APC Microbiome Ireland - Doctoral Theses

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    Unravelling the role of Bacteroides and Phocaeicola in the human gut: exploring evolutionary dynamics, mother-to-infant vertical transmission, and functional characteristics
    (University College Cork, 2023) da Silva Morais, Emilene; Stanton, Catherine; Ross, R. Paul; Science Foundation Ireland; IFF - International Flavors & Fragrances
    The human gut microbiota is composed of trillions of microrganims and plays a major role in human health and disease by performing a series of physiological functions, such as protection against pathogens, energy production, maintenance of intestinal barrier integrity, and regulation of the immune system. Bacteroides is one of the major genera present in the human gut. Bacteroides colonise the gut soon after birth in vaginally born babies and its abundance increases after weaning. However, different studies have shown that Bacteroides are missing or present in lower relative abundance in infants born by caesarean section (CS). Lower Bacteroides relative abundance has been associated with higher risk of atopic sensitization, especially in children of Asian heritage. Bacteroides and Phocaeicola have been linked to both health and disease. In the gut, Bacteroides is often associated with health benefits, but it can become an opportunistic pathogen in other body sites. Herein, this thesis explored a number of research topics aimed at gaining a better understanding of the role of Bacteroides and Phocaeicola in the human gut, such as production of metabolites, genetic and evolutionary features, vertical transfer, persistence and relative abundance of Bacteroides and Phocaeicola in early life. Characterization of a new species of Bacteroides and a potential Phocaeicola probiotic strain were also carried out. In Chapter 1, the current knowledge about the association of Bacteroides and Phocaeicola with health and disease was explored. Metabolic conditions associated with altered levels of these species, as well as their role as opportunistic pathogens and the challenges associated with the use of Bacteroides and Phocaeicola as probiotic were investigated. Numerous pre-clinical studies showed the benefits of strains of Bacteroides and Phocaeicola in various conditions, however, more clinical research is needed to evaluate if these benefits extend to humans. Chapter 2 explores Bacteroides and Phocaeicola in early life. Metagenomic analysis was carried out to compare the relative abundance of these species in vaginally delivered (VD) and CS-born infants during the first year of life. The presence of vertically transferred strains and strains that were persistent across different time points were also evaluated. Whole genome sequence (WGS) was carried out and the average nucleotide identity (ANI) was used to determine whether different isolates were the same strain. A blast comparison of the whole genome was also carried out. Metabolomics analyses and γ-aminobutyric acid (GABA) production was carried out in a subset of the isolates. Metagenomic analysis showed that Bacteroides are absent in CS children who were not exposed to antibiotics before birth, but present in CS-born children whose mothers were exposed to antibiotics during pregnancy and VD infants. Five groups of isolates that were vertically transferred and/or persistent across different timepoints were identified. The metabolomics profile showed that Bacteroides and Phocaeicola strains were able to produce organic acids and indole. On Chapter 3 a comparative analysis of the genomes of P. dorei and P. vulgatus were performed. Nearly 4000 assemblies were used to build the pan-genome of these species and to evaluate functional differences, like Carbohydrate-Active enZymes (CAZyme) content, antimicrobial resistance profile and mobile genetic elements. Both species have an open pan-genome and a similar antimicrobial resistance profile, but different CAZyme content, indicating they might have evolved to occupy different niches. We also hypothesised that P. dorei evolved from a larger than average subclade of P. vulgatus. Chapter 4 describes a novel Bacteroides species, Bacteroides sp. nov. (strain MSB163), which was isolated from a healthy mother 4 weeks after giving birth. The WGS, metabolomic profile, respiratory quinones, polar lipids, growth conditions and fermentation of different carbohydrate sources were analysed. Apart from differences in the genome, the main differences between MSB163 and its closest relatives, B. cellulosilyticus and B. intestinalis are the presence of phosphatidylglycerol on its cell membrane and the ability to ferment melezitose and sorbitol. In chapter 5, we characterize strain MSB026 (P. dorei), a bacteriocin producer and potential probiotic candidate. MSB026 was isolated from a VD infant, not exposed to antibiotics. WGS, growth conditions, 2’FL utilization, and production of compounds of interest were analysed. The production of health beneficial compounds, like GABA and SCFAs, associated with the strain’s technological robustness make it a potential probiotic candidate. Bacteroides and Phoceicola play a major role in the prevention and onset of different health conditions. They are present in the gut from the first days of life, producing numerous metabolites and interacting with the host in differen ways. The results outlined in this thesis expand on the knowledge of two important commensal genera of the human gut that have an important role in host health.
