UMD Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/3
New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a given thesis/dissertation in DRUM.
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Item FOOD SAFETY IN THE ERA OF NEXT-GENERATION SEQUENCING: GENOMIC CHARACTERIZATION OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI AND METAGENOMIC SURVEILLANCE OF IRRIGATION SURFACE WATER(2023) Huang, Xinyang; Meng, Jianghong; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In this study, we first utilized high-throughput next-generation sequencing (NGS) and bioinformatic analyses to characterize potential public health threats posed by non-top-7 Shiga toxin-producing Escherichia coli (STEC). NGS allowed us to detect virulence (n = 46) and antimicrobial resistance (AMR) (n = 27) factors within the genomes of the STEC strains, to make genome-wide comparisons with published human clinical isolates, and to characterize three novel O-antigen gene clusters. We found that the distribution of 33 virulence genes and 15 AMR determinants exhibited significant differences among serotypes (P < 0.05), and that 47 strains were genetically related to human clinical strains inferred from a pan-genome phylogenetic tree. We secondly developed a web tool, PhyloPlus, that allowed users to generate customized bacterial and archaeal phylogenies, which can be incorporated into their own microbial community studies. We also utilized two public datasets (human microbiome, n = 60; fermented food metagenomes, n = 62) to illustrate how application of phylogeny improved our analyses. We showed that the integration of phylogenies introduced alternative phylogeny-based diversity metrics and allowed more conservative null model constructions, thereby reducing potential inflation of type I errors. Finally, we employed deep metagenomic shotgun sequencing, and our developed web tool, to investigate on a collection of 404 surface water samples collected from four regions in Latin America. We reported the high detection rates of pathogenic and contaminant bacteria in these samples, including Salmonella (29.21%), Listeria (6.19%), and E. coli (35.64%), necessitating the monitoring and proper treatment on these surface waters. We also described the regional differences in terms of sample taxonomic composition and the resistome, and further presented key factors that drove the separation patterns for each sampling region. We utilized recent metagenomic assembly and binning algorithms to report the construction of 1,461 de-replicated metagenome-assembled genomes (MAGs) that were of at least medium quality. The incorporation of the MAGs into the taxonomic classifier Kraken2’s database led to a 12.85% increase in classifiable sequence reads. Additionally, we conducted network analysis on AMR genes and the genus-level taxonomy, based on assembled contigs, to provide information to better understand the dynamics of the transferring of AMR genes.Item A MULTI-OMICS APPROACH TO CHARACTERIZING THREE HEALTH RELEVANT FUNCTIONS OF THE HUMAN GUT MICROBIOME(2022) Braccia, Domenick James; Hall, Brantley; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The human gut is home to trillions of microorganisms that routinely interact with their human host in both beneficial and detrimental ways. The advent of next-generation sequencing and high-throughput “omics” technologies has created new opportunities to examine the role that the human gut microbiome plays on human health, especially in regard to gastrointestinal diseases such as Inflammatory Bowel Disease and colorectal cancer. In my dissertation, I utilize genomic, transcriptomic, metabolomic, and protein sequence datasets to characterize three health-relevant functions of the human gut microbiome. First, I performed a multi-omic, bioinformatic analysis to identify the bacterial enzyme, bilirubin reductase. While bilirubin reduction to urobilinogen and stercobilinogen is a well-known function of the human gut microbiome, the enzyme(s) responsible for the conversion of bilirubin to non-toxic reduced products have yet to be fully characterized. In this chapter, I review how I leveraged publicly available metabolomic, metagenomic, and metatranscriptomic data to explore over 2 million putative reductase genes and identify a candidate operon encoding bilirubin reductase. Second, I examined sources of microbial hydrogen sulfide (H2S) production by bacteria of the human gut microbiome. H2S is a sulfuric gas produced by various bacterial phyla of the human gut microbiome and is implicated in the etiology of gastrointestinal diseases such as Inflammatory Bowel Disease and colorectal cancer. In this chapter, I show via bioinformatic analysis that the capacity to produce H2S via cysteine degradation is ubiquitous in the human gut. Third, I explored bacterial prodrug