Microbiology - Doctoral Theses
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Item Human bifidobacterial isolates with a focus on glycan degradation and bacteriocin production by Bifidobacterium pseudocatenulatum(University College Cork, 2023) Sanchez Gallardo, Rocio; van Sinderen, Douwe; Cotter, Paul; Science Foundation Ireland; H2020 Marie Skłodowska-Curie ActionsBifidobacteria are commensal bacteria which inhabit the gastrointestinal tract of humans and other mammals, and to which various probiotic or health-promoting benefits have been attributed. Bifidobacterial strains can metabolize both host- and diet-derived glycans, and these metabolic abilities are critical for their establishment in the gut. Bifidobacteria are among the first colonisers of the neonatal gut, partially due to their ability to digest human milk oligosaccharides (HMOs), present in human milk. Members of the species Bifidobacterium breve, Bifidobacterium bifidum and Bifidobacterium longum are commonly isolated from infant faecal samples and are among the most abundant species found in early life. As the infant changes its diet during weaning the microbiota evolves and therefore the bifidobacterial abundance, prevalence and composition will be subject to change. Infant-associated species become less abundant and other species such as Bifidobacterium adolescentis start to play a more prominent role as they are capable of digesting dietary plant-glycans, therefore being adult-associated strains. Some studies have indicated that Bifidobacterium pseudocatenulatum is a bifidobacterial species that remains constant across the lifetime of a person. Thus, it is expected of this species possesses an extensive enzymatic machinery to adapt to dietary changes. Chapter II of this thesis focuses on the isolation and genomic characterisation of bifidobacterial strains from breast milk. Optimization of a cultivation protocol allowed the recovery of viable Bifidobacterium from frozen human milk samples. Furthermore, the genomic characterization allowed a preliminary analysis of the potential of these strains as probiotic. Chapter III of this thesis describes the isolation and characterisation of 36 B. pseudocatenulatum strains from mother-infant dyads. Following an initial comparative genomic analysis of all strains we focussed on a selection of the B. pseudocatenulatum strains so as to explore their genomic diversity and functionality. Growth abilities of these strains on more than 30 carbohydrate substrates facilitated the correlation between the presence of specific GH-encoding genes and their ability to degrade certain glycans. A large proportion of the genomic diversity observed in these strains was shown to correspond to the presence of prophage-like elements. Chapter IV details the metabolism of LNT in the species B. pseudocatenulatum. Using a combination of transcriptomic and molecular biology techniques, the enzymatic machinery responsible for the degradation of LNT in B. pseudocatenulatum was elucidated. Furthermore, genes were identified that were shown to be responsible for certain B. pseudocatenulatum strains to degrade particular fucosylated HMOs, believed to be important for infant health. Chapter V focussed on the extracellular degradation of particular plant-glycans, in particular xylan and starch, by specific strains of B. pseudocatenulatum. The extracellular enzymes responsible for the degradation of these complex carbohydrates were identified and characterised in this set of novel strains. Furthermore, the distribution across the species of homologs of these key genes was analysed. Chapter VI describes the identification and characterisation of a gene cluster responsible for the production of a novel antimicrobial peptide, designated Pseudocin 196, by B. pseudocatenulatum MM0196. Pseudocin 196 was purified and tested for its antimicrobial activity against a range of bacteria. Pseudocin 196 is the first reported bacteriocin produced by a B. pseudocatenulatum species of human origin and was shown to inhibit clinically relevant pathogens. The work presented in this thesis expands the knowledge of Bifidobacterium pseudocatenulatum taxon, being in particular focussed on various genomic and metabolic capabilities of this species. The findings of this thesis have generated insights into the physiological and ecological roles played by this bacterial species as a common member of the human microbiome. Understanding the symbiotic relationship between this bacterial species and its human host may generate rational approaches through the development of novel functional food products that help to support and maintain the gut homeostasis.Item 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 & FragrancesThe 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.Item 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, CatherineOne 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.Item Impact of food, environmental and pharmaceutical antimicrobials on the gut microbiome(University College Cork, 2024) Walsh, Lauren; Ross, R. Paul; Hill, Colin; Science Foundation IrelandThis thesis is concerned with antimicrobials (both protein, peptide and chemical based) and their role in the gut microbiome from a functional and compositional perspective, as well as the isolation and development of new bacteriocins against pathogenic bacteria of interest. Pharmaceutical antimicrobials such as antibiotics, bacteriocins, phages and their endolysins are discussed in chapters 1, 3, 4 and 5. Chapter 1 describes antibiotic alternatives that could potentially be used to treat nosocomial methicillin resistant Staphylococcus aureus (MRSA) infection. Some alternative options include bacteriocins, phages and phage lysins. In Chapter 5, the antibiotics fidaxomicin and vancomycin were compared with the two bacteriocins, thuricin CD and nisin, as potential therapeutics to combat CDI and to assess their overall impact on the gut microbiome. Chapters 3 and 4 specifically discuss the isolation of novel bacteriocin producing bacteria. Chapter 3 describes the isolation of two bacteriocin producing strains termed AS1 and AS2. Chapter 4 outlines the isolation of