observed altered intestinal immune markers and subtle behavioural changes following this intervention. Taken together, these results provide novel insights on time sensitive critical windows for the gut microbiome, and its impact in behaviour and immunity outcomes in the host. While further investigation into the mechanisms underlying these effects is crucial, these findings highlight the involvement of gut microbial signalling on host behaviour and immunity. This research paves the way for the future development of therapeutic options that target the gut microbiome to modify these age-dependent behavioural and metabolite alterations.
The Sudabiome: oral and gut microbiome parameters of the Sudanese population including dietary and cultural [Toombak] metagenomics
(University College Cork, 2022-12-15) Sami, Amel; Ross, R. Paul; Stanton, Catherine; , Imad; Science Foundation Ireland; Esther Alliance Group
The Sudabiome is a unique metagenomic project focusing on several critical areas of the Sudanese population. It highlights a pivotal framework in understanding novel aspects of Sudanese health that include oral and gut health, cultural metagenomics, nutritional trends, migratory travel impacts, and the susceptibility or protection against diseases. Toombak is a fermented smokeless tobacco produced from the Nicotiana Rustica tobacco plant, utilised predominantly by Sudanese males. The Toombak is ‘dipped’ in the oral cavity and replaced several times a day. The microbiome, mycobiome, phylogenetics and metabolomics of 21 pre-prepared and ready to buy Toombak samples purchased from different regions in Khartoum city were assessed, as well as the pH, chemical and microscopical composition of the product. The heavy metals, chromium, cobalt, and copper were high in the pre-prepared form of Toombak, while iron, tobacco-specific nitrosamines [TSNAs], formaldehyde and acetaldehyde were considerably elevated in both types compared to EU regulated smokeless tobacco products. The phyla, Firmicutes and Actinobacteria dominated all samples of Toombak. Virgibacillus was found to be significantly increased in the pre-prepared form [q=3.6314e-8] compared to the ready to buy forms, while Corynebacterium casei [q=1.6417e-5], Atopococus tabaci [p=0.05], and Staphylococcus gallinarum [p=0.01] were the genera abundant in the ready to buy Toombak in comparison to the pre-prepared form. The mycobiome of the ready to buy Toombak was found to be enriched with Aspergillus [85.54%] with the species, Aspergillus heterocaryoticus [43.27%] and austwikii [39.48%] dominating the samples. Furthermore, utilising PICRUSt, the ready to buy samples were found to harbour an increased activity of metal transport systems [K02006, K02013 and K02015] and an antibiotic transport system [K09687]. The Toombak had a large non-homogenous and irregular particle distribution with increased sodium, while the pH of samples was in the alkaline range [8.73-9.92]. TSNA, formaldehyde and acetaldehyde levels observed in Toombak were found to be some of the highest compared to other smokeless tobaccos from around the world such as snus from Sweden and moist smokeless tobacco from the USA. These findings highlight that the final composition of Toombak is affected by its preparation method, with Toombak use having the potential to impart many negative consequences on the health of its users. Dependency on Toombak use was examined through the utilisation of the Fagerstrom test for nicotine dependence smokeless tobacco questionnaire. Nineteen chronic Toombak users had an 85% dependence score. In addition, stress level over a 3-month retrospective period was evaluated through ‘scalp-side’ hair cortisol analysis. Mean cortisol levels were significantly lower [p=0.023] amongst Toombak users [9.7 pg/ml] compared to non-users [19.4 pg/ml]. While use of Toombak may initially bring anxiolytic effects to its users, blunting of the hypothalamic-pituitary axis chronically ensues in Toombak users, affecting cortisol release. This is likely due to the continued high levels of nicotine exposure and toxic effects that many Toombak compounds cause to body organs, including the adrenal glands. The Sudan has a rich heritage of food fermentation that involves a vast array of raw starting materials and methodologies of production that have been preserved for centuries. Forty-six Sudanese fermented foods were sourced from six food categories that included, crop [sorghum and millet], plant [Cassia obtusifolia and sesame], fish, animal, and dairy sources to establish these foods metagenomic composition, some of which were explored for the first time. 16S rRNA sequencing was undertaken, while a subset of foods underwent internal transcriber [ITS] sequencing for mycobiome analysis. Beta diversity [p=0.001] was significantly varied between the categories of Sudanese fermented foods. Animal, fish, and plant-based foods exhibited the highest alpha diversity richness [p=8.7229e-07] while fish and plant-based foods had an elevated Shannon index [p=0.001]. Crop-based foods [sorghum and millet] were found to be enriched in the genus Acetobacter [96.89%], plant-based foods in Aeriscardovia [99.14%], dairy-based foods in Enhydrobacter [85.03%], animal-based foods in Bacteroides [98.22%] and fish-based foods in Sporosarcina [99%]. A loss of Lactobacillus abundance was observed between the preparatory [26.91%] and final [4.38%] stages of sorghum and millet fermented foods. The Kawal food, obtained from the fermentation of the Cassia obtusifolia plant was found to harbour a comprehensive microbiome [Bacillus, Lactobacillus, Pediococcus, Citrobacter and Bifidobacterium] and mycobiome [Candida, Aspergillus, Cladosporium and Malassezia] which could potentially harbour unknown but novel probiotic benefits to consumers. Sudanese fermented foods were however also found to be a source for pathogenic bacteria that included Escherichia-Shigella found abundantly in the plant-based foods [46.8%] and Wohlfahrtiimonas which were found to be abundant in animal-based foods [79.07%]. Intra-food categorical analysis found Lactobacillus to be enriched in the dairy food Mish or deep yoghurt [86%] compared to Gergosh or the dairy/legume biscuit [24%] and Garis or fermented camel milk [10%]. Extensive screening of the oral microbiome of users and non-users of Toombak was undertaken through 415 samples detecting salivary [n=72], plaque [n=71], tongue, buccal cheek, floor of the mouth and palatal mucosal [n=272] microbiome variations. In a subset of salivary samples, the mycobiome [ITS] was also assessed. The microbiome of oral squamous cell carcinoma and premalignant samples [n=46] from formalin-fixed paraffin-embedded tissue was further evaluated from another cohort of users and non-users of Toombak. Alpha diversity [richness] was low for tongue microbiome compared to other mucosal locations [p=3.6849e-34] and Beta diversity was found to be significant between the four oral mucosal locations [p<0.001]. Fusobacteria and Patescibacteria in saliva and Actinomyces in the saliva [p=0.0045], tongue [p=0.013] and palate [q=0.001], were correlated with Toombak use. Skin-associated microbiome were increased amongst the oral cavity of Toombak users and included Staphylococcaceae [q=0.037] in the saliva and Cutibacterium [q=0.04] in the buccal cheek and floor of the mouth. In Toombak users, utilising LEfSe plotting, the genus Peptostreptococcus was most distinct for Toombak use. Non-users of Toombak were found to have abundances in Prevotella [buccal, p=0.04] and Bifidobacterium [tongue, q=0.0049] while Scardovia was discriminant of non-user’s tongue microbiome. Virulent strains such as Prevotella nigrescens and Streptococcus equinus [tongue, p=0.043] were found in the oral microbiome of Toombak users while Prevotella salivae and Streptococcus sobrinus [tongue, q=0.023] were found in non-users of Toombak. There was a three-fold enrichment of oral Aspergillus [78.93%] in the saliva of Toombak users compared to non-users [21.07%] with reduced Candida abundance in Toombak users [4.33%] compared to non-users [95.67%]. Virulent fungal species were also found in smokeless tobacco users [Candida tropicalis] compared to non-users [Candida albicans]. Premalignant lesion microbiome composition included Rothia [p=1.5e-10] and Peptostreptococcus [p=6.5e-07] agreeing with the literature. The oral cancer microbiome in Toombak users harboured genera favouring a poor survival and metastasis that included Stenotrophomonas, [p=0.043] and Schlegelella, [p=0.048], compared to genera found in oral cancer samples from non-users of Toombak such as Lactobacillus, [p=0.024]. The genus Corynebacterium_1 was found to be common to the Toombak product, the oral cavity [saliva, p=0.0054, floor of the mouth, p=0.0028] and oral cancer samples [p=0.044] of Toombak users. These findings highlight that several oral distinctions