Browsing by Subject "Metagenomics"

Now showing 1 - 7 of 7
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    Development And Application Of Targeted Dna Sequencing Tools To Profile Microbiome-Wide Antimicrobial Resistance And Pathogens Of Public Health Importance
    (2024-02) Slizovskiy, Ilya
    Antimicrobial resistance (AMR) poses critical health challenges as drivers of frequent and severe global outbreaks. In the U.S. alone, AMR accounts for one infection every eleven seconds, and one death every fifteen minutes. Bacterial antimicrobial resistance genes (ARGs) are the underpinning determinants of AMR, and traditionally prevention and surveillance efforts have focused on cultivating and studying resistant pathogens harboring DNA-encoded ARGs, isolated from human, animal, food, and environmental sources. However, across most compartments of the biosphere, bacteria reside as community members of complex microbial ecosystems with diverse ecological interactions and defined niche profiles. The perspective of the community-level composition and processes that lead to AMR rise and dissemination is rarely accounted for. Though culture-independent methods like PCR have been used for decades, recent advances in the field of metagenomics offers the possibility of directly sequencing the entire genetic milieu across the total microbiota within a sample (i.e. the ‘metagenome’). This technique offers an ecosystem-wide glimpse into bacterial community members, their genes, and their functional potential. However, metagenomic sequencing is rarely adopted in public health surveillance and tracking of pathogens, as well as risk assessment of AMR. The resulting sequencing data offers a low resolution and fragmented view of AMR hazard potential within microbial communities which is not conducive to motivating quality evidence-based decision-making for clinicians, public health practitioners, food producers, and policy makers. This dissertation consists of four integrated studies that attempt to: (1) Formalize the major impediments precluding informative metagenomic sequencing and data analysis in the study of AMR and its hazard potential; (2) Demonstrate an improved metagenomic approach to elucidate epidemiological trends in a major public health context of AMR; (3) Innovate and implement a novel metagenomic sequencing platform and associated bioinformatic tools to address impediments to metagenomic sequencing and enhance risk characterization of AMR; and (4) Extend technical metagenomic innovations for deployment in public health surveillance and monitoring activities. All metagenomic methods involved the use of unique human, animal, environmental, and food safety-related samples, and all studies were conducted using systems in vivo, in vitro, and / or in silico.
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    From functional metagenomics to unique synthetic expression strategies in iron-reducing bacteria.
    (2012-05) Gonzalez, Tanhia Denys
    Cellulose and hemicellulose are renewable sources of fermentable sugars. The use of fermentable sugars for the production of alternative energy sources (i.e. ethanol, butanol, etc.) is an attractive solution to alleviate the shortage and high prices of petroleum. Cellulases and hemicellulases are the two groups of glycosyl hydrolases responsible for breaking down the polysaccharide component of biomass into their respective sugar moieties. The enzymatic hydrolysis of cellulose and hemicellulose has relied on enzymes originally produced by culturable organisms. This thesis describes the use of metagenomics coupled to high-throughput screening techniques to identify glycosyl hydrolases originally encoded by uncultured organisms. The findings of this thesis include the identification and biochemical characterization of a unique endoglucanase. Besides catalyzing the hydrolysis of soluble and insoluble cellulosic substrates, this endoglucanase exhibited a domain architecture that has not been previously reported in the literature. This thesis also describes two different strategies to engineer the surface of (Fe+3)-reducing bacteria. These expression systems are a valuable tool for studying the cellular respiration of Geobacter and Shewanella. Furthermore, they have practical applications in the area of whole-cell biocatalysis in microbial fuel cells. The first strategy involved using an autodisplay system to engineer the cell envelope of Geobacter and Shewanella. The autodisplay system translocated a functional β-galactosidase enzyme to the cell envelope of G. sulfurreducens and S. oneidensis. Furthermore, this system proved to be an effective tool for catalyzing reactions in electrochemical cells using biofilms of G. sulfurreducens cells. The second strategy exploited the use of in-frame fusions with the c-type cytochrome OmcZ to translocate a recombinant protein to the outer membrane and extracellular matrix of Geobacter sulfurreducens. This is the first time that the c-type cytochrome OmcZ has been used to engineer biofilms of Geobacter sulfurreducens.
