Microbiology - Doctoral Theses

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    Development of a ribosome profiling protocol to study translation in the yeast Kluyveromyces marxianus
    (University College Cork, 2023-04-25) Fenton, Darren; Baranov, Pavel V.; Morrissey, John P.; Horizon 2020 Framework Programme
    During mRNA translation, the ribosome protects ~28 nt of mRNA within its mRNA tunnel. Ribosome profiling is a method which takes advantage of this protected mRNA fragment, commonly referred to as the ribosome protected fragment (RPF). This method uses endonucleases to digest unprotected mRNA, purifying the RPF and generating a cDNA library. Deep sequencing of these cDNA libraries can reveal the locations of all translating ribosomes in vivo. These data can be used to study aspects of mRNA translation including recoding (e.g. +1 frameshifting), ribosome stalling and differential gene expression between multiple conditions. In addition, as RPFs can be mapped back to the genome, novel translated ORFs may be discovered including novel protein coding genes and regulatory elements of mRNA translation such as upstream open reading frames present in the 5’ leaders (uORFs). Ribosome profiling was initially developed in the model yeast S. cerevisiae, but has since spread to human and bacterial models. The first results chapter of this thesis describes the development of a ribosome profiling protocol to study translation in the yeast Kluyveromyces marxianus, which has not previously be developed. This protocol includes detailed steps to carry out the wet lab protocol as well as some aspects on computational processing. This protocol is accompanied by the release of the K. marxianus genome and transcriptome to a publicly available genome (GWIPS-viz) and transcriptome browser (Trips-Viz) which are tailored specifically for ribosome profiling data. Together, these protocols and browsers will allow others in the K. marxianus community to generate ribosome profiling data and upload/analyse data via these genome browsers. The second results chapter describes the use of a combination of multiple methods including ribosome profiling, RNA-seq and transcript start site sequencing in what is described as “multiomics”. Using these multiomic data, the transcriptional and translational landscape is explored revealing a wide range of features including N-terminal extensions, upstream open reading frames and frameshifting. In addition, these data were used to generate a more complete and accurate genome annotation for K. marxianus by incorporating previously unannotated genes as well as a large number of gene annotation corrections such as splicing errors and start codon corrections. The third results chapter describes using the developed ribosome profiling protocol to study how K. marxianus adapts to a rapid increase in temperature in an effort to understand how thermotolerant yeast adapts to such a stress. These data include both ribosome profiling and RNA-seq with multiple timepoints. Interestingly, the response to heat shock included a large and rapid response whereby cellular respiration is immediately upregulated, supported by both ribosome profiling, RNA-sequencing and biochemical assays.
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    The Sudabiome: oral and gut microbiome parameters of the Sudanese population including dietary and cultural [Toombak] metagenomics
    (University College Cork, 2022-12-15) Sami, Amel; Ross, R. Paul; Stanton, Catherine; , Imad; Science Foundation Ireland; Esther Alliance Group
    The Sudabiome is a unique metagenomic project focusing on several critical areas of the Sudanese population. It highlights a pivotal framework in understanding novel aspects of Sudanese health that include oral and gut health, cultural metagenomics, nutritional trends, migratory travel impacts, and the susceptibility or protection against diseases. Toombak is a fermented smokeless tobacco produced from the Nicotiana Rustica tobacco plant, utilised predominantly by Sudanese males. The Toombak is ‘dipped’ in the oral cavity and replaced several times a day. The microbiome, mycobiome, phylogenetics and metabolomics of 21 pre-prepared and ready to buy Toombak samples purchased from different regions in Khartoum city were assessed, as well as the pH, chemical and microscopical composition of the product. The heavy metals, chromium, cobalt, and copper were high in the pre-prepared form of Toombak, while iron, tobacco-specific nitrosamines [TSNAs], formaldehyde and acetaldehyde were considerably elevated in both types compared to EU regulated smokeless tobacco products. The phyla, Firmicutes and Actinobacteria dominated all samples of Toombak. Virgibacillus was found to be significantly increased in the pre-prepared form [q=3.6314e-8] compared to the ready to buy forms, while Corynebacterium casei [q=1.6417e-5], Atopococus tabaci [p=0.05], and Staphylococcus gallinarum [p=0.01] were the genera abundant in the ready to buy Toombak in comparison to the pre-prepared form. The mycobiome of the ready to buy Toombak was found to be enriched with Aspergillus [85.54%] with the species, Aspergillus heterocaryoticus [43.27%] and