follow Toombak use, allowing for a microbiome that could potentiate disease and destabilise the normal oral microbial flora while our findings also harbour the potential for microbiome biomarkers to be employed in the future screening of oral carcinoma in Sudan. In 141 participants, an inter-continental metagenomics study was achieved on residents from Africa [Sudan] and Europe [Ireland] concerning diet frequency, microbiome composition and metabolome activity of stool samples. The European Prospective Investigation into Cancer and Nutrition Norfolk Food Frequency Questionnaire [EPIC - Norfolk FFQ] was modified and employed to compare food groups, nutrient and energy intakes between three cohorts, the Irish living in Ireland [Irel], the Sudanese living in Ireland [SudIrel] and the Sudanese living in Sudan [Sud]. Cereals and cereal products, fats and oil, fish and fish products, fruits and vegetables, meat and meat products, sugar preserves and snacks were consumed at a reduced intake in the Sud cohort with a high strength of association and large effect size [eta2 = >0.14]. Egg and egg products [eta2 =0.04], nuts and seeds [eta2=0.05] and non-alcoholic beverages [eta2=0.108] were also consumed highly in Sud, albeit with low and medium effect sizes. The average energy intake [measured in kcal/day] between groups was statistically different [p <0.001] and was found to be lowest in Sud [1077 kcal/day], compared to SudIrel [2729 kcal/day] and Irel [3442 kcal/day]. The diet of the Sud cohort was thus found to be low in calcium [p=0.05], carbohydrates, fibre, protein, fat, potassium, sodium, and vitamins A, C, B1, B2, B6, B12 and D [p<0.001] but high in vitamin B5 [pantothenic acid] and intermediate in Vitamin K levels. These results highlight the variations of dietary consumption between the two populations; Sudan and Ireland and helps set a fundamental previously unmet base for future research to evaluate dietary trends in the Sudanese population, including children. Microbiome Beta diversity plotting [q=0.001] highlighted a distinct ‘sandwiched’ presentation to the cohort SudIrel compared to Irel and Sud groups, with R2 = 63%. Alpha diversity measures however were found to be non-significant between groups [Shannon index or evenness, p=0.74]. Microbiome genera such as Prevotella, [q=3.169e-06], Megasphaera [q=4.61e-05], Klebsiella [q=3.9067-e], Bifidobacterium [q=7.023e-03] and Lactobacillus [q=2.918e-09] were found to be depleted in the Irel cohort compared to both Sud and SudIrel while in the Irel cohort, the microbiome was instead enriched in Lachnospira [q=1.623e-09] and Bacteroides [q=3.056e-05]. Collinsella was found to be four times more abundant in Sud and SudIrel [44%] compared to Irel [11%]. Principal coordinate analysis of metabolomic compounds from stool samples highlighted 27 metabolites to be significantly varied between Irel and Sud including proline [p=0.003], adipic acid [0.04], and phosphocholine [p=0.02], 21 metabolites to be significantly varied between Sud and SudIrel including sugar alcohol [p=0.04] and N-acetylneuraminic acid [p=0.01] and no significant metabolomic variations between Irel and SudIrel. The short-chain fatty acids, isobutyric [p=0.0] and isovaleric acid [0.002] were significantly lower in the stool samples from those in the Irel cohort. LEfSe plotting of PICRUSt KEGG orthology pathways indicated multi-drug resistance activity pathways in the Irel cohort [K09686], mineral and heavy metal transport activity in Sud [K06199] and in SudIrel, carbohydrate metabolism pathways were prominent [K02794, K02796, K02775, K02773, K02774]. From these results, a migratory impact on the traditional microbiome of the Sudanese population occurs that has not been previously explored which may allow for both the susceptibility and prevention of disease in this population. Those who migrate from the Sudan to Ireland [SudIrel], carry both tradition microbiome preservation [i.e., continued Prevotella abundance] and modern microbial carriage [i.e., Bacteroides enrichment]. Through these studies, there has been a novel and in depth uncovering of human and environmental microbiome features associated for the first time with the Sudanese population in comparison to Irish controls. Through fermentation techniques for example, cultural [Toombak] and dietary [fermented foods] produces may carry health benefits but also numerous predicaments such as the increased risk of