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    Investigating Bi-directional Impacts of the Microbiome and Drinking Water Quality in Drinking Water Distribution System Water Mains and Storage Facilities
    (2022-07) Gomez, Christa Kimloi
    The microbial communities that live in the biofilms of drinking water distribution system (DWDS) environments can exert significant impacts on drinking water quality before it reaches the consumer. The relatively recent advent and accessibility of powerful culture-independent techniques, such as high-throughput sequencing, have enabled characterization of diverse microbial communities; however, the difficulties of accessing DWDS infrastructure has hindered many efforts to study the health-relevant DWDS microbiome. In this work, high-throughput sequencing and quantitative real-time polymerase chain reaction (PCR) techniques were leveraged to characterize the biofilm communities of simulated and full-scale water mains, as well as the in situ suspended and biofilm communities of elevated water storage towers and underground reservoirs in a chloraminated DWDS. Seasonal variability and drivers of community composition were assessed in the simulated DWDS biofilms and in full-scale drinking water storage facilities. Among other examined drivers of community, the presence and concentration of disinfectant was an important selective pressure that impacted community composition. Communities in the simulated and full-scale DWDS biofilms were generally dominated by bacteria that live preferentially in, and form biofilms, exhibit increased resistance to disinfectant concentrations, or display versatility in substrate-utilization. These included genera that contain opportunistic pathogens, such as Mycobacterium, Pseudomonas, and Stenotrophomonas, genera implicated in microbiologically-caused corrosion of infrastructure (sulfate-reducing Desulfovibrio), - as well as ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) that catalyze nitrification processes and can cause reoccurring, problematic decreases in chloramine residual concentrations. Species-level taxonomic resolution of DWDS Mycobacterium, achieved by sequencing a mycobacterial heat shock protein gene, indicated that the bulk of these bacteria were not disease-associated strains. Early stages of community succession occurred rapidly for biofilms on new surfaces that were in proximity to more established biofilms – within a month, the biofilms on new surfaces exhibited similar compositions to neighboring, older biofilms. Apart from early changes in composition indicative of an initialization stage, biofilm communities in water storage facilities were temporally stable, although somewhat spatially heterogeneous. In contrast, suspended communities showed seasonal changes and were heavily influenced by water chemistry. Additionally, suspended communities were spatially homogeneous within a facility, and even at different facilities within the DWDS. In storage facilities that experienced problematic nitrification episodes and decreases in chloramine concentrations, suspended AOB concentrations increased as chloramine concentrations decreased. Notably, decreases in disinfectant were not accompanied by increases in the growth of other bacteria. Rather, as AOB concentrations increased, the total biomass of suspended communities actually decreased. During nitrification events, biofilm and suspended community compositions were most similar, lending further support to the concept that biofilms may act as reservoirs for nuisance and pathogenic bacteria in the DWDS. Abundant taxa were consistent with other studies of DWDSs that maintain chloramine, which provided support for the applicability of these findings to other systems, especially as there are no studies to compare to, to-date, of the microbiome in elevated storage towers.
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    Metagenomic Survey Of Denitrifying Woodchip Bioreactors: Carbon And Nitrogen Cycling Under Varying Temperature And Flow Regimes
    (2020-02) Pauleon, Aaron
    The denitrifying woodchip bioreactor (WCBR) is a promising edge of field technology used as a biofilter of nitrate pollutants. These reactors have diminishing performance under low temperature and high flow conditions. In this study the taxonomic, nitrogen metabolism, and glycoside hydrolase profiles of meso-scale biochar-amended denitrifying woodchip bioreactors (WCBRs) are assessed and compared through shotgun metagenomic sequencing with reads aligned to protein coding sequences. Four treatment conditions: 14.5oC+12Hr hydraulic residence time (HRT), 14.5oC+4Hr, 6oC+12Hr, and 6oC+4Hr were analyzed in triplicate for the effects of temperature and flow rate (HRT) on the removal of nitrate from synthetic agricultural runoff water. The experimental design offered greater flow and temperature controls than field scale reactors while offering greater size and realism than most lab-scale reactors. Temperature and flow conditions had significant impacts in every category of analysis. The warm (14.5oC) and slow (12Hr HRT) WCBRs removed the greatest percentage of nitrate (75% of 30mg/L influent), were the most microbially abundant, and the most diverse. These reactors also had greatest average metagenomic potential for plant matter degrading enzymes. The taxonomic and functional analyses indicate bacterial dominance among extracted DNA, although ascomycete fungi were present across all treatments (0.7%-5.2%). By estimates, most bacteria across WCBRs were atypical denitrifiers (>50%) while a minority were typical denitrifiers (<12%). Large portions (78%) of the core nitrogen metabolism were attributed to the creation or assimilation of ammonia with nitrogen fixation appearing unexpectedly enriched (26%). Comparisons to bacteria-dominant midwestern corn soil reveal relatively high fungi representation, low archaea representation, and lower microbial diversity in the WCBR samples. The overall metagenomic commitments to nitrogen cycling and glycoside hydrolases were higher in the WCBRs befitting a concentrated nitrate and polysaccharide environment. The findings of this study highlight the otherwise unreported taxonomic and metabolic patterns of WCBRs, revealing topics for future study and potential avenues for further engineering to inform and enhance the use of denitrifying woodchip bioreactors moving forward.
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    Pearl in the mud: Genome assembly and binning of a cold seep Thiomargarita nelsonii cell and associated epibionts from an environmental metagenome
    (2014-01) Fliss, Palmer Scott
    As the study of microbes and their impact on the environment grows, so too does the desire to understand the genetic basis of the physiologies that make possible interactions between microbial cells and their environment. Since it is now much more cost-effective to sequence bacterial genomes, environmental metagenomic assembly is a very attractive option for obtaining the genetic blueprints of bacterial physiologies. Bacteria of the genus Thiomargarita (Greek; theio-: sulfur; margarites: pearl), pose a particularly interesting quandary. The genus includes the world's largest bacteria, but as uncultured organisms, their physiologies and basis for their gigantism are not well understood. In order to investigate the genetic basis for these modes, a single cell MDA amplification approach was used on T. nelsonii cells collected at the Hydrate Ridge methane seep off of the coast of Oregon. These particular cells were derived from a gastropod-attached epibiont community. Next-generation sequencing produced a metagenomic product representing both T. nelsonii and attached bacteria (epibionts). These reads were assembled into contigs, binned using the tetranucleotide frequency of the resultant contigs, and finalized using a more stringent secondary assembly. The resulting draft genome shows evidence in Thiomargarita nelsonii for a complete denitrification pathway not previously known in large, vacuolated, sulfur-oxidizing bacteria. Additionally, the genes necessary for polyphosphate metabolism were observed. Polyphosphate metabolism is thought to play a role in the formation of phosphatic minerals that serve as important reservoirs in the marine phosphorous cycle.
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    Use of metagenomics and metatranscriptomics to determine the influence of organic sulfur compounds on the sulfur cycle in the sediment of a low-sulfate freshwater system
    (2020-08) Hyde, Emily
    The sulfur cycle is an important and complex biogeochemical cycle involving both inorganic and organic species in both oxic and anoxic environments. However, due to the lack of research regarding the sulfur cycle in freshwater systems, the contributions of organic sulfur compounds to the sulfur cycle are underappreciated. Recent studies have suggested organic sulfur compounds likely fuels sulfate reduction, especially in low-sulfate oligotrophic freshwater systems, through a possible cryptic sulfur cycle. To determine the contributions that organic sulfur compounds may have in this environment, we used Lake Superior sediment to analyze for the presence of and expression of sulfur cycling genes. In these metagenomes, we found genes for sulfur reduction, oxidation and organic sulfur compound degradation. Metabolic pathway analysis showed presence of not only organic sulfur compounds contributing to the sulfur cycle, but tetrathionate, thiosulfate, and polysulfides playing a role as well. Using Lake Superior sediments, we also conducted sediment incubations to measure the biotransformation capability of sulfur-containing amino acids, sulfonates, and an analog for a common sulfolipid. Taurine and sodium dodecyl sulfate produced higher sulfate values in incubations, suggesting that microbes prefer sulfonates over sulfur-containing amino acids, in addition to a possible partiality towards oxidized organic sulfur compounds over reduced forms regarding sulfate production. The preference of sulfonates is supported by the commonality of taurine genes present as well as the low, but present transcription values of sulfoacetaldehyde degradation. While sulfur-containing amino acids do not produce sulfate values near that of sulfonates or sulfolipids, there are still present and transcriptionally active genes that can contribute to sulfate reduction in the system. Regarding methyl-sulfurs, metatranscriptomic data shows that methyl-mercaptan (intermediate within dimethyl sulfide and methionine degradation) degradation is transcriptionally active across genomes. By combining biotransformation incubation data, metagenomics, and metatranscriptomics, we analyzed how methylated sulfurs, sulfur-containing amino acids and sulfonates can fuel a sulfur cycle in a low-sulfate environment, informing us on how pathways may have operated in our Earth’s geologic past.
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    Wild Primate Gut Microbiota Protect Against Obesity
    (2017-04) Sidiropoulos, Dimitrios, N; Clayton, Jonathan; Al-Ghalith, Gabe; Shields-Cutler, Robin; Ward, Tonya; Blekhman, Ran; Kashyap, Purna; Knights, Dan
    The gastrointestinal tract hosts trillions of bacteria that play major roles in metabolism, immune system development, and pathogen resistance. Although there is increasing evidence that low dietary fiber in Westernized societies is associated with dramatic loss of natural human gut microbiome diversity, the role of this loss in obesity and inflammation is not well understood. Non-human primates (NHPs) can be used as model systems for studying the effects of diet and lifestyle disruption on the human gut microbiome. Captive primates are typically exposed to low-fiber diets and tend to have human-associated microbiota in place of their native microbiota. In order to explore interactions between the gut microbiota and dietary fiber, we transplanted captive and wild primate gut microbiota into germ-free mice and then exposed them to either a high- or low-fiber diet. We found that the group receiving low-fiber diet and captive primate microbiota became obese and had high levels of circulating inflammatory cytokines, while mice receiving high-fiber diet and wild primate microbiota remained healthy. Mice with the wild primate microbiota and low-fiber diet acquired intermediate levels of obesity, demonstrating an interaction between dietary fiber and the microbiota. These results show that the modern human gut microbiome interacts with low-fiber diets to cause inflammation and obesity, and suggest a possible clinical role for manipulation of the microbiota in the treatment of obesity.