austwikii [39.48%] dominating the samples. Furthermore, utilising PICRUSt, the ready to buy samples were found to harbour an increased activity of metal transport systems [K02006, K02013 and K02015] and an antibiotic transport system [K09687]. The Toombak had a large non-homogenous and irregular particle distribution with increased sodium, while the pH of samples was in the alkaline range [8.73-9.92]. TSNA, formaldehyde and acetaldehyde levels observed in Toombak were found to be some of the highest compared to other smokeless tobaccos from around the world such as snus from Sweden and moist smokeless tobacco from the USA. These findings highlight that the final composition of Toombak is affected by its preparation method, with Toombak use having the potential to impart many negative consequences on the health of its users. Dependency on Toombak use was examined through the utilisation of the Fagerstrom test for nicotine dependence smokeless tobacco questionnaire. Nineteen chronic Toombak users had an 85% dependence score. In addition, stress level over a 3-month retrospective period was evaluated through ‘scalp-side’ hair cortisol analysis. Mean cortisol levels were significantly lower [p=0.023] amongst Toombak users [9.7 pg/ml] compared to non-users [19.4 pg/ml]. While use of Toombak may initially bring anxiolytic effects to its users, blunting of the hypothalamic-pituitary axis chronically ensues in Toombak users, affecting cortisol release. This is likely due to the continued high levels of nicotine exposure and toxic effects that many Toombak compounds cause to body organs, including the adrenal glands. The Sudan has a rich heritage of food fermentation that involves a vast array of raw starting materials and methodologies of production that have been preserved for centuries. Forty-six Sudanese fermented foods were sourced from six food categories that included, crop [sorghum and millet], plant [Cassia obtusifolia and sesame], fish, animal, and dairy sources to establish these foods metagenomic composition, some of which were explored for the first time. 16S rRNA sequencing was undertaken, while a subset of foods underwent internal transcriber [ITS] sequencing for mycobiome analysis. Beta diversity [p=0.001] was significantly varied between the categories of Sudanese fermented foods. Animal, fish, and plant-based foods exhibited the highest alpha diversity richness [p=8.7229e-07] while fish and plant-based foods had an elevated Shannon index [p=0.001]. Crop-based foods [sorghum and millet] were found to be enriched in the genus Acetobacter [96.89%], plant-based foods in Aeriscardovia [99.14%], dairy-based foods in Enhydrobacter [85.03%], animal-based foods in Bacteroides [98.22%] and fish-based foods in Sporosarcina [99%]. A loss of Lactobacillus abundance was observed between the preparatory [26.91%] and final [4.38%] stages of sorghum and millet fermented foods. The Kawal food, obtained from the fermentation of the Cassia obtusifolia plant was found to harbour a comprehensive microbiome [Bacillus, Lactobacillus, Pediococcus, Citrobacter and Bifidobacterium] and mycobiome [Candida, Aspergillus, Cladosporium and Malassezia] which could potentially harbour unknown but novel probiotic benefits to consumers. Sudanese fermented foods were however also found to be a source for pathogenic bacteria that included Escherichia-Shigella found abundantly in the plant-based foods [46.8%] and Wohlfahrtiimonas which were found to be abundant in animal-based foods [79.07%]. Intra-food categorical analysis found Lactobacillus to be enriched in the dairy food Mish or deep yoghurt [86%] compared to Gergosh or the dairy/legume biscuit [24%] and Garis or fermented camel milk [10%]. Extensive screening of the oral microbiome of users and non-users of Toombak was undertaken through 415 samples detecting salivary [n=72], plaque [n=71], tongue, buccal cheek, floor of the mouth and palatal mucosal [n=272] microbiome variations. In a subset of salivary samples, the mycobiome [ITS] was also assessed. The microbiome of oral squamous cell carcinoma and premalignant samples [n=46] from formalin-fixed paraffin-embedded tissue was further evaluated from another cohort of users and non-users of Toombak. Alpha diversity [richness] was low for tongue microbiome compared to other mucosal locations [p=3.6849e-34] and Beta diversity was found to be significant between the four oral mucosal locations [p<0.001]. Fusobacteria and Patescibacteria in saliva and Actinomyces in the saliva [p=0.0045], tongue [p=0.013] and palate [q=0.001], were correlated with Toombak use. Skin-associated microbiome were increased amongst the oral cavity of Toombak users and included Staphylococcaceae [q=0.037] in the saliva and Cutibacterium [q=0.04] in the buccal cheek and floor of the mouth. In Toombak users, utilising LEfSe plotting, the genus Peptostreptococcus was most distinct for Toombak use. Non-users of Toombak were found to have abundances in Prevotella [buccal, p=0.04] and Bifidobacterium [tongue, q=0.0049] while Scardovia was discriminant of non-user’s tongue microbiome. Virulent strains such as Prevotella nigrescens and Streptococcus equinus [tongue, p=0.043] were found in the oral microbiome of Toombak users while Prevotella salivae and Streptococcus sobrinus [tongue, q=0.023] were found in non-users of Toombak. There was a three-fold enrichment of oral Aspergillus [78.93%] in the saliva of Toombak users compared to non-users [21.07%] with reduced Candida abundance in Toombak users [4.33%] compared to non-users [95.67%]. Virulent fungal species were also found in smokeless tobacco users [Candida tropicalis] compared to non-users [Candida albicans]. Premalignant lesion microbiome composition included Rothia [p=1.5e-10] and Peptostreptococcus [p=6.5e-07] agreeing with the literature. The oral cancer microbiome in Toombak users harboured genera favouring a poor survival and metastasis that included Stenotrophomonas, [p=0.043] and Schlegelella, [p=0.048], compared to genera found in oral cancer samples from non-users of Toombak such as Lactobacillus, [p=0.024]. The genus Corynebacterium_1 was found to be common to the Toombak product, the oral cavity [saliva, p=0.0054, floor of the mouth, p=0.0028] and oral cancer samples [p=0.044] of Toombak users. These findings highlight that several oral distinctions follow Toombak use, allowing for a microbiome that could potentiate disease and destabilise the normal oral microbial flora while our findings also harbour the potential for microbiome biomarkers to be employed in the future screening of oral carcinoma in Sudan. In 141 participants, an inter-continental metagenomics study was achieved on residents from Africa [Sudan] and Europe [Ireland] concerning diet frequency, microbiome composition and metabolome activity of stool samples. The European Prospective Investigation into Cancer and Nutrition Norfolk Food Frequency Questionnaire [EPIC - Norfolk FFQ] was modified and employed to compare food groups, nutrient and energy intakes between three cohorts, the Irish living in Ireland [Irel], the Sudanese living in Ireland [SudIrel] and the Sudanese living in Sudan [Sud]. Cereals and cereal products, fats and oil, fish and fish products, fruits and vegetables, meat and meat products, sugar preserves and snacks were consumed at a reduced intake in the Sud cohort with a high strength of association and large effect size [eta2 = >0.14]. Egg and egg products [eta2 =0.04], nuts and seeds [eta2=0.05] and non-alcoholic beverages [eta2=0.108] were also consumed highly in Sud, albeit with low and medium effect sizes. The average energy intake [measured in kcal/day] between groups was statistically different [p <0.001] and was found to be lowest in Sud [1077 kcal/day], compared to SudIrel [2729 kcal/day] and Irel [3442 kcal/day]. The diet of the Sud cohort was thus found to be low in calcium [p=0.05], carbohydrates, fibre, protein, fat, potassium, sodium, and vitamins A, C, B1, B2, B6, B12 and D [p<0.001] but high in vitamin B5 [pantothenic acid] and intermediate in Vitamin K levels. These results highlight the variations of dietary consumption between the two populations; Sudan and Ireland and helps set a fundamental previously unmet base for future research to evaluate dietary trends in the Sudanese population, including children. Microbiome Beta diversity plotting [q=0.001] highlighted a distinct ‘sandwiched’ presentation to the cohort SudIrel compared to Irel and Sud groups, with R2 = 63%. Alpha diversity measures however were found to be non-significant between groups [Shannon index or evenness, p=0.74]. Microbiome genera such as Prevotella, [q=3.169e-06], Megasphaera [q=4.61e-05], Klebsiella [q=3.9067-e], Bifidobacterium [q=7.023e-03] and Lactobacillus [q=2.918e-09] were found to be depleted in the Irel cohort compared to both Sud and SudIrel while in the Irel cohort, the microbiome was instead enriched in Lachnospira [q=1.623e-09] and Bacteroides [q=3.056e-05]. Collinsella was found to be four times more abundant in Sud and SudIrel [44%] compared to Irel [11%]. Principal coordinate analysis of metabolomic compounds from stool samples highlighted 27 metabolites to be significantly varied between Irel and Sud including proline [p=0.003], adipic acid [0.04], and phosphocholine [p=0.02], 21 metabolites to be significantly varied between Sud and SudIrel including sugar alcohol [p=0.04] and N-acetylneuraminic acid [p=0.01] and no significant metabolomic variations between Irel and SudIrel. The short-chain fatty acids, isobutyric [p=0.0] and isovaleric acid [0.002] were significantly lower in the stool samples from those in the Irel cohort. LEfSe plotting of PICRUSt KEGG orthology pathways indicated multi-drug resistance activity pathways in the Irel cohort [K09686], mineral and heavy metal transport activity in Sud [K06199] and in SudIrel, carbohydrate metabolism pathways were prominent [K02794, K02796, K02775, K02773, K02774]. From these results, a migratory impact on the traditional microbiome of the Sudanese population occurs that has not been previously explored which may allow for both the susceptibility and prevention of disease in this population. Those who migrate from the Sudan to Ireland [SudIrel], carry both tradition microbiome preservation [i.e., continued Prevotella abundance] and modern microbial carriage [i.e., Bacteroides enrichment]. Through these studies, there has