Microbiology - Doctoral Theses

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Molecular characterisation of a conjugative Bifidobacterium megaplasmid
(University College Cork, 2023) Dineen, Rebecca L.; van Sinderen, Douwe; O'Connell Motherway, Mary; Science Foundation Ireland
Bifidobacterium species are highly abundant autochthonous bacteria of the human gut microbiota, particularly during host infancy. Various members of the Bifidobacterium genus have been associated with a plethora of health-promoting attributes, among which maintaining gut homeostasis, limiting pathogenic bacterial invasion as well as their purported ability to modulate host immune responses are notable examples. Due to their positive association with human health, this genus has received substantial scientific attention and commercial interest. Plasmids were once considered an atypical feature of Bifidobacterium and those identified within this genus were primarily small cryptic plasmids which were presumed to replicate by a so-called rolling circle mechanism. However, the recent availability of long-read single-molecule sequencing technologies precipitated the resolution of the first reported bifidobacterial megaplasmid isolated from the common and abundant inhabitant of the human gut, Bifidobacterium breve JCM7017. The discovery of this >190 kb conjugative megaplasmid, denoted pMP7017, and the subsequent identification of pMP7017 homologs in several B. longum subsp. longum strains, highlights the prevalence of this megaplasmid family within this genus, representing an unexplored feature. Conjugative plasmids such as pMP7017 play a central role in bacterial evolution and have the potential to significantly influence the activity of the microbiome community and, by extension, impact human health and physiology. The research described in this thesis covers the replication functions of megaplasmid pMP7017 and exploits these functions for the development of important molecular tools that facilitate the genetic engineering of these genetically recalcitrant bacteria. As pMP7017 represents the first and, thus far, only experimentally validated conjugative plasmid of bifidobacterial origin, research performed within the context of this thesis also concerned examination of the conjugative functions of this megaplasmid. While the molecular characterisation of pMP7017 represents an opportunity for the development of much needed molecular tools and provides a starting point in the understanding of bifidobacterial DNA transfer systems, an integrated approach of comparative analyses and metagenomic data mining has generated highly relevant and insightful information concerning the biology and distribution of pMP7017 and related megaplasmids.
Development of synthetic biology tools for Kluyveromyces marxianus and their application to produce aromatic molecules in this yeast
(University College Cork, 2023) Akinola, Joel Abidemi; Morrissey, John P.; Horizon 2020; Eli Lilly and Company
The experimental work in this thesis was carried out as part of the European Union-funded Horizon 2020 project CHASSY (https://www.ucc.ie/en/eri/projects/chassy.html). CHASSY was a collaborative research project made up of industry and academic partners who set out to unlock the potential of yeasts as microbial cell factories for producing valuable compounds. While Saccharomyces cerevisiae is the most studied yeast for which there are many genetic engineering tools available and is thus commonly used for synthetic biology (SynBio) applications, it has certain limitations for specific applications. This has in recent times led to the exploration of the industrially attractive non-saccharomyces yeast, Kluyveromyces marxianus for various synthetic biology applications. This yeast has a QPS/GRAS status that drives its application in the food industry, it is thermotolerant, able to metabolise and grow on several feedstocks and SynBio tools for achieving heterologous gene expression and genome engineering by CRISPR technologies have been developed for it. Those recent advancements have contributed to research on this yeast in regard to improving our understanding of its physiology and for SynBio applications. To further improve genome editing tools for the yeast, a counter-selectable amidase (amdS) gene from bacteria was developed in this thesis for marker-dependent integration. Close to 100% of the transformants obtained had an amdS marker cassette integrated when the cassette was transformed. A CRISPR-based marker-free system was also developed and optimised for simultaneous editing of more than one locus in the yeast’s genome. This system is highly useful because it eliminates genetic engineering restrictions that arise as a result of the need for markers, and it was made possible thanks to the yeast’s homologous recombination machinery which allows homology directed repair (HDR). Thus, the system was optimised by improving HDR through the inactivation of non-homologous end-joining, the expression of recombinases and chemical synchronisation of yeast cells into the S/G2 phase of cell cycle. The optimisation allowed efficient marker-free deletion of specific genes, integration at specific loci and multi-loci integration at repeat sequences that are distributed around the yeast’s genome. The developed tools in this work are entirely based on the well-established yeast toolkit standard which uses Golden Gate cloning technique for DNA assembly, making it possible to use them in combination with pre-existing tools and they are transferable between SynBio applications. Therefore, the tools were used for unprecedented engineering of K. marxianus for heterologous production of a group of plant aromatic compounds called phenylpropanoids. The biosynthesis of these compounds takes the aromatic amino acids (AAA), phenylalanine, and tyrosine as precursors. Therefore, the key enzymes of the shikimate pathway which is responsible for AAA biosynthesis and the glycolytic and pentose phosphate pathways which supply phosphoenolpyruvate and erythrose 4-phosphate as precursors of the shikimate pathway were overexpressed in order to increase AAA production. The phenylpropanoid pathway begins with the conversion of tyrosine to coumaric acid by means of a single step reaction catalysed by tyrosine ammonia lyase or phenylalanine in two reaction steps where phenylalanine ammonia lyase first converts phenylalanine to cinnamic acid and then cinnamic acid 4-hydroxylase and cytochrome P450 reductase convert cinnamic acid into coumaric acid. Given that coumaric acid is an important phenylpropanoid from which many other phenylpropanoids are produced, its production was first engineered into K. marxianus in with the expectation that it will be possible to further engineer coumaric acid producers to produce more complex phenylpropanoids. This work resulted in the production of coumaric acid at a yield of over 40mg/g glucose in 5L batch bioreactors with 2.5L working volumes of minimal glucose medium. Subsequently, plant genes that catalyse the derivatisation of coumaric acid into an important flavonoid called naringenin (coumaric acid:coenzyme A ligase, chalcone synthase and chalcone isomerase) were expressed in a coumaric acid producer. This however, only produced very low amounts of naringenin with minor decrease in coumaric acid yield, giving rise to a study in which the depletion of coumaric acid was the objective. Since the derivatisation requires coenzyme A and malonyl-CoA, increased biosynthesis of both metabolites was engineered by overexpressing acetyl-CoA carboxylase, acetyl-CoA synthase, and the entire coenzyme A biosynthetic pathway, leading to naringenin yield of 1231µg/g glucose. Due to the fact that freely available intracellular AAAs are catabolised by the Ehrlich pathway in yeasts, a study was conducted to identify the genes that contribute K. marxianus phenylpyruvate decarboxylase activity, a key player that is responsible for driving flux through the pathway. That study revealed that ARO10 contributed most of the enzyme activity with some coming from PDC1 and provided information that enabled the reduction of the activity so that AAAs are available to enter the heterologous phenylpropanoid pathway. Together, this work demonstrates the potential of K. marxianus as a cell factory for producing phenylpropanoids, bringing us closer to the production of these compounds from diverse feedstocks.
