The building plumbing microbiome has important implications, especially in terms of its role as a reservoir and conduit for the spread of opportunistic pathogens (OPs), such as Legionella pneumophila. This dissertation applied next-generation DNA sequencing tools to survey the composition of building plumbing microbiomes and assessed hypothetical factors shaping them. A challenge to identifying key factors shaping building plumbing microbiomes is untangling the relative contributions of influent water quality, provided by drinking water utilities, and those of building-level features, such as pipe materials. To this end, standardized pipe rigs were deployed at the treatment plants and in distal portions of the water distribution system at five water utilities across the eastern U.S. Source water and treatment practices appeared to be the overarching factors shaping the microbial taxonomic composition at the tap, with five key water chemistry parameters identified (total chlorine, pH, P, SO42- and Mg2+). Hot water plumbing is of particular interest because OPs tend to proliferate in warm water environments and can be inhaled in aerosols when showering. Two identical lab-scale recirculating hot water rigs were operated in parallel to examine the combined effects of water heater temperature set point, pipe orientation, and water use frequency on the hot water plumbing microbiome. Our results revealed distinct microbial taxonomic compositions between the biofilm and water phases. Importantly, above a threshold of 51 °C, water heater temperature, pipe orientation, and water use frequency together incurred a prominent shift in microbiome composition and L. pneumophila occurrence. While heat shock is a popular means of remediating L. pneumophila contamination in plumbing, its broader effects on the microbiome are unknown. Here, heat shock was applied to acclimated lab-scale hot water rigs. Comparison of pre- versus post- heat shock samples indicated little to no change in either the microbial composition or L. pneumophila levels at the tap, where both water heater temperature and water use frequency had the most dominant effect. Overall, this dissertation contributes to advancing guidance regarding where to most effectively target controls for OPs and also advances research towards identifying the features of a 'healthy' built environment microbiome. / PHD / Drinking water is often misconceived to be “sterile,” whereas in reality the water distribution and plumbing systems that convey the water to the consumer represent a robust microbial habitat. While it is not possible, or even desirable, to kill all of the microbes present in drinking water, the Safe Drinking Water Act in the U.S. enforces measures to purify and disinfect water at the treatment plant and keep bacterial numbers low in water mains and up to the consumer property line. However, current regulatory frameworks are designed to protect against fecal- (e.g., raw sewage and manure) derived pathogens, whereas recently opportunistic pathogens (OPs), including Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa have come to the forefront as the leading source of tap-water related illness in the U.S. and other developed countries. In contrast to traditional fecal pathogens, building plumbing systems are a natural habitat for OPs, where they can readily proliferate. Currently there are no provisions within the Safe Drinking Water Act or other regulations to protect consumers specifically from OPs. There are also no “silver bullet” remedial measures that consistently and reliably defend against OPs colonizing building building plumbing, particularly when aiming to protect against multiple types of OPs. A major challenge in preventing and remediating OP proliferation in building plumbing is that they tend to be protected from disinfectants, such as chlorine, inside amoeba hosts and within the slimy layer that forms on the surface of pipe walls called “biofilm”. With the recent advent over the past decade of next-generation DNA sequencing, there are new reasons to take interest in the microbial composition of tap water. In particular, next-generation DNA sequencing has provided new insight into the composition of the human microbiome, e.g., the microbes that naturally inhabit our skin, gut, and lungs, and has revealed striking relationships with human health (e.g., obesity, diabetes, asthma, autism, allergies). The question naturally arises with respect to the factors shaping the human microbiome, with role of the “built environment” being of fundamental interest. The built environment; including homes, offices, schools, hospitals, and vehicles, is where most humans in developed countries spend > 90% of their time. Tap water is likely an important feature shaping the microbiome of the built environment, serving as a conduit for microbes into tiny droplets called aerosols, which can be inhaled into the lungs or otherwise inoculate the skin during showering or be transferred onto food during food preparation. Thus, there is interest in mapping out the microbiome of tap water and the factors that shape it, not only because of its potential to harbor OPs, but because of its potential general effect on built environment and human microbiomes. Long-term research could lead towards identifying which microbes serve a beneficial, or “probiotic,” role in preventing pathogen growth and benefiting human health. The purpose behind the body of research described in this dissertation was to apply newly available next-generation DNA sequencing tools towards mapping out the microbial composition characteristic of tap water, with emphasis on implications for preventing proliferation of OPs. Of particular interest was the relative role of what water utilities and building operators can do to protect public health. To this end, the DNA sequencing approach was applied to carefully controlled and replicated field- and laboratory-scale plumbing rigs to gain insight into the relative roles and interactions of the water quality provided by drinking water utilities and practical building-level engineering controls. Specific factors investigated included: stagnation (i.e., the tendency of water to sit unused in pipes in 8 hour cycles), pipe material (e.g., metallic versus plastic), pipe configuration (i.e., up or down flow to induce convective mixing vs stratification, respectively), water heater temperature set point (i.e., balancing hotter temperatures needed to kill pathogens versus lower temperatures desirable to save energy or prevent scalding), and heat-shock treatment (i.e., temporarily elevating the water heater temperature and flushing the system to kill off pathogens). There were several general findings that can be highlighted based on this research. First, based on comparison of standardized plumbing rigs installed at five water utilities in the U.S., the nature of the water provided by the local water utility was the overarching factor shaping the microbiome composition at the tap, moreso than pipe material or stagnation. Second, there exists an ideal threshold water heater temperature setting (51 °C based on the conditions of this study) above which there is a concordant shift in microbiome composition and decrease in L. pneumophila occurrence. Third, consistent water heater temperature setting above this threshold has a stronger long-term influence on the microbiome composition and L. pneumophila control than temporarily elevating the temperature for heat-shock treatment. Finally, biofilm and bulk water microbial compositions are extremely diverse in composition (e.g., thousands of species of microbes in each) and functional markers, and distinct from one anaother in terms of their characteristics under different operational conditions. In sum, this study takes a step towards better understanding building plumbing microbiome and identifies several promising engineering and control factors that can ultimately inform intentional engineering of the building plumbing microbiome, particularly with respect to protecting public health against OPs and potentially other microbiome-related ailments in the future.

