Soils harbor a huge diversity of microorganisms, which are dominated by bacteria and fungi. These soil microorganisms, collectively termed as the soil microbiome, are major contributors to soil biodiversity and play essential roles in soil functions (e.g. soil fertility and plant nutrition, organic matter degradation and nutrient cycling, and soil formation). Therefore, many studies in recent decades have explored soil microbial diversity in order to unravel driving forces of its variations. Hence, this thesis reports on spatial and temporal variations of the soil microbiome in response to site specificities, i.e. local climate as well as soil physico-chemistry, and host tree parameters. To avoid effects of intraspecific genetic variations, the pedunculate oak clone DF159 (Quercus robur L.) generated by the project TrophinOak-PhytOakmeter of the Soil Ecology Department at the Helmholtz Centre for Environmental Research (UFZ) was used as phytometer system. In the PhytOakmeter project of which this thesis is a part, saplings regenerated from microcuttings of DF159 were out-planted in grassland, forest and urban field sites in Central Germany and along a European North-South transect. The overall goal of the project is to analyze how the clone adapts to and performs under different regional climatic contexts and changing environment conditions. Pedunculate oak was chosen as a model tree species because it is engaged in highly complex and diverse multitrophic interactions, including soil microorganisms. Q. robur displays an endogenous rhythmic growth with alternating growing flushes in shoot and root, which can be repeated two to four times along a vegetation period. These alternating flushes have been shown to impact on variations of biological activities in soil zones close to the tree roots. Based on the above-described background, the current PhD study investigated changes in the soil microbial communities associated to the pedunculate oak phytometer outplanted in grassland sites at two different spatial scales: (1) the local scale by comparing the soil microbiomes associated to the phytometer in sites located within a close geographic space of Central Germany with similar climatic conditions; and (2) continental scale by making a similar comparison among sites along a European North-South transect, which encompasses a wide range of climatic and soil physico-chemical conditions. Moreover, temporal scale was considered, whereby the variability of the microbiomes intra-annually along a vegetation period was analyzed. Soil samples were taken not only in the tree root zone (RZ), i.e. soil zone containing living roots of the tree, but also in the tree root-free zone (RFZ), i.e. soil zone out of reach of any tree roots, but within the same field plot, to access also the local microbial pools. The analyses used a PCR-based Illumina MiSeq amplicon sequencing approach targeting bacteria and fungi, to assess their diversity, community structure and functionality after assignment of their OTUs to functional groups. In addition to Chapter 1, which introduces the whole work of this PhD research, the findings are presented within Chapters 2-4, of which two studies were already published in international peer-reviewed journals, while another study was published as a conference paper. The thesis is closed by the synopsis Chapter 5 that integrates discussion of all the publication chapters together with an outlook section. Chapter 2 “Tree root zone microbiome: exploring the magnitude of environmental conditions and host tree impact” published in Frontiers in Microbiology investigates the relative contribution of abiotic environmental and host tree parameters among four sites characterized by homogeneous climatic conditions in Central Germany, two years after the tree out-plant. We first compared at each field site the composition of the bacterial and fungal communities between the RZ of the oak clone, called PhytOakmeter in this chapter, and the tree RFZ. The chapter further evaluates the diversity and structure of the microbial communities within the tree RZ among the sites. The results revealed different microbial compositions between the tree RZ and RFZ, whereby the tree RZ-associated microbiome included numerous ectomycorrhizal fungi of the genera Hebeloma, Exophiala, Scleroderma, Tomentella, Trichophaea, and Tuber. This quick recruitment of specific beneficial microbial taxa from the local microbial pool seems to be among the tree strategies to acclimate to local site conditions. However, the overall tree contribution to shape soil microbial communities was lower than the impact of abiotic environmental parameters. The results revealed also a similar level of microbial diversity within the tree RZ among the sites for both the bacteria and fungi, an outcome attributed to the homogeneous climatic conditions within the sites and the common genetic identity of the host trees. In contrast, structure of the microbial communities was site-specific. Chapter 3 “Balance between geographic, soil, and host tree parameters to shape soil microbiomes associated to clonal oak varies across soil zones along a European North-South transect” published in Environmental Microbiology, also examines the relative impact of geographic, soil physico-chemical, and pedunculate oak clone parameters on the variability of the soil microbiome, but at a larger spatial scale from Lapinjärvi (Finland) to Bordeaux (Southwestern France), which is characterized by a broad range of geographic and soil physico-chemical conditions. In addition to the tree RFZ total microbiome and the tree RZ total microbiome, this chapter introduces a new sub-microbiome