Paenibacillus ottowii FAA_942_34, which demonstrated activity against IBD-associated bacteria. Environmental antimicrobials and food antimicrobials were examined in chapters 2, 6 and 7. Chapter 2 is a review focusing on the herbicide glyphosateTM, specifically focusing on the compositional and functional changes that glyphosate elicits in the gut microbiome. In chapter 6, glyphosate and four food preservatives were analysed for their effect on the gut microbiome. In Chapter 7, the heavy metal cadmium was used as a selective agent to identify transformants acquiring pJOS01. Following electroporation of pJOS01 into Staphylococcus aureus RN4220, a smaller 21 Kb plasmid termed pJOS02 was recovered in transformants. Formation of pJOS02 from pJOS01 is thought to result from the presence of inverted repeat regions at either end of pJOS02. Overall, the results of this thesis outline a variety of antimicrobials and their effect on the gut microbiome and their potential as therapeutics. This research builds on the growing amount of knowledge around the antimicrobials, the gut microbiome and the effect antimicrobials have on the composition and functionality of the gut microbiome.Item The gut microbiome in inflammatory bowel disease and its confounders(University College Cork, 2023) Eckenberger, Julia; Claesson, Marcus; Science Foundation IrelandInflammatory bowel disease (IBD), encompassing Crohn's disease (CD) and ulcerative colitis (UC), is a chronic remittent-relapsing inflammatory disorder of the gastrointestinal tract that affects millions of people worldwide. Despite progress in disentangling the pathogenesis of this disease, the exact cause of IBD remains unknown. As with other chronic inflammatory disorders, the tissue damage is immune mediated and arises from an interaction of genetic susceptibility factors, environmental triggers and indigenous gut microbiota. The gut microbiota play a central role in IBD pathogenesis. However, despite consistent reports of alterations in gut microbial composition, a coherent microbial signature for IBD remains elusive. Therefore, this work investigated the influence of IBD on the gut microbiota with a particular focus on the multitude of factors, both external and internal, that can potentially confound the distinctions between healthy and diseased states. Our investigation uncovered significant compositional disparities, especially in the case of CD, when comparing individuals with diseases to the control group. Furthermore, longitudinal analyses revealed reduced temporal stability in the microbiota of IBD patients, especially those experiencing fluctuations in disease activity. Geographic location emerged as one of the strongest drivers of microbiota variance, only second to a diagnosis with CD, followed by a history of surgical resection and a diagnosis with UC. Other life style factors also exerted an influence, however, the majority of the compositional variance remained either unexplained or was stochastic in nature. In view of the increasing evidence that commonly prescribed, non-antibiotic drugs interact with the gut microbiome, we re-examined the microbiota variance in IBD to determine the degree to which medications might account for compositional differences between disease-subtypes and geographic location. Although there were variations in medication profiles among individuals from different countries, treatments accounted for a relatively small proportion of the geographic contribution to microbiome. With that said, the cumulative effects of multiple medications significantly contributed to the microbiome differences between patients with UC and CD. Cognizant of the crucial role that microbial metabolites play as molecular messengers facilitating communication between the gut microbiota and the host, we next conducted an investigation into the role of microbial metabolites in patients with CD within the context of the liver-bile acid-microbiota axis. Our findings indicate that the typical signalling from the gut to the liver is disrupted in patients with CD compared to healthy controls, which led to excessive hepatic bile acid (BA) synthesis in a subset of patients. Moreover, variations in hepatic BA synthesis and BA reabsorption within the CD patient group were associated with the resection status. As a result of this disruption, we observed specific microbial changes among CD patients marked by an increase in bile-resistant and a decrease in bile-sensitive genera. This suggests that changes in BA metabolites significantly contribute to the observed differences in microbial composition between health and disease as well as between patients with CD. Collectively, these findings underscore the dynamic nature of microbiomes, highlighting their capacity to adapt to changing environmental conditions while also being subject to host-driven regulation. This added complexity, as well as the increasing data volumes, underscores the need for innovative analytical methodologies that can effectively capture all available information, especially considering the unique characteristics of microbiome data. Hence, we assessed machine learning algorithms, including Support Vector Machines (SVM), Extreme Gradient Boosting (XGB), and Random Forest (RF), for their ability to classify IBD phenotypes using gut microbiome data. All of the tested models successfully differentiated between IBD and non-IBD controls and, to a lesser degree, between IBD subtypes across studies conducted in diverse geographic locations. Importantly, all three algorithms exhibited variations in the selection of taxonomic features considered significant for the classification task, underscoring the need for caution when applying machine learning to tasks aimed at understanding underlying biological aspects rather than solely achieving precise phenotype predictions. In conclusion, the multifaceted approaches undertaken in this body of work yielded valuable insights into the complex interplay of lifestyle, medication, and microbial metabolites in the context of IBD, emphasising the importance of personalized approaches for host heterogeneity and environmental factors in the pursuit of precision medicine.