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    Nutritional psychiatry, stress and the microbiota-gut-brain axis: focus on dietary fiber
    (University College Cork, 2024) Lachmansingh, David Antoine; Clarke, Gerard; Lavelle, Aonghus; Cryan, John
    Dietary fiber has an assortment of health benefits and the gut microbiota has been shown to be an integral regulator of brain function and behavior. The interface between these features may have implications from a therapeutic as well as a neurobiological perspective in terms of mental health function, especially in circumstances where stress is considered a significant risk factor for anxiety and depressive symptoms. A prodigious amount of research has helped to shape the narrative of a possible relationship between stress and the microbiota-gut-brain axis. However, the nature of the relationship between dietary fiber, stress and the microbiota-gut-brain axis needs to be explored, especially with efforts focused on eliciting and understanding probable mechanisms. We demonstrated that dietary fiber has the ability to influence gut-brain modules (GBMs) including SCFA synthesis via the gut microbiota, by devising a systematic review protocol followed by its execution and deployment across studies examining the impact of different dietary fiber and compositional alterations with publicly available datasets. These datasets were re-analyzed via consistent GBM bioinformatic pipelines with the results illustrating a high probability that dietary fiber favors an increased abundance of SCFA-producing taxa and GBMs for SCFA synthesis. We also showed via a cross-sectional analysis, that dietary fiber was associated with reduced state anxiety in healthy individuals. The combination of these results have furthered our understanding in the nutritional psychiatry field, indicating that dietary fiber may recruit SCFA production in reducing anxiety. Such findings could allow for further research focusing on SCFAs impacting anxiety. Additionally, this platform may also allow for other GBMs impacted by dietary fiber to be evaluated in the role of reducing anxiety symptoms, as well as other symptoms relating to stress and depression. Taken together, our findings have confirmed that the microbiota-gut-brain axis and dietary fiber are implicated in the way we feel and our mental health.
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    Characterisation and applications of bacteriocinogenic Isolates from the deep-sea fish microbiome
    (University College Cork, 2024) Uniacke-Lowe, Shona; Ross, R. Paul; Hill, Colin; Stanton, Catherine
    One of the major grand challenges confronting humanity in the coming decades is the increased risk of infection, stemming from the alarming surge in antimicrobial resistance among pathogenic bacteria. Consequently, there is an urgent need to uncover new antimicrobials and mechanisms, including antimicrobial bacterial strains, which could potentially substitute for some of the widely employed antibiotics. In this respect, the main goal of this thesis work was initially to explore deep-sea fish microbiomes as a source of antimicrobials and their prospective clinical applications. Our hypothesis was that these microbiomes represent some of the last bastions of microbial communities that have been negligibly impacted by human activity and as such are untapped for this purpose. Although still very limited, there is an accumulating number of studies where marine fish microbiomes are being characterised and assessed for their bacteriocinogenic potential (Chapter 1). In many cases these microbiomes are dominated by Gram-negative Pseudomonadota and Gram-positive Bacillota and Actinomycetota. They can be important hosts to uncommon species that are not found in the surrounding waters, as well as to novel taxa. Of particular interest, in terms of their antimicrobial production, are bacilli, lactic acid bacteria and actinobacteria. Already, a limited number of novel bacteriocins from marine fish gut isolates have been identified and characterised. These include Formicin, a class I (lanthipeptide) bacteriocin from mackerel, several class IIa bacteriocins, and CAMT6, a very recently identified class IId