The plant nectar microbiome (NMB) primarily composed of fungi and bacteria can qualitatively and quantitively affect floral rewards and ultimately impact plant-pollinator interactions and plant reproductive success. Evidence suggests that changes in microenvironmental conditions across spatial gradients can induce changes in the floral nectar microbiome, leading to microbiome variation within species. Specifically, changes in biotic and abiotic conditions across an elevation gradient (i.e. pollinator community composition, temperature, UV exposure, nutrient availability) have the potential to induce variation NMB composition and abundance. However, this has been little explored. For instance, we could expect that the diversity and abundance of the NMB will decrease with elevation as the growing conditions become less favorable (e.g. lower temperatures). In this study, we evaluate spatial variation in nectar microbiome composition, diversity, and abundance in populations of Rhododendron catawbiense at high (H) and low (L) elevations separated by over 1000ft in Roan Mountain, TN. Nectar samples were collected, plated, quantified and isolated. Fungal ITS sequences were obtained from individual colonies by Sanger sequencing and directly from nectar samples by Nanopore NGS. Preliminary results suggest that elevation can affect fungal abundance and composition in the NMB. We found 10 fungal species inhabiting the nectar of R. catawbiense (H= 4; L= 9). We also observed variation in nectar yeast abundance with more than 30x as many CFUs (colony forming units) (H= 1.89 ±10.64; L=38.43 ±1.39.12) and twice as many RPKMs (reads per kilobase matched) (H= 2.8x107 ± 2.46x107; L=5.5x107 ±3.6x107) on average at the lower elevation. Interestingly, R. catawbiense NMB in the lower elevation had a broader diversity of species (Simpson Diversity Index: H= 0.52; L= 0.95), perhaps as a result of more favorable growing conditions. This trend is largely associated with higher abundance of Metschnikowiaceae yeast OTUs and lower abundance of potentially pathogenic Basidyomycete OTUs. Overall, results indicate that elevation can mediate changes in the composition and abundance of microorganisms in the NMB, which in turn can lead to differences in pollinator community composition and plant reproductive success. These results highlight the need evaluate within-species variation in NMB at large spatial scales and its potential consequences for plant reproductive success, plant-pollinator interactions and plant community dynamics. This is particularly important in the face of human-mediated environmental disturbances that can alter plant-microbe interactions.

Fecal Fermentation Profile, Nutrient Proximate Analysis and Well-being of Aerobic versus Anaerobically Trained Female Athletes Lindsey Stallworth1, Anna K Whitlock1, Sonja J Lafollette2, Laiken Lewis2, Kristy Thomas1,3, Amy E Wahlquist MS4, W. Andrew Clark PhD, RD, LDN1,3, Mary Andreae MS, RD, LDN1,3, Michael Stone PhD5 College of Clinical and Rehabilitative Health Science, Department of Rehabilitative Science1; College of Arts and Science, Department of Biological Science2; Quillen College of Medicine, Department of Biomedical Science3; Department of Epidemiology and Biostatistics, College of Public Health; Clemmer College of Education4, Department of Sport, Exercise, Recreation, and Kinesiology5, East Tennessee State University, Johnson City Introduction: The human gut microbiome serves a role in health and disease prevention. The composition of intestinal bacteria and their metabolites (fecal fermentation profile, FFP) can influence mood, sleep, immune response, inflammation and the ability to digest and absorb nutrients. Current studies show that populations who are more physically active have a more diverse microbiome compared to sedentary groups, resulting in higher adaptability to physical exertion. There is a limited amount of research focusing on the microbiome of physically active groups with different training regimens. Therefore, we investigated if athletes under aerobic versus anaerobic based training express differences in fecal fermentation profile, nutrient proximate analysis and measures of well-being. We hypothesize that aerobically trained athletes will express a more diverse microbiome as measured by FFP. Materials and Methods: Members from the ETSU Women’s Track and Field Team were recruited to join a research study evaluating physiological differences between aerobically (n=9) and anaerobically (n=3) trained athletes. Research participants read and signed an informed consent document (ETSU