been a novel and in depth uncovering of human and environmental microbiome features associated for the first time with the Sudanese population in comparison to Irish controls. Through fermentation techniques for example, cultural [Toombak] and dietary [fermented foods] produces may carry health benefits but also numerous predicaments such as the increased risk of oral cancer development and systemic cortisol imbalance from Toombak use and the contraction of food-borne diseases from contamination during Sudanese fermented food production. The studies on Sudanese human oral and gut health have further provided insight into how the oral and gut microbiome may respond to modern challenges such as migration and economic, dietary and cultural influences. This project further give light on the merging of next generation sequencing science with economic and population challenges. Continued microbiome research in Sudan should continue to be geared towards sharper preventative disease strategies, management, and treatment outcomes.
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    Leveraging community-based sequencing approaches to characterise the dairy microbiome
    (University College Cork, 2022) Yap, Min; Cotter, Paul; O'Toole, Paul W.; O'Sullivan, Orla; Irish Dairy Levy; Science Foundation Ireland; Department of Agriculture, Food and the Marine, Ireland; Horizon 2020
    Microorganisms exist in communities along the food chain and contribute, positively and negatively, to the safety and quality of food products downstream. While traditional culture-based methods usually investigate specific groups of microorganisms, advances in high-throughput DNA sequencing have enabled the study of entire microbial communities in food or food-related samples. In this thesis, shotgun metagenomic sequencing was employed to investigate microbial communities in bulk tank milk. First, shotgun sequencing was used to determine the impact of farm cleaning methods on the microbiome. The results revealed that seasonality and geography significantly impacted the bulk tank milk microbiota but that the use of chlorine-based relative to chlorine-free cleaning methods did not result in differences. Next, further sampling was performed to determine if interactions exist between the bulk tank milk microbiota, raw milk chemical composition and climatic variables. Analysis of shotgun sequencing data from bulk tank milk over 12-months revealed a stable core microbiota and showed a significant and obvious influence of season and location on the raw milk microbiota. Associations were detected between pathogenic and mastitis-related taxa and chemical composition and climate/environmental variables. The interconnected nature of these variables suggests there is merit in considering the use of chemical and/or climate variables as markers for food safety and spoilage threats in bulk tank milk. While useful information can be obtained through shotgun metagenomic sequencing, the untargeted nature of this approach involves the sequencing of all DNA present in samples, including host-derived reads that are present in high proportions in milk (~90%), as well as both live and dead cells, which could alter inferred community structure or diversity. To overcome these challenges, two studies were completed to improve current shotgun metagenomic sequencing methodology. First, three methods were evaluated to assess their relative ability to deplete host DNA or enrich microbial DNA. The MolYsis™ Complete5 kit yielded significantly higher proportions of microbial reads and improved sequencing depth, allowing for further characterisation of the bovine and human milk microbiomes through the recovery of metagenome-assembled genomes (MAGs). Tackling the issue of microbial viability, 4 methods were explored to compare their relative efficacy at distinguishing between viable and non-viable cells with a five-strain live and heat-inactivated model microbial community spiked into a bovine milk matrix and sequenced on Illumina and Oxford Nanopore Technologies (ONT) platforms. The results showed that all methods were relatively accurate, but significant differences between library type (DNA and RNA) and sequencing technologies (Illumina and ONT) were found. Variations between library types were due to the differing molecular targets, while sequencing depths, error rates and bioinformatic pipelines likely contributed to the differences between sequencing technologies. This pilot study provides insights into the potential enhancement of sequencing-based approaches to better characterise and differentiate live and dead microbes in food-related environments, but more work is required to optimise these methods before more widespread use. Overall, the use of shotgun metagenomics provided valuable insights into the bulk tank raw milk microbiota, improving our current understanding of the factors that influence the quality and safety of raw milk. Building on the work performed during this thesis, further improvements can enhance existing community-based sequencing methods to allow for better understanding of food microbiomes, that in turn would be beneficial for food safety, quality, and public health.
