Browsing by Author "Metcalf, Jessica, committee member"
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Item Open Access Characterization of the resistome and microbiome of retail meats processed from carcasses of conventionally and naturally raised cattle(Colorado State University. Libraries, 2019) Thomas, Kevin, author; Belk, Keith, advisor; Morley, Paul, committee member; Metcalf, Jessica, committee memberConcern over human exposure to antimicrobial resistance (AMR) via consumption of meat products has raised questions about use of antimicrobial drugs in food-animal production. This concern has led to an increase in consumer demand for meat products from naturally-raised cattle, or those raised without use of antimicrobials. While previous studies have assessed AMR gene presence in cattle and throughout the beef supply chain, very little work has surveyed the resistome on retail meats available for consumer purchase. The objective of this study was to determine the extent of antimicrobial resistance and characterize the microbiome in retail ground beef products from naturally-raised (raised without antibiotics) and conventionally-raised cattle utilizing 16S rRNA and targeted shotgun metagenomic, high-throughput sequencing techniques. Differing in packaging types and lean points, samples of ground beef derived from carcasses of cattle that were conventionally-raised (n = 50) or naturally-raised cattle (n = 50) were purchased from retail outlets in six major metropolitan cities throughout the United States. Samples were shipped to Colorado State University and processed following 48 hours of refrigeration at 4°C. Thirty-gram portions of each sample were removed and subjected to DNA extraction procedures via DNeasy PowerFecal Microbial Kit. Cell lysates were composited by production system and city before being subjected to paired-end 16S rRNA gene sequencing and targeted shotgun metagenomic sequencing using an enrichment system developed in our laboratory. Microbiome analysis was performed from 16S data with QIIME2 v.2018.4 by utilizing many of the available plugins. Resistome analysis of enriched metagenomic data was performed using a modified AMRPlusPlus pipeline. Microbiome alpha diversity analysis indicated that ground beef processed from conventionally-raised animals had a greater (P < 0.05) species richness than natural ground beef products. Microbiome composition differed (P < 0.05) between samples of differing production systems based on abundance weighted UniFrac distances. Additionally, when analyzed using unweighted UniFrac distances, microbial composition differed (P < 0.05) between samples from different cities. Differences in product packaging availability between cities may have caused these differences detected in microbiome composition, as well as environmental contamination or product handling in distribution. Targeted shotgun sequencing yielded a total of 4.6 trillion reads across all 60 composite samples, with only 58 samples containing hits to AMR. Of these 58 samples, 10.1 million reads were assigned to: 520 groups, 101 mechanisms of resistance, and 22 classes of antibiotics. The three most abundant classes of resistance detected included tetracyclines (56% of assigned reads), multi-drug resistance (21% of reads), and beta-lactams (7% of reads). An analysis of similarity on samples ordinated using Euclidian distances suggested that the overall resistome differed (P < 0.05) by production system, likely driven by greater antimicrobial resistance group variation among conventional retail samples. Results from this study profiled resistance and characterized microbial composition of retail beef products from two major production practices. While the results do not discredit concern over imprudent use of antibiotics in beef production, differing management techniques in cattle production do not appear to have a direct impact on the resistome or microbiome of final retail products available to consumers.Item Open Access Demystifying viruses: understanding the role of river viruses on microbial community structure and biogeochemical cycling through a multi-omic lens(Colorado State University. Libraries, 2023) RodrÃguez-Ramos, Josué, author; Wrighton, Kelly, advisor; Hall, Edward, committee member; Metcalf, Jessica, committee member; Wilkins, Michael J., committee memberViruses are the most abundant entity on the planet, with estimates of up to 1031 viral particles dispersed across the globe in every ecosystem that can sustain life. Today, as the world responds to the COVID-19 pandemic, the word "virus" often evokes a negative response because of their impacts on human health and disease. Yet, most viruses that exist in the world can only infect bacteria and archaea. In fact, it has long been estimated that for every 1 bacterial or archaeal cell, there are 10 viruses that can infect it. While bacteria and archaea are long regarded as essential to overall ecosystem health and functionality, the roles of viruses in natural systems are much less understood and appreciated. Due to a scarcity of genome-resolved multi-omic studies, this lack of understanding is compounded in river ecosystems, which play critical roles modulating global carbon