IRB-0122.15s-ETSU), completed 2 surveys (anthropometric, well-being), provided fecal (stool) and salivary cortisol samples for analysis. A 1g aliquot of the stool sample was collected and stored at -80 C for future microbiome analysis (UT, Knoxville, TN). The remainder of the stool sample was frozen at -80 C, lyophilized and ground to a fine powder for FFP and nutrient proximate analysis (NPA). FFP isolated short chain volatile fatty acids from the stool were identified via gas chromatography (Shimadzu). NPA of the stool included total calories, total nitrogen, dry matter. Mental well-being in regard to training were assessed via an athlete well-being survey, sport training survey, and salivary cortisol test (taken the morning of providing fecal sample). Dietary habits were assessed using a Food Frequency Questionnaire (FFQ). Anthropometric data including height, weight, hydration status and lean body mass (Bio impedance Analysis (BIA)). Results: No statistical differences were noted between groups for FFP, dry matter, total calories or total nitrogen of the stool sample. Anthropometric measures of percent body fat (p < 0.0058), percent skeletal muscle (p < 0.0086) and body mass index (p < 0.0106) were lower for aerobically trained versus anaerobically trained athletes. Not enough surveys were completed for measures of well-being to be statistically analyzed. Conclusion: The hypothesis is rejected since there was no difference in the diversity of short chain volatile fatty acids. The data set is skewed because of the low number of anaerobic athlete participants to fairly evaluate the hypothesis. Additional samples are being sought to balance the data and data for salivary cortisol, food frequency questionnaire and fecal microbiome will be collected later.

No description available.

High altitude is one of the most extreme environments inhabited by endotherms, where extreme cold temperatures and low O2 availability (hypoxia) can constrain aerobic heat production (thermogenesis) to maintain body temperature (Tb). Recent findings suggest that the gut microbiome contributes to whole-body thermogenesis, but the significance of this mechanism for coping in cold environments is unknown. We examined whether the gut microbiome contributes to adaptive variation in thermogenic performance in deer mice at high altitude. Mice from populations native to high altitude and low altitude were born and raised in common conditions. Adults from both populations were acclimated to warm (25C) normoxia or cold (5C) hypoxia (~12 kPa O2 for 6 weeks) in a full factorial design, and a subset of mice in each group were treated with antibiotics to deplete the gut microbiome. Thermogenic endurance was then measured as the duration that Tb and metabolism could be maintained during acute cold challenge. In lowlanders, antibiotics had only modest effects on thermogenic endurance. In highlanders, in stark contrast, antibiotic treatment led to pronounced reductions in thermogenic endurance in both environments. These effects could not be explained by impairments in aerobic heat production by host thermogenic tissues, because antibiotic treatment had no effects on cold-induced increases in O2 consumption or UCP1 content of brown adipose tissue. These results suggest that the gut microbiome plays an increased role in thermogenesis in high-altitude mice. Thermogenic performance contributes to fitness at high altitude, suggesting that changes in host-microbe interactions contribute to high-altitude adaptation. / Thesis / Master of Science (MSc) / High altitude is one of the most extreme environments inhabited by endotherms. Recent findings suggest that the gut microbiome contributes to thermogenesis, but the significance of this mechanism for coping in cold environments is unknown. We examined whether the gut microbiome contributes to adaptive variation in thermogenic performance in deer mice at high altitude. Adult mice from low and high-altitude populations were acclimated to warm normoxia or cold hypoxia, and a subset of mice in each group were treated with antibiotics to deplete the gut microbiome. Thermogenic endurance was measured as the duration that Tb and metabolism could be maintained during acute cold challenge. In lowlanders, antibiotics had modest effects on thermogenic endurance but in highlanders, it led to pronounced reductions in thermogenic endurance in both environments. These results show that thermogenic performance contributes to fitness at high altitude, suggesting that changes in host-microbe interactions contribute to high-altitude adaptation.