called tree RZ affine microbiome. The latter was defined as a subset of the RZ bacteria and fungi, significantly enriched in this zone compared to the tree RFZ. The results demonstrated an interplay among abiotic environmental and host tree parameters in shaping bacterial and fungal communities of the tree RZ along the European transect. These parameters showed a descending order of magnitude of their impact on the tree RZ total microbiome: geographic > soil physico-chemical > host tree parameters. However, for the variability of the RZ affine microbiome alone, the impact of the abiotic environmental parameters decreased, while the tree influence was strongly increased, particularly for fungi. Another important result was the highest proportion of the tree RZ affine microbial OTUs shared among all four sites, which was here designated as the tree “core” microbiome. These bacteria and fungi with significant affinity to the host tree, and shared by all the sites because of their ability to cope with diverging environmental conditions across the transect, may be playing a crucial role in supporting the wide distribution of Q. robur across Europe. Interestingly, we found no members of the RZ affine microbiome to be exclusive of only one particular site. Chapter 4 “Temporal changes and alternating host tree root and shoot growth affect soil microbiomes” published in Proceedings as conference paper after “The 1st International Electronic Conference on Microbiology”, considers a temporal scale, and here the variability of the tree RZ and RFZ total microbiomes was analyzed along a vegetation period in two sites of Central Germany. The soil was sampled at different time points coinciding with the tree alternating root and shoot growth, and the fall senescence that concludes the vegetation period. The results show a directional change over time along a vegetation period for the bacterial communities. However, the fungal communities did not show such temporal changes; they rather displayed a fine spatial scale partitioning closely linked to host plant individuals. In addition to the effect of temporal succession, deeper analyses of the generated data set will enable us to specify the impact of the alternating root and shoot growth characteristic of the tree endogenous rhythmic growth in the near future. These further analyses will include for example zooming in the tree RZ affine microbiome and in individual microbial functional groups. The results presented in this thesis evidence the quick impact of pedunculate oak tree clone on the soil microbiome within a two-year time span after the tree out-plant. Also, to different extents, geographic, soil physico-chemical, and host tree concurrently shape the soil bacterial and fungal communities. This thesis shows different spatial and temporal responses to the abiotic environmental and tree parameters between the soil bacterial and fungal communities. The use of tree clonal phytometer to study the tree-related parameters on soil microbiomes was proved to be a promising tool, to unravel the hierarchy of different abiotic and biotic factors in shaping the soil microbiome associated to long live trees. Finally, this work represents a first step toward establishing a long term monitoring of the dynamics of soil microbiomes associated to trees, as a strategy to unravel how these microorganisms participate to the long term acclimation of these long live plants to diverse and changing environments.

The aetiology of major depressive disorder (MDD) is poorly understood. Current evidence suggests immune activation and gut microbiota may play a role. Recent studies demonstrated that behavioural traits can be transferred through microbiota transplantation into germ-free (GF) mice. Here we study whether microbiota from patients with MDD can induce depressive-like behaviour. Methods: GF NIH Swiss mice were colonized with stool microbiota from a patient with MDD with elevated faecal β-defensin 2, or a healthy donor (HC). After three weeks, behaviour was assessed using standard tests. Expression of neuroimmune markers was assessed in the gut and brain using gene expression profiling and immunohistochemistry. Microbiota composition was assessed by 16S rRNA sequencing. Results: Microbiota profiles differed between the two groups of mice (p=0.001). Mice colonised with microbiota from a single characterised MDD patient (MDD1), exhibited lower preference for sucrose (p=0.002) and more emotionality (p=0.003) than mice with HC microbiota, however other MDD mice did not display abnormal behaviour. Abnormal MDD1 behaviour was associated with lower BDNF expression in the dentate gyrus of the hippocampus (p=0.02). Mice colonised with another characterised MDD patient (MDD4 mice) did not have differences in BDNF expression in the same region (p=0.20). MDD1 and MDD4 mice had altered hippocampal and gut gene expression for genes associated with the immune and nervous system. In summary, GF mice colonized with MDD1 microbiota exhibit depression-like behaviors. This appears to be accompanied by changes in intestinal permeability and neuroimmune function. These results suggest that gut microbiota has the capacity to influence the expression of MDD in some patients. / Thesis / Master of Science (MSc)

Irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) are two of the most commonly diagnosed chronic digestive disorders in Western countries with increasing prevalence among Canadians. However, the etiology of IBS and IBD remain poorly understood due to a complex interplay of genetic, psychosocial and environmental factors, which hampers efforts at early detection/screening, accurate diagnosis and effective treatments notably in children. This thesis aims to reveal new biochemical insights into the pathophysiology underlying IBS and IBD when using an untargeted metabolite profiling (i.e., metabolomics) approach on urine and stool specimens based on multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Chapter I reviews brief history and current challenges in diagnosis and treatment, as well as current metabolomics literature of IBS and IBD. Chapter II first develops a robust method for high throughput profiling of anionic metabolites in human urine samples when using MSI-CE-MS. For the first time, we demonstrate that incidental capillary fractures are caused by irreversible aminolysis of the outer polyimide coating due to the frequent use of volatile ammonia based buffers under alkaline conditions (pH > 9) in electrospray ionization-MS. Chapter III subsequently applies this validated method to investigate differentially excreted urinary metabolites between adult IBS patients and healthy controls, which indicated significantly accelerated rates of collagen degradation and cell turn-over in IBS patients. Chapter IV later develops a novel stool extraction protocol for characterization of the fecal metabolome together with meta-genomic data for elucidating complex host-gut microflora interactions from a cohort of pediatric IBD patients, including Crohn’s disease and ulcerative colitis. In this pilot study, a panel of discriminating metabolites in urine is shown to allow for differential diagnosis of major pediatric IBD sub-types as an alternative to colonoscopy and histopathology that are invasive, expensive and prone to ambiguous test results. Finally, Chapter V involves a longitudinal metabolomics study that aims to identify metabolic trajectories that predict treatment responses of a cohort of pediatric Crohn’s disease patients following initiation of exclusive enteral nutrition (EEN) therapy. In the end, Chapter VI highlights major outcomes of thesis and future direction of metabolomics in IBS and IBD with a specific focus on improved stool specimen collection and validation of biomarker specificity relative to other related gastrointestinal disorders. In summary, this thesis has demonstrated metabolic processes that are associated with exacerbation of symptoms or remission in subset of IBS and pediatric IBD patients. With follow up studies with larger cohort of patients, potential biomarkers identified in this thesis will contribute the development of more accurate and non-invasive decision making process for diagnosis and treatment, resulting in long-lasting remission and improved quality of life of patients suffering from chronic digestive disorders. / Thesis / Doctor of Science (PhD)

MSc Thesis - The effect of wastewater effluent on the gut microbiome of rainbow darter / The microbiome plays an important role in host physiology and can be influenced by species, diet, and environment. Municipal wastewater effluent contains a mixture of chemicals including antibiotics and antimicrobials that may affect the gut microbiome of fish living downstream of these discharges. Thus, this study examines the effect of wastewater treatment plant (WWTP) effluent on the gut microbiome of wild rainbow darter (Etheostoma cearuleum), and examines how the gut microbiome of wild fish changes in the lab. Fish were collected from sites upstream and downstream of 2 major WWTPs along the central Grand River and gut contents were aseptically sampled. After extracting gDNA, nested PCR of the V3-V4 region of the 16S rRNA gene, and Illumina sequencing were performed. The gut microbiome of exposed fish had increased bacterial diversity and was dominated by Proteobacteria, which has been linked to altered health outcomes in mammals. Next, rainbow darters were collected from a reference site on the Grand River. Fish were sampled in the field, after a 14 day lab acclimation, and after a 28 day exposure to environmental stressors (WWTP effluent or triclosan, an antimicrobial found in WWTP effluent). Surprisingly, there were no changes in the microbiome after exposure to environmental stressors. Major changes were observed between the field and laboratory fish suggesting that environment and diet are important factors influencing the gut microbiome. Changes in the gut microbiome continued up to 42 days in the lab, indicating longer acclimation periods may be needed. This study showed that effluents altered the gut microbiome of fish in the field, but not in the laboratory for unknown reasons. Laboratory studies indicated that transitioning to a new environment may require greater than 14 days before achieving a stable microbiome. / Thesis / Master of Science (MSc) / Wastewater is the largest source of pollution affecting Canada’s aquatic ecosystems; effluents contain antibiotics and antimicrobials that can affect fish and other aquatic life. The gut microbiome of fish is influenced by host species, its diet, and the environment, and thus contaminants released via wastewater effluents may alter the gut microbiome of fishes in receiving waters. This study found that the gut microbiota of rainbow darter fish exposed to wastewater effluents in the central Grand River (Waterloo/Kitchener, Ontario) were dominated by Proteobacteria and had increased diversity. Wild fish transitioned to the lab were dominated by Firmicutes and had decreased bacterial diversity in the gut compared to those in the wild. Altogether, these results suggest that wild fish exposed to wastewater effluents had altered gut microbiomes; transitions to new environments and laboratory acclimation periods are important considerations when studying the fish gut microbiome.

