Fusobacterium nucleatum is a Gram-negative bacterium that serves as a bridging organism in polymicrobial biofilms within the oral cavity. Although the bacterium is abundant in healthy gingival tissue, recent studies have found that F. nucleatum is associated with a wide-spectrum of human diseases which include periodontal disease, preterm birth, endocarditis, colorectal cancer, and pancreatic cancer. Previous studies of F. nucleatum virulence have uncovered two surface adhesins, Fap2 and FadA, that interact with the surface of human cells; however, the study of new virulence factors was previously limited as there was no gene deletion system available to functionally analyze F. nucleatum proteins. Interestingly, F. nucleatum has a diverse landscape of structurally unique surface adhesins called Type 5c secreted trimeric autotransporter adhesins (TAAs), which are a family of proteins that are historically known for their contributions to bacterial pathogenesis. This dissertation encompasses the use of recombinant protein expression systems and newly developed gene deletion technology to provide a foundational understanding of the contribution of Type 5c secreted proteins in F. nucleatum pathogenesis. Our results show that the presence of TAAs on the surface of F. nucleatum contribute to the bacterium's ability to bind and invade human cells, establishing the need to characterize other F. nucleatum surface proteins. Additionally, our studies analyzed the proinflammatory landscape induced by F. nucleatum through the identification of specific cytokines that are being secreted during in vitro infections of human cells. Cytokine signaling is a critical aspect of the host cell immune response as it promotes the recruitment of immune cells to the site of infection for efficient clearance of bacterial pathogens. While it has been well established that F. nucleatum modulates the secretion of IL-8, our studies identified that the bacterium also promotes the secretion of CXCL1, which is an important signaling protein that promotes tumor metastases. Overall, the work provided in this dissertation has delivered the initial characterization of TAAs in F. nucleatum virulence, a framework for future studies of Type 5c secreted proteins in Fusobacterium pathogenesis, and the role of Fap2 and FadA in promoting pro-inflammatory and pro-metastatic signaling from colorectal cancer cells. / Master of Science in Life Sciences / Fusobacterium nucleatum is a Gram-negative bacterium that serves as a bridging organism in polymicrobial biofilms within the oral cavity. Although the bacterium is abundant in healthy gingival tissue, recent studies have found that F. nucleatum is associated with a wide-spectrum of human diseases which include periodontal disease, preterm birth, endocarditis, colorectal cancer, and pancreatic cancer. Previous studies of F. nucleatum virulence have uncovered two surface adhesins, Fap2 and FadA, that interact with the surface of human cells; however, the study of new virulence factors was previously limited as there was no gene deletion system available to functionally analyze F. nucleatum proteins. Interestingly, F. nucleatum has a diverse landscape of structurally unique surface adhesins called Type 5c secreted trimeric autotransporter adhesins (TAAs), which are a family of proteins that are historically known for their contributions to bacterial pathogenesis. This dissertation encompasses the use of recombinant protein expression systems and newly developed gene deletion technology to provide a foundational understanding of the contribution of Type 5c secreted proteins in F. nucleatum pathogenesis. Our results show that the presence of TAAs on the surface of F. nucleatum contribute to the bacterium’s ability to bind and invade human cells, establishing the need to characterize other F. nucleatum surface proteins. Additionally, our studies analyzed the proinflammatory landscape induced by F. nucleatum through the identification of specific cytokines that are being secreted during in vitro infections of human cells. Cytokine signaling is a critical aspect of the host cell immune response as it promotes the recruitment of immune cells to the site of infection for efficient clearance of bacterial pathogens. While it has been well established that F. nucleatum modulates the secretion of IL-8, our studies identified that the bacterium also promotes the secretion of CXCL1, which is an important signaling protein that promotes tumor metastases. Overall, the work provided in this dissertation has delivered the initial characterization of TAAs in F. nucleatum virulence, a framework for future studies of Type 5c secreted proteins in Fusobacterium pathogenesis, and the role of Fap2 and FadA in promoting pro-inflammatory and pro-metastatic signaling from colorectal cancer cells