and nitrogen biogeochemistry. The overarching aims of this dissertation are to harness genome-resolved, multi-omic datasets to 1) decipher the impact that viruses can have on river microbial communities and biogeochemical cycling, and 2) to explain how viral ecology can enhance our understanding of river ecosystem function. To define the role that viral and microbial communities have on river function, I first set out to understand what is currently known of river viral ecology. In Chapter 1, I provided a background primer on viruses and their impacts on natural ecosystems. I then zoomed in on viral roles exclusively within rivers and described the current state of river viral ecology. I also highlighted some of the knowledge gaps addressed specifically by my thesis. My literature review revealed that while there are publicly available metagenomic datasets, there is a drastic underutilization of genome-resolved strategies which are critical for constraining microbial metabolism and viral impacts into informative units. Further, these datasets are largely unused because the data is collected in an un-coordinated manner, leading to the lack of similar sampling methods, and ultimately an inability to make results interoperable. Together, in this chapter I present compelling evidence for the need of genome-resolved, virus-host paired multi-omic analyses that are pivotal to our understanding of river ecosystems and lay the groundwork for the questions I will address throughout my dissertation. After identifying that there was a gap studies that leverage metagenome assembled genomes (MAGs) and viral metagenome assembled genomes (vMAGs), for Chapter 2 I focused on using a genome-resolved lens to uncover the microbial and viral metabolic underpinnings responsible for the biogeochemical cycling of carbon and nitrogen in the Columbia River system. This chapter used a dataset that was spatially resolved at the centimeter scale for three sediment cores across two transects of the Columbia River and included 33 samples, all of which had metagenomes that were paired to metaproteomes, biogeochemistry, and metabolites. Using this dataset, I created the first river microbial and viral database genome-resolved database called Hyporheic Uncultured MAG and vMAG (HUM-V). Leveraging metaproteomics paired to HUM-V database, I built a conceptual model outlining microbial and viral contributions to carbon and nitrogen biogeochemistry in these river sediments. With this metabolic reconstruction, I showed an intertwined carbon and nitrogen cycle that can likely contribute to the fluxes of nitrous oxide. Specifically, I demonstrated that well recognized river microbes like those of the phyla Nitrososphaeraceae as well as other less recognized phyla like Binatia encode and express genes for denitrification. I also showed that the clade II nosZ gene, which is responsible for nitrous oxide production, could possibly act as a nitrous oxide sink without contributing to its production. Linking viral members to microbial hosts demonstrated that viruses may be key modulators of carbon and nitrogen cycling. Specifically, I presented evidence that viruses can infect key nitrifying organisms (i.e., Nitrospiraceae) as well as key polymer degrading organisms (i.e., Actinobacteria). Highlighting their potential roles, linear regression analyses consistently identified viral organisms as key predictors of ecosystem biogeochemistry. Chapter 2 of my thesis yielded insights that uncovered some of the microbial contributions that were thought to occur but were poorly defined in river sediments (e.g., nitrogen mineralization), and presented a genome-resolved, virus-host paired strategy that I could then use to directly assess how viruses impacted host metabolism and ecosystem function. Ultimately, Chapter 2 highlights the power of genome-resolved database strategies to reduce existing predictive uncertainties in river corridor models. Having provided a genome-resolved view of metabolic processes in Chapter 2, for Chapter 3 I set out to expand upon our understanding of river viruses by providing insights into their temporal and spatial dynamics. For this, I worked with a finely tuned temporal dataset from an urban stream near Berlin, Germany called the Erpe River. The Erpe River dataset is a metagenomic timeseries where samples were collected every 3 hours for a total of 48 hours across both the surface water (SW) and pore water (PW) compartments. In addition to metagenomes, Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) and biogeochemistry were collected for each sample. Using this dataset, I created a database consisting of 1,230 vMAGs and 125 MAGs. Only 1% of our vMAGs clustered to known taxonomic representatives, highlighting the underrepresentation of river viruses in public databases. Due to this underrepresentation, I supplemented my viral taxonomic analyses with over 20,000 vMAGs spanning different publicly available studies that were relevant to rivers and wastewater treatment plants and showed that nearly half of the novel genera identified were cosmopolitan in aquatic ecosystems. I also characterized the spatial and temporal dynamics of the river microbiomes across the surface water (SW) and pore water (PW) compartments. Both the viral and microbial communities were distinct between the SW and PW samples and were both driven by the same chemical drivers. Given that these compartments had distinct communities, I set out to understand how they were changing over time. By employing multiple temporal statistical methods, I show that SW communities are more persistent and more stable relative to the PW communities, likely resulting from the homogeneous selection pressures of the SW, and the heterogeneity within the sediment. In addition to resolving these temporal dynamics, I highlight some specific virus and host genomes that influence biogeochemical cycling. In summary, my third chapter shows how river viral and microbial communities change across spatial and temporal gradients, and highlights how genome-resolved metagenomics enhances our interpretation of microbiome data. The final chapter of this dissertation (Chapter 4) summarizes the key findings of my thesis and provides future perspectives to inspire research in environmental river viral ecology. This section also showcases several publications that I have worked on throughout my doctoral degree that span multiple ecosystems like mouse guts, human guts, soils, and the development of the computational tool Distilled and Refined Annotation of Metabolism (DRAM). This final chapter also highlights a manuscript that I was involved in that showcases a new scientific framework: Interoperable, Open, Coordinated, and Networked (ICON). I further highlight this framework to address how these ICON strategies are beginning to be implemented in other fields and propose that in order to move the discipline of river microbial ecology forward, we need to implement ICON frameworks and the standardization and coordination of sampling collection. In summary, the aims of this dissertation were to summarize what is known in the field of river viral ecology (Chapter 1), to investigate viral roles that viruses play on river organic nitrogen and carbon processing (Chapter 2), to interrogate the temporal and spatial dynamics of viruses within rivers (Chapter 3), and to summarize how this dissertation has added to the understanding of river viral ecology, and what the next big questions for the field should be (Chapter 4). Ultimately, these works shine a spotlight on the viruses found in river ecosystems and shows that they likely play key roles in the regulation of microbial biogeochemical cycles.Item Open Access Effects of potential Tylosin substitutes on Salmonella prevalence and the microbiome of subiliac lymph nodes of beef feedlot cattle(Colorado State University. Libraries, 2017) Holzer, Katlyn, author; Martin, Jennifer, advisor; Belk, Keith, committee member; Metcalf, Jessica, committee member; Schountz, Tony, committee memberTylosin, a macrolide antibiotic, is fed to feedlot cattle for liver abscess prevention. Tylosin alternatives are currently being investigated as pressures to reduce the amount of antibiotics used in livestock increase. Understanding effects of various feeding strategies on the safety of beef products is a priority as alternatives are investigated. This study investigated the effect of Tylosin, and two Tylosin alternatives on the prevalence of Salmonella in subiliac lymph nodes (SLN) and the microbiome of SLN from feedlot cattle. Salmonella harborage in the lymph node is a challenge for the beef industry as ground beef is made from beef trimmings that commonly contain lymph nodes. Consumption of contaminated ground beef is one of the possible foodborne routes of Salmonella infection. SLN (n=600) were collected from feedlot cattle (n = 5,481) at the time of slaughter. Overall 84.6% of the SLN were positive for Salmonella and the treatment did not influence prevalence (P > 0.8402). Samples from each pen of feedlot cattle (15 SLN/pen) were composited for microbiome analysis using 16s rRNA amplicon sequencing. Samples were analyzed using the open-source software Quantitative Insights Into Microbial Ecology (QIIME). The treatment did not influence the microbiome of the SLN (P = 0.223; P= 0.267). The top three phyla present were Proteobacteria (67.3%), Actinobacteria (10.2%), and Acidobacteria (9.5%). Although Salmonella was culturally isolated, it was not identified in the microbiome analysis because the genus could not be resolved for 18.9% of the Enterobacteriaceae family. Understanding why Salmonella is detectable in the lymph nodes may hold the key for prevention, and characterizing the microbiome is crucial for this process.Item Open Access Equine fecal microbiota changes associated with anthelmintic administration(Colorado State University. Libraries, 2018) Kunz, Isabelle, author; Coleman, Stephen, advisor; Hess, Tanja, committee member; Hassel, Diana, committee member; Metcalf, Jessica, committee memberThe equine gastrointestinal tract contains a complex ecosystem comprised of microorganisms and intestinal helminth parasites. Increasing evidence suggests that horses rely on their gastrointestinal microbial populations for many aspects of metabolic and immune function. Furthermore, interactions between the microbes, helminths, and their host may impact these functions, which are vital