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- Title
- CARBON-MEDIATED ECOLOGICAL AND PHYSIOLOGICAL CONTROLS ON NITROGEN CYCLING ACROSS AGRICULTURAL LANDSCAPES
- Creator
- Curtright, Andrew James
- Date
- 2022
- Collection
- Electronic Theses & Dissertations
- Description
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The sustainable intensification of agriculture relies on the efficient use of ecosystem services, particularly those provided by the microbial community. Managing for these ecosystem services can improve plant yields and reduce off-site impacts. For instance, increasing plant diversity is linked to positive effects on yield, and these beneficial effects are often mediated by the microbial community and the nutrient transformations it carries out. My dissertation has aimed to elucidate the...
Show moreThe sustainable intensification of agriculture relies on the efficient use of ecosystem services, particularly those provided by the microbial community. Managing for these ecosystem services can improve plant yields and reduce off-site impacts. For instance, increasing plant diversity is linked to positive effects on yield, and these beneficial effects are often mediated by the microbial community and the nutrient transformations it carries out. My dissertation has aimed to elucidate the mechanisms by which plant diversity improves agricultural production. In particular, I have focused on how changes to the amount and diversity of carbon (C) inputs affects soil microorganisms involved in the nitrogen (N) cycle. My work spans multiple scales of observation: from a global meta-analysis to mechanistic studies utilizing denitrification as a model system.In a global meta-analysis, I found that increasing plant diversity through intercropping yields a net increase in extracellular enzyme activity. This effect varied by plant species and soil type suggesting that increases in the quality of nutrient inputs mediates these positive effects on microbial activity. Then, I looked at how intercropping cover crops into corn affects soil nutrient pools and microbial activities in a field experiment. No effect of interseeding cover crops into corn was found on soil nutrient pools or microbial activities. However, by analyzing differences in relationships between nutrient pools and microbial activities at two locations throughout Michigan, I was able to describe how the availability of dissolved organic C (DOC) drives differences in microbial N-cycling processes. I then investigated how C availability drives activity in microbial hotspots within the soil by comparing differences in denitrification potential in bulk soil versus the rhizospheres of corn and interseeded cover crops. Here, I found that denitrification rates were increased in the rhizospheres of all plant types, and this effect varied depending on the species of plant. I was able to further differentiate the impact of DOC and microbial biomass C on the rhizosphere effect and found that C availability was the primary driver of differences in denitrification rates between rhizospheres. Since plants provide many different forms of C to soil microbes, it is important to understand how the chemistry of C inputs affects microbial activity. I used a series of C-substrate additions to determine how C chemistry affects denitrifiers. I found that amino acids and organic acids tended to stimulate the most nitrous oxide (N2O) production and reduction. Although management and site affected overall rates of denitrification, C-utilization patterns of microbes were mostly similar between locations. To identify the mechanisms responsible for these effects, I performed a final experiment to track how denitrifiers utilized different C compounds. The C substrates that stimulated the most complete reduce of N2O also were utilized with the lowest C-use efficiency (CUE). This suggests possible trade-offs between N2O reduction and CUE, with important implications for how to manage microbial communities. Overall, my work demonstrates that land management can impact microbial community activity by influencing the identity of soil C inputs. While the importance of increasing soil C inputs has been known, this dissertation supports the notion that the chemical identity of C inputs can exert significant controls on microbial activity. Moreover, by comparing microbial traits I highlight the importance of trade-offs in how microbially mediated C- and N cycling are coupled.
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- Title
- CHEMICAL BIOLOGY AND GENETIC STUDIES TARGETING THE MYCOBACTERIAL CELL ENVELOPE
- Creator
- Williams, John Tison
- Date
- 2021
- Collection
- Electronic Theses & Dissertations
- Description
-
Mycobacterium tuberculosis is one of the leading causes of death due to a single infectious pathogen. The evolution and spread of drug resistant strains requires new antibiotics to control the TB pandemic. Over the last decade, the lipid flippase MmpL3 has been identified as a potential drug candidate based on its essential nature for cell viability and repeated identification as the lead target of small molecule inhibitors of Mtb growth. Using a combined untargeted and targeted whole cell...
Show moreMycobacterium tuberculosis is one of the leading causes of death due to a single infectious pathogen. The evolution and spread of drug resistant strains requires new antibiotics to control the TB pandemic. Over the last decade, the lipid flippase MmpL3 has been identified as a potential drug candidate based on its essential nature for cell viability and repeated identification as the lead target of small molecule inhibitors of Mtb growth. Using a combined untargeted and targeted whole cell phenotypic screen I identified novel inhibitors of this valued target. A combination of lipid profiling and an innovative competitive binding assay supported MmpL3 as the target of these inhibitors. Cross resistance profiling of MmpL3 inhibitors against twenty-four unique mmpL3 Mtb mutants demonstrated that the level of resistance is associated with the proximity of resistant mutants to essential residues for protein function. Further, these resistance profiles suggested that MmpL3 inhibitors fall into separate clades depending on their chemical scaffolds. The results of this screen led to the development of novel potent analogs for one of the identified MmpL3 inhibitors, HC2099. These analogs were active against clinically relevant drug resistant Mtb strains that cause treatment failure in patients. Active analogs were able to kill Mtb inside of infected macrophages, an infectious niche of Mtb, without inducing cytotoxicity against these important immune cells. One of these analogs, MSU-43085, was orally bioavailable and successfully inhibited Mtb growth in infected mice, supporting further development and highlighting the therapeutic potential of this series. High throughput screens are often used to identify new inhibitors of Mtb growth. However, prioritized hits form these screens often identify similar targets such as MmpL3, lipid synthesis enzymes, redox cyclers, as well as inhibitors of the electron transport chain. Follow up studies of these inhibitors are often time consuming, costly and result in the rediscovery of previously identified targets. While this is not necessarily detrimental to Mtb drug discovery, as these reoccurring targets have therapeutic potential. The continued prioritization of inhibitors for these common metabolic pathways potentially limits the identification of inhibitors for novel targets. Therefore, additional steps that identify inhibitors of these common pathways could reshape how high throughput screen hits are prioritized. By applying the targeted mutant screen used to identify MmpL3 inhibitors to a non-prioritized library of hits from a high throughput screen, we identified more than fifty new potential MmpL3 inhibitors. Using an iterative strategy of applying additional mutants of commonly identified targets, this strategy promises to lead to parallel follow-up studies of inhibitors with known and unknown mechanisms of action. The ability of Mtb to enter into quiescent states in response to host stresses is one of the leading causes for the extended time to cure and evolution of drug resistance. These states can be induced by several environmental stresses including acidic pH, hypoxia, and others. In an effort to study this adaptation in the rapidly growing mycobacterial species M. smegmatis, we identified a lethal sodium citrate phenotype. Transcriptional profiling and genetic screening of mutants tolerant to sodium citrate indicated that this phenotype was due to the combined action of both chelation and osmotic stresses. Cell viability could be reduced from sodium citrate killing by cation and osmoprotectant supplementation. From these experiments we propose a model that can be applied to study carbon source uptake and probe the role of genes identified from the forward genetic screen with unknown function
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- Title
- COMPUTATIONAL APPROACHES TO COMPLEX BIOLOGICAL PHENOMENA
- Creator
- Franklin, Joshua Luke
- Date
- 2021
- Collection
- Electronic Theses & Dissertations
- Description
-
Biological systems can be difficult to understand due to a vast array of interacting phenom-ena. The result is that some seemingly "easy" questions go unanswered. For example, we have long known that bacteria utilize many distinct flagellar configurations, but in most cases it remains unclear why they do so. We know that cell differentiation is critical to many biological processes, yet we still do not fully understand how such spatiotemporal patterning occurs. Despite mutation being one of...