Emergent infectious diseases are a major driver of the accelerated rates of biodiversity loss that are being documented around the world. Global losses of amphibians provide evidence of this, especially those associated with chytridiomycosis, a lethal skin disease caused by the fungus Batrachochytrium dendrobatidis (Bd). Amphibian skin can harbor diverse bacterial communities that, in some cases, can inhibit the growth of Bd. Thus, there is interest in using skin bacteria as probiotics to mitigate Bd infections in amphibians. However, experiments testing this conservation approach have yielded mixed results, suggesting a lack of understanding about the ecology of these microbial communities. My dissertation research aimed to assess basic ecological questions in microbial ecology and to contribute to the development of probiotics using amphibian skin bacteria. First, to assess whether environmental conditions influence the function of amphibian skin bacterial communities, I conducted a field survey across low and high elevation populations of an amphibian host to assess their skin bacterial communities and metabolite profiles. I found that similar bacterial communities produced different metabolites at different locations, implying a potential functional plasticity. Second, since culturing is critical for characterizing bacteria, I aimed to identify the culture media (low vs high nutrient concentration) that recovers the most representative fraction of the amphibian skin bacterial community. I found that media with low nutrient concentrations cultured a higher diversity and recovered a more representative fraction of the diversity occurring on amphibian skin. I also determined that sampling more individuals is critical to maximize culture collections. Third, I assessed the diversity of the amphibian skin fungal community in relation to Bd infection across eight amphibian species. I determined that amphibian species was the most important predictor of fungal diversity and community structure, and that Bd infection did not have a strong impact. My dissertation highlights the importance of environmental conditions in the function of amphibian skin bacteria, expands our knowledge of the understudied fungal component of the amphibian skin microbiome, and complements current efforts in amphibian conservation. / Ph. D. / In light of the global losses of amphibian diversity due to, in part, the skin disease chytridiomycosis (caused by the fungus Batrachochytrium dendrobatidis [Bd]); the discovery that some amphibian-skin bacteria can inhibit Bd growth provides hope for amphibian conservation via their use as probiotics to control Bd infections. However, experiments testing these bacteria have yielded inconsistent results, suggesting a limited understanding about the factors influencing the diversity of amphibian-skin microbes and their ability to inhibit Bd. Also, efforts to identify effective candidates for probiotic therapy are still premature. Thus, my dissertation had an ecological emphasis and focused on complementing conservation efforts focused on probiotics. First, I assessed whether environmental conditions influence bacteriallyproduced products, which can have antifungal properties. Specifically, I surveyed low and highelevation populations of an amphibian species to assess the skin-bacteria and their products. I determined that, while skin bacterial communities were similar across an environmental gradient, their products differed, suggesting potential different antifungal properties. Second, I assessed the ability of different culture media types (low vs high nutrient concentrations) to grow a high portion and most representative fraction of the amphibian-skin bacteria. I found that culture media with low nutrient concentrations allowed the growth of a higher diversity of the bacteria occurring on the amphibian-skin, including the abundant members, and also determined that including a large number of amphibians is the best way to improve culture collections. Third, I assessed the fungal diversity occurring in the skin of different amphibian species and how it might response to Bd infections, and examined whether skin-fungi interact with co-occurring bacteria. I found that the amphibian species was the most important driver of the fungal diversity, and that Bd infection did not influence the diversity of these communities. Moreover, I identified the most diverse fungal phyla occurring in the amphibian-skin and determined that these fungi might interact with co-occurring bacteria. My dissertation contributes to our understanding about the influence of the environmental conditions in the amphibian-skin bacteria, expands our limited knowledge on the amphibian-skin fungi, and complement current amphibian conservation efforts.