oral cancer development and systemic cortisol imbalance from Toombak use and the contraction of food-borne diseases from contamination during Sudanese fermented food production. The studies on Sudanese human oral and gut health have further provided insight into how the oral and gut microbiome may respond to modern challenges such as migration and economic, dietary and cultural influences. This project further give light on the merging of next generation sequencing science with economic and population challenges. Continued microbiome research in Sudan should continue to be geared towards sharper preventative disease strategies, management, and treatment outcomes.
From youth to old age: therapeutic potential of microbiota interventions in the gut-brain-axis
(University College Cork, 2022-08-17) Minuto, Chiara; Dinan, Timothy G.; Cryan, John; Clarke, Gerard
There is a growing recognition of the involvement of the gut microbiota in the regulation of certain physiological and metabolic parameters across the life cycle of animals. A greater understanding of the microbiota changes throughout the life cycle may provide a novel therapeutic target for treating various age-related disorders from autism in childhood to dementia in old age. The increase in the elderly population with a rapid rise in dementia, cardiovascular and metabolic disease, leads to the growing demand for new interventions to decelerate the senescent decline. However, if the microbiota is to become a therapeutic target a far greater understanding of the structural changes which take place throughout the lifespan is required. In this thesis, we focus attention on microbiota-targeted interventions in young, middle age and old animals. The studies examine behaviour, physiology, and metabolism, exploring effects both in the periphery and in the brain, across a range of treatments from prebiotics to polyphenols. We highlight the physiological differences between the various age groups. Intestinal permeability and neuronal plasticity were the only modifiable parameters in the elderly. In younger animal prebiotics impact metabolism and inflammation. Polyphenols rich diets (grape pomace and rafuma) significantly alter intestinal permeability reversing the damage to the intestinal barrier due to ageing. The effect was not directly linked to changes in microbial diversity of the gut and did not significantly ameliorate the impaired immune system of the aged animals’ intestine or hippocampus. Only a significant increase in the number of dendritic cells emerged in conjunction with a reduced level of TNF-α in the blood. Neither did the polyphenols reverse some of the behavioural deficits associated with ageing. Cumulatively, these data show the specific effect of polyphenols in the elderly. We next investigated whether inulin, the most widely studied prebiotics, could improve host metabolism in middle-aged mice. We tested 52 metabolic markers involved in hypothalamic-pituitary-adrenal (HPA) axis regulation, cholesterol and glucose homeostasis and immune regulation. Interestingly, the liver, the brain and the mesenteric fat did not show any modification in middle-age animals. Inulin had a greater impact in young animals and stimulated the lipid and glucose metabolism, increasing the level of Sirtuin1, a fundamental metabolic sensor, without impacting transcriptional factor changes in the liver. The study also uncovered a range of changes in the HPA. In fact, inulin decreased the level of the glucocorticoids receptors (Nr3c1) and corticotropin-releasing hormone (Crhr1) receptors in the amygdala of young mice. In an attempt to understand the mechanisms through which microbiota-targeted interventions exert the anti-aging effects, we used a model of premature ageing (ApoE deficient mice). The effect of two different strains of lactobacillus (L.reuteri and L.mucosae) and two prebiotics derived from plants (plant sterol esters and oat b- glucan) were examined. Neural plasticity was restored in the Prefrontal cortex after 24 weeks of prebiotic administration and the level of claudin 5 increased in the hippocampus following consumption of L. mucosae. Overall, these results provide novel insights regarding the impact of a portfolio of microbiota-targeted interventions on age-related alterations in physiology and brain function and suggest the importance of using target-specific therapeutic options during particular time windows for maximum gain.
Evaluating substrate utilisation to target newly identified health-promoting gut bacteria
(University College Cork, 2021-12) Lordan, Cathy; Cotter, Paul; Ross, R. Paul; Teagasc
Promoting the growth and/or activity of potentially beneficial human gut microorganisms through the provision of specific substrates has been the subject of many studies over recent decades. Such substrates can include prebiotics, which promote growth in a targeted manner, but prebiotics can also be combined with other less target-specific nutrients to further enhance the growth of these targets. This field has continued to advance in recent years and has also expanded in response to the identification of new target species, such as Akkermansia muciniphila, Eubacterium rectale, Faecalibacterium prausnitzii, Roseburia inulinivorans, and Ruminococcus bromii, many of which produce metabolites that can contribute to host health. These developments have been in part due to improvements in culture-based techniques, advances in DNA sequencing-based approaches and improved computational pipelines. The combined use of these tools has enormous potential with respect to elucidating substrates that can be applied to specifically target microbes of interest and is the focus of this thesis. Chapter 1 reviews the literature relating to prebiotics, and potential prebiotics, with a focus on more recently identified health-promoting gut bacteria. Building on this, in Chapter 2 we applied both in silico and in vitro techniques to predict and assess substrate utilisation for seven strains across five species of interest. The bioinformatics-based component of Chapter 2 is expanded in Chapter 3 through the analysis of publicly available microbial genomes of the same species of interest and, through the creation of metabolic models, predicting the substrates that these microorganisms could consume. Finally, in Chapter 4 we employ an ex vivo model to evaluate, through shotgun metagenomic sequencing, the impact different substrates, including simple sugars, oligosaccharides, and whey protein concentrate had on the taxonomic composition and functional potential of a colonic microbiome. Thus, facilitating the design and testing of a functional prototype beverage comprised of some substrates assessed here. Overall, this thesis explores different substrates that could be applied to target the growth and/or activity of recently identified health modulating microbes within the human gut. Combining in silico, in vitro, and ex vivo approaches have the potential to identify and assess a variety of substrates that may be applied to bring about microbiome-mediated enhancements of host health.
Towards a translational understanding of colonic bacteria in Crohn's disease pathology
(University College Cork, 2022-03) O'Donoghue, Keith; Claesson, Marcus; Joyce, Susan; Melgar Villeda, Silvia; Science Foundation Ireland
Crohn’s disease (CD) is a chronic inflammatory condition of unknown aetiology, with the microbiota thought to be a major contributor to the disease. This thesis aimed to investigate the potential role of the gut microbiota in this context. The approaches taken were to examine CD associated Bacteroides. We choose 3 Bacteroides species (B. fragilis, B. vulgatus, B. thetaiotaomicron) to examine here, for their ability to impact gut related functions via a range approach from cell culture to metabolite production and in application to relevant animal models. We found subtle, but significant effects in co-cultures with cells via supernatants that could induce changes to gene expression to impact cell proliferation and maturation as well as immune response modulation. The effects were strain dependent, and they were impactful. The work revealed alterations to signalling metabolites, bile acids, again in a strain dependant manner. These effects did not translate to huge changes in metabolites in vivo, in normal mice and in the genetic CD model. However, gender specific changes to inflammation modulation, BAs, the faecal microbiota and in signalling were determined in female mice related to the antibiotic cocktail issued to them and altered in male mice colonized by B. vulgatus. The work provides several different impacts towards future direction, this work has also been, in part, published.

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