activation required for the activation of immune system modulators such as sulfasalazine. After curating amino acid sequences of known azoreducing genes and performing a protein sequence search across the Unified Human Gastrointestinal Genomes (UHGG) collection containing 4,644 genomes, I identified putative azoreducing and non-azoreducing bacterial strains to be experimentally validated. Together, these results highlight a successful mult-omic approach to characterizing three diverse but health-relevant functions of the human gut microbiome.Item METHOD VALIDATION AND DEVELOPMENT FOR THE METAGENOMIC EXPLORATION OF MICROBIAL COMMUNITIES(2022) Commichaux, Seth; Pop, Mihai; Rand, Hugh; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Our world is inhabited and shaped by diverse and complex microbial communities which we are only beginning to characterize and understand. With the advent of affordable high-throughput sequencing, the study of the genomic content of microbial communities, metagenomics, has accelerated our understanding of their impact on human and environmental health. The increasing number of datasets produced by metagenomic studies provide many opportunities for novel bioinformatic analyses and for the development of computational methods. However, careful benchmarking and validation are also important undertakings to ensure the integrity of methods and research in such a rapidly developing field. Here, we explored several problems in metagenomics by benchmarking existing methods and technologies, developing new methods, recommending best practices, and highlighting opportunities for future work. First, microbial gene catalogs document and organize the genes found in microbial communities and provide a reference for the standardized analysis of metagenomic data. Although commonly used to explore the intersection between microbiomes, humans, and ecosystems, the methods used for their construction and effectiveness for metagenomic analyses had not been critically evaluated. Our analysis highlighted important limitations of gene catalogs, opportunities for future research, and allowed us to recommend best practices. Second, we assessed if nanopore long read sequencing could expedite the accurate reconstruction of a pathogen genome from a microbial community. The investigation of foodborne illness outbreaks routinely uses short-read whole genome sequencing of pure culture pathogen colonies. However, culturing is a bottleneck and short reads cannot span all bacterial genomic repeats, often leading to fragmented assemblies. Our results showed that the integration of long-read sequencing could expedite the public health response by reconstructing complete pathogen genomes from a microbial community after limited culturing. Additionally, our evaluation of state-of-the-art assembly tools identified biases and areas for improvement. Third, we describe taxaTarget, a supervised learning approach for the taxonomic classification of microeukaryotes in metagenomic data. Metagenomics has been underutilized for microeukaryotes due to the many computational challenges they present. Existing tools often implement universal sequence similarity cutoffs which ignore that sequences can evolve at different rates and, thus, have different discriminatory power. We show that a data-driven approach to determining classification thresholds can result in higher sensitivity and precision than existing tools. Fourth, we explored the use of horizontally transferred plasmids to relate an outbreak strain to the microbiome of a suspected environmental source. The investigation of the 2020 red onion outbreak recovered the outbreak strain from patients but not the farms implicated as the likely source of contamination. Our analysis identified highly similar plasmids in the outbreak strain and environmental isolates collected from the farms, which supported a connection between the outbreak strain and the implicated farms. Additionally, we highlighted the need for more detailed and accurate metadata, more extensive environmental sampling, and a better understanding of plasmid molecular evolution before such analyses can be added to the public health response.Item Assessing 16S rRNA Marker-Gene Survey Measurement Process Using Mixtures of Environmental Samples(2018) Olson, Nathan D; Corrada Bravo, Héctor; Computer Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Microbial communities play a fundamental role in environmental and human health. Targeted sequencing of the 16S rRNA gene, 16S rRNA marker-gene surveys, is used to measure and thus characterize these communities. The 16S rRNA marker- gene survey measurement process includes a number of molecular laboratory and computational steps. A rigorous measurement assessment framework can evaluate measurement method performance, in turn improving the validity of marker-gene survey study conclusions. In this dissertation, I present a novel framework and mixture dataset for assessing 16S rRNA marker-gene survey bioinformatic methods. Additionally, I developed software to facilitate working with 16S rRNA reference sequence databases and 16S rRNA marker-gene survey feature data. Computational steps, collectively referred to as bioinformatic pipelines, combine multiple algorithms to convert raw sequence data into a count table, which is subsequently used to test biological hypotheses. Algorithm choice and parameters can significantly impact pipeline results. The assessment framework and software developed for this dissertation improve upon existing assessment methods and can be used to evaluate new computational methods and optimize existing pipelines. Furthermore, the assessment framework presented here can be applied to other microbial community measurement methods such as shotgun metagenomics.Item REDUCED CAMPYLOBACTER INFECTION AND ENHANCED PERFORMANCE IN POULTRY WITH BIOACTIVE PHENOLICS THROUGH EPIGENETIC MODULATION OF THE GUT MICROBIOME(2017) Salaheen, Serajus; Biswas, Debabrata; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Campylobacter jejuni, a major enteric pathogen and a natural resident in the poultry gut, causes gastrointestinal illness followed by severe post-infection complications, including Guillain-Barré syndrome, reactive arthritis, myocarditis, and ulcerative colitis in humans. Risk assessment studies have projected a 30-fold reduction in human campylobacteriosis cases with only a 100-fold reduction in the number of C. jejuni colonizing the poultry gut. Current commercial poultry production practices involve use of antibiotic growth promoters (AGP); modulation of gut microbiota with AGPs for food safety and enhanced performance in poultry can be justified until acquisition of antibiotic resistance in zoonoses through inter-bacterial transfer of antibiotic resistance genes (ARGs) in a complex microbial community is considered. As an alternative, natural phenolics extracted from by-products of berry juice industry, with antimicrobial, anti-inflammatory, anticarcinogenic, antioxidant and vasodilatory activities, demonstrate promising prospects. In this study, we adopted mass-spectrometry, microbiological, phylogenetic, and metagenomic approaches to evaluate bioactive phenolic extracts (BPE) from blueberry (Vaccinium corymbosum) and blackberry (Rubus fruticosus) pomaces as AGP alternative. We detected that major phenolics in BPE included, but were not limited to, apigenin, catechol, chlorogenic acid, cinnamic acid, coumarin, ellagic acid, eugenols, flavan, gallic acid, gingerol, glucosides, glucuronides, myricetin, phenols, quercetin, quinones, rhamnosides, stilbenol, tannins, triamcinolone, and xanthine. BPE reduced C. jejuni growth and motility in vitro, resulting in lower adherence and invasiveness to chicken fibroblast cells. Anti-inflammatory effects of BPE significantly reduced the expression of pro-inflammatory cytokine genes in chick macrophage cell line ex vivo. Furthermore, BPE reduced the colonization of C. jejuni in broiler cecum by 1 to 5 logs while increasing broiler weight by 6% compared to 9.5% with commercial AGPs. Metagenomic analysis of broiler gut indicated that BPE caused an AGP-like pattern in bacterial communities with a comparative increase of Firmicutes and a concomitant reduction of Bacteroidetes in broiler ceca. AGP supplementation clearly caused phage induction and a richer resistome profile in the cecal microbiome compared to BPE. Functional characterization of cecal microbiomes revealed a significant variation in the abundance of genes involved in energy and carbohydrate metabolism. Our findings established a baseline upon which mechanisms of plant based antimicrobial performance-enhancers in regulation of animal growth can be investigated.Item The skin microbiome of woodland salamanders and its association with hosts' taxonomy, environment and health status(2016) Muletz Wolz, Carly Rae; Lips, Karen R.; Behavior, Ecology, Evolution and Systematics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Microbial communities play central roles in animal health. Host species, environmental conditions and presence of pathogens can affect the diversity and composition of animal-associated microbiomes. Amphibians form integral and functionally important symbioses with microbes. The amphibian microbiome interacts with pathogens, and can protect hosts from disease, including the disease chytridiomycosis, caused by skin infection by Batrachochytrium dendrobatidis (Bd). The implications of amphibian-microbiome associations are difficult to predict because little is known about the factors shaping bacterial communities or their functional traits, such as anti-Bd properties. I used culture-dependent and culture-independent methods to characterize the skin microbiome of Plethodon salamanders in field and laboratory studies. I hypothesized that the evolutionary history, environmental conditions, and health status of the hosts shape skin bacterial community assemblages. In a field study, I sampled sympatric, congeneric salamander species (Plethodon cinereus, P. glutinosus, P. cylindraceus) across three localities to quantify the distribution of both anti-Bd bacteria and the entire bacterial community. I identified 50 anti-Bd bacterial OTUs and 480 bacterial OTUs overall on the salamander skin, with high prevalence and abundance of anti-Bd bacterial genera Pseudomonas, Acinetobacter and Stenotrophomonas. Within a locality, co-occurring salamanders generally had similar microbiome diversity and composition patterns, but these differed among sites. This indicates that environment is more influential in shaping skin microbiome patterns than differences in host properties in these species. I sampled P. cinereus along an elevational gradient, as a proxy for environmental variables that co-vary with elevation. Microbiome diversity and composition changed with elevation, in which compositional changes were related to soil pH. In a laboratory experiment, I quantified the responses of P. cinereus and the skin microbiome to temperature (13, 17, 21 °C) and pathogen (Bd+, Bd-) exposure to determine whether the native microbiome affected survival at natural temperatures. Temperature changed the microbiome, but this did not prevent host mortality from Bd. Instead, Bd exposure changed the microbiome and caused 78% mortality. My results demonstrate that environmental conditions and pathogen presence are important factors determining skin microbiome structure in Plethodon salamanders. These findings contribute to our understanding of animal-microbial symbioses, microbial community ecology, and amphibian disease ecology.Item MICROBIAL INTERACTIONS AND ECOLOGY WITHIN BLOOMS OF THE TOXIC DINOFLAGELLATE KARENIA brevis ON THE WEST FLORIDA SHELF.(2013) Meyer, Kevin Anthony; O'Neil, Judith M; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The dinoflagellate Karenia brevis is capable of significant ecological and economic impacts in Florida waters where blooms typically occur. Blooms and cultures of K. brevis were sampled to determine the composition, production, and possible ecological function of bacteria and virus communities associated with K. brevis. Bacterial communities on the West Florida Shelf (WFS) were similar inside and outside K. brevis blooms, but primary and secondary (bacterial) production and bacteria and virus abundances were different depending on bloom stage. Bloom stages need to be identified so that discrete sampling events can be combined to characterize an entire bloom event. Within an initiating bloom bacterial production and mortality was high and viral abundance was low, suggesting that viral genomes were either within host cells or bacterial mortality was due to mixotrophic grazing by K. brevis or heterotrophic nanoflagellates. In a maintenance phase bloom the bacterial community was metabolically stressed, subject to increased viral infection, and most likely not being subjected to mixotrophic grazing. Bacterial communities associated with healthy K. brevis were dominated by the Cytophaga-Flavobacterium-Bacteroides (CFB) complex. As K. brevis shifted to stationary or senescing growth communities had higher proportions of Alphaproteobacteria. The SAR406 group, typically found in deep waters, was present in the surface waters of the WFS which supports existing K. brevis bloom formation hypotheses involving upwelling of deep waters from the mid to outer shelf. The CFB complex of bacteria also need to be further investigated as the consistent presence of CFB bacteria in both blooms and cultures of K. brevis suggest CFB bacteria are capable of numerous interactions with K. brevis. Furthermore, such interactions may be a vector of bloom control through viral infection; a high proportion of CFB bacteria would be ideal for density-dependent viral infection which could disrupt interactions between bacteria and K. brevis. Inoculating cultures of K. brevis, which included associated bacteria, with viral concentrates from the WFS showed differences in bacterial production and growth which indicate viruses are acting upon the bacterial community and not the dinoflagellates. Interactions between bacteria and K. brevis need to be further elucidated and explored for a better understanding of the role of each in dynamics of this harmful algal species. There may be a natural community succession amongst bacteria during blooms: utilizing certain indicator species to indicate bloom stage and transition between stages may aid in bloom forecasting and detection efforts.