Microbiology - Doctoral Theses
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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 Exploring early-life microbiome transfer and therapeutic applications in bovines and humans(University College Cork, 2023) Linehan, Kevin; Stanton, Catherine; Ross, R. Paul; APC Microbiome Institute; Science Foundation IrelandThe microbiome consists of intricate microbial communities, including bacteria, archaea, eukarya, viruses, bacteriophages, and their associated products. These dynamic entities establish symbiotic relationships with their bovine and human hosts, exerting direct or indirect influences on physiology throughout life, impacting both health and disease outcomes. The early-life microbiome exerts a profound impact on developmental trajectories and long-term health. The extent to which different maternal microbial sources and perinatal factors contribute and shape the initial colonisation, development, and functionality of the neonatal microbiome is a topic of ongoing research. Understanding these factors is crucial for comprehending the early establishment of the microbiome. Given the current antibiotic resistance crisis, there is significant importance in leveraging host-microbiome interactions to develop microbiome-based therapeutics. This thesis explores a number of research foci with a view to gain a better understanding of (1) the influence of different maternal microbial sources and perinatal factors on the initial establishment of the human infant gut microbiome, (2) harnessing the bioactive composition of bovine colostrum for bovine and human health applications, (3) characterising the virome of bovine colostrum and the influence of perinatal factors on its composition, and (4) the potential of microbiome-based therapeutics for disease treatment in bovines and humans. Chapter 1 discusses the impact of perinatal factors, including maternal nutrition, antibiotic use, gestational age, and mode of delivery, on the initial colonisation, development, and function of the human neonatal gut microbiome. The elucidation of the precise extent to which these factors influence gut microbiome establishment and identification of those with the most decisive effects on colonisation are essential for improving infant health. In Chapter 2, the diverse array of bioactive components in bovine colostrum suitable for the development of functional foods, nutraceuticals, and pharmaceuticals with veterinary and human health applications are discussed. The processing techniques used to produce high-value colostrum-based products, and recent studies utilizing bovine colostrum for veterinary and human health are also outlined. In Chapter 3, using a cohort of 18 healthy mother-infant dyads, the microbial composition of three potential maternal sources of microbial transmission (oral, vaginal and placental) to the microbiota of their new-born infant (oral and meconium microbiota) were characterised. This allowed investigation of the contribution of numerous transmission routes and the impact of various perinatal factors on the initial establishment of the infant gut and oral microbiome. The results of this study consolidate and corroborate recent findings surrounding the existence of a meconium microbiome and the absence of a placental microbiome. Furthermore, the study shows that significant vertical transfer, primarily from the maternal oral cavity to the infant oral cavity occurs in early life. In Chapter 4, a reproducible, low cost and high-throughput virome extraction method was developed for bovine colostrum. Shotgun sequencing and viral specific metagenomics bioinformatics were performed on samples from 72 dairy cows, given dry cow therapy (n=48) or naturally dried off (n=24). The impact of farm level variables (location and parity) were also assessed. Phages carrying multidrug resistance genes (smeS, lfrA, kdpE and baeS) were identified. Antibiotic treatments significantly impacted virome composition and the presence of resistance genes specific to the administered antibiotic. This study provides novel insights into disease development and transmission in animals and humans, and the contribution of viruses to the spread of global antimicrobial resistance. In Chapter 5, two novel Staphylococcus aureus bacteriophage species from the genus Phietavirus were isolated. Phages were lytic against several human and bovine mastitis causing strains of Staphylococcus aureus (including MRSA). Phages displayed excellent characteristics for in vivo experiments, with no resistance genes present, stability to variations in pH (4 to 9), temperature (up to 60 °C), chloroform resistant and capable of replicating in mastitic milk. Finally, in Chapter 6, a field trial was undertaken to investigate the efficacy of emulsion based postbiotic and live-biotherapeutic formulations of Lactococcus lactis DPC3147, producer of the bacteriocin lacticin 3147, as alternative therapeutics for bovine mastitis. Twenty eight cows with