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    Characterization and engineering of sugar transporters in the yeast Kluyveromyces marxianus for biotechnological applications
    (University College Cork, 2022-06-30) Donzella, Lorena; Morrissey, John P.; Sousa, Maria Joao; H2020 Marie Skłodowska-Curie Actions
    Sustainable and efficient use of lignocellulosic hydrolysates in yeast-based biorefineries requires the simultaneous consumption of all the sugars present in this raw material, hexoses and pentoses. This represents a key bottleneck because of the low number of specific pentose transporters identified and the slow rate of pentose uptake into the yeast cell when glucose is present. Kluyveromyces marxianus is a non-conventional yeast that has attracted considerable attention due to its traits of industrial interest, like the ability of naturally assimilating pentoses, high thermotolerance and fast growth rate. In this thesis, the main goal was to investigate and engineer pentose uptake in K. marxianus to obtain a transporter that would allow co-consumption of glucose and xylose for the application in biorefineries. In order to identify novel pentose transporters, a K. marxianus screening platform (ΔPT) was constructed by deleting 12 genes that encoded transporters of the major facilitator superfamily (MFS). Using this ΔPT platform, which was unable to grow on pentoses, we identified several native K. marxianus transporters that recovered the native strain’s ability to grow on either xylose and/or arabinose. Kinetic studies on the most promising candidates using 14C-labelled sugars were carried on. One member of these transporters showed high affinity for both xylose and glucose, though the affinity for glucose was still 150-fold higher than for xylose. Using in silico analysis we selected target amino acids of this transporter for site-directed mutagenesis in an attempt to convert it into a specific xylose transporter. Several mutants were constructed and characterised by radioactive uptake and growth assays in bioreactors on mixture of sugars. One of these mutants decreased its ability to transport glucose but still transported xylose with even higher affinity kinetics. The successful reprogramming of this transporter to increase its preference for xylose over glucose was used to facilitate efficient co-consumption of hexose and pentose sugars in a novel K. marxianus strain, significantly shortening the time of the whole process. Keywords: yeast, sugar transporters, xylose, biorefineries, Kluyveromyces spp.
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    Evaluating substrate utilisation to target newly identified health-promoting gut bacteria
    (University College Cork, 2021-12) Lordan, Cathy; Cotter, Paul; Ross, R. Paul; Teagasc
    Promoting the growth and/or activity of potentially beneficial human gut microorganisms through the provision of specific substrates has been the subject of many studies over recent decades. Such substrates can include prebiotics, which promote growth in a targeted manner, but prebiotics can also be combined with other less target-specific nutrients to further enhance the growth of these targets. This field has continued to advance in recent years and has also expanded in response to the identification of new target species, such as Akkermansia muciniphila, Eubacterium rectale, Faecalibacterium prausnitzii, Roseburia inulinivorans, and Ruminococcus bromii, many of which produce metabolites that can contribute to host health. These developments have been in part due to improvements in culture-based techniques, advances in DNA sequencing-based approaches and improved computational pipelines. The combined use of these tools has enormous potential with respect to elucidating substrates that can be applied to specifically target microbes of interest and is the focus of this thesis. Chapter 1 reviews the literature relating to prebiotics, and potential prebiotics, with a focus on more recently identified health-promoting gut bacteria. Building on this, in Chapter 2 we applied both in silico and in vitro techniques to predict and assess substrate utilisation for seven strains across five species of interest. The bioinformatics-based component of Chapter 2 is expanded in Chapter 3 through the analysis of publicly available microbial genomes of the same species of interest and, through the creation of metabolic models, predicting the substrates that these microorganisms could consume. Finally, in Chapter 4 we employ an ex vivo model to evaluate, through shotgun metagenomic sequencing, the impact different substrates, including simple sugars, oligosaccharides, and whey protein concentrate had on the taxonomic composition and functional potential of a colonic microbiome. Thus, facilitating the design and testing of a functional prototype beverage comprised of some substrates assessed here. Overall, this thesis explores different substrates that could be applied to target the growth and/or activity of recently identified health modulating microbes within the human gut. Combining in silico, in vitro, and ex vivo approaches have the potential to identify and assess a variety of substrates that may be applied to bring about microbiome-mediated enhancements of host health.