Show moreBiological systems can be difficult to understand due to a vast array of interacting phenom-ena. The result is that some seemingly "easy" questions go unanswered. For example, we have long known that bacteria utilize many distinct flagellar configurations, but in most cases it remains unclear why they do so. We know that cell differentiation is critical to many biological processes, yet we still do not fully understand how such spatiotemporal patterning occurs. Despite mutation being one of the driving forces of evolution, we still have a hazy understanding of how organisms respond and adapt to high mutation rates. However, ad- vances in technology, modeling, and experimental techniques have enabled us to investigate the small and nuanced effects that can answer these questions. In this dissertation, I have used modern computational tools and statistical techniques to investigate evolutionary and behavioral processes. Agent-based models of evolution and flagellar inheritance have allowed me to investigate the evolution of mutational robustness and trade-offs associated with flagellar motility, respectively. By writing Bayesian mixed- effect models, I have been able to precisely quantify the metabolic cost of producing flagella and describe spatiotemporal patterns of cell differentiation within fruiting bodies of Myxo- coccus xanthus. Careful quantitative modeling of biological phenomena can help cut through the complexity of these systems. As computational power continues to grow and software continues to become more sophisticated, these computational approaches will both become more powerful and easier to use. Computational approaches will not replace experiments in most cases; instead, computational models can direct experiments, which can themselves direct new modeling efforts, in an iterative loop of progressing knowledge.
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- Title
- Characterization of Broadly Conserved AvcID Toxin-Antitoxin System and Its Mechanism to Inhibit Phage by Disrupting Nucleotide Metabolism
- Creator
- Hsueh, Brian Yifei
- Date
- 2022
- Collection
- Electronic Theses & Dissertations
- Description
-
ABSTRACTCHARACTERIZATION OF BROADLY CONSERVED AVCID TOXIN-ANTITOXIN SYSTEM AND ITS MECHANISM TO INHIBIT PHAGE BY DISRUPTING NUCLEOTIDE METABOLISMByBrian Yifei HsuehThe prevalence of antiphage defense systems, which have recently been shown to be located on mobile genetic elements in bacteria, have sparked interest to understand the coevolutionary arms race of bacteria and bacteriophage (phage). Bacteria and phages have coexisted for billions of years, and phages are widely distributed in...
Show moreABSTRACTCHARACTERIZATION OF BROADLY CONSERVED AVCID TOXIN-ANTITOXIN SYSTEM AND ITS MECHANISM TO INHIBIT PHAGE BY DISRUPTING NUCLEOTIDE METABOLISMByBrian Yifei HsuehThe prevalence of antiphage defense systems, which have recently been shown to be located on mobile genetic elements in bacteria, have sparked interest to understand the coevolutionary arms race of bacteria and bacteriophage (phage). Bacteria and phages have coexisted for billions of years, and phages are widely distributed in different environmental niches populated by their bacterial hosts, including the human intestine and marine environment. The evolutionary pressure imposed by phages have led bacteria to evolve diverse strategic systems to protect themselves from phage predation, including CRISPR-Cas, restriction-modification, and abortive infection. Recent studies have begun to reveal that toxin-antitoxin (TA) system are associated with antiphage defense systems. Vibrio cholerae El Tor, the causative agent of current cholera pandemics, has acquired two unique genomic islands of unknown origins, known as Vibrio Seventh Pandemic Islands 1 & 2 (VSP-1 & 2). It is hypothesized that the acquisition of VSP islands increase environmental fitness of El Tor. While both islands encode approximately 36 open reading frames, yet many remain largely uncharacterized. In this work, I characterize a novel TA antiphage system encoded on VSP-1 of V. cholerae, here named AvcID. Chapter 2 describes the biological function of AvcD toxin by which it possesses deoxycytidylate deaminase (DCD) activity and produces dUMP as the final product. Further experiments identify the AvcI antitoxin as a small RNA and determine that it post-translationally inhibits the activity of AvcD. Moreover, AvcD consists of two domains—a N-terminal P-loop NTPase and a C-terminal DCD—and mutations in conserved features of each domain abrogate its activity. AvcD is widely conserved across kingdoms, and virtually all bacteria that encode AvcD also have AvcI homologs. Notably, chromosomal AvcID can solely be activated by transcriptional shutoff in V. cholerae, demonstrating that AvcID is a type III TA system. Unlike canonical type III TA systems, in which the toxin is an endoribonuclease, the AvcD toxin is a deaminase. Importantly, the AvcID system provides antiphage defense in Escherichia coli that lacks this system by corrupting nucleotides for phages to utilize to reduce coliphage replication efficiency. In Chapter 3, I explore the activation mechanism of the AvcID system as well as the consequences to phages after encountering AvcID. During infection, virtually all lytic phages induce transcription shutoff of the host by hijacking host transcription machinery to make virion progeny. I uncover that phage-induced transcriptional shutoff leads to turnover of labile AvcI antitoxin and concomitantly activates the deaminase activity of AvcD, leading to a disruption of nucleotide levels. This disruption of nucleotide levels is shown in both susceptible phages (ex. T5) and resistant phages (ex. T7). Through an unknown mechanism, AvcID also increases the abundance of defective phages that are susceptible to AvcID. In summary, this work has made contributions in the field of TA systems and its association with the antiphage defense paradigm by uncovering the biological function and mechanism in response to phage infection.
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- Title
- Characterization of map3773c, ferric uptake regulator protein, in iron metabolism of mycobacterium avium subsp. paratuberculosis
- Creator
- Miyagaki Shoyama, Fernanda
- Date
- 2020
- Collection
- Electronic Theses & Dissertations
- Description
-
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD) in ruminants, a chronic inflammation of the intestines characterized by persistent diarrhea that leads to malnutrition and muscular wasting (Rathnaiah et al., 2017). Unfortunately to date, reliable JD diagnostics are lacking. Culture of MAP from feces has been the most reliable method for diagnosis of JD, however MAP requires eight to sixteen weeks to produce colonies in culture, presenting a...