Lower respiratory tract infectious diseases are a world-wide healthcare burden with bacterial pathogens accounting for a large portion of primary and secondary infections. The human respiratory tract is home to hundreds of species of microbes that comprise the human airway microbiome. These commensals play a crucial role in human health in part by providing colonization resistance against pathogens. In a previous study from the Surette lab it was shown that specific bacterial isolates from the respiratory microbiome inhibits the growth of pathogens aerobically. This included an isolate of Staphylococcus aureus which inhibited the growth of Enterococcus faecium. This activity was further characterized in this thesis and the underlying mechanism was explored through comparative genomics. As well, this observation provided proof-of-concept for a large-scale screen for additional isolates which inhibit pathogen growth. I hypothesized that the respiratory tract microbiota included many other bacteria capable of inhibiting the growth of respiratory tract pathogens in both aerobic and anaerobic environments, and that anaerobic conditions will identify new activities not detected aerobically. To examine and identify potential beneficial bacteria, I have screened ~5000 respiratory tract bacteria from the Surette lab’s airway isolate collection against four pathogens: Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae. The respiratory tract commensals were pinned onto the pathogen-lawn and their interaction was expressed as zones of clearing or altered growth phenotypes of the pathogen. The results of the screen showed that anti-pathogen activity was a common feature of respiratory tract commensals. In particular, S. pneumoniae was inhibited by taxonomically diverse members of the microbiota representing three phyla (Proteobacteria, Firmicutes and Actinobacteria). Many of the facultative anaerobes that inhibited S. pneumoniae expressed their activity in anerobic conditions. / Thesis / Master of Science (MSc) / The human respiratory tract harbours commensal and pathogenic bacteria, and the latter cause most of the lower respiratory tract infections. The commensal bacteria help to train the immune system and impede the growth of pathogens through colonization resistance. A previous study by the Surette lab identified bacterial isolates from the respiratory tract that inhibit the growth of select pathogens, among them, a particular strain of Staphylococcus aureus. Based on the results of the earlier study, I hypothesized that the respiratory tract bacteria is a good source of commensals that can inhibit the growth of S. aureus and other respiratory pathogens, such as Streptococcus pneumoniae, Pseudomonas aeruginosa and Klebsiella pneumoniae. To find potential therapeutic bacteria, I screened ~5000 respiratory tract isolates from the Surette lab’s strain collection for the ability to impair growth of target pathogens. Additionally, I further characterized the activity of the previously identified S. aureus strain against various Lactobacillalles strains and used comparative genomics to identify potential biosynthetic genes required for biosynthesis of molecules with antibacterial activity within the genome of S. aureus. The research reported in this thesis demonstrates that many commensal bacteria that live within our airways have the ability to inhibit the growth of bacterial pathogens. This work may provide a new source of antibiotics against respiratory infections and new strategies to reduce susceptibility to infections in vulnerable populations.

Necrotic enteritis (NE) is a major enteric disease in commercial poultry and manifests itself in clinical and subclinical forms. Despite years of research, NE is still among the leading diseases with the greatest economic impact on poultry production. Subclinical forms lead to poor performance (reduced feed intake, weight gain and eventually higher feed conversion ratio) but usually occurs with low mortality rates. The use of antibiotic growth promoters (AGPs) is proving to be an effective tool in maintaining gut health and modifying gut microbiota, thus improving broiler performance and reducing NE. Removal of AGPs has led to an increase in NE occurrence, particularly the subclinical forms. The lack of alternative strategies to control NE is mainly due to limited insight into the relationship between NE pathogenesis, the host microbiome and its immune responses. Therefore, key to overcoming NE is to define the cellular and molecular mechanisms that are involved in the progression of the disease, especially with regard to mucosal immune responses and gut microbiome. Also, assessing the impact of these changes on gut cell metabolism and function is of great importance. This information would be a valuable guide to prevent the onset or alleviate the negative impact of NE on bird's health and performance. In a series of experiments conducted for this project, the effect of single or multi-strain probiotics as well as multi-component additives were tested during NE challenge in order to define the cellular and molecular mechanisms that are involved in the progression of the disease. Results of these experiments revealed that challenging broilers with a 'naturally occurring' NE led to differential expression of tight junction (TJ) proteins in the jejunum compared to