The microbiome has become a focus of research, particularly in the field of human health and precision medicine, due to its role in human development, immunity, and nutrition. Microbiome profiling studies have become more tractable and advanced in large part thanks to advancements in metagenomics. One such study is the Black Women’s Health Study (BWHS), which aims to better understand health risks and disease development specific to Black women, who are more susceptible to certain health conditions. However, a major obstacle for reproducibility of microbiome research is the high sensitivity of microbial compositions to external factors and batch-to-batch technical variability, resulting in batch effects that often hinder analysis of factors of interest. While batch effect adjustment methods have been developed for other biomedical data, they do not appropriately account for two unique features of microbiome data: 1) its compositional nature, and 2) extreme overdispersion and zero-inflation. My dissertation addresses these challenges by evaluating and improving batch effect correction methods for microbiome data and then applies these approaches to data from BWHS. First, I evaluated ComBat-Seq, along with existing microbiome-specific tools, in removing batch effects from both simulated 16S rRNA and real-world shotgun metagenomic sequencing data while preserving effects belonging to biological factors of interest. Second, I applied ComBat-Seq in an epidemiological study in which I identified several oral health-related genera among adult Black women to be associated with the host’s geographic location in the US. Finally, I introduced an extension to ComBat-Seq that improves its performance in batch effect correction on rare taxa with outliers via imputation. I demonstrated that, by replacing zeroes with predicted non-zero read counts that follow the observed compositional structure of the data, imputation effectively reduced the number of problematic cases in which outliers were intensified after batch effect correction. Collectively, my thesis demonstrates that 1) when the specific features of microbiome data are accounted for, batch effect correction methods offer a promising solution to address batch effect in microbiome data and improve microbiome profiling studies and 2) it is important to consider social/environmental factors associated with the host’s physical location when studying the oral microbiome.

The global burden of mental health disorders is rising with the world health organization recently having recognized major depressive disorder as the leading cause of disability worldwide. Nearly one in five Canadians are now estimated to struggle with a mental health disorder and Generalized Aanxiety Ddisorder (GAD) Mmajor Ddepressive Ddisorder (MDD) and Bbipolar Ddisorder are three of the most prevalent. Despite significant research into the disorders’ cause, the pathophysiology and underlying etiology of these diseases remains largely undiscovered. Recent research has highlighted the potential role of the gut microbiota in mental health, in particular in connection with anxiety. Our research aims to investigate this link in a cohort of GAD, MDD and bipolar patients recruited through the Anxiety Treatment and Research Centre at St. Joseph’s Healthcare in Hamilton, as well as through the University Health Nnetwork in Toronto. 71 GAD, 18 MDD, 17 euthymic MDD and 23 Bipolar patients provided fecal samples from which DNA was extracted, following which the V3 region of the 16S rRNA gene was amplified and sequenced using the Illumina Miseq platform. Sequencing data was analyzed through an in-house pipeline to construct community profiles of patients and age and sex matched healthy controls. My work involved analyzing the data to identify signature organisms that might identify putative disease associated microbial communities for further hypothesis generation about possible roles in disease. Furthermore, an extensive culturing effort was undertaken to identify and characterize some of the Bacteroides strains which were enriched in the GAD patient population. This study presents novel insights into some of the organisms that may be markers for a number of different diseases as well developing a better understanding of the Bacteroides that were correlated with anxiety. / Thesis / Master of Science (MSc) / Mental hHealth disorders including Ggeneralized Aanxiety Ddisorder (GAD), Mmajor Ddepressive Ddisorder (MDD) and Bbipolar Ddisorder (BD) affect somewhere between five and ten percent of Canadians, with the Wworld Hhealth Oorganization recently labeling MDD the leading cause of disability worldwide. Despite decades of research, on these disorders we still have a poor understanding of what factors may contribute to causing these disorders their underlying mechanisms of actionetiology. Another hallmark of many mental health conditions are increased rates of gastrointestinal symptomsor digestive distress relative to the healthy populatioindividualsn. Recently, there has been a great deal of research about how the trillions of bacteria that live in the digestive tract play a role in neuronal brain development and behaviour. This study aimed to better understand which the organisms reside iinn the digestive tract of mental health patients in hopes of better understanding how some of these organisms could be contributing to patients’ poor mental health. Additionally, through better understanding the nature of these communities, recommendations could potentially be made about therapeutic interventions to restore a healthy community.