Ischemic heart disease (IHD) is a leading cause of global mortality. Emerging evidence suggests a potential role of the gut microbiome in IHD development. The present study explored the association between alpha diversity, representing microbial richness and evenness, and IHD, as well as the relationship between specific gut microbial species and IHD risk. The study uses a longitudinal design with data from the two cohorts within the Swedish Infrastructure for Medical Population-based Life-course and Environmental Research (SIMPLER). Survival analysis is employed to calculate the hazard rate for all associations. Because of thecompositionality of the gut microbiome species-level data a centered log-ratio (CLR) transformation is applied before the main analysis. We find no associations between alpha diversity and risk of IHD in the age and sex-adjusted model as well as in the main model adjusted for age, sex, smoking, education, physical activity, hypertension, high cholesterol, and intakes of fish, red and processed meat, fermented dairy, fruit and vegetables and alcohol. Five species are found to be statistically significantly associated with IHD in the main model before correction for multiple testing, which is statistically non-significant after the correction.

Honeybees (Apis mellifera L.) are the major insect pollinators of many different crops. A drastic decline in the honey bee populations has been reported over the past decade. While many factors have contributed to this decline, pesticides, poor nutrition, and Varroa mites are the most common concerns noted by scientists and beekeepers. Aside from direct toxicity from pesticides, it has been observed that sublethal pesticide doses have effects on honey bee physiology and behavior such as oxidative stress, disruption of foraging and homing, and changes to honey bee neurophysiology. The primary objectives of this project were to examine honey bee gut microbiome, physiology, and pesticide exposure along an agricultural intensification gradient and to examine the interactive impacts of pesticide exposure and poor nutrition on honey bees in a controlled laboratory cage setting. Sixteen honey bee colonies were placed in four locations across Mississippi with varying degrees of natural forage availability.