non-challenged birds. Supplementation of certain additives reduced NE lesion scores, improved performance and increased mRNA abundance of claudin-3, a key epithelial TJ protein. Multi-strain probiotics and multi-component additives (including a symbiotic and a product containing probiotics, prebiotics and essential oils) were more effective than single-strain probiotics or prebiotics. The aforementioned additives were also more effective in modulating immune responses to NE, especially by decreasing the mRNA abundance of IFN-γ and IL-10 in the jejunum. Furthermore, supplementation of these additives led to an increase in the expression of nutrient transporters (SGLT-1) and regulators of energy metabolism (PGC-1α, mTOR and AMPK); thus, improving nutrients absorption and metabolism. Microbial profiling using 16S rRNA sequencing showed that supplementation of each specific additive led to a signature-like microbiome in the ileal scrapings of broilers. However, supplementation of multi-component additives (including a symbiotic and a product containing probiotics, prebiotics and essential oils) modified the ileal microbiome in association with lower NE lesion scores, better performance and modulated immune responses. These additives reduced the relative abundance of bacteria such as ASF356, Bacteroides, Clostridium sensu stricto 1, Faecalibaculum, Lachnospiraceae UCG-001, Muribaculum, Oscillibacter, Parabacteroides, Rikenellaceae RC9 gut group, Ruminococcaceae UCG-014, and Ruminiclostridium 9 and increased the relative abundance of Lactobacillus compared to NE challenged birds. Collectively, these data indicate that during a subclinical naturally occurring NE, the use of multi-strain probiotics or multi-component additives, compared to single-strain probiotics or prebiotics, would be a more promising strategy in alleviating the effect of this enteric disease. / Doctor of Philosophy / Necrotic enteritis, an enteric disease, is one of the major diseases that negatively impacts the poultry industry and producers' profitability. The growing ban on the use of antibiotics that were used to prevent this disease has increased the number of necrotic enteritis outbreaks worldwide. Having a better understanding of the cellular and molecular mechanisms that are involved in the onset of this disease is of crucial importance and could lead to finding more effective ways to control this disease without drugs. The gut is the site of digestion and absorption of nutrients so any damage would lead to poor bird performance. In a series of experiments conducted for this project, several combinations of beneficial bacteria and nutrient sources that help bacterial growth in the gut (prebiotics) improved gut health leading to better performance during the grow-out period (days 0-42) when birds reach market age. These supplements protected the gut lining and reduced damages due to necrotic enteritis with less severe lesions. Barrier function of the gut was also improved by supplementing the diet with combination of beneficial bacteria and nutrients that help their growth in the gut. There are special types of proteins (called tight junctions) that seal up the space between intestinal cells (enterocytes) and prevent pathogens in the gut lumen from entering the body, thus preventing inflammation and disease. This helps the body to use the absorbed nutrients for growth rather than spending energy to fight pathogens, which collectively results in better growth performance. Concurrent supplementation of beneficial bacteria plus nutrients that help their growth balanced the immune responses in the gut by increasing the copy number of cytokines. Cytokines are proteins that orchestrate immune responses that the host mounts against pathogens. Certain cytokines regulate such responses by preventing the immune system from overreacting and mounting unnecessary reactions, thus preserving energy and nutrients for growth while reducing inflammation. Nutrient uptake from the gut lumen is facilitated by nutrient transporter proteins that reside on intestinal cells (enterocytes). Birds concurrently supplemented with beneficial bacteria and nutrients that help their growth in the gut increased the abundance of these proteins, resulting in improved nutrient uptake and performance compared to the control birds. Co-supplementation of beneficial bacteria and nutrient sources that help their growth modified the type and number of bacteria that are present in the gut lumen. The modified bacterial community were able to produce metabolites such as butyrate and propionate, which are beneficial for the health and growth of the intestinal cells, thus improving the bird's health and its performance. Overall, compared to beneficial bacteria alone, co-supplementation of beneficial bacteria with the nutrients that help their growth in the gut significantly reduced intestinal lesions and improved performance of broiler chickens during the production period. Moreover, dietary addition of these supplements improved gut barrier function by regulating the gene expression of tight junction proteins and gut mucosal immune responses as well as modifying the bacterial community of the gut. Therefore, such combination supplements hold promise in controlling necrotic enteritis in poultry and sustain good overall performance that translates into higher profitability to producers.