Show moreMycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD) in ruminants, a chronic inflammation of the intestines characterized by persistent diarrhea that leads to malnutrition and muscular wasting (Rathnaiah et al., 2017). Unfortunately to date, reliable JD diagnostics are lacking. Culture of MAP from feces has been the most reliable method for diagnosis of JD, however MAP requires eight to sixteen weeks to produce colonies in culture, presenting a major hurdle to diagnosis (Bannantine et al., 2002). Currently, no current vaccine can protect animals against JD or prevent shedding of MAP (Garcia and Shalloo, 2015). These challenges establish a need for a better understanding of the MAP physiology during infection. Unlike other mycobacteria, MAP has a unique requirement for supplementation of an iron-binding siderophore (mycobactin J) for optimal growth in laboratory media. A whole genome sequence of MAP K10 revealed a truncation of the mbtA gene that was speculated to have led to its mycobactin dependency (Li et al., 2005). Zhu et al (2008) showed that MAP is able to transcribe all mycobactin synthesis genes in an intra-macrophage environment. Furthermore, several genes responsible for iron acquisition in infected tissues, including genes responsible for mycobactin biosynthesis have been shown to be upregulated in naturally infected tissues (Janagama et al., 2010). Its known that iron plays an important role in vital biological processes; however high intracellular concentration of free iron can lead to toxicity to the bacteria. As such, bacteria activate expression of specific group of genes that are controlled by a metal-sensing regulatory transcription factor. In 2009, Janagama and others identified and characterized the iron dependent regulator (IdeR) in MAP. In addition, in a MAP-specific genomic island, MAP carries three putative ferric uptake regulator (Fur) boxes, an iron regulated transcriptional control motif, (Stratmann et al., 2004). To date, nothing is known about the role of Fur in MAP. To elucidate the mechanisms of iron homeostasis in MAP, we investigated LSP15 using a transposon mutant MAP3776c. We demonstrated a phenotype for LSP15 genes in a culture model of cell entry and survival and suggest a function in epithelial cell pathogenesis, however further functional analysis with complementation by a MAP3776c::Tn strain would be necessary to confirm these findings. Additionally, full characterization of a Fur-like protein (MAP3773c) was performed. Using PRODORIC for computational analysis, 23 different pathways that were likely regulated by MAP3773c were identified. These findings were confirmed using a chromatin immunoprecipitation assay followed by high-throughput sequencing (ChIP-seq), that revealed 58 regions where Fur binds under iron replete and deplete conditions. From those, three were directed related to iron regulation MAP3638c (hemophore-like protein), MAP3736c (Fur box) and MAP3776c (ABC transporter). Using the Fur box consensus sequence, we confirmed binding specificity and Mn2+ availability by a chemiluminescent electrophoresis mobility shift assay (EMSA). A transcriptional profile of the parent MAP K10, deletion mutant of MAP3773c and the complemented strains was developed under iron replete and depleted conditions. However, under the current experimental conditions, we are unable to conclude if the lack of transcriptional responses in our study was indicative of a lack of FUR activity.
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- Title
- Characterization of the development of the tonsillar microbiome in pigs
- Creator
- Peña Cortes, Luis Carlos
- Date
- 2017
- Collection
- Electronic Theses & Dissertations
- Description
-
"Pig tonsils are identified as a potential reservoir for many bacterial and viral pathogens that can survive asymptomatically in this location and may have a high potential of being zoonotic. It has been suggested that the microbiome plays a significant/ substantial role in host colonization by pathogenic microorganisms and also exerts regulatory roles in the resistance to infection. Despite the important role that the tonsillar microbiome could play in the colonization and persistence of...
Show more"Pig tonsils are identified as a potential reservoir for many bacterial and viral pathogens that can survive asymptomatically in this location and may have a high potential of being zoonotic. It has been suggested that the microbiome plays a significant/ substantial role in host colonization by pathogenic microorganisms and also exerts regulatory roles in the resistance to infection. Despite the important role that the tonsillar microbiome could play in the colonization and persistence of pathogens in the host, there are no in-depth studies characterizing the development of tonsillar microbiome in pigs or how this microbiome is structured over time. Surprisingly a similar study is also absent in humans. There was a clear need to investigate the development of the tonsillar microbiome in pigs to lay the basis for future studies focused on more complex subjects such as the relationship between the normal tonsillar microbiome and pathogens in the tonsils. Understanding the development of the pig tonsillar microbiome over time and the role of the tonsillar microbiome in the acquisition and persistence of a pathogenic microorganism will lay the basis for the design of novel intervention strategies to control the presence of the pathogen and reduce the risk of transmission to other animals or humans. Moreover, these studies are expected to provide an animal model to test hypotheses generated by microbiome data that cannot be tested in humans. The goal of this study was to characterize tonsillar microbiome development in pigs, and how this microbiome is structured and how the structure changes through different times in the life of pigs. The chapters in this thesis will present pertinent data related with the composition of the pig tonsillar microbiome and how it alters through the life of pigs, possible maternal sources for some of the identified members of this microbiome, as well as the microbiome structure and progressive change through their life. Furthermore, the results will show that challenges associated with management procedures typically present in swine farms generate prominent changes in the microbiome composition and abundance of diverse bacterial families. Finally, the study will show the microscopic structure of the tonsillar epithelium and crypts and the presence of diverse bacterial communities on the surface of pig tonsils throughout different time points of their life. The final chapter will also describe the morphological changes of the tonsillar surface in pigs that are seen and are associated with changes in the microbial communities observed through different time points in their life. Taken together, the results presented here demonstrate that there is a temporal succession in the development of the pig tonsillar microbiome through the life of pigs."--Pages ii-iii.
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- Title
- Characterizing single-cell behavior of flagellated pathogenic bacteria in mucus and visco-elastic environments
- Creator
- Nguyen, Nhu Thi Quynh
- Date
- 2020
- Collection
- Electronic Theses & Dissertations
- Description
-
Flagellar motility is required for some enteric pathogens to colonize the intestine. Understanding how these pathogens overcome the mucus layer protecting the epithelial tissue is necessary for disease prevention. In this thesis project I examined bacterial motility in mucus to understand factors that facilitate mucus penetration and contrasted this with the motility in different visco-elastic materials.In this thesis, I used single cell tracking to characterize the flagellar motility of...
Show moreFlagellar motility is required for some enteric pathogens to colonize the intestine. Understanding how these pathogens overcome the mucus layer protecting the epithelial tissue is necessary for disease prevention. In this thesis project I examined bacterial motility in mucus to understand factors that facilitate mucus penetration and contrasted this with the motility in different visco-elastic materials.In this thesis, I used single cell tracking to characterize the flagellar motility of Vibrio cholerae and Salmonella enterica in different visco-elastic environments. First, I tested if V. cholerae and S. enterica were still motile in unprocessed pig intestinal mucus. Second, I studied factors that supported their motility in mucus. The first factor I investigated was the effect of pH on V. cholerae motility. I also studied the role of curvature in V. cholerae motility within mucus and some other visco-elastic environments, including liquid and agar. Last, I began investigation of the role of flagellar number in S. enterica motility. In these studies, I focused on analyzing the effective diffusion of bacteria and factors promoting the diffusion in each environment. My findings demonstrate that V. cholerae and S. enterica are able to swim in mucus, and that the torque, and the curvature of V. cholerae, and the flagellar number of S. enterica, play a significant role in bacteria motility in mucus and liquid. Moreover, my findings help elucidate the significance of motility in pathogenesis.
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- Title
- Combination of physical and chemical interventions for reduction of loosely and tightly associated bacteria on broiler carcass skin
- Creator
- Singh, Pranjal
- Date
- 2016
- Collection
- Electronic Theses & Dissertations
- Description
-
The study was conducted to develop processing interventions to reduce pathogen load on broiler carcasses and to understand patterns of bacterial association to broiler skin with the following purposes: 1) assess the effect of hot water spray (HWS, 71oC, 1 min) on broiler carcasses for reduction of loosely, intermediately, and tightly associated bacteria, 2) quantify loosely and tightly associated bacteria on broiler skin using different sampling protocols, 3) assess the effect of trisodium...