Research on adolescent drinking shows that younger people are at greater risk of developing behavioral deficiencies that can be detrimental to their social relationships and health over time. Recent research has shown that changes within the microbiome-gut-brain axis (MGB) can affect social behavior. These changes involve microbiota populations that influence addictive behaviors after prolonged substance intake through neurochemical shifts that extend through the nervous, circulatory, and immune system. Using Massey's biosocial model, I aim to expand on the relationship between the MGB axis, social behavior, and adolescent alcohol use disorder through a meta-theoretical approach. I explore the strengths and shortcomings of Massey's biosocial model of segregation and stratification and its use of the allostatic load model, telomere length, and gene exposure to develop a stronger theoretical concept using the micro-gut-brain axis as a conceptual foundation. Can the MGB-axis model be used to identify potential pathways in which alcohol use disorder (AUD) persists from adolescence to adulthood? I find that adolescent drinking leads to changes in microbiota populations that are known to influence AUDs and increases the development of diseases such as liver disease and its effects on social behavior. The MGB axis can help us understand the effects of substance and dietary habits on disease and illness by connecting life science knowledge and sociological perspectives. With this modern application of cognitive sociology, I have shown that future research on addictive behaviors should consider the application of biomarker data to further expand on new theoretical and methodological approaches in the study of disease and addiction.

Emerging clinical evidence implicates cellular senescence in the pathogenesis of various inflammatory conditions including inflammatory bowel diseases (IBDs), demonstrating that the intestinal stem cell crypts of patients with early Crohn’s disease exhibit markers positive for cell cycle inhibitor proteins. This phenomenon coupled with chronic systemic inflammation, a term coined “inflammaging," triggers many age-related pathologies and accelerates mortality. Our research evaluates the efficacy of interventions that target these death-resistant senescent cells to improve overall health and vitality. Particularly, we investigated the effects of Fisetin, a potent flavanoid with senolytic properties, in a dextran sodium sulfate (DSS) induced mouse model of colitis. Our findings reveal that Fisetin significantly inhibits senescence and inflammation in the colon while simultaneously enhancing the relative abundance of beneficial microbes, especially Akkermansia muciniphila, showcasing its potential for managing IBDs. Additionally, given the profound restoration of the microbiome and the central role of resident microbes in the production of metabolites essential for facilitating immunomodulation, we extended our investigations to further explore the effects of fecal microbiota transplant (FMT) from long-living Ames dwarf mice, characterized by low inflammatory status, into normal mice. Our results show notable shifts in microbial diversity, indicating that FMT may combat dysbiosis, a precursor to several conditions, including autoimmune, metabolic, and neurodegenerative diseases. Lastly, our exploration of potential anti-aging pharmacological interventions including Metformin (MF) and Trodusquemine (MSI-1436) during the postnatal window has demonstrated robust transcriptomic alterations of key biomarkers in the GH/Igf1 axis, such as Pi3k, Akt, and Mtor, suggesting delayed aging and improved liver function in young mice. These epigenetic changes underscore that early-life pharmacological interventions may forestall the onset of age-related metabolic disorders. All in all, there remains an urgent need for breakthroughs that can enhance healthspan to ensure that the rapidly growing population of older adults enjoys life in these extended years