to maintaining gastrointestinal homeostasis. The use of anthelmintic drugs is a common practice of intestinal helminth parasite control in domestic horse health management. The use of anthelmintic drugs is very effective for controlling the burden of parasitic infection and associated clinical signs, however anthelmintic administration has also been associated with gastrointestinal disturbances in the horse. The potential effects of anthelmintic drug administration on the equine gastrointestinal microbiota and its' role in homeostasis is not yet understood. The objective of research presented in this thesis is to investigate the impact of anthelmintic administration on the equine gastrointestinal microbiota. This goal was addressed in two main studies, which used 16S rRNA amplicon sequencing to identify and describe the microbial populations present in equine fecal samples. The first study described is a pilot project which aimed to characterize the impact of anthelmintic treatment on the fecal microbiota of horses without an observable helminth infection. The results of this study indicated that subtle differences in the microbial community composition and structure are detectable between samples collected before and after anthelmintic treatment. A second study was then conducted as a follow-up to the pilot which included horses with varying detectable levels of helminth infection as determined by fecal egg counts. Results of the second study further demonstrated microbiota changes following anthelmintic treatment, while also suggesting that the most notable effects of anthelmintic treatment may be observed in fecal samples between 48 and 72 hours post-treatment. The results of these studies suggest that anthelmintic treatment may be associated with changes in the equine fecal microbiota.Item Open Access Evaluating Bouteloua gracilis cultivars' performance after drought; The role of the soil microbiome(Colorado State University. Libraries, 2024) Donne, Carina, author; Smith, Melinda, advisor; Havrilla, Caroline, committee member; Trivedi, Pankaj, committee member; Metcalf, Jessica, committee memberDrought has affected the Great Plains throughout history, most notably during the Dust Bowl of the 1930's. While most drought events are not as severe as the Dust Bowl, they still cause significant agricultural losses every year. As research has begun to uncover the mechanisms and responses of drought, there are still unanswered questions. For instance, the mechanisms of ecosystem recovery after drought ends remain relatively unexplored. It is possible that intervention methods such as reseeding will need to be done to help restore ecosystem structure and function after drought. After the Dust Bowl, it was a common practice to reseed native grasses, such as Blue Grama (Bouteloua gracilis), in sites severely impacted by the drought. Given forecasts of droughts on par or even more severe than the Dust Bowl, reseeding may need to be employed more frequently in the future to enhance post-drought recovery. However, with reseeding efforts, it is imperative to understand the adaptability of cultivars to the environmental conditions in which they are planted. One aspect of environmental conditions that has rarely been examined the soil microbiome. Here, I used a common garden experiment that included two cultivars of B. gracilis that were planted with soil microbial inocula extracted from either previously droughted or non-droughted soils. These soils were collected from a recently ended four-year drought experiment in the shortgrass steppe of northeastern Colorado, which caused the widespread loss of B. gracilis. The goal of the greenhouse experiment I conducted was to examine whether the post-drought legacy of altered soil microbial communities affected the growth and performance of two common cultivars of B. gracilis. I assessed plant performance by measuring weekly height to estimate relative growth rate and at the end of the experiment, I measured plant above- and belowground biomass. I found no significant differences in relative growth rate or plant biomass, and minimal differences in the bacterial community composition between the two cultivars. These results suggest that the post-drought legacy of altered soil bacterial communities did not differentially affect growth and performance of the two common B. gracilis cultivars evaluated in this study, and that the growth of these cultivars did not differ in their effects on the soil bacterial communities found under ambient vs. previously droughted conditions. Overall, both cultivars may be suitable for reseeding in the shortgrass steppe grassland after extreme drought, yet further studies are needed to examine a broader range of B. gracilis cultivars and whether soil bacterial communities previously exposed to extreme drought would allow for improved growth and performance of different cultivars to future drought conditions.Item Open Access Gene expression analysis before and after the pelvic flexure in the equine hindgut(Colorado State University. Libraries, 2024) Moss, Cameron D., author; Coleman, Stephen J., advisor; Engle, Terry, committee member; Metcalf, Jessica, committee member; Landolt, Gabriele, committee