No description available.

Premise plumbing, the pipes and fixtures at the building level, present a unique challenge for maintaining drinking water quality. Of particular concern are opportunistic pathogens, including Legionella pneumophila which can regrow in premise plumbing and cause disease in immunocompromised populations. The goal of this work was to explore engineering methods for control of L. pneumophila and total regrowth. The first line of study involved a series of experiments with simulated glass water heaters (SGWHs) to investigate interactions between specific water chemistry factors and L. pneumophila regrowth, and the second used laboratory grade purified water to investigate the limits of a nutrient control approach for biological stability. Several water chemistry factors including assimilable organic carbon (AOC) content, granular activated carbon (GAC) biofiltration, plumbing materials, copper concentrations and temperature were investigated using SGWHs. AOC is the carbon available for bacteria growth in drinking water. Results indicated that AOC reduction may be a promising method for controlling L. pneumophila and total bacteria regrowth, but there may be a point at which AOC reduction is no longer effective. Prior GAC biofiltration removed organic carbon and was effective in controlling total bacterial regrowth in SGWHs, but actually encouraged L. pneumophila regrowth. A wide variety of materials typically encountered in premise plumbing was investigated and only had limited effect on proliferation of L. pneumophila and total bacteria. The effects were dynamic, even with long-term studies. Copper pipes held promise for control of L. pneumophila, as did copper concentration across a range of pHs. Aqueous copper concentration released from pipes was dependent on temperature, however, and thus this control method may not be applicable in all hot water lines. The peak temperatures for L. pneumophila proliferation fell between 41 and 45 °C, temperatures which could be encountered in a hot water distribution system when the water heater is set to 48 °C, as is often recommended with scalding and energy concerns. A constant temperature of 53 °C seemed to provide control of L. pneumophila, but recolonization is possible even at these high temperatures. Work with laboratory grade water indicated that extreme control of nutrients was not enough to completely control regrowth in premise plumbing. With stagnation in the cleanest conditions, a 2-log increase of a diverse group of bacteria was observed within 10 days. As drinking water can never achieve such nutrient removal, this study presents the limits of nutrient removal as a strategy for regrowth control. This work explored both the potential and the limitations of several mechanisms for controlling regrowth in premise plumbing. Understanding how these water chemistry factors affect L. pneumophila and total bacterial regrowth is critical to identifying the most effective engineering controls. / Master of Science

Enteric microbiota influences enteric viral infections, and host response to these pathogens and vaccines. Using gnotobiotic (Gn) pigs transplanted with human gut microbiota (HGM), we studied the effects of HGM on the immune response to oral rotavirus vaccination and rotaviral disease. We also used HGM transplanted Gn pigs to determine the effects of HGM on human norovirus infection. Despite commercially available vaccines, human rotavirus is a leading acute gastroenteritis in children, especially those in developing countries. Human norovirus (HuNoV) is a leading cause of acute gastroenteritis in all age groups worldwide, and no vaccines are commercially available. Further understanding of how enteric microbiota influences these viral diseases may identify therapeutic targets. In our rotavirus study, pigs were colonized with HGM from an infant with low fecal concentrations of enteropathy biomarkers and responded well to their first dose of oral rotavirus vaccine (healthy human gut microbiota "HHGM"); or pigs were colonized with HGM from an infant with high fecal concentrations of enteropathy biomarkers and a poor response to the first dose of oral rotavirus vaccine (unhealthy human gut microbiota "UHGM"). HHGM colonized pigs had stronger cell-mediated and mucosal immune response to oral rotavirus vaccine compared to UHGM pigs based on the number of rotavirus-specific IFN-γ producing T cells in the ileum, spleen, and blood, and trends towards higher rotavirus specific antibody titers in intestinal contents, respectively. Significant correlations between multiple Operational Taxonomic Units (OTUs) of bacteria and frequencies of IFN-γ producing T cells at the time of human rotavirus challenge existed, suggesting that certain members of the microbiota influenced the immune response