Viruses, and particularly bacteriophages, are key players in many microbial ecosystems and can profoundly influence the human microbiome and its impact on human health. While the bacterial and archaeal fraction of the human microbiome can now be profiled at an unprecedented resolution via cultivation-free metagenomics, viral metagenomics is still extremely challenging. The lack of universal viral genetic markers limits the de-novo discovery of viral entities, and the low number of available viral reference genomes from cultivation studies does not cover well the phage diversity in human microbiome samples. Viral-like particle (VLP) purification has been proposed as a set of experimental tools to concentrate viruses in samples prior to sequencing, but it remains unclear how efficient and reproducible such tools are in practice. In this thesis we aim to address some of these challenges and better exploit the potential of viral metagenomics in the context of the human microbiome. First, we performed and studied the performance of VLP procedures on freshwater and sediment samples. We found that bacteria can still be abundant at the end of the filtration process, thus lowering the efficiency of the enrichment. Analyzing samples with a low enrichment may lead to inconsistent conclusions, as the residual bacterial contamination might misdirect the computational analysis. To better quantify the extent of non-viral contamination in VLP sequencing, we designed ViromeQC, a novel open-source tool able to assess and rank viromes by their viral purity directly from the raw reads. In ViromeQC, rRNA genes and bacterial single-copy proteins are used as a proxy to estimate non-viral contamination. With the ViromeQC, we conducted the largest meta-analysis on the degree of enrichment of thousands of viral metagenomes, and concluded that the vast majority of them are three-fold less enriched than a standard metagenome. ViromeQC was then used to select the human gut viromes that had the highest enrichment as a starting point for a novel reference-free pipeline for the discovery of previously uncharacterized viral entities. The approach included metagenomic assembly of the enriched viromes as well as extensive mining of many thousands of assembled metagenomes, and led to a catalog of 162,876 sequences of highly-trusted viral origin. Most of these predicted viral sequences had no match against any known virus in RefSeq even though some of them showed a prevalence in gut metagenomes of up to 70%. Our analyses and publicly available tools and resources are helping to uncover the still hidden virome diversity and improve the support for current and future investigations of the human virome.

Antimicrobial resistance poses a significant challenge to human health and is also a pressing One Health concern. The routine use of antibiotics as growth promoters in agricultural animals has contributed to the emergence of antibiotic resistance, which can subsequently affect human populations. Discontinuing this practice has led to a surge in infections and therapeutic antibiotic use in these animals. This increased susceptibility to infections may be linked, at least partially, to the loss of colonization resistance resulting from alterations in the microbiome. This study focuses on poultry, as the consumption of chicken meat can introduce antibiotic-resistant microbes into the human population. The overarching hypothesis for this research project is that a rationally designed consortium of microbes sourced from healthy chickens will increase colonization resistance and decrease susceptibility to infections as an alternative to growth-promoting antibiotics. The first goal was to analyze the broiler chicken’s gut microbiome and to establish a comprehensive culture collection of microorganisms from healthy chickens. Culture-enriched and culture-independent 16S sequencing was applied to assess the cultivability of the samples and to analyze their microbial profiles. Isolates were identified using MALDI-TOF and 16S rRNA gene sequencing. Frozen samples (from antibiotic-free farms) had a greater microbial diversity than fresh samples (from a university research facility). However, a greater proportion of the microbiome was recovered by culture from the fresh compared to the frozen samples. A strain collection of 1121 isolates representing 121 species was constructed. In Aim 2, I carried out a functional screen to identify isolates from the culture collection that inhibited the growth of the predominant poultry pathogens, E. coli and C. perfringens. Several isolates were identified that inhibited one or the other pathogens and a small number of isolates killed both pathogens. These microbes form the basis of therapeutic consortia to increase colonization resistance in chickens. / Thesis / Master of Science (MSc) / In the poultry industry, antibiotics have been used to promote chicken’s growth. This has contributed to the spread of antibiotic resistance to animal/human pathogens. When the use of growth-promoting antibiotics is stopped, the chickens become more susceptible to infections. These chickens have possibly lost protective bacteria that help fight pathogens. I thought that bacteria from healthy chicken’s intestine could help fight pathogens. To do this, I isolated a large collection of chicken gut’s good bacteria from healthy birds after individually separating them from the mixture using growing methods and sequencing. I separated bacteria from frozen and fresh mixtures, found that more bacteria grow from fresh mixtures. I then tested individual bacteria from this collection to see if they stop pathogenic bacteria like E. coli and C. perfringens from growing. I found that many bacteria could do this which may be used to develop a therapeutic community of good bugs to colonize chickens to make them more disease resistant.