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 Ireland
The 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.
The use of lactic acid bacteria as methane mitigation strategies in ruminants
(University College Cork, 2023) Doyle, Natasha; Stanton, Catherine; Ross, R. Paul; Hill, Colin; Horizon 2020
Lactic acid bacteria (LAB) have often been cited as potential strategies for the reduction of methane emissions from ruminants, however more research is needed to identify whether LAB can be an effective methane mitigation option in ruminant livestock. The aim of this thesis was to investigate the potential of LAB as methane mitigation strategies in ruminants, by determining the effect of LAB on methane production, animal health and productivity. Chapter 1, a literature review, addresses the topic of greenhouse gas (GHG) emissions in agriculture, including methane, nitrous oxide and carbon dioxide. Methane production from ruminants is influenced by a wide variety of factors, including feed additives such as lactic acid-utilizing bacteria, LAB and yeast. This chapter focuses on the use of direct fed microbials (DFMs) for the reduction of GHG emissions in agriculture. The strengths and challenges associated with each strategy are also explored. Chapter 2 focuses on the use of LAB specifically for methane mitigation in ruminants. Enteric fermentation is the largest anthropogenic source of agricultural methane and, as such, requires innovative solutions for the reduction of methane from livestock. This chapter reviews current literature and provides a comprehensive analysis on the potential use of LAB as methane mitigation strategies. To determine the methane mitigation potential of LAB, several species of methanogens (methane producing microorganisms) were cultivated. In Chapter 3, a total of 6 methanogenic species of both human and ruminant origin were cultivated. Methanobrevibacter ruminantium and Methanobrevibacter gottschalkii were chosen for cultivation as they comprise ~75% of the archaeal population within the rumen. Several human methanogens were also chosen for cultivation to allow for comparison of cultivation techniques required for these methane producing microorganisms and for the determination of methane production from several methanogenic strains. Gas chromatography methods determined that methanogens of rumen origin produced almost 4000 times more methane (90g/L) in vitro, when compared to human methanogen strains (0.025g/L). Establishment of methanogen cultivation techniques were essential to allow for further research to be carried out in Chapter 4. Methane inhibition trials assessed the potential of LAB and various bacterial families, including Staphylococcus, for the reduction of methane in vitro. Staphylococcus capitis APC 2918 resulted in the best methane inhibition of 54% and 69% in Methanobrevibacter ruminantium and Methanobrevibacter gottschalkii, respectively. Commercially available LAB strains (provided by a commercial partner), Lactobacillus plantarum LP58 and Lactococcus lactis subsp. lactis SL242, demonstrated methane reduction up to 28% against Mbb. gottschalkii (P <0.05). The screening process reported in chapter 4 resulted in a biobank of 20 strains with bacteriocin capabilities and possible anti-methanogenic potential. Both of the commercially available LAB strains, Lactobacillus plantarum LP58 and Lactococcus lactis subsp. lactis SL242, with demonstrated methane reduction effects of 9-28% in vitro, were chosen as silage co-inoculants for use in an animal intervention study. Chapter 5 determined the effects of these LAB co-inoculants on silage quality. LAB when administered as co-inoculants resulted in good quality silage in terms of pH (4.27, SD 0.653), dry matter percentages (26%, SD 1.532) and overall appearance. However, when compared to control silage with no silage inoculant, little statistical difference was seen. This LAB inoculated silage was subsequently used in a 7 week animal intervention study in Chapter 6. Animal intervention studies resulted in reduced methane emissions of 5.7% (P < 0.01) in 30 late-lactating Holstein dairy cattle. Ruminal content of cattle fed LAB inoculants resulted in a 2-log reduction of Proteobacteria (P = 0.006), a major phylum of gram-negative bacteria. Cyanobacteria were also significantly reduced by ~3 logs in treatment cattle (P = 0.007). Cattle treated with LAB inoculated silage produced milk with high lactose content (4.6%, P<0.05), reduced milk urea nitrogen between weeks 2 and 5 of the animal intervention (P< 0.05), and greater free fatty acids (9.92%, P <0.05). Taken together, the results of this thesis demonstrate that LAB provide an effective solution for the reduction of methane emissions in ruminants, while simultaneously improving animal health and maintaining productivity. Through the knowledge gathered, it can be said that LAB have the potential to provide methane mitigation solutions which will aid in the development of a competitive and sustainable Agri-food sector.
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.

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