The gut-brain axis describes a paradigm wherein the trillions of microorganisms inhabiting the gastrointestinal tract engage in bidirectional communication with the host central nervous system. Adaptive immunity represents an important intermediate in this dynamic crosstalk; previous work in our lab has demonstrated that T-lymphocytes, a main class of immune effector cells, contribute to neurodevelopmental processes and behavioral outcomes across the lifespan. Parallels between the phenotype of T-cell deficient and germ free mice led us to hypothesize that bidirectional T-cell-microbe communication is critical for normal neurodevelopment, and that T-cell deficiency impacts the neural circuitry underpinning behavior via disruption of the gut-brain axis. The main objective of this thesis was to elucidate the mechanisms by which T-cells mediate developmental gut-brain signalling. The first installation examined the gut microbiome, gut metabolome, and neurochemical profile in wild-type and T-cell deficient mice from adolescence to adulthood, demonstrating that absence of T-cells impacts the developmental trajectory of functional microbiome output and levels of neuroactive molecules in the brain. Experiment two investigated the impact of T-cell deficiency on gut-brain communication through the lens of host gene expression in the parenchyma and the intestine. T-cell deficient mice showed significant changes in genes related to intestinal immunity and barrier function, in addition to decreases in microglia-related genes in the prefrontal cortex during early life. The final experiment transitioned into a wild-type model to measure the co-evolution of T-cell subsets in mucosal and central immune compartments with composition and diversity of the microbiota. We demonstrated a parallel diversification of the gut microbiome and the functional T-cell repertoire, whereby emergence and proliferation of specific T-cell subsets is linked to compositional shifts in dominant microbial communities across development. Together, our results demonstrate the importance of T-cells for normal development of the holo-organism, with implications for the developmental wiring of functional brain circuitry. / Thesis / Doctor of Philosophy (PhD) / Modern medicine has increasingly placed emphasis on the mind-body connection. This has been exemplified by a series of recent discoveries surrounding the importance of the gut microbiome in maintaining our physical and mental health. One of the key channels through which the microbiome communicates with the host is through the immune system, an equally complex network of cells and proteins that protect the body against invading pathogens. Indeed, these systems evolve alongside each other and engage in constant crosstalk throughout the lifespan, with downstream impacts on the developing brain. This thesis sought to further explore the role of T-cells, a key component of the adaptive immune system, in coordinating gut-microbiome-brain interactions across development. The first experiment examined the microbiome as well as small molecules in the gut and brain of normal mice and mice lacking T-cells. The second experiment built on this work to examine how T-cells influence the expression of different genes in the gut and brain. Finally, the third experiment mapped different populations of T-cells and microbiome composition from the first week of life to adulthood, to better understand how they interact at different stages of development. This work will offer insight into how T-cells talk to the microbiome and how they transmit signals from the gut to the brain, with implications for understanding neurodevelopmental disorders and how they arise.

Symbiont intestinal microbiomes contribute to host immunity, but may also contribute to autoimmune diseases. Segmented filamentous bacteria (SFB), designated Candidatus Arthromitus, are one of the key players in the gut microbiome. They have a unique cell cycle and are thought to play a role in immune establishment in infancy.  This research explored the feasibility of non-human primate infants as animal models for elucidating SFB function by analyzing previously published 16S rRNA gene sequencing data with the Divisive Amplicon Denoising Algorithm 2 (DADA2) algorithm and statistical methods.  Consequently, non-human primate infants as animal models for SFB investigation were seen as a potential option. However, the study also brought up questions about the species-specificity and transmission modes of SFB, thus additional research is needed.