to the vaccine. After the vaccinated pigs were challenged with human rotavirus, HHGM pigs had less severe and shorter duration of viral shedding and diarrhea compared to UHGM pigs, suggesting that HHGM facilitated development of stronger protective immunity. These results demonstrated that composition of the enteric microbiota influenced host immune response to oral vaccination. In the norovirus study, Gn pigs were colonized with HHGM to determine the effects of microbiota on HuNoV infection. Colonized pigs shed more virus for a longer duration than non-colonized pigs, and also had higher viral titers in the duodenum and distal ileum. Diarrhea was more severe 4-10 days post-infection and lasted longer in colonized compared to non-colonized pigs. Twenty-seven genes related to the immune system were highly upregulated in HuNoV infected, colonized pigs compared to non-colonized controls. These result showed that HHGM influenced infectivity of HuNoV in the Gn pig model and altered host gene expression related to the immune system. These studies showed that HHGM can improve the host immune response and efficacy of rotavirus vaccine, but it can also enhance infection and clinical disease in HuNoV infected Gn pigs. Depending on the virus, gut microbiota may be beneficial or detrimental to the host. Those developing future treatments aimed at altering microbiota to prevent or ameliorate one viral pathogen need to consider the potential for enhancing a different pathogen. These studies demonstrated the usefulness of HGM transplanted Gn pigs for evaluation of microbiota influence on infection and immunity of enteric viral pathogens. / Ph. D. / Gut microbiota influences intestinal viral infections, and host response to these pathogens and vaccines. Using gnotobiotic (Gn) pigs transplanted with human gut microbiota (HGM), we studied the effects of HGM on the immune response to oral rotavirus vaccination and rotaviral disease. We also used HGM transplanted Gn pigs to determine the effect of HGM on human norovirus infection. Despite commercially available vaccines, human rotavirus is a leading acute gastroenteritis in children, especially those in developing countries. Human norovirus (HuNoV) is a leading cause of vomiting and diarrhea in all age groups worldwide, and no vaccines are commercially available. Further understanding of how gut microbiota influences these viral diseases may identify therapeutic targets. In our rotavirus study, pigs were colonized with HGM from an infant without evidence of intestinal disease based on fecal analysis, and who responded well to the first dose of oral rotavirus vaccine (healthy human gut microbiota “HHGM”); or pigs were colonized with HGM from an infant with evidence of potential intestinal dysfunction and a poor response to the first dose of oral rotavirus vaccine (unhealthy human gut microbiota “UHGM”). HHGM colonized pigs had a stronger immune response to the oral rotavirus vaccine compared to UHGM pigs. Significant correlations between multiple Operational Taxonomic Units (OTUs) of bacteria and frequencies of rotavirus-specific immune cells at the time of human rotavirus challenge existed, suggesting that certain members of the microbiota influenced the immune response to the vaccine. After the vaccinated pigs were challenged with human rotavirus, HHGM pigs had less severe and shorter duration of viral shedding and diarrhea compared to UHGM pigs, suggesting that HHGM enhanced vaccine efficacy. These results demonstrated that composition of the gut microbiota influenced host immune response to oral vaccination. In the norovirus study, GN pigs were colonized with HHGM to determine the effects of microbiota on HuNoV infection. Colonized pigs shed more virus for a longer duration than non-colonized pigs, and also had higher viral titers in sections of small intestine. Diarrhea was more severe 4-10 days after infection and lasted longer in colonized compared to non-colonized pigs. Twenty-seven genes related to the immune system were highly upregulated in HuNoV infected, colonized pigs compared to controls. These result showed that HHGM influenced infectivity of HuNoV in the Gn pig model and altered host gene expression related to the immune system. These studies showed how HHGM improved the host immune response and efficacy of rotavirus vaccine, but conversely enhanced infection and clinical disease in HuNoV infected pigs. Depending on the virus, gut microbiota may be beneficial or detrimental to the host. Those developing future treatments aimed at altering microbiota to prevent or ameliorate one viral pathogen need to consider the potential for enhancing a different pathogen. These studies showed the usefulness of HGM transplanted Gn pigs for evaluation of microbiota influence on infection and immunity of intestinal viruses.