Targeted gene manipulation in a complex microbial community is an enabling technology for precise microbiome editing. This thesis introduces a new microbial therapeutic system dubbed Bacterial CRISPR-Transposase Reduction of Virulence In Situ (BACTRINS). BACTRINS is an in-situ microbiome engineering platform designed for efficient and precise genomic insertion of a desired payload and simultaneous knockout of target genes. When applied against a Shiga toxin-producing pathogen in the gut, this system delivers a CRISPR-associated transposase by bacterial conjugation for site-specific inactivation of the Shiga toxin gene and integration of a nanobody therapeutic payload to disrupt pathogen attachment. A single dose of this therapy resulted in high efficiency Shiga gene inactivation and improved survival in a murine infection model of Shiga-producing pathogen. This work establishes a new type of live bacterial therapeutic capable of reducing gut infections by transforming toxigenic pathogens into commensal protectors.

Alzheimer’s Disease is a devastating disease and a major medical challenge of the 21st century without preventative treatment available and absence of a cure. Due to the profound clinical impact on patients with the disease, and emotional and financial impact on their loved ones, there has been extensive research into therapeutic agents. Unfortunately, many of the studies are abandoned or fail due to a lack of complete understanding of the complex pathophysiology of Alzheimer’s Disease, and ineffective drug targets. While the etiology of disease remains controversial, the accumulation of both amyloid beta protein and neurofibrillary tangles has been the most supported theory for the past thirty years. Also in recent years, the gut microbiome has gained much attention for its unexplored potential and role in health and disease, and the Mediterranean Diet has been deemed the “healthiest” diet for the sixth consecutive year by health experts. This study reviews the current understood pathophysiology of AD and standard diagnostic criteria. This thesis aims to provide an overview of the neuroprotective effects of the Mediterranean Diet, the alterations of the gut microbiome that are associated with the Mediterranean Diet, and the alterations of the gut microbiome associated with Alzheimer’s Disease. Based on the analysis of the large number of studies presented, this thesis concludes that the Mediterranean Diet optimally modifies the ratio of Firmicutes:Bacteroidetes in the gut of people with Alzheimer’s Disease and reduces pathological and symptomatic progression of disease.

Ecosystems comprise large groups of highly interdependent organisms. Cnidarians, such as sea anemones and corals, are keystone species in many marine ecosystems, especially coral reefs. Each individual cnidarian also constitutes an ecosystem unto itself, a "holo- biont", consisting of the host animal and accompanying microbial symbionts. To interro- gate cnidarian holobionts, I used computational approaches to analyze the transcriptomes of three cnidarians and build mechanistic models of their microbial symbionts. In par- ticular, I analyzed and annotated the transcriptomes of the cauliflower coral Pocillopora damicornis, the lined sea anemone Edwardsiella lineata, and the starlet sea anemone Ne- matostella vectensis, providing information about the molecular functions expressed by these organisms, and allowing development of a corresponding set of public databases: PocilloporaBase, EdBase, and an updated version of StellaBase, that facilitate access to the corresponding datasets. Additionally, I developed a method to infer the phylogenetic antiquity of transcripts. This method also allowed me to identify transcripts from other organisms (e.g., microbes) belonging to the anemone or coral holobiont. In parallel – in order better to understand the microbial symbionts that share envi- ronments with cnidarian hosts, I also developed new computer-simulation approaches for modeling metabolic interactions between different microbial species. These approaches are based on genome-scale stoichiometric reconstructions of metabolic networks and on Flux Balance Analysis (FBA). In addition to contributing to the development and testing of a new FBA-based platform for modeling communities in structured environments (Compu- tation Of Microbial Ecosystems in Time and Space, or COMETS), I used this platform for specific in silico experiments on microbial symbiosis. In particular, I computed all pairwise interactions between 582 different prokaryotic models, and identified global patterns of pu- tative positive (cross-feeding) vs. negative (food competition) interactions in this matrix of species pairs. I found that about 7% of the pairs yielded a greater biomass when grown together than when grown separately as monocultures. Despite existing challenges, such as the limitations of gap-filling steps in model construction and the need for a better knowl- edge of nutrient composition in natural environments, this approach could in the future help forecast shifts in the coral holobiont under likely scenarios of marine environmen- tal changes. In general, this work demonstrates how the integration of high-throughput sequencing technology and mechanistic systems-biology simulations, can provide unique tools to analyze interactions between microbes, and to mitigate or reverse adverse changes in marine ecosystems.