The microbiome has long been an alluring target to study and recent advancements in microbial detection and omics-technologies has further revolutionized our view of how human diseases are impacted by the microbiome. A member of the human microbiome that has garnered such attention is Fusobacterium nucleatum, a Gram-negative, anaerobic bacterium, that normally inhabits the human oral cavity. Interestingly, F. nucleatum is highly invasive into surrounding cells and tissues of the periodontal pocket (below the gymline) and capable of disseminating throughout the entire body. Because of this, F. nucleatum is associated with a wide variety of diseases, most recently and strikingly, colorectal cancer. Despite the pathogenic potential of F. nucleatum, there is limited knowledge about the molecular mechanisms contributing to the invasive nature and virulence of this oral bacterium. This gap in knowledge can be attributed to the absence of genetic tools and resources to investigate and study host-pathogen interactions of Fusobacterium. Progress in dissecting the role of Fusobacterium in disease has been hindered by a lack of fully sequenced and annotated genomes, and the absence of genetic systems to generate target virulence gene deletions to validate mechanisms contributing to host-pathogen interactions. Breakthroughs discussed in this work focus on developing and expanding the genetic toolkits and resources available for studying F. nucleatum interactions in relation to human health and disease. As part of this work, herein, I introduce FusoPortal, an online database of fully sequenced and annotated Fusobacterium genomes, that enabled the bioinformatic annotation and correction of large protein encoding reading frames, that were previously misannotated. This database features a custom basic local alignment search tool (BLAST) server that establishes this resource as a powerful tool for identifying potential virulence factors that contribute to Fusobacterium pathogenesis. Most notably, FusoPortal facilitated my discovery of DNA uptake machinery involved in natural competence and transformation in F. nucleatum. This work is the first to characterize natural competence in a Fusobacterium species, and also enables the expansion of Fusobacterium genetics utilizing the newly found competence mechanism. The findings within this dissertation encompass a paradigm shift in efficient and robust tools to study F. nucleatum biology and pathogenesis. By creating tools for identifying key genes, proteins, and mechanisms involved in Fusobacterium induced or accelerated diseases, there is the potential to accelerate the development of novel therapeutics and vaccines against the emerging 'oncomicrobe' Fusobacterium nucleatum. / Doctor of Philosophy / The trillions of microbes living on or in the human body, collectively called the microbiome, has long been a captivating target to study and understand its role in human health and disease. Recent advances in technology have revolutionized our view of the individual components of the human microbiome, which has led to a renaissance in understanding how specific bacterial species could be used to modulate human health and fight a myriad of diseases. A member of this microbiome that has garnered such attention is Fusobacterium nucleatum, a bacterium that lives in oxygen free pockets along the gumline in the human mouth. A striking feature of F. nucleatum is its ability to invade surrounding tissue, driving bacterial spread throughout the entire body. This bacterium is associated with a wide variety of diseases, most importantly colon cancer. Although F. nucleatum is implicated in these diseases, we still know very little about the mechanisms used by Fusobacterium to promote disease. This roadblock in studying F. nucleatum can largely be attributed to the lack of molecular tools and resources to investigate and study the interactions between the bacteria and its human host. Therefore, research discussed in this work revolved around developing and resources available for studying F. nucleatum interactions in relation to human health and disease. One such resource developed was FusoPortal, an online website with fully sequenced and annotated genomes. This resource was critical in the bioinformatic annotation and correction of large proteins that were previously misannotated. This website features a tool that allows one to search this complete set of genes for a specific sequence establishing this resource as an important tool for identifying key genes and mechanisms that could influence F. nucleatum ability to infect and cause disease. Most notably, FusoPortal provided the means to discover a bacterial system that can import DNA and integrate it into the bacterial genome, a process called natural competence. This work is the first to characterize natural competence in a Fusobacterium species, and has allowed me to utilize the newly found natural competence mechanism to enhance Fusobacterium genetics. In summary, the findings within this dissertation brings about a new horizon for studying F. nucleatum biology, thereby, providing the framework for creating future therapeutic strategies to treat diseases including colorectal cancer.

This dissertation documents efforts to evaluate metabolic variables and the fecal microbiome in adult horses fed grass hay. In the first study, eight Arabian geldings limit-fed an 18% vs. 12% non-structural carbohydrate (NSC) hays in a cross-over design during two 28-day periods were included to evaluate the influence of grass hay NSC on serum insulin and plasma glucose concentrations. Serum insulin concentrations was higher in geldings fed the 18% NSC hay; however, this difference was only detected on day 7 and none of the geldings developed hyperinsulinemia. Blood glucose concentrations did not differ between hay groups. The second and third studies were extensions of the first and were conducted to use denaturing gradient gel electrophoresis (DGGE) and real-time PCR in evaluating the effect of forage carbohydrates on equine fecal bacteria diversity and abundance, respectively. Fecal microbiomes were similar (80.5-87.9%) between geldings. The abundance of bacteria belonging to the Firmicutes phylum increased (p = 0.02) in the feces of geldings fed 12% NSC hay (mean 8.06 range [8.03-8.11] log10 copies/g feces) compared to the feces of the same geldings when fed the 18% NSC hay (7.97 [7.97-7.98] log₁₀ copies/g feces). The Firmicutes (43.7%), Verrucomicrobia (4.1%), Proteobacteria (3.8%), and Bacteroidetes (3.7%) phyla dominated the fecal microbiomes. This work was the first to report the presence of the Actinobacteria, Cyanobacteria, and TM7 phyla in the equine fecal or gut microbiome. There was a high abundance (38%) of unclassified bacterial sequences in the gelding fecal microbiome. In the fourth study, 5 overweight adult mixed-breed mares and 5 adult mixed-breed mares in moderate condition, limit-fed a grass hay, were used to evaluate the effect of body condition on diet digestibility, plasma and fecal volatile fatty acid (VFA) concentrations, and fecal bacterial abundance. Hay, fecal, and blood samples were taken daily for 4 days after a 10 day adaptation period. A difference in hay digestibility, fecal VFA concentration, or bacterial abundance was not detected between overweight mares and mares in moderate condition. Plasma acetate, a product of microbial fermentation of fiber, was higher in the overweight mare group. / Ph. D.

Neurodevelopmental disorders, such as autism spectrum disorders, schizophrenia, and attention deficit hyperactivity disorder, are a heterogeneous set of developmental disorders affecting the central nervous system. Studies into their etiology remain challenging, as neurodevelopmental disorders frequently present with a wide range of biological, behavioral, and comorbid symptomologies. Increasing epidemiological reports of antibiotic use during pregnancy as a significant correlate of subsequent mental disorder diagnosis in children suggest a mechanism of influence via the maternal gut-fetal brain axis. Importantly, antibiotics cause dysbiosis of the gut microbiome and disrupt the delicate composition of the microbial inoculum transferred from mother to child, which is critical for development of the immune system and holds implications for long-term health outcomes. The research objective of this dissertation is to reveal a causal mechanism of maternal microbial influence on neurodevelopment by examining the brain's resident immune cells, microglia, and corresponding behavioral outcomes in a mouse model of antibiotics-driven maternal microbiome dysbiosis (MMD). We identify early gross motor deficits and social behavior impairments in offspring born to MMD dams, which paralleled hyperactivated microglia in brain regions specific to cognition and social reward. The MMD microglia also exhibited altered transcriptomic signatures reflective of premature cellular senescence that support evidence of impaired synaptic modeling found in MMD brains. We report that these deficits are rescued in the absence of Cx3cr1, a chemokine receptor expressed ubiquitously on microglia, to highlight a pathway in which maternal microbiota may signal to neonatal microglia to undergo appropriate neurodevelopmental actions. Finally, we characterize Lactobacillus murinus HU-1, a novel strain of an important gut bacterium found in native rodent microbiota, and demonstrate its use as a probiotic to restore microglial and behavioral dysfunction in MMD offspring. / Ph. D. / Population studies on neurodevelopmental disorders, such as autism spectrum disorders, schizophrenia, and attention deficit hyperactivity disorder, highlight antibiotic use during pregnancy as a major correlate of subsequent diagnoses in children. These findings support a growing body of evidence from animal and human studies that the microbial ecosystems (“microbiome”) found in and on our bodies play significant roles in mental health, including mood, cognition, and brain function. Importantly, antibiotics during pregnancy create an imbalance of the gut microbiome (“dysbiosis”) and disrupt the microbial inoculum transferred from mother to child, which is critical for maturation of the infant immune system and holds implications for long-term health outcomes. Thus, the research objective of this dissertation is to identify a mechanism of influence from the mother’s gut to the neonate’s brain by examining the brain’s resident immune cells (“microglia”) in a mouse model of antibiotics-driven maternal microbiome dysbiosis (MMD). We uncover autism-like behavioral deficits and dysfunctional microglia in MMD offspring, and characterize signaling cues specific to microglia by which improper neurodevelopment may be taking place. We also reveal that the detrimental effects of MMD are reversed in mice born to mothers pretreated with a probiotic candidate, Lactobacillus murinus HU-1, to suggest maternally-derived Lactobacillus may help to mediate proper neurodevelopment.