memberThe equine hindgut is the primary site of the horse's nutrient breakdown, absorption, and energy production. More than 60% of the horse's energy comes from hindgut fermentation. In this process, commensal microbes in the hindgut aid in the digestion of plant materials to create volatile fatty acids that can be used by host cells to make energy. Many severe health issues- such as colic, laminitis, or colonic impactions- often occur in the equine hindgut, making it an important site to study to provide better management, treatment, and prevention options for horses suffering from gastrointestinal disease. Although much research exists focusing on the microbiome and overall physiology of the equine hindgut, relatively little addresses the role of gene expression in maintaining a complex yet essential homeostatic balance within the gastrointestinal tract. Previous from our lab found major differences in the microbial content of gastrointestinal compartments of the equine hindgut, separated by the pelvic flexure. The pelvic flexure is a short, narrow, horseshoe-shaped loop in the equine large colon. It defines the ventral and dorsal segments of the colon and is a common site of colonic impaction in horses. Although the pelvic flexure cannot and should not act as a "barrier," something "barrier-like" may be occurring around this region as it pertains to the hindgut microbiome. The mechanism for this action is not defined. As a result, this thesis aims to investigate gene expression in the intestinal epithelial cells of the ventral colon, pelvic flexure, and dorsal colon regions of a healthy hindgut to determine what differences exist. The insight gained from this analysis will provide a baseline for comparison to understand how gene expression patterns in these tissues adapt to changes in the microbiome and external factors like diet. The results of this thesis are the first steps towards a better understanding of homeostasis in the equine hindgut.Item Open Access Machine learning methods to discover patterns in microbially driven soil carbon sequestration(Colorado State University. Libraries, 2020) Thompson, Jaron, author; Munsky, Brian, advisor; Metcalf, Jessica, committee member; Chan, Joshua, committee memberUnderstanding how microbiomes function is a major area of research, as microorganisms play a significant role in environments spanning nearly every corner of the earth. Recent advances in DNA sequencing technology have made it possible to profile microbial communities, yet noise and sparsity in microbiome data makes it difficult to identify consistent patterns in microbial community behavior. In this thesis, we apply a host of machine learning methods to elucidate the role of the soil microbiome in mediating soil carbon sequestration. We demonstrate that broad characteristics of the soil microbiome such as richness and biomass can be used to forecast abundance of dissolved organic carbon (DOC) in soil. We also show that feature selection analysis using a host of machine learning and standard statistical techniques identifies a consensus set of significant taxa that predict DOC abundance. Finally, we demonstrate how these feature selection techniques can be used to explore more advanced probabilistic models that assign accurate estimates of prediction confidence. The methods proposed in this thesis could be used to design optimized microbial communities that combat climate change by promoting increased levels of carbon storage in soil.Item Open Access The effect of tylosin exposure or exclusion on liver abscess prevalence, fecal pathogen populations, and the microflora of finished beef products from feedlot cattle(Colorado State University. Libraries, 2018) Weissend, Carla Jane, author; Martin, Jennifer, advisor; Belk, Keith, committee member; Bryant, Tony, committee member; Metcalf, Jessica, committee member; Weir, Tiffany, committee memberThere is great pressure to reduce use of, and find alternatives to, antibiotics in animal production. More than 70% of feedlot cattle in 1000+ head lots are currently exposed to tylosin phosphate, a macrolide antibiotic used for the reduction and prevention of liver abscesses. As such, its potential removal from cattle feeding strategies could have a marked impact on both the economics of the beef industry and food safety. Additionally, little is known about the effect of tylosin exposure or exclusion on the microbiome of finished beef products. In light of these facts, a blinded, randomized, controlled field trial was conducted to evaluate the impact on the prevalence of liver abscesses and the characterization of the microbiome of feces, liver abscesses, carcasses, and finished beef products through 16S rRNA gene sequencing and shotgun sequencing from feedlot cattle with and without exposure to tylosin. Overall, liver abscess rates were lower among cattle exposed to tylosin. However, there were no differences among treatment groups for any of the sample types, suggesting that removal of tylosin from current feeding strategies will not upend the safety of the beef supply. The information gained in this study will provide valuable insight as the search for alternative feeding strategies to antibiotics continues.