chronic mastitis were treated with emulsion-based formulations containing either viable L. lactis DPC3147 cells (n=15) or heat-killed L. lactis DPC3147 cells (n=13). The efficacies of the two formulations in stimulating a localised immune response (measuring interleukin-8 concentrations in milk) and cure rates (somatic cell counts reductions and pathogen absence) were evaluated. This study demonstrated that the presence of heat-inactivated bacteria (a postbiotic) was as effective as the live bio-therapeutic in eliciting a localised immune response in cows with chronic mastitis. The results outlined in this thesis provide valuable insights into the intricate dynamics of early-life microbiome transfer and outline novel microbiome-based therapeutics for applications in bovines and humans.Item Development of knowledge base and methodology for the rational microbiome modulation in irritable bowel syndrome(University College Cork, 2023) Das, Anubhav; O'Toole, Paul W.; Baranov, Pavel V.; Jeffery, Ian B.; 4D Pharma Cork Ltd.The gut microbiota typically comprises a steady-state community whose composition and functions are governed in part by factors including diet, disease, medications, ethnicity, geographic location, host genotype, and other lifestyle and clinical factors. There is a large body of research that supports the role of the microbiota in disease, host gene regulation and maintenance of host physiology. My thesis focuses on Irritable Bowel Syndrome (IBS), a chronic functional gastrointestinal disorder, associated with alterations in microbiota composition and function. IBS is marked by symptoms like pain and distension of the abdomen, abnormal bowel habits, leading to social disablement. Patients with IBS are mainly classified into four clinical sub-types based on symptoms, viz., IBS-C (Constipation predominant), IBS-D (diarrhoea predominant), IBS-M (mixed), and IBS-U (unclassified), but there is heterogeneity within IBS at various levels including symptoms as well as microbiome composition. Attributed by some investigators as a disorder of the gut-brain axis, the aetiology of IBS remains unclear. There has been growing interest in the development of efficient microbiome based diagnostic tools and therapeutic products that rely on robust and biologically relevant biomarkers which can be identified through a deeper understanding of the disorder. Considering this, the goals of my thesis include investigation of alterations in microbiome composition, function, and structure in IBS, which could be applied to diagnose or stratify IBS patients. My research also focused on understanding inter-microbial interaction patterns, and development of statistical models which can aid in screening of potential live bio-therapeutic products (LBPs) and augment transformative therapeutics for microbiome related disorders like IBS. To work toward the goals of my thesis, I analyzed a cohort of 80 IBS patients and 65 matched Control subjects. Using state of the art bioinformatics methods, along with statistical, and systems biology-based approaches, I explored the various microbiome data types searching for insights into the underlying biology of IBS pathophysiology. Firstly, I took a multi-omics analysis approach to investigate alterations in microbiome community composition and structure, predicted functionality, and faecal and urine metabolome, along with dietary habits in IBS. Subsequently, I analyzed the non-bacterial components of the microbiome (mycobiome and virome) to further understand their potential roles in IBS progression. An integrative, inter-kingdom analysis of the microbiome was also conducted to explore the differences in inter-kingdom interactions in IBS as well as comparison of the ability of these components to predict IBS. Finally, I performed a systems biology-based metabolic modelling of microbiomes of the samples to evaluate changes in metabolic events, and inter-microbial metabolic interactions in IBS, followed by identification of species predicted as being capable of modulating the overall metabolic output of the microbiome community of IBS and Controls. The findings of my analysis not only corroborated previously reported observations, but also provided novel insights which have clinical implications. I observed significant differences in faecal microbiome and metabolome of patients with IBS and Bile Acid Malabsorption (BAM) and developed predictive models to stratify IBS patients. I also observed reduced agreement within microbiome, and between omics datasets, in datasets from subjects with IBS. Inter-kingdom analyses also indicated a lack of concordance between bacteriome and non-bacterial components in IBS. Metabolic modelling analysis showed differences in predicted reaction rate profiles between IBS and Controls, along with lack of co-operation within the bacterial community, and provided a distinct taxonomic-metabolomic signature of dysbiosis in IBS. Based on perturbation analysis of the metabolic models, I could identify species acting as potent modulators of the community metabolic output. Given the stochasticity associated with the microbiota of IBS, there is scope for these modulating species to directionally perturb the microbial community towards a more favourable structure (i.e., one promoting gut health), thus representing a therapeutic target.Item Characterization of two prevalent yet distinct conjugation systems among lactococci(University College Cork, 2022-12-08) Ortiz Charneco, Guillermo; van Sinderen, Douwe; Mahony, Jennifer; Science Foundation Ireland; DSM Food and BeverageLactic acid bacteria are Gram-positive, non-motile, lactic acid-producing microorganisms, with Lactococcus lactis and Lactococcus cremoris representing species that are extensively applied as starter bacteria in dairy fermentations. Starter culture providers continuously aim to expand their culture catalogues and enhance their existing cultures to maintain pace with the ever-changing consumer demand for products with diverse organoleptic attributes. Plasmids present in L. lactis and L. cremoris species harbor genes associated with many important technological traits, such as bacteriophage resistance, lactose metabolism and exopolysaccharide (EPS) production. There are practical limitations associated with the isolation of new strains, while there are regulatory hurdles associated with the application of genetically-modified organisms (GMOs). Consequently, the enhancement of existing starter strains through natural gene transfer methods including conjugation can be a highly advantageous approach, though with its challenges as (plasmid) conjugation efficiencies between lactococcal strains reported in literature have been very low. This thesis describes the optimization and characterization of two plasmid-encoded conjugation systems that are commonly found in L. lactis and L. cremoris strains. Chapter II details an optimized protocol for conjugation with the lactococcal conjugative plasmid pNP40, which represents a highly prevalent conjugation system among lactococci. A combined approach of in silico and mutational analyses facilitated the functional assignment of many conjugation-associated genes including a transcriptional repressor of this system. Furthermore, certain non-conjugative plasmids were observed to co-mobilise together with the conjugative plasmid. Chapter III details the mutational and functional analysis of the pUC11B conjugation gene cluster, which was demonstrated to be capable of self-transfer using the optimized conjugation protocol described in Chapter II. This conjugation system, along with those encoded by plasmids pMRC01 and pAF22, represents the second most prevalent type of conjugation system among lactococcal plasmids in public databases. Within the conjugation gene cluster, a transcriptional repressor and an anti-restriction modification (RM) system were identified. Interestingly, the anti-RM system promotes more efficient conjugation of this plasmid into recipient strains with Type II or Type III RM systems. Chapter IV focuses on the transcriptional analysis of the conjugation gene clusters of pNP40 and pUC11B, revealing the presence of three and five promoters, respectively, as well as their precise transcriptional start sites. Moreover, regulation of several of these promoters appears to be mediated by the individual or concerted action of the corresponding relaxase and transcriptional repressor associated with each conjugation system. In Chapter V, pNP40- and pUC11B-mediated mobilisation of smaller, non-conjugative plasmids present in the same donor strains is reported. The origin of transfer (oriT) sequences of pNP40 and pUC11B were determined. Sequences similar to these oriT sequences present in other, non-conjugatable plasmids had been reported to be required for their mobilisation. Moreover, the relaxases of both conjugation systems presented specific nickase activity to their respective oriT-containing sequences. Finally, the presence of additional copies of the auxiliary gene mobC in the same donor strain was shown to confer a high-frequency mobilisation phenotype. Chapter VI details the identification and characterization of surface adhesins encoded within the two prevalent lactococcal conjugation gene clusters. The surface adhesion functions of both pNP40 and pUC11B conjugation systems appear to be interchangeable between these two otherwise divergent systems. Additionally, overexpression of these proteins promoted significant cell clumping phenotypes, reminiscent of their cell adhesin function. In summary, the thesis provides a wide range of novel findings which facilitated comprehensive insights into the functionality, prevalence and diversity of lactococcal conjugation systems and the means by which conjugation is regulated. Understanding these systems and the enhancement of conjugation processes is invaluable to the rapid development of robust and technologically desirable starter cultures for sustainable food production systems.