Aquaculture, the cultivation of aquatic organisms in a controlled environment, offers both economic and nutritional benefits to human society. As there is an increased demand to feed a growing human population, many wild-caught fisheries have struggled due to the overexploitation of resources. Currently, production relies heavily on wild-caught fish to produce fishmeal to feed farm-raised fish. The demand for alternative materials in fish feeds has grown rapidly as fishmeal resources have become limited. Antibiotic resistance emergence in aquaculture systems is another area of concern. Reducing antibiotic use via alternate prophylactic measures to increase host health is an essential area of research; modulation of the host intestinal bacterial community via prebiotics is one possibility. Prebiotics refer to non-digestible food ingredients that are thought to stimulate the growth of beneficial bacteria, consequently benefiting host health by indirectly reducing the possibility of bacterial pathogen proliferation. This occurs through various measures such as competition for space and resources. The intestinal bacterial community has a significant impact on a variety of host factors that include host development, physiology, immunity, and nutrient acquisition. In turn, there are multiple factors impacting the bacterial community, including the presence of pathogens and/or antibiotics, environmental conditions, host genetics, and the diet consumed. To promote environmental sustainability and improve production and animal health in aquaculture, a collaboration was created with Anheuser-Busch of the brewing industry and Maltento, a functional ingredient company. With breweries around the globe, Anheuser-Busch produces consistent, food grade byproducts that are safe for human consumption. Two of the most prevalent brewery byproducts are brewer's spent yeast (BSY) and brewer's spent grain (BSG). BSY contains a variety of beneficial nutrients such as proteins, essential amino acids, and carbohydrates. BSG is high in fiber but low in protein; however, black soldier fly larvae can be cultured on BSG to convert the low-value product into insect biomass to be used in fish feed, as insects themselves are full of beneficial lipids and proteins. The objective of the work presented in this thesis was to evaluate the efficacy of using low-value brewery waste products, converted into high-value feed additives, for aquaculture practices. Specifically, the effects of dietary feed additives on the production, health, and intestinal bacterial community of aquaculture-raised rainbow trout were examined. Inadvertently, benefits of the feed additives on fish subjected to chronic and acute thermal stress were also assessed. Overall, the results of the study found that the feed additives did not significantly change the production efficiency of the rainbow trout, though some increase in growth was observed. When subjected to chronic thermal stress conditions, fish fed the experimental diets outperformed those fed the control diet regarding growth parameters. The intestinal bacterial community of the fish was significantly altered from the beginning of the trial compared to the end of the trial, though differences were not attributed to the feed additives. Instead, the resulting intestinal dysbiosis is believed to have stemmed from the physiological response of the fish to thermal stress conditions. When the fish underwent an acute thermal stress event, causing mortality, fish fed three of the five experimental diets were found to have higher survival rates compared to the control. Ultimately, results of this project suggest that the BSY and BSG-fed insect feed additives may have increased the health and robustness of the fish during a period of thermal stress. However, further research under controlled conditions is needed to evaluate if the observed host health benefits can directly be attributed to the feed additives. / Master of Science / Aquaculture refers to the method of rearing aquatic organism such as fish and shellfish under controlled conditions. Within the food industry, aquaculture is one of the fastest growing sectors, and provides important economic and nutritional benefits to humans. Additionally, aquaculture is an important alternative to fisheries that rely on catching fish from the natural environment. Wild-caught fisheries have struggled due to the overfishing, and unfortunately, many aquaculture practices still rely on wild-caught fisheries to produce fishmeal used in feed for carnivorous fish. Research into alternate protein sources to use in fish feed has been on the rise. Additionally, as the emergence of multi-drug resistant bacteria continues to increase, reducing antibiotic use has become a priority across all fields whether it be healthcare or the food industry. Within aquaculture, using alternative prophylactic measures such as prebiotics to increase animal health and disease resistance could lead to the overall reduction of antibiotic use. Prebiotics are non-digestible food ingredients believed to help the beneficial bacteria within the intestinal track to grow. In turn, the increased numbers of beneficial bacteria reduce the possibility of pathogenic