Microbial communities play important roles in human health and disease, are essential components of terrestrial and marine ecosystems, and are crucial for producing commercially valuable molecules in industrial processes. These communities consist of hundreds of species involved in complex interactions. Mapping the interrelationships between different species in a microbial community is vital for understanding and controlling ecosystem structure and function. Advances in sequencing and other omics technologies have led to thousands of datasets containing information about microbial composition, gene expression, and metabolism in microbial communities associated with human hosts and other environments. These provide valuable information in understanding how microbes interact with each other and how their interactions affect the health of their host (e.g., human or plant). Furthermore, understanding these interactions paves the way for the rational design and modulation of synthetic communities for producing antibiotics, biofuels, and pharmaceutical products. The first part of my thesis is focused on improving the workflow for the inference of microbial co-occurrence relationships from abundance data. Toward this goal, we developed Microbial Co-occurrence Network Explorer (MiCoNE), a pipeline that infers microbial co-occurrences from 16S ribosomal RNA (16S rRNA) amplicon data. This pipeline involves numerous complex steps that require specific tools and parameter choices, posing open questions about the robustness and uniqueness of the inferred networks. Through MiCoNE, we systematically analyzed how these choices of tools affect the final network and proposed guidelines on appropriate tool selection for a particular dataset. We envisage that this pipeline could be used to integrate multiple datasets and generate comparative analyses and consensus networks that can guide our understanding of microbial community assembly in different biomes. The second part of my thesis focuses on microbe-host interactions rather than microbe-microbe associations. In particular, we sought to predict the effects of microbial metabolites on human receptors and their associated regulatory pathways. We specifically focus on the Aryl hydrocarbon receptor (AHR), a ligand-mediated transcription factor involved in tumorigenesis. In this project, we aimed to systematically predict the binding of diverse microbial metabolites secreted from microorganisms found in the human oral microbiome to the AHR to identify links between the microbiome and cancer initiation. We further build a mathematical model of the AHR regulatory pathway and model the effects of ligand binding on downstream molecules. We envision that these methods could be used to predict the impact of microbial dysbioses on human regulatory pathways. In the final part of my thesis, we turn to the question of whether computational algorithms can help control microbial community growth to achieve specific objectives. In particular, we describe the development of a reinforcement learning algorithm to learn optimal environmental control strategies to steer a microbial community towards a certain goal, such as reaching a specific taxonomic distribution or producing desired metabolites. We train the reinforcement learning framework through community-level simulations of genome-scale metabolic models (GEMs) for different microbial species in bioreactor systems. In this project, we simulate a simple case study with two auxotrophic mutants to verify the algorithm's validity. Ultimately we aim to simulate the implementation of the algorithm in experimental bioreactor systems. Overall, the work presented in this thesis demonstrates how microbe-microbe and microbe-environment (including microbe-host) interactions represent plastic system-level properties whose understanding can help unravel the role of microbial communities in specific diseases. Correspondingly, manipulating these interactions, e.g., by appropriately modifying environmental conditions, can serve as a promising strategy for steering communities towards desired states, including producing valuable molecular products. / 2024-11-06T00:00:00Z