Microbiology - Doctoral Theses

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    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.
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    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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    Impact of antibiotics on the gut and milk microbiome
    (University College Cork, 2023) Patangia, Dhrati; Stanton, Catherine; Ross, R. Paul; Science Foundation Ireland
    While antibiotics are lifesaving, they come at the cost of antimicrobial resistance (AMR) and collateral damage to microbiome. Using single-sample and longitudinal study designs, this thesis aimed to study antibiotic resistance genes (ARGs) spread using shotgun metagenomics sequencing. ARGs developed in animals can enter humans through the food chain. We examined the effect of single antibiotic use on microbiome and resistome in milk from dairy cows longitudinally. The microbial diversity increased from colostrum to later time-points in the antibiotic groups which was not prominent in the no-ab group. Further, microbial composition in all groups was different, leading to distinct clustering of no- antibiotic from antibiotic groups. All groups had a wide resistome profile, with antibiotic groups showing higher ARG abundance. Also, high abundance of mastitic pathogens was absent in the no-ab group. The results show that prophylactic antibiotics during DCT is not essential. We next longitudinally studied microbiome and resistome in infants divided into three groups [CSab (C-section/antibiotic), CSnoab (C-section/antibiotic naive) and VDnoab (Vaginal delivered/antibiotic naive)] based on delivery mode and antibiotic use during early life. CSab group showed low initial microbial diversity, which increased gradually and slowly. CSab group demonstrated significant associations to antibiotic classes corresponding to the antibiotic administered to infants in this group. Taxa belonging to Gammaproteobacteria were dominant carriers of ARGs, most being non persisters. The results show that early antibiotic exposure can have immediate and long-term effects on the infant microbiome. Next, publicly available shotgun-sequencing data was downloaded, analysed, resulting in a catalogue of early-life gut genomes of infants below three years of age which was used to study the global resistome. Gram-negative bacteria such as Escherichia, Enterobacter, Citrobacter, Klebsiella had highest ARG abundance with Glycopeptides, fluoroquinolone, macrolides, tetracyclines being most abundant classes. High abundance of ARGs was positively related to the socioeconomic status and healthcare access index of a country. These results confirm infant gut microbiota as ARG reservoir. Lastly, the gut microbial and resistome profile of adult cystic fibrosis (CF) patients was studied due to their chronic antibiotic use. The CF group had a microbial composition significantly distinct from controls with higher abundance of ARGs and virulence factors. CF group showed strong association to antibiotic classes administered to individuals in this group. Our results demonstrate the need to investigate the resistome and functional profile in this patient group; as antibiotic overuse can lead to MDR, aggravating the health status. This thesis sheds light on the microbial and resistome profile in previously unexplored manners and provides a baseline for researchers and policy makers to design pre-emptive and proactive measures to maximise health restoration and minimise ARG development.
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    LAB as probiotics and silage inoculants for enteric methane inhibition in ruminants
    (University College Cork, 2022-10-19) Mbandlwa, Philiswa; Stanton, Catherine; Ross, R. Paul; Hill, Colin; FACCE-ERAGAS
    There is a constant need for antimicrobial agents and producers that can be used in animal feed and other applications in the agricultural industry. This need justifies the research on novel antimicrobial alternatives such as bacteriocins. Data showed the agricultural environment as a source of LAB, dominated by Lactobacillus species with bacteriocin-producing abilities. Subsequently, an investigation on using CFS and bacteriocins from LAB to reduce methane production in Methanobrevibacter found that L. plantarum (LP58) was most effective in reducing methane from M. gottschalkii while individual strains of L. lactis affected methane production in Methanobrevibacter differently. Purified bacteriocins nukacin and lacticin 3147 did not influence in vitro methane production. The nutritive and fermentative properties of L. plantarum and L. lactis were detailed as silage co-inoculants and these inoculants enhanced the beneficial microbes in the silage and resulted in silage with significantly higher lactic acid concentration. Finally, an animal feeding trial showed significant methane reduction in the dairy cattle fed LAB-treated silage. Gaining insight into the different ruminotypes can enable more targeted approaches for methane mitigation. An investigation of the role of LAB in modifying the rumen microbiome and metabolome showed that LAB positively impacts the rumen by significantly increasing acetate and butyrate production while significantly decreasing Proteobacteria. Livestock farming is a significant sector in the Irish economy as it creates jobs, enables export, and is an intricate part of Irish culture. Although it will be challenging for Irish agriculture to meet its climate targets, concerted efforts, such as using LAB as enteric methane mitigators, can enable Ireland to reach its target of reducing atleast 10% of its GHG emissions by 2030. If overall methane emissions are lowered, the national dairy herd can be maintained or increased, and jobs can be created while continuing to supply meat and milk to the growing world population. Future research will focus on finding even more potent methane reducing LAB and characterisation and elucidating the mechanism of methane inhibition in vitro using LAB CFS.