Host-associated microbial communities, also known as microbiomes, are essential to the health of their hosts, and disturbance of these communities can negatively impact host fitness. The honey bee gut microbiome is a relatively simple host-associated community that makes an excellent model system for studying microbiome stability. In addition, honey bees are essential agricultural pollinators, so factors that impact their health are important for food security. The presented research focused on the stability of the honey bee gut microbiome in response to disturbance from the antibiotic tetracycline. Tetracycline was chosen because it is the most commonly used antibiotic in beekeeping, and may have negative effects on bees through the disruption of their gut microbiomes. The first study presents a new fecal sampling method for studying the honey bee gut microbiome of individual bees over time. This method accurately represented bacterial community structure in the gut microbiome as determined with 16S rRNA gene amplicon sequencing, as fecal and whole gut samples did not differ significantly for individual bees. The fecal sampling technique was then used to examine changes to individual honey bee gut bacterial communities before and after tetracycline exposure. Minimal differences in gut community structure were detected prior to and five days after tetracycline treatment. However, there was variability in how individual gut microbiomes were affected by tetracycline treatment, highlighting the importance of intraspecific variation in response to disturbance. The second study investigated whether the timing of disturbance during a host's life impacts microbiome community stability. Newly emerged bees were treated with tetracycline, returned to their hive, and recollected 7 or 14 days later. The gut communities of the bees were then characterized using 16S rRNA gene amplicon sequencing. Gut microbiome structure of bees treated with tetracycline at emergence differed from controls both 7 and 14 days after emergence, with the antibiotic-treated bees having lower community richness overall. This study showed that early life disturbance of host-associated microbial communities can influence microbiome structure later in life. The final study describes the occurrence of antibiotic resistance genes (ARGs) in honey bee gut bacterial symbionts from hives across the US. Honey bee gut metagenomes were sampled from hives at 13 apiaries located in a transect from Virginia to Washington, and ARG presence was assessed across the sites. We also specifically quantified the abundances of two common tetracycline resistance genes (tet(B) and tet(M)) across apiaries. ARGs, both for antibiotics used in beekeeping and unrelated antibiotics, were detected in honey bee gut bacteria from all apiaries. Tetracycline resistance genes were the most common across all apiaries, and the abundance of two tetracycline resistance genes varied by apiary. Members of the honey bee gut microbiome contained different proportions of ARGs, but taxa within a single family contained similar proportions, possibly indicating phylogeny plays a role in ARG accumulation. In particular, Gilliamella and Frischella, both in the family Orbaceae, contained the highest percentages of ARGs. The results from this study suggest honey bee bacteria act as reservoirs of ARGs. Overall, the presented research contributes to the field of biology by highlighting the importance of intraspecific variation in host-associated microbial communities and presenting a new method for studying honey bee gut microbiome variation at the individual-level, showing that early life events in honey bees influence microbiome development, and suggesting that honey bee bacterial symbionts have adapted to deal with antibiotic disturbance through the accumulation of ARGs. / Doctor of Philosophy / Nearly all animals, including honey bees, have communities of bacteria that live on and in them. These communities, called microbiomes, are often essential to the health of their hosts. For instance, communities of gut bacteria can be important for breaking down food for digestion. Honey bees have approximately 10 bacterial species that consistently live in their guts and provide these types of services to their host. As with many bacterial communities, these beneficial bacteria can be impacted by exposure to antibiotics, even though antibiotics can also be important for treating or preventing dangerous bacterial infections. In honey bee hives, the antibiotic tetracycline is used to prevent bacterial disease. However, tetracycline may simultaneously be negatively impacting colony health through disruption of the honey bee gut microbiome. The goal of the presented work was to understand how tetracycline impacts the honey bee gut microbiome. In my first chapter, I demonstrate a new fecal sampling method that will allow us to understand how gut microbiomes from individual bees change over time. I first compared the bacteria found in fecal samples to those in the whole guts of bees and found that the bacterial communities of the fecal samples and guts were very similar, indicating that fecal sampling is a good method for studying the honey bee gut microbiome. I then used my fecal sampling method to determine how individual honey bee gut microbiomes respond to antibiotic disturbance over time. I collected fecal samples from adult bees prior to treatment, treated the bees with tetracycline, and after five days of being maintained in the lab, recollected fecal samples. My results showed few changes to the bacterial communities before and after treatment, suggesting some honey bee gut microbiomes may be resistant to tetracycline. In my second chapter, I addressed whether exposure to antibiotics early in life had long-term impacts on the gut microbiome. I treated bees at the start of adulthood with tetracycline, returned the bees to their hive for 7 or 14 days, and assessed their microbiome. Tetracycline treatment at the beginning of adulthood changed the gut microbiome later in life, as the microbiomes of tetracycline-treated bees and controls differed from one another both 7 and 14 days after exposure. This chapter shows that disturbances to microbiomes during early life can also affect microbiomes later. My third chapter addressed how honey bee bacteria have adapted to antibiotic use by identifying antibiotic resistance genes (ARGs) in honey bee gut bacteria from 13 hives located in a transect across the US from the state of Washington to Virginia. I found a variety of antibiotic resistance genes in honey bee gut bacteria, both associated with beekeeping and likely environmental contamination. The prevalence of antibiotic resistance genes in honey bee bacteria may help us track antibiotic resistance in the environment. Ultimately, my dissertation contributes to our understanding of how antibiotic use affects honey bees by changing their gut microbiome.