bacteria invading and establishing a presence in the intestinal track. The intestinal microbiome refers to the various organisms, such as bacteria, viruses, and fungi, that live commensally within the host digestive tract. The bacterial community within the intestinal microbiome has many important roles, including effects on host development, physiology, immunity, and nutrient acquisition. Many factors also impact the bacterial community, including the presence of pathogens and/or antibiotics, environmental conditions, host genetics, and the diet consumed. To promote environmental sustainability and improve production and animal health in aquaculture, a collaboration was created with Anheuser-Busch of the brewing industry and Maltento, a functional ingredient company. With breweries around the globe, Anheuser-Busch produces consistent, food grade byproducts that are safe for human consumption. Two of the most prevalent brewery byproducts are brewer's spent yeast (BSY) and brewer's spent grain (BSG). These low-value waste products can consequently be converted into high-value feed additives for use in aquaculture. The objective of the work presented in this thesis was to evaluate the effects of BSY and BSG-fed insect dietary feed additives on the production, health, and intestinal bacterial community of aquaculture-raised rainbow trout. Unintentionally, benefits of the feed additives on fish subjected to chronic and acute high-temperature thermal stress were also explored. Overall, the results of the study found that while the feed additives did not significantly increase the growth of the rainbow trout, benefits were still observed. When subjected to chronically high-water temperatures, fish fed the experimental diets outperformed those fed the control diet regarding growth parameters. The intestinal bacterial community of the fish was significantly altered from the beginning of the trial compared to the end of the trial, though differences are not believed to be caused by the feed additives. Instead, the resulting shift in the bacterial community is believed to have stemmed from the stress-response of the fish triggered by high water temperature. When the fish underwent an acute thermal stress event, which caused mortality, fish fed three of the five experimental diets were found to have higher survival rates compared to the control. Ultimately, results of this project suggest that the feed additives may have increased the health and robustness of the fish while undergoing thermal stress. However, further research under controlled conditions is needed to evaluate if the observed host health benefits can be attributed directly to the feed additives.

The tumor microenvironment is a well-recognized contributor to cancer progression in solid tumors. Cancer cell interactions with abnormal extracellular matrix, tumor associated immune and stromal cells, and aberrant fluid flow all contribute to cancer progression. Breast tumors are often characterized by a dense collagenous stroma and a hypoxic core. A recently identified and little understood component of the breast tumor microenvironment is the breast microbiome. The work described here elaborates on the importance of the tumor microenvironment in cancer progression and demonstrates the importance of studying cancer-microbiome interactions in the context of tumor microenvironmental stimuli. / Master of Science / One of the major barriers to effective cancer treatment is the environment is which cancer grows. Tumors insulate themselves in a thick protein structure that leads to a stiffening of the breast tissue. In addition, irregular tumor-associated blood vessels lead to poor blood flow, and therefore a lack of oxygen, in the center of the tumor. These and other characteristics of tumors create an environment in which cancer cells are resistant to current anti-cancer therapies and thereby allows them to flourish. It was recently discovered that, contrary to previous belief, there are resident bacteria present in normal and cancerous breast tissue. The role they play in controlling cancer development and progression in the tumor is unknown. The work described here elaborates on the tumor environmental barriers to current anti-cancer therapies and shows how one bacterial produced compound may interact with other features of the tumor environment in order to control breast cancer survival.

No / This review focuses on treatment targets for the most common inflammatory skin diseases, eczema and psoriasis with an emphasis on cytokines expressed in the uppermost layer of the skin which is easily accessible for diagnostic and therapeutic approaches. Recently, a significant body of research has highlighted the influence of the skin barrier and the patients’ microbiome on skin inflammatory responses and we will comment on their impact on mediator regulation. Itch is a prominent dermatology symptom which is influenced by cytokines and can via itch–scratch cycle impact on the skin barrier and mediator expression associated with damage. Taking the contribution of pruritus and superficial skin damage into account, we address cytokines as targets for stratified treatment approaches in subgroups of eczema and psoriasis.