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    Potential of cheddar cheese to impact on the gut microbiota
    (University College Cork, 2023-04-28) Leeuwendaal, Natasha Kim; O'Toole, Paul W.; Stanton, Catherine; Beresford, Tom P.; Teagasc
    There is growing evidence in the scientific literature relating to the potential of food to programme gut microbiota and, in so doing, impact positively on gut-health. Fermented foods and in particular cheese, support a rich microbial population and are a well-recognized source of indigenous lacticaseibacilli, referred to as Non-Starter Lactic Acid Bacteria (NSLAB) that grow to high numbers in the cheese during ripening. Species such as Lacticaseibacillus paracasei and Lacticaseibacillus rhamnosus are common members of the NSLAB microbiota and are also recognised as an important component of a healthy gut microbiome. Indeed, many of the commercially successful probiotic bacteria include these species. Fermented foods are also highly nutritious and offer the potential to influence directly the growth of the indigenous gut microbiota. The hypothesis tested in this project was that cheese, along with its natural NSLAB populations, has the potential to positively affect the gut microbiota and thus be considered a food with added human health benefits. To explore this hypothesis, the potential of NSLAB to survive gastric transit was investigated by exposing the bacterial populations separated from the cheese matrix to Simulated Stomach and Duodenum Passage (SSDP) followed by plating on MRS medium. Populations of NSLAB, up to 107 CFU/g of cheese were recovered following this treatment suggesting that significant populations may survive gastric transit and thus, gain entry to the small and large intestine. A selection of 240 isolates surviving SSDP were characterised including examination for a number of typical probiotic characteristics. Arising from this, two strains, Lacticaseibacillus paracasei DPC 7150 and Lacticaseibacillus rhamnosus DPC 7102, were selected that demonstrated probiotic potential. To further investigate how these strains survive in cheese and to explore whether a cheese or fermented milk matrix provided best protection during simulated digestion, these strains were added during the manufacture of both products. Potential to survive gastric transit was investigated using the INFOGEST2.0 static digestion model and strains were identified using Pulse-Field Gel Electrophoresis (PFGE). The data obtained demonstrated that Lb. paracasei DPC 7150 and Lb. rhamnosus DPC 7102 persisted in the cheese during ripening and that the Cheddar cheese matrix provided significantly more protection during simulated gastric transit. In order to determine the effects of these lacticaseibacilli-containing Cheddar cheeses in vivo, a short-term feeding study using Black 6 mice was designed to assess cheese intake on the gut microbiome, as well as other parameters of interest. While groups consuming cheese did not differ from the control chow diet in body weight, organ mass, adipose tissue mass, or total plasma cholesterol, differences were observed in plasma triglyceride levels (with those of the Cheddar-consuming groups being significantly higher). The alpha and beta diversity of the gut microbiome, including between certain dietary groups and when compared with their microbial populations prior to commencement of the feeding trial differed significantly. Thus, consumption of Cheddar cheese containing the lacticaseibacilli as adjuncts caused shifts in intestinal microbial populations in comparison in mice even when administered for only 3 weeks. However, in a human study whereby a pre-diabetic cohort was instructed to consume 120 g of commercial Cheddar cheese not containing the lacticaseibacilli adjuncts per day for a 6-week period, no significant differences were observed between the gut microbiome groups prior and post cheese-enriched diet. The overall conclusions from this study are that Cheddar cheese contains a diverse NSLAB population, a significant portion of which can survive gastric transit and display probiotic characteristics. These strains survive during manufacture and ripening of Cheddar cheese and fermented milk and the Cheddar cheese matrix offers good protection during gastric transit. Cheese containing selected strains of lacticaseibacilli as adjuncts can affect the mouse gut microbiome.