Microbiology - Doctoral Theses
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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.Item Marine bacteria as a source of polyester-degrading enzymes with biocatalytic potential(University College Cork, 2023) Carr, Clodagh M.; Dobson, Alan; Clarke, David J.; Science Foundation Ireland; Synthesis and Solid State Pharmaceutical CentreThe issue of plastic waste continues to surge, as does our collective awareness of this global problem and interest in finding sustainable solutions for its control. Bacteria, which are among the earliest known forms of life, have evolved over millions of years to degrade organic matter found in the environment by producing enzymes that can catalyse the breakdown of various compounds for energy and nutrient gain. With biotechnological advances in recent decades, bacterial enzymes have emerged as a tool for the catalysis of chemical reactions, where they can aid in the development of safer, more efficient, and more environmentally friendly processes. The conventional recycling of plastic waste has typically consisted of a thermo-mechanical process, where waste is ground down, melted, and reformed into new, but lower-quality products that are less likely to be recycled multiple times. While alternative chemical methods can improve recyclability by facilitating recovery of the raw materials used to make plastic, enzyme-based treatments enable the same process to be conducted under milder, less energy-intensive reaction conditions without the use of hazardous reagents and solvents. Enzymes that are specialized to break apart ester bond-containing compounds in nature can be employed for the degradation of polyester-based plastics, allowing for the sustainable recycling of these materials after use. Polyester-degrading enzymes (termed polyesterases) have been studied with a particular focus on the recycling of polyethylene terephthalate (PET), a synthetic polyester which is mass-produced for use in food and beverage packaging or as a fiber in the textile industry. By an enzymatic hydrolysis mechanism, the PET polymer may be degraded into its short-chain oligomeric intermediates MHET and BHET and/or its constituent monomers ethylene glycol (EG) and terephthalic acid (TPA) which can subsequently be recycled into PET or upcycled into value-added products. In chapter 1, a literature review was conducted on microbial PET hydrolase enzymes to assess existing knowledge in the field, identify key challenges, and determine important areas for future research. Thermophilic bacteria from the phylum Actinomycetota such as Thermobifida fusca emerged as a major source of PET-hydrolyzing enzymes as well as those with fungal and metagenomic origins, while Ideonella sakaiensis, a mesophilic bacterium isolated from a PET-contaminated site served as a model system featuring both PET and MHET hydrolases predicted to work in tandem. Relatively few PET-hydrolyzing enzymes were reported from marine environments, an aspect which we hoped to expand upon. In chapter 2, activity screening and genome mining of marine bacterial isolates led to the identification of a polyesterase, BgP, from a deep-sea, marine sponge-derived Brachybacterium sp. isolate. BgP was explored as a structural homolog of cutinase-like enzymes, such as Cut190, LCC, and TfCut2, which had previously been reported for efficient hydrolysis of PET and its hydrolytic activity was confirmed on the PET model substrate polycaprolactone (PCL). In chapter 3, PET-hydrolyzing activity was confirmed for SM14est, a marine sponge-derived polyesterase from Streptomyces sp. SM14 with this enzyme exhibiting a preference for high-salt conditions and moderate temperatures (up to 45°C). In chapter 4, MarCE, a marine carboxylesterase was found encoded in the genome of a Maribacter sp. isolated from a sea sponge sample collected at Lough Hyne. MarCE was shown to hydrolyze polycaprolactone diol and putative binding of PET oligomers was demonstrated by molecular docking analysis. The work presented on BgP, SM14est, and MarCE makes a case for the continued exploration of marine-derived bacteria, in particular those found within the unique marine sponge ecosystem, as a source of potentially novel polyesterases with relevance for the biological degradation of synthetic polyesters among other biocatalytic applications.Item Shotgun metagenomics-based exploration of kefir microbiomes(University College Cork, 2023) Walsh, Liam; Cotter, Paul; O'Toole, Paul W.; TeagascFermentation is among the world’s oldest forms of food processing, having been applied for millennia to preserve or enhance foods and still accounts for a significant component in the human diet. One such fermented food is milk kefir, which is regularly consumed in eastern European countries and is becoming increasingly popular in western society as a functional food, with numerous scientific studies and reviews associating health benefits with its consumption. Water kefir represents another fermented food of considerable interest, which is increasingly being perceived as a non-dairy alternative to milk kefir. In this thesis, we demonstrate that metagenomic analysis is a valuable tool to (i) expand our, and the general public’s understanding on the microbiology of kefir fermentations, (ii) determine the potential functionality of specific microbes therein and (iii) investigate the impact of milk kefir on the host gut microbiome. In chapter 1, we critically analyse the tools and pipelines that have been used, or that could be applied, to the analysis of metagenomic and metatranscriptomic data relating to fermented foods. In addition, we critically analyse a number of studies of fermented foods in which these tools have previously been applied to highlight the insights that these approaches can provide. Chapter 2 is focused on the shotgun metagenomics-based analysis of 256 kefir milk samples produced from milk kefir grains sourced from various parts of the world. This chapter provides considerable insight into the heterogeneity of these populations, while also uncovering conserved features such as the presence of Lactococcus lactis and Lactococcus cremoris, which may help to define the minimal components required for a fermented milk product to be considered a milk kefir. In chapter 3, we show that shotgun metagenomics, when used alongside metabolomics, can provide evidence that milk kefir modulates the gut microbiome. We show that daily consumption of kefir in a healthy cohort has a subtle impact on the urinal metabolome and gut microbiome. The principle change to the gut microbiome was the detection of Lactococcus raffinolactis post kefir consumption. The detection of Lactococcus raffinolactis is particularly notable given its low relative abundance across kefir metagenomes generated in chapter 2. In chapter 4, we describe Kefir4All, a citizen science project designed to provide the general public with an opportunity to expand their awareness, knowledge and practical skills relating to microbiology, introduced from the perspective of producing a fermented food, i.e., milk kefir or water kefir. In chapter 5, we highlight how research relating to the milk kefir and water kefir microbiome was greatly extended through the efforts of the Kefir4All citizen scientists through the investigation of compositional, functional and evolutionary change in milk and water kefir microbial communities over 21 weeks of repeat regular fermentation by citizen scientists. Overall, this thesis highlights that bioinformatic analysis of high throughput sequencing datasets can expand our knowledge of microbial communities associated with fermented foods and in the host following consumption, while also highlighting the merits of employing fermented food-related studies to raise awareness, knowledge and interest in microbiology and fermentation.Item Molecular characterisation of a conjugative Bifidobacterium megaplasmid(University College Cork, 2023) Dineen, Rebecca L.; van Sinderen, Douwe; O'Connell Motherway, Mary; Science Foundation IrelandBifidobacterium 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.