bacteriocin. These novel bacteriocins cover a range of molecular structures, with varying spectra of activity – some of which were capable of targeting important pathogenic strains, such as Clostridium difficile and Listeria monocytogenes. Bacteriocins from marine fish microbiota also have diverse applications, not only for therapeutic, but also as food preservatives and probiotics. This provides great scope for the potential of finding novel bacteriocins from deep-sea fish microbiomes. Chapter two outlines an exploration of bacterial isolates from the gut and skin of deep-sea fish for antimicrobial production. In vitro screening was initially used to screen for antimicrobial producers with activity against various indicators including foodborne pathogenic bacteria. Subsequent in silico screening of the genomes of selected producers revealed the presence of a wide range of secondary metabolite biosynthetic gene clusters (BGCs) as well as potentially novel bacteriocins, including class I (lanthipeptide), class IIc (circular) and class IId bacteriocins. Genomic screening also revealed the almost complete absence of antimicrobial resistance genes. Chapter three outlines the description of a novel species, Winogradskyella bathintestinalis APC 3343, which was isolated from the intestine of a deep-sea dragon fish, Malacosteus niger, as part of the screening study. This yellow pigment-producing strain was differentiated from related type strains of Winogradskyella species based on a combination of 16S, genomic, biochemical, and phenotypic characterisation. This study represents one of first to characterise a novel Winogradskyella species isolated from a deep-sea fish. Chapter four describes the characterisation of a novel Planococcus species, isolated from the skin of a deep-sea ‘snub nose spiny eel’ (Notacanthus chemnitzii), for which the name Planococcus notacanthi was proposed. This strain, APC 4016, had been shown to inhibit the growth of Gram-positive target strains and was subsequently found encode a potentially novel lanthipeptide with similarity to cerecidin of Bacillus cereus. Based on phylogenetic analysis and its distinctive phenotypic characteristics, such as a tolerance of a wide range of growth conditions and the presence of menaquinone 5 as a respiratory quinone, Planococcus notacanthi was deemed a novel species, with APC 4016 as the type strain. We also explored the application of these deep-sea isolates and their DNA as controls for determining relative and/or absolute quantification of bacteria, in terms of cell numbers, in human metagenomic sequencing experiments. Quantified genomic DNA from two deep-sea species (crucially that have not been identified in human microbiome samples) was applied to human faecal samples and subsequently extracted and detected using 16S rDNA sequencing and qPCR, alongside the microbiome DNA. The preliminary data shows that the “spiked-in” deep-sea DNA has the potential to be used as a marker for relative abundance of bacterial cell numbers. Chapter five outlines the identification of an agarolytic (agar-degrading) isolate from the skin of a deep-sea fish and the subsequent molecular characterisation of one β-agarase and one putative B agarase encoded by the isolate. Sequence analysis of the two agarase proteins, Aga4017A and Aga4017B, showed that they shared structural similarities and key characteristics to GH16 and GH50 family β-agarases, respectively. Recombinant pCR2.1-TOPO vectors encoding aga4017A or aga4017B were created and cloned into E. coli TOP10. Transformants containing this pCR2.1-TOPO_aga4017A vector were able to express the agarase protein and exhibited agarolytic activity. The predicted 3D model of Aga4017A was also elucidated using Alphafold2 and revealed it to be a globular protein with distinctive modules corresponding to a signal peptide, a catalytic GH16 β-agarase domain and a ricin B lectin -like carbohydrate-binding domain. Overall, this work demonstrates the biodiscovery potential of the microbiome of deep-sea fish, in terms of its microbials inhabitants, the antimicrobials they produce, and also their functionalities, which may have applications for utilisation in the field of microbiome research.
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    The role of the gut microbiota in the function and integrity of brain barriers
    (University College Cork, 2023) Knox, Emily G.; Cryan, John; O'Driscoll, Caitriona M.; Rodriguez Aburto, Maria; Clarke, Gerard
    The barriers of the brain help maintain an optimal environment for central nervous system (CNS) function and homeostasis. There are many instances such as during age related disorders and neuropsychiatric disorders in which these barriers are under threat. Importantly, the gut-microbiota can interact with the brain barriers, thereby altering barrier physiology and communication with the brain. This bidirectional communication is known as the microbiota-gut-brain axis. Modulation of the gut microbiota can thereby alter functions in the brain, potentially through influence of the brain barriers. In this thesis we focus on the interface of both brain barriers; the blood-cerebrospinal fluid barrier (CSF) and blood-brain barrier (BBB) in microbiota-gut-brain axis communication. In particular, using germ-free mice which lack a gut microbiota, we investigated the contributing role of the gut microbiota in regulation of the blood-CSF barrier. Following confocal microscopy for visualization of the tight junction protein, ZO-1, germ-free mice were found to have a disruption of the tight junction protein networks in isolated choroid plexus tissue. Similar assessment of the choroid plexus capillaries revealed no difference between germ-free and conventional mice. Since microbial metabolites are some of the known contributing factors of the gut microbiota’s manipulation of the barriers, we next investigated the effects of physiological concentrations of short chain fatty acids (SCFAs), butyrate and propionate, with and without induced barrier dysfunction (LPS) on the actin cytoskeleton and tight junction protein dynamics using an in vitro BBB model. We found that the butyrate and propionate altered filamentous actin directionality, increased tight junction protein spikes, and protected from LPS induced decrease in mitochondrial footprint and barrier integrity measured by trans-endothelial electrical resistance (TEER). While we do not yet understand the dynamics of the BBB following acute stress, we know that microbial metabolites can interact with BBB function, so we next assessed the potential for acute-stress induced metabolites to regulate BBB integrity using the same in vitro model. Following TEER assessment of a broad range of concentrations, we found that microbial metabolites from different classes; SCFAs, bile acids, and tryptophan metabolites impact BBB integrity. Specifically, butyrate, propionate, acetate, 3β-hydroxy-5-cholenic acid, and tryptamine exerted protective effects while acetate and cholic acid exerted detrimental effects at various concentrations. Lastly, since diet is a major modulator of the gut microbiota and especially in infancy, we assessed the impact of infant formulations fermented with probiotic strains of Bifidobacterium uniquely isolated from infant stool samples for potential effects on BBB integrity. Cell-free supernatants from the fermentation process which would contain a pool of microbial metabolites were then collected and exposed to the same in vitro model for assessment of barrier integrity with and without LPS disruption. We found that in each of the four infant formulations, there was protection from LPS disruption following fermentation with some of the Bifidobacterium strains. Overall, these results provide valuable insight into the interphase of brain barriers in microbiota-gut-brain axis communication. We highlight the role of another brain barrier in this interface, the blood-CSF barrier and further elucidate the microbial metabolite interaction in BBB physiology. Further, using two of the major modulators of the gut microbiota, stress and diet, we reveal that there is scope for the impacted metabolites or metabolic pools to influence brain barrier interactions.
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    The gut microbiome of the wild great tit (Parus major): drivers and fitness consequences
    (University College Cork, 2023) Somers, Shane Edmond; Quinn, John; Ross, R. Paul; Stanton, Catherine; Irish Research Council for Science, Engineering and Technology; European Research Council; Science Foundation Ireland
    The gut microbiome plays a vital role in its host’s ecology. Clinical studies have shown gut microbes increase host health and fitness by providing digestive and immune functions, as well as aiding development. Natural variation in the microbiome is widely believed to affect host fitness in the wild but we are lacking experimental studies to test this. The microbiome varies with both host and environmental factors but most studies to date have focussed on individual factors and not adequately addressed the multiple overlapping and hierarchical drivers of microbiome variation working at environmental, host and microbial scales. This thesis investigates the role of the gut microbiota in host fitness, and how this is affected by and varies across contexts. Additionally, we address sources of variation in the gut microbiota at a host and environmental level, accounting for host ecology and drivers at different scales. We find that the host’s weight is correlated with microbiome diversity during development but that the direction of this relationship is context dependent. This shows that the microbiome interacts with the environment to determine host fitness and is important because it helps explain the contradictory findings linking diversity to weight. We also show that the interaction between the host, its microbiome and environment change with developmental stage. Specifically, we found that the microbiome of developed individuals is remarkably resilient to environmental perturbation, while developing individuals are much more sensitive, with important implications for future experiments. We developed a novel method for experimentally perturbing the microbiome that will allow microbiome researchers to begin testing hypotheses linking the microbiome to host ecology and evolution in natural settings. Finally, we show that welfare measures, such as environmental enrichment may interact with the gut microbiota to impact on host health and behaviour. In summary, I show that variation in the microbiome is linked to host ecology and that this variation is linked to host fitness.