APC Microbiome Ireland - Doctoral Theses

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    Shotgun metagenomics-based exploration of kefir microbiomes
    (University College Cork, 2023) Walsh, Liam; Cotter, Paul; O'Toole, Paul W.; Teagasc
    Fermentation 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.
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    The gut microbiome of the wild great tit (Parus major): drivers and fitness consequences
    (University College Cork, 2023) Somers, Shane Edmond; Quinn, John; Ross, R. Paul; Stanton, Catherine; Irish Research Council for Science, Engineering and Technology; European Research Council; Science Foundation Ireland
    The gut microbiome plays a vital role in its host’s ecology. Clinical studies have shown gut microbes increase host health and fitness by providing digestive and immune functions, as well as aiding development. Natural variation in the microbiome is widely believed to affect host fitness in the wild but we are lacking experimental studies to test this. The microbiome varies with both host and environmental factors but most studies to date have focussed on individual factors and not adequately addressed the multiple overlapping and hierarchical drivers of microbiome variation working at environmental, host and microbial scales. This thesis investigates the role of the gut microbiota in host fitness, and how this is affected by and varies across contexts. Additionally, we address sources of variation in the gut microbiota at a host and environmental level, accounting for host ecology and drivers at different scales. We find that the host’s weight is correlated with microbiome diversity during development but that the direction of this relationship is context dependent. This shows that the microbiome interacts with the environment to determine host fitness and is important because it helps explain the contradictory findings linking diversity to weight. We also show that the interaction between the host, its microbiome and environment change with developmental stage. Specifically, we found that the microbiome of developed individuals is remarkably resilient to environmental perturbation, while developing individuals are much more sensitive, with important implications for future experiments. We developed a novel method for experimentally perturbing the microbiome that will allow microbiome researchers to begin testing hypotheses linking the microbiome to host ecology and evolution in natural settings. Finally, we show that welfare measures, such as environmental enrichment may interact with the gut microbiota to impact on host health and behaviour. In summary, I show that variation in the microbiome is linked to host ecology and that this variation is linked to host fitness.
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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.
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    Mapping the microbiota in hidradenitis suppurativa
    (University College Cork, 0022) McCarthy, Siobhán; O'Toole, Paul W.; Shanahan, Fergus; Murphy, Michelle
    Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease characterized by the formation of nodules, abscesses, and fistula at intertriginous sites. Pain, pruritus, malodour, and suppuration have a significant impact on quality of life for HS patients. The skin-gut axis is an area of emerging research in inflammatory skin disease and is a potential contributory factor to the pathogenesis of HS. Skin microbiome alterations in HS have become an area of expanding research with its role in the pathogenesis of HS becoming clearer. Gut microbiome alterations have been described in many inflammatory diseases including inflammatory bowel disease, rheumatoid arthritis, and cancer, but little is known about the gut microbiome in HS. Our hypothesis is that gut, as well as skin, microbiota alterations and their metabolites, may be responsible for skin inflammation in HS. The level of pain experienced by 150 patients with HS was measured using the visual analogue scale and EQ-5D-5L questionnaires. 59 patients with HS provided faecal samples, nasal and skin swabs of affected sites for analysis. These 59 patients also provided serum samples and completed questionnaires including the Dermatology Life Quality Index (DLQI), Morisky Medication Adherence Scale, International Physical Activity Questionnaire, and Food Frequency Questionnaire. 30 healthy controls provided fecal samples and 20 healthy controls provided nasal and skin swabs. We performed bacterial 16S rRNA gene amplicon sequencing on total DNA derived from the samples. Calprotectin was determined by enzyme-linked immunoassay (CALPROLAB™ Calprotectin ELISA (HRP). Complement C5a levels were assessed using enzyme-linked immunosorbent assay (Abcam ab193695 Complement C5a Human ELISA Kit). Pain was widely reported in 134 responders to the visual analog scale questionnaire. 82.1% reported some level of pain and 35.8% reported high levels of pain. In the EQ-5D-5L questionnaire, 81.4% of responders indicated that they felt pain or discomfort, and 31.0% expressed “severe” or “extreme” pain/discomfort. Weak correlation was seen between levels of pain and one marker of gut microbiota alpha diversity (Chao1), but this failed to be repeated for all indices of microbiota species richness. A significant impact on quality of life with a mean DLQI score of 12.15 (SD 7.67) was observed in patients with HS, which is in line with other severe dermatoses. 59% of patients reported a large or extremely large impact on their quality of life. The DLQI score did not correlate with gut microbial diversity. In this study, over 40% of patients with HS also reported some difficulty remembering to take their medications with 20% reporting that they forgot to take their medication in the previous 2 weeks. Patients with HS also reported moderate-to-high levels of physical activity; however this may be overestimated. Alterations in gut microbiota diversity did not correlate with levels of physical activity. Microbiome alpha diversity was significantly lower in the faecal, skin and nasal samples of individuals with HS which may be secondary to disease biology or related to antibiotic usage. Ruminococcus gnavus was more abundant in the faecal microbiome of individuals with HS, which is also reported in Crohn’s disease (CD), suggesting comorbidity due to shared gut microbiota alterations. No significant difference in faecal microbiota composition or overall habitual diet was seen in the patients with HS and diet was not associated with disease severity. Diet in HS was significantly different to controls, and similar to patients with CD. Finegoldia magna was over-abundant in HS skin samples relative to healthy controls. It is possible local inflammation is driven by F. magna through promoting the formation of neutrophil extracellular traps (NETs). 27.1% of patients with HS had a raised faecal calprotectin level of >50mg/kg with markedly elevated levels of >150mg/kg in 8.5% suggesting occult gastrointestinal inflammation. Median complement C5a level in patients with HS was elevated at 47 ng/ml (IQR 30.55 ng/ml) which is in keeping with prior studies. These alterations in both the gut and skin microbiome in HS warrant further exploration, and therapeutic strategies including faecal microbiota transplant or bacteriotherapy could be of benefit. Faecal calprotectin is a simple test which should be considered in patients with HS, with further investigations warranted in patients with elevated levels. C5a may provide a therapeutic target in patients with HS and serum C5a may act a biomarker of the disease.
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    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.