Yes / Background: Oral administration of purified omega-3 (ω-3) PUFAs is associated with changes to the fecal microbiome. However, it is not known whether this effect is associated with increased PUFA concentrations in the gut. Objectives: We investigated the luminal bioavailability of oral ω-3 PUFAs (daily dose 1 g EPA and 1g DHA free fatty acid equivalents as triglycerides in soft-gel capsules, twice daily) and changes to the gut microbiome, in the ileum. Methods: Ileostomy fluid (IF) and blood were obtained at baseline, after first capsule dosing (median 2 h), and at a similar time after final dosing on day 28, in 11 individuals (median age 63 y) with a temporary ileostomy. Fatty acids were measured by LC–tandem MS. The ileal microbiome was characterized by 16S rRNA PCR and Illumina sequencing. Results: There was a mean 6.0 ± 9.8-fold and 6.6 ± 9.6-fold increase in ileal EPA and DHA concentrations (primary outcome), respectively, at 28 d, which was associated with increased RBC ω-3 PUFA content (P ≤ 0.05). The first oral dose did not increase the ileal ω-3 PUFA concentration except in 4 individuals, who displayed high luminal EPA and DHA concentrations, which reduced to concentrations similar to the overall study population at day 28, suggesting physiological adaptation. Bacteroides, Clostridium, and Streptococcus were abundant bacterial genera in the ileum. Ileal microbiome variability over time and between individuals was large, with no consistent change associated with acute ω-3 PUFA dosing. However, high concentrations of EPA and DHA in IF on day 28 were associated with higher abundance of Bacteroides (r2 > 0.86, P < 0.05) and reduced abundance of other genera, including Actinomyces (r2 > 0.94, P < 0.05). Conclusions: Oral administration of ω-3 PUFAs leads to increased luminal ω-3 PUFA concentrations and changes to the microbiome, in the ileum of individuals with a temporary ileostomy.

Microbiome research has become a ubiquitous component of contemporary clinical research, with potential to uncover associations between microbiome composition and disease. With microbiome data becoming more prevalent, the need to understand how to analyse such data is increasingly important. One complicating property of microbiome data is that it is inherently compositional and thus constrained to simplex-space; because of this, it cannot be analysed directly using conventional statistical methods. In this paper, we transform the compositional data in order to lift the simplex-constraint, and then investigate the viability of applying conventional statistical methods to the data. Using a high-dimensional data set containing gut-microbiome samples from Parkinson's- and control patients, we first transform the raw data to centred log-ratio scale, and then use permutational multivariate analysis of variance (PERMANOVA) to test if there are differences between the two groups with respect to bacterial abundances. We then employ three machine learning classifiers -- Logistic regression, XGBoost, and Random Forest -- and evaluate their performance on the transformed data. The results from PERMANOVA indicate that gut-microbiome composition in the patients with Parkinson's disease indeed differ from that in the control individuals. The Random Forest method achieves the highest classification accuracy, followed by XGBoost, while logistic regression performs poorly, questioning its viability in analysis of high-dimensional compositional microbiome data. We find four bacterial species of high importance for the classification: Prevotella copri, Prevotella sp. CAG 520, Akkermansia muciniphila, and Butyricimonas virosa, where the first three have been previously mentioned in the Parkinson's literature.

No / There is an ongoing debate about the reasons for and factors contributing to healthcare-associated infection (HAI). Different solutions have been proposed over time to control the spread of HAI, with more focus on hand hygiene than on other aspects such as preventing the aerial dissemination of bacteria. Yet, it emerges that there is a need for a more pluralistic approach to infection control; one that reflects the complexity of the systems associated with HAI and involves multidisciplinary teams including hospital doctors, infection control nurses, microbiologists, architects, and engineers with expertise in building design and facilities management. This study reviews the knowledge base on the role that environmental contamination plays in the transmission of HAI, with the aim of raising awareness regarding infection control issues that are frequently overlooked. From the discussion presented in the study, it is clear that many unknowns persist regarding aerial dissemination of bacteria, and its control via cleaning and disinfection of the clinical environment. There is a paucity of good-quality epidemiological data, making it difficult for healthcare authorities to develop evidence-based policies. Consequently, there is a strong need for carefully designed studies to determine the impact of environmental contamination on the spread of HAI.