Show moreThe study was conducted to develop processing interventions to reduce pathogen load on broiler carcasses and to understand patterns of bacterial association to broiler skin with the following purposes: 1) assess the effect of hot water spray (HWS, 71oC, 1 min) on broiler carcasses for reduction of loosely, intermediately, and tightly associated bacteria, 2) quantify loosely and tightly associated bacteria on broiler skin using different sampling protocols, 3) assess the effect of trisodium phosphate dip, hot water dip, and their combinations with/without brushing on reduction of loosely- and tightly-associated bacteria on broiler carcasses and related structural changes in the skin, and 4) assess the characteristics of Salmonella association and penetration to broiler skin with and without stomaching. In study 1, broiler skin was quantitatively assessed for loosely, intermediately, and tightly associated bacteria by rinsing, stomaching, and grinding the same skin. Hot water spraying (71oC, 1 min) of broiler carcasses followed by water immersion chilling reduced mesophilic aerobic bacteria (MAB) populations by 2.07, 1.84, and 2.04 log CFU/g and Salmonella prevalence by 65, 65, and 73% at loose, intermediate and tight association levels, respectively. Hot water spraying and chilling reduced loosely associated Campylobacter prevalence by 45% but no reduction was observed at intermediate and tight association levels. Broiler carcasses suffered from an undesirable partially cooked appearance after the hot water spray treatment. In study 2, the left and right side skin (10 X 7 cm) of broiler carcasses were sampled by 10 swabings and 10 stomachings, and finally ground. Sixty five and 83 % of MAB populations on broiler skin could not be recovered after 10 consecutive stomachings and swabbings, respectively, but were recovered after grinding. The first stomaching or swabbing recovered most of the loosely associated bacteria but not tightly associated bacteria, which were recovered by grinding. In general, stomaching was more effective than swabbing for bacteria recovery from broiler skin. In study 3, microbial reductions and structural changes in broiler skin were evaluated after trisodium phosphate dip (TWD/TSP), hot water dip (TWD/HWD), and their combination (TSP/HWD) with (TSP/HWD/B)/without brushing. Microbial analysis, scanning electron microscopy and histological staining showed that TSP/HWD caused greater reduction in MAB, E. coli, total coliforms, and Salmonella with deeper penetration into the stratum compactum layer of the skin dermis than single application of TSP or hot water dip. TSP/HWD/B yielded lowest skin population of MAB whereas penetration into the dermis was similar to TSP/HWD but with sloughing off of part of stratum compactum in some areas. Both TSP/HWD and TSP/HWD/B changed skin color, with an increase in yellowness being the most prominent. In study 4, loose and tight associated GFP tagged Salmonella were quantified and imaged after refrigerated incubation of inoculated broiler skins for up to 48 h. GFP tagged Salmonella were not able to associate tightly with broiler skin during exposure for up to 48 h at 4oC as application of two stomachings (1 min each) removed almost 88% of the Salmonella cells from the inoculated skin. Confocal imaging of broiler skins, either dipped (2 min) or stomached (2 min) in inoculated buffer, revealed that Salmonellae were present on the surface and inside crevices at average depths of 10 and 68 μm, respectively, after dipping, with some cells pushed to average depths of 62 and 132 μm, respectively, after stomaching. Trapped water seemed to hold Salmonellae deep inside the crevices.
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- Title
- Crop microbiomes and the search for effective biocontrol of Fusarium graminearum on wheat
- Creator
- MacCready, Kristi Gdanetz
- Date
- 2018
- Collection
- Electronic Theses & Dissertations
- Description
-
"Manipulation of naturally occurring microbial communities to reduce plant diseases or increase crop yields requires a thorough understanding of interactions within the phytobiome, in particular, how microbial communities change as plants age, across plant species and organs, and under different land management regimes. Plants were sampled from a wheat-maize-soybean crop rotation site that implements four different land management strategies (conventional, no-till, reduced inputs, and organic...
Show more"Manipulation of naturally occurring microbial communities to reduce plant diseases or increase crop yields requires a thorough understanding of interactions within the phytobiome, in particular, how microbial communities change as plants age, across plant species and organs, and under different land management regimes. Plants were sampled from a wheat-maize-soybean crop rotation site that implements four different land management strategies (conventional, no-till, reduced inputs, and organic). The fungal and bacterial communities of leaves, stems, and roots of wheat, maize, and soybean throughout the growing season were analyzed using fungal internal transcribed spacer and bacterial 16S rRNA gene amplicon sequencing. Analysis of sequence-based fungal communities has some limitations due to the unreliable phylogenetic resolution of DNA sequence alignments. To improve this deficiency, a tool that improved phylogenetic resolution was developed. This tool increases the number of operational taxonomic units which are identified at genus and species levels. Endophytes were isolated from the wheat plants used for microbial community analysis and tested for antagonistic activity toward the wheat pathogen Fusarium graminearum during wheat seedling and head infection. Endophytes on crops can be developed to manage disease, and endophyte-based biocontrols could solve current limitations in F. graminearum disease control. Additionally, functional analysis of F. graminearum secondary metabolite genes provides insight into the function of their gene products for this fungal pathogen. Microbial community structure is affected by various genetic factors of the host plant, environmental factors, and interactions with other organisms. Understanding community responses to these factors is necessary for targeted manipulation of communities to reduce plant disease."--Pages ii-iii.
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- Title
- DEVELOPMENT OF 3D BIOACTIVE AND ANTIBACTERIAL SILICATE-BASED SCAFFOLDS FOR BONE TISSUE REGENERATION IN LOAD-BEARING APPLICATIONS
- Creator
- Marsh, Adam Christoph
- Date
- 2022
- Collection
- Electronic Theses & Dissertations
- Description
-
Current gold-standard approaches to addressing the needs of bone defects in load-bearing applications entail the use of either autographs or allographs. Both solutions, however, are imperfect as both autographs and allographs carry the risk of additional trauma, threat of disease transmission, and potential donor rejection respectively. Porous 3D scaffolds are attractive alternatives, illuminating a potential path towards achieving the ideal scaffold for targeting bone tissue regeneration in...
Show moreCurrent gold-standard approaches to addressing the needs of bone defects in load-bearing applications entail the use of either autographs or allographs. Both solutions, however, are imperfect as both autographs and allographs carry the risk of additional trauma, threat of disease transmission, and potential donor rejection respectively. Porous 3D scaffolds are attractive alternatives, illuminating a potential path towards achieving the ideal scaffold for targeting bone tissue regeneration in load-bearing applications, usurping autographs to become the new gold-standard. To unlock the full healing potential of 3D scaffolds, such scaffolds must be multifunctional such that (1) their mechanical performance meets the requisite requirements as dictated by the mechanical performance characteristics of interest for native bone tissue, (2) they stimulate the necessary biological responses for bone tissue regeneration, and (3) they exhibit antibacterial characteristics to combat the threat of infection. To date, no reports document 3D scaffolds exhibiting all three performance characteristics. The aim of this dissertation, therefore, is to deliver 3D scaffolds that are mechanically competent, possess and exhibit inherent and advanced antibacterial characteristics, and are successful at providing the needed biological characteristics for bone tissue regeneration. To achieve this, this dissertation implements a multidisciplinary approach, utilizing comprehensive structural characterization across a wide range of scales to elucidate process – performance relationships to execute scientifically driven modifications to engineer and deliver a 3D scaffold to successfully target bone tissue regeneration in load-bearing applications. A silver-doped bioactive glass-ceramic (Ag-BG) composition was selected as the material for scaffold synthesis due to its inherent and attractive antibacterial and biological performance characteristics. Two fundamentally different processing approaches were utilized for synthesizing Ag-BG scaffolds: the polymer foam replication technique and fused filament fabrication (FFF). The Ag-BG scaffolds studied herein were found to exhibit advanced antibacterial performance characteristics against methicillin-resistant Staphylococcus aureus (MRSA), a common pathogen implicated in osteomyelitis development, able to combat MRSA both in planktonic and biofilm forms. Ag-BG scaffolds demonstrated the ability to form an apatite-like layer when immersed in simulated body fluid (SBF), an indicator that Ag-BG scaffolds will induce the necessary mineralization for bone tissue regeneration, in addition to exhibiting attractive cell viability, proliferation, and differentiation characteristics when studied in vitro. The mechanical performance of Ag-BG scaffolds reported herein saw progressive improvements in each iteration of Ag-BG scaffold synthesis, achieving desirable mechanical competency and reliability as a result of the multidisciplinary approach formulated. In addition to the exploration of developing 3D antibacterial and biological silicate-based scaffolds capable of targeting bone tissue regeneration in load-bearing applications, foundational work towards the development of class II hybrid scaffolds comprised of gelatin methacryloyl (GelMA) and Ag-BG for targeting softer tissue regeneration. The novel syntheses applied to the successful molecular coupling of GelMA and Ag-BG presents an attractive class II hydrogel showing great promise as a compatible ink for 3D bioprinting cell-laden scaffolds capable of targeting tissue regeneration of more sophisticated systems.
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- Title
- DYNAMIC DESERTS : THE SYNERGISTIC EFFECTS OF FUNGI, SUNLIGHT, AND NON-RAINFALL MOISTURE ON PLANT LITTER DECOMPOSITION IN DRYLANDS
- Creator
- Logan V, James Robert
- Date
- 2021
- Collection
- Electronic Theses & Dissertations
- Description
-
Drylands cover well over one third of the Earth’s land and are an important part of the global carbon cycle. Despite this, most models underestimate carbon turnover in arid and semi-arid systems, limiting our ability to predict how they will respond to changing climates. This is partly because many models are driven by rainfall, assuming little to no decay between precipitation events. Unlike in many wetter systems though, plant litter decomposition in drylands is largely controlled by non...
Show moreDrylands cover well over one third of the Earth’s land and are an important part of the global carbon cycle. Despite this, most models underestimate carbon turnover in arid and semi-arid systems, limiting our ability to predict how they will respond to changing climates. This is partly because many models are driven by rainfall, assuming little to no decay between precipitation events. Unlike in many wetter systems though, plant litter decomposition in drylands is largely controlled by non-rainfall processes including photodegradation and biotic decomposition supported by non-rainfall moisture (fog, dew, and water vapor; “NRM”). Despite their importance however, few studies have examined how these drivers interact with one another and with fungal communities to influence carbon turnover. In this dissertation, I demonstrate how photodegradation, NRM, and fungal decomposers interact to accelerate carbon turnover in drylands. To do this, I leveraged a natural gradient of NRM frequency in the Namib Desert that receives intense solar radiation. In one study, I used a reciprocal transplant design to show that moisture regime exerts a strong influence on litter-associated fungal communities and show that the relationship between NRM and litter decay rates depends on the composition of the decomposer community. In another study, I manipulated solar radiation for three years and found that photodegradation of the plant cuticle allows litter to absorb more water during NRM events, accelerating biotic decomposition. By examining litter-associated fungal communities under these same radiation treatments, I also show that fungi are largely insensitive to radiation stress and that photodegradation mainly affects decomposition rates in this system through photochemical changes in litter that increase subsequent biotic decomposition. Finally, to quantify the relationship between NRM and carbon turnover on multi-year timescales, I measured mass loss for 30 months along a moisture gradient spanning an order of magnitude of NRM frequency. By coupling these data with continuous meteorological measurements over the same period, I show that accounting for NRM and temperature sensitivity substantially improves the performance of a simple exponential litter decay model. These findings build on previous work demonstrating the importance of solar radiation and NRM as crucial drivers of litter decomposition and point a way forward for future studies to examine how these two processes may interact under future climate scenarios. Drylands are undergoing significant changes from anthropogenic climate change and understanding the drivers of litter decomposition allows us to better predict how these ecosystems are responding to global change.
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- Title
- ECOPHYSIOLOGY OF (PERI)ORAL BACTERIA AND IMPACT OF OTIC COLONIZATION
- Creator
- Jacob, Kristin Marie
- Date
- 2022
- Collection
- Electronic Theses & Dissertations
- Description
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The middle ear is typically assumed to be sterile in health due to its secluded location, closed off from external forces by the tympanic membrane (ear drum) and from the naso/oropharynx by a collapsed Eustachian tube. However, the periodic opening of the Eustachian tube to the naso/oropharyngeal space, which releases pressure across the eardrum and drains otic fluids, could introduce bacteria. Previous studies have tested for the presence of bacteria in the uninfected otic cavity using...
Show moreThe middle ear is typically assumed to be sterile in health due to its secluded location, closed off from external forces by the tympanic membrane (ear drum) and from the naso/oropharynx by a collapsed Eustachian tube. However, the periodic opening of the Eustachian tube to the naso/oropharyngeal space, which releases pressure across the eardrum and drains otic fluids, could introduce bacteria. Previous studies have tested for the presence of bacteria in the uninfected otic cavity using samples collected via invasive surgeries (through or around the eardrum). Findings from these studies are controversial due to contradictory results between studies, lack of critical experimental controls, and sampling of participants with underlying ailments (i.e., cochlear implant surgery) that could impact the microbiology of the otic mucosa. The studies reported herein bypass these limitations by using samples of otic secretions collectively non-invasively (through the mouth) in a cohort of healthy young adults. This dissertation describes cultivation-dependent methods to investigate the microbiology of the middle ear in health. The study used an IRB-approved protocol (#17-502) to collect otic secretions in order to 1) sequence their microbiome (contribution by Dr. Joo-Young Lee) and 2) recover in pure culture otic bacteria for further characterization (my contribution). As controls, we also collected buccal (top palate and inside of cheeks) and oropharyngeal swabs from each participant. Of the collected secretions, samples from 19 individuals were used for culture independent studies, while samples from the remaining 3 participants were subjected to culture dependent studies. 16S rRNA-V4 sequencing detected a diverse and distinct microbiome in otic secretions comprised primarily of strictly anaerobic bacteria belonging to the phyla Bacteroidetes, Firmicutes and Fusobacteria, and to a lesser extent facultative anaerobes (Streptococcus). I recovered from the otic, oropharyngeal, and buccal secretions 39 isolates of predominantly facultative anaerobes belonging to Firmicutes (Streptococcus and Staphylococcus), Actinobacteria (Micrococcus and Corynebacterium), and Proteobacteria (Neisseria) phyla, and used partial 16S rRNA amplicon sequences to demonstrate the distinct phylogenetic placement of otic streptococci compared to the oral ancestors (Chapter 2). This finding is consistent with the ecological diversification of oral streptococci once in the middle ear microenvironment. The recovery of streptococci and transient migrants (Staphylococcus, Neisseria, Micrococcus and Corynebacterium) from otic secretions prompted us to study the adaptive responses that give the streptococcal migrants a competitive advantage during the colonization of the middle ear (Chapter 3). For these studies, I sequenced and partially assembled the genomes of the otic isolates and used the full length 16S rRNA sequences for taxonomic demarcation at the species levels. Phylogenetic analyses demonstrated the oral ancestry of the otic streptococci, which retained from the otic adaptive traits critical for growth and reproduction in the middle ear mucosa (biofilm formation, mucolytic and proteolytic activity, robust growth under redox fluctuations, and fermentative production of lactate, a key metabolic intermediate in the otic trophic webs). These adaptive traits give oral streptococci a colonization advantage over competing (peri)oral migrants such as Staphylococcus. Furthermore, the otic streptococci inhibited the growth of otopathogens, including Staphylococcus aureus. These antagonistic interactions give streptococci a competitive advantage during the colonization of the middle ear and suggest a role for these commensals in promoting mucosal health. The ability of staphylococcal migrants to breach the middle ear mucosal barrier and cause infections prompted us to study the environmental factors that facilitate the spreading of staphylococci from the nasal to the middle ear mucosa. Allergies, respiratory maladies (cold, flu), or (peri)oral bacterial infections (sinus, adenoids, tonsils, etc.) lead to inflammation of the Eustachian tube and changes in the rheological properties of the otic mucus that increase the risk of infections. Thus, we examined the spreading of staphylococci on mucus-like viscous surfaces (semisolid agar plates). In Chapter 4, I show that mucins, the mucosal glycoproteins that control the viscosity and wettability of the mucus layer, induce the rapid spreading and dendritic expansion of clinical isolates closely related to S. aureus and Staphylococcus epidermidis but not of Staphylococcus hominis. Mucin glycosylation controlled the hydration of the mucoid surface and the ability of the cells to spread rapidly, in a process that was dependent on the secretion of surfactant-active, phenol-soluble modulins via the agr-quorum sensing two-component system. These results provide a plausible explanation for the rapid spreading of staphylococcal otopathogens from the nasopharynx to the middle ear through a swollen, and mucin-rich Eustachian tube. The work described in this dissertation provides much needed understanding of the adaptive responses that allow (peri)oral bacteria to colonize the middle ear. The studies add to the accumulating evidence that the middle ear mucosa is not sterile but rather harbors a commensal microbiota in health. These commensal community shares many metabolic similarities with ancestors in oral biofilms and retain adaptive traits critical for growth in the otic mucosa and inhibition of otopathogens. Additionally, this work identifies environmental factors that could contribute to staphylococcal virulence, broadening the understanding of newly identified motility phenotypes in the genus that could provide novel pharmaceutical targets.
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- Title
- EFFECTS OF PLACENTAL LISTERIA MONOCYTOGENES INFECTION ON FETAL NEURODEVELOPMENT
- Creator
- Lee, Kun Ho
- Date
- 2022
- Collection
- Electronic Theses & Dissertations
- Description
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Maternal infection can lead to adverse pregnancy outcomes. Numerous epidemiological studies have demonstrated an association between prenatal infection and neuropsychiatric disorders, including autism spectrum disorder (ASD). Different prenatal infections are associated with distinct neurological pathologies, necessitating studies of the diversity of prenatal pathogens and their consequences. Listeria monocytogenes (Lm) is a foodborne pathogen that causes listeriosis, which typically affects...
Show moreMaternal infection can lead to adverse pregnancy outcomes. Numerous epidemiological studies have demonstrated an association between prenatal infection and neuropsychiatric disorders, including autism spectrum disorder (ASD). Different prenatal infections are associated with distinct neurological pathologies, necessitating studies of the diversity of prenatal pathogens and their consequences. Listeria monocytogenes (Lm) is a foodborne pathogen that causes listeriosis, which typically affects immunocompromised individuals, including pregnant mothers. Prenatal infection with Lm can cause detrimental pregnancy outcomes, such as miscarriages, stillbirths, preterm labor, and death in newborns. However, neurological outcomes of maternal listeriosis have not been characterized. Here, I sought to investigate whether placental infection with Lm is associated with altered neurodevelopment by using a bioluminescence strain of Lm and a murine model of pregnancy-associated listeriosis. I show that placental infection affects neurodevelopment during pregnancy and behavior in the offspring.To investigate how placental infection with Lm dysregulates fetal brain development, I performed RNA-seq on fetal brains to quantify the enrichment of genes that were associated with the infection during gestation. The findings of RNA-seq analysis illustrated that placental infection with Lm altered fetal brain transcriptome and showed sexually dichotomous gene expression profiles. I further assessed the effects of different traits, including Lm exposure, the intensity of placental infection, and sex on the fetal transcriptome using systems biology. The genes were grouped into co-expression modules. Notably, maternal infection and its intensity measured by bioluminescence imaging signal were significantly associated with specific modules, suggesting these traits are the main factors driving these transcriptional changes. Lastly, I showed that placental Listeria infection enriched ASD-associated genes. These results demonstrate that maternal listeriosis dysregulates fetal brain transcriptome during gestation. Neurodevelopment is a complex process influenced by various environmental factors during pregnancy. To examine whether prenatal infection with Lm affects cortical lamination and neural activity, I performed hematoxylin and eosin staining and immunohistochemistry. Gross anatomy of the brain structure analysis showed that placental infection with Lm affected cortical lamination in a localized manner. Furthermore, increased neural activity was observed in Lm- exposed male offspring. These results illustrate that placental infection with Lm induces morphological changes in brain tissue during neurodevelopment. Behavioral symptoms of neuropsychiatric disorders are an important component of the diagnosis. Animal behavioral assays and tools have been developed to examine animal behavior such as social interactions, anxiety, and repetitive behaviors. I examined behavior tests that resembled ASD to determine if mouse offspring born following placental infection displayed abnormal behavior. Lm-exposed offspring exhibited altered behaviors and showed sex-dependent behavioral changes. Overall, my work highlights the impact of maternal listeriosis on brain development during pregnancy and its effects on offspring’s behavior and contributes to the understanding of the spectrum of fetal neurodevelopment.
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- Title
- ENVIRONMENTAL DRIVERS AND EVOLUTIONARY CONSEQUENCES OF HORIZONTAL GENE TRANSFER IN SOIL BACTERIA
- Creator
- Kittredge, Heather
- Date
- 2021
- Collection
- Electronic Theses & Dissertations
- Description
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Horizontal gene transfer (HGT) is a driving force in bacterial evolution and could drive rapid adaptation in bacterial communities. Natural transformation is one mechanism of HGT that allows bacteria to pick up extracellular DNA (eDNA) from the environment and integrate it into their genome. But the rate of HGT in natural environments, and the role this process plays in facilitating rapid adaptation remains unknown. As climate change threatens the stability of environments worldwide,...
Show moreHorizontal gene transfer (HGT) is a driving force in bacterial evolution and could drive rapid adaptation in bacterial communities. Natural transformation is one mechanism of HGT that allows bacteria to pick up extracellular DNA (eDNA) from the environment and integrate it into their genome. But the rate of HGT in natural environments, and the role this process plays in facilitating rapid adaptation remains unknown. As climate change threatens the stability of environments worldwide, understanding how quickly bacteria can adapt to novel environments is essential. My dissertation research characterizes the environmental drivers and evolutionary consequences of natural transformation in a highly transformable model soil bacterium Pseudomonas stutzeri.Despite decades of research on understanding HGT at the molecular level, less is known about the ecological drivers of HGT. To understand the soil conditions relevant for transformation, I first measured eDNA in the field over a short-term drying rewetting disturbance (Ch. 2). I found that eDNA increased in response to the rewetting disturbance but quickly disappeared from soil, suggesting a small portion of this eDNA could be transformed by bacterial cells recovering from the disturbance. To test the efficiency of transformation under the conditions in which eDNA disappeared, I created a novel microcosm system for quantifying transformation in soil (Ch. 3). Here, I inoculated soil with live antibiotic-susceptible, and dead antibiotic-resistant P. stutzeri. I then tracked the evolution of antibiotic resistance over a range of soil conditions and eDNA concentrations. Transformation drove the evolution of antibiotic resistance across a wide range of soil moistures and increased in response to larger inputs of dead cells (eDNA source), with antibiotic resistance repeatedly appearing in antibiotic free soil. Despite the prevalence of transformation across bacterial species, the evolutionary origins and consequences of transformation are still largely unknown. Transformation presumably provides a fitness benefit in stressful or continuously changing environments, but few studies have quantified changes in transformation in response to adaptive evolution. Here, I evolved P. stutzeri at different salinities and tested how the growth rate and transformation efficiency changed in response to salt adaptation (Ch. 4). Overall, the growth rate increased in response to adaptation, but the transformation efficiency declined, with only ~50% of the evolved populations transforming eDNA at the end of experiment – as opposed to 100% of ancestral populations transforming eDNA. Overall, my dissertation research elucidates the factors driving transformation in soil, setting the stage for future experiments to scale up estimates of transformation to the whole community level. I find that transformation occurs under most soil conditions and allows genetic variants to arise at low frequencies in the absence of selection. I also report novel experimental evidence that transformation efficiency can change dramatically, and in a highly variable manner, over just ~330 generations. Taken together, this body of research highlights a role for transformation in many natural systems of ecological significance, and points to dead cells as an important but often overlooked source of genetic diversity.
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- Title
- EPIDEMIOLOGY OF ANTIBIOTIC RESISTANT SHIGA TOXIN-PRODUCING ESCHERICHIA COLI (STEC) AND NON-TYPHOIDAL SALMONELLA (NTS) IN MICHIGAN
- Creator
- Mukherjee, Sanjana
- Date
- 2018
- Collection
- Electronic Theses & Dissertations
- Description
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The enteric pathogens, Shiga toxin-producing E. coli (STEC) and non-typhoidal Salmonella (NTS), are leading causes of foodborne infections in the US, resulting in 265,000 and 1.2 million illnesses every year, respectively. The emergence of antibiotic resistance in these pathogens has been documented and is of great concern due to negative patient health outcomes and the possibility of transfer of resistance genes to other clinically relevant pathogens. However, there is a scarcity in...
Show moreThe enteric pathogens, Shiga toxin-producing E. coli (STEC) and non-typhoidal Salmonella (NTS), are leading causes of foodborne infections in the US, resulting in 265,000 and 1.2 million illnesses every year, respectively. The emergence of antibiotic resistance in these pathogens has been documented and is of great concern due to negative patient health outcomes and the possibility of transfer of resistance genes to other clinically relevant pathogens. However, there is a scarcity in information about frequencies of antibiotic resistant and factors associated with resistant STEC and NTS infections in Michigan. It is necessary to have a complete understanding about the of emerging antibiotic resistance and factors driving the rise of resistance in STEC and NTS to help develop effective control strategies. In this dissertation, 980 STEC isolates collected from patients in Michigan between 2001 and 2014 were examined for resistance to clinically relevant antibiotics. The examination of STEC strains for resistance, revealed high frequencies of resistance to ampicillin and trimethoprim-sulfamethoxazole, with significant increases in antibiotic resistance rates observed over this 14-year period. Multivariate logistic regression analysis identified non-O157 serotypes to be independently associated with antibiotic resistance. The recent increase in incidence of non-O157 serotypes observed in the US, coupled with the high frequencies of antibiotic resistance observed in this study, suggest the emergence of antibiotic resistant non-O157s as important human pathogens. Additionally, antibiotic resistant STEC isolates from patients in recent years (2010-2014) were more likely to cause hospitalizations than pansusceptible STEC isolates, suggesting that resistant STEC infections may result in adverse patient outcomes. Using whole genome sequencing, we also identified chromosomal mutations and 33 horizontally acquired genes present in the genomes of non-O157 STEC, likely conferring resistance. Importantly, by creating a co-occurrence network of these genes, we identified the co-occurrence of certain resistance genes, which are possibly present on the same mobile genetic element, thus resulting in multi-drug resistance. In addition to examining resistance in STEC, a total of 198 clinical NTS isolates collected between 2011 and 2014 were also examined for antibiotic resistance in this dissertation. Resistance to tetracycline, trimethoprim-sulfamethoxazole and ampicillin were commonly observed. Concerningly, high frequencies of multidrug resistant NTS were also observed with significant increases in their prevalence observed between 2011 and 2014. These high multidrug resistant rates have important implications on patient care as the efficacy of multiple antibiotics is reduced. Antibiotic resistant NTS isolates were also found to result in significantly longer mean hospital stays compared to pansusceptible NTS. Serovar specific differences in frequencies of antibiotic resistance were observed; S. Enteritidis were observed to have lower resistance frequencies than other serovars. Lastly, to better understand the role that cattle reservoirs play in harbouring antibiotic resistant STEC strains, we examined 121 STEC isolates collected in 2012 from six cattle farms in Michigan for antibiotic resistance. While high resistance frequencies to tetracycline and trimethoprim-sulfamethoxazole were observed in certain herds, no resistance to ampicillin was observed, unlike what was observed in STEC isolates collected from patients. While different populations of resistant STEC may be circulating in the clinical and agricultural environments, continuous monitoring of resistance in the cattle reservoir is warranted to determine if animal reservoirs can serve as potential sources of resistant infections in humans.
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- Title
- Ecological and evolutionary consequences of exometabolites for microbial interactions
- Creator
- Chodkowski, John Luke
- Date
- 2021
- Collection
- Electronic Theses & Dissertations
- Description
-
Interspecies interactions have fundamental roles in shaping microbial communities. Microbial community members can produce and release a diverse set of extracellular small molecules, collectively referred to as exometabolites. Interspecies interactions that result from exometabolites can alter the response and behaviors of microbial community members. This dissertation work demonstrates the establishment of a synthetic community system that facilitates the study of exometabolite-mediated...
Show moreInterspecies interactions have fundamental roles in shaping microbial communities. Microbial community members can produce and release a diverse set of extracellular small molecules, collectively referred to as exometabolites. Interspecies interactions that result from exometabolites can alter the response and behaviors of microbial community members. This dissertation work demonstrates the establishment of a synthetic community system that facilitates the study of exometabolite-mediated interspecies interactions. This system was then used to understand the consequences of exometabolite-mediated interactions in a 3-member synthetic microbial community over stationary phase using a multi-omics approach. Lastly, an experimental evolution showed the consequences of long-term exometabolite interspecies interactions on the evolution of antibiotic resistance. This work advances knowledge on the dynamic response and behaviors of microbial community members' during non-growth states. This work also demonstrates how bacterial-bacterial interactions in a simple environment can lead to the emergence of antibiotic resistance.
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- Title
- Ecology of calliphorid larval masses and postmortem colonization estimate variability
- Creator
- Weatherbee, Courtney R.
- Date
- 2016
- Collection
- Electronic Theses & Dissertations
- Description
-
"The objectives of this work were to first, classify the insect and microbe interactions associated with carrion decomposition; and second, evaluate the variability in post-colonization interval (PCI) estimates among temperature sources and Calliphoridae species. Replicate (N = 6) swine carcasses were placed in a field and sampled regularly throughout decomposition. The ambient temperature at varying distances from the carcasses was recorded, and samples of larvae and their associated...
Show more"The objectives of this work were to first, classify the insect and microbe interactions associated with carrion decomposition; and second, evaluate the variability in post-colonization interval (PCI) estimates among temperature sources and Calliphoridae species. Replicate (N = 6) swine carcasses were placed in a field and sampled regularly throughout decomposition. The ambient temperature at varying distances from the carcasses was recorded, and samples of larvae and their associated microbiomes were collected. Microbial communities were characterized using 16s amplicon sequencing. I hypothesized that insect and microbial community composition would shift over time and that different microbial communities would become more similar to each other over time. Additionally, I predicted that different Calliphoridae species would produce different accumulated degree hour (ADH) ranges and the closest temperature data set to the carcass would result in a more accurate PCI estimate. Larval masses consisted of three Calliphoridae species (Cochliomyia macellaria, Lucilia coeruleiviridis, and Phormia regina) whose relative abundances shifted over time. Microbial communities of the carcass surface, maggot mass, and internal larvae also changed over time as well as among the different locations. The closest temperature source produced the greatest ADH range but the estimated PCI was most similar to ADH ranges generated from local and regional weather station data. These findings are relevant for both carrion ecology and the forensic sciences by contributing to the understanding of the effect of carrion within ecosystems as well as advancing a new direction of forensic microbiology and the quantification of variability in forensic entomology"--Page ii.
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- Title
- Effects of dietary fiber sources on the gastro-intestinal microbiota, fermentation metabolites, and listeria monocytogenes in vivo and in vitro
- Creator
- Walker, Ryan (Ryan Adam)
- Date
- 2019
- Collection
- Electronic Theses & Dissertations
- Description
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Dietary fiber consumption influences the gastro-intestinal microbiota, gastro-intestinal function, and health. This research investigated the effects of dietary fiber sources on bacterial composition, short-chain fatty acid (SCFA) production, gastro-intestinal barrier function, and Listeria monocytogenes challenge in mice and human colon cells. L. monocytogenes causes life-threatening illness in humans and animals. Some dietary fibers protect animals against illness in models of infection,...
Show moreDietary fiber consumption influences the gastro-intestinal microbiota, gastro-intestinal function, and health. This research investigated the effects of dietary fiber sources on bacterial composition, short-chain fatty acid (SCFA) production, gastro-intestinal barrier function, and Listeria monocytogenes challenge in mice and human colon cells. L. monocytogenes causes life-threatening illness in humans and animals. Some dietary fibers protect animals against illness in models of infection, while others enhance infection. Therefore, it is unclear if consuming certain dietary fiber sources confers protection against foodborne illnesses. The objectives of this research were: 1) describe how dietary fiber sources affect bacterial composition in vivo and in vitro; 2) quantify SCFAs produced by bacterial fermentation; 3) determine if fiber sources differentially affect L. monocytogenes infection in vivo; and 4) determine if bacterial metabolites promote or inhibit L. monocytogenes infection by affecting in vitro epithelial barrier function.In this project, dietary fiber sources differentially affected gastro-intestinal bacterial composition in vivo and in vitro, L. monocytogenes infection in mice, and enhanced barrier integrity in vitro. The overall results of this project demonstrate that dietary fiber sources differentially influence certain gastro-intestinal bacterial populations, measures of diversity, and that resulting compositional changes are important in the pathogenicity of L. monocytogenes. Additionally, fermentation end products enhance gastro-intestinal barrier function in this in vitro model, which may be an important factor in the prevention of enteric infection in humans. In summary, both the gastro-intestinal microbiota and its metabolites are important factors for maintaining gastro-intestinal health.
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- Title
- Environmental Microbial Surveillance : From Source Tracking in Watersheds to Pathogen Monitoring in Sewersheds
- Creator
- Flood, Matthew Thomas
- Date
- 2022
- Collection
- Electronic Theses & Dissertations
- Description
-
Understanding of the connections between water and health, through the use of water quality monitoring, surveys and surveillance, can help to address the impacts of anthropomorphic changes on the environment. This study sought to understand these connections through the water quality monitoring within watershed basins as well as pathogen surveillance within sewersheds. Specifically, this dissertation sought to 1) understand the sources of pollution and their connections with land use in the...
Show moreUnderstanding of the connections between water and health, through the use of water quality monitoring, surveys and surveillance, can help to address the impacts of anthropomorphic changes on the environment. This study sought to understand these connections through the water quality monitoring within watershed basins as well as pathogen surveillance within sewersheds. Specifically, this dissertation sought to 1) understand the sources of pollution and their connections with land use in the various subsections of watersheds; 2) to find a cost-effective way to surveil the spread of SARS-CoV-2 using wastewater surveillance; and 3) to understand the differences in wastewater surveillance between communities. Water quality monitoring using microbial source tracking (MST) was performed with a survey of five mixed-use watersheds in Michigan. Through the use of spatial clustering, it was found that temporal contamination was primarily driven by precipitation and its associated variables (e.g., streamflow, turbidity, overland flow), while spatial contamination is driven by land uses (e.g., septic tank density, tile drain proportions, and tillage). Additionally, porcine fecal contamination was more often correlated with nutrients in streams than either bovine or human contamination. The development of a cost-effective workflow for the detection and quantification of SARS-COV-2 in wastewater was undertaken. Wastewater from communities around Michigan were collected and analyzed along with viral surrogates for SARS-CoV-2 to investigate different workflow options. The Pseudomonas phage Phi6 was seeded in different wastewater matrices to test concentration and recovery by ultrafiltration-based method and polyethylene glycol (PEG) precipitation. The PEG method provided better virus recovery than the ultrafiltration-based methods as measured using RT-ddPCR. The comparison of two communities (A and B) wastewater results for SARS-CoV-2 analyzed against case data was undertaken. These results were significantly correlated with cases in both communites, but the level of correlation differed based on spatial (e.g., zipcode vs county level cases) and temporal (e.g., date of symptom(s) onset vs. the referral date for cases) resolution. Wastewater surveillance was more representative of higher spatial resolution (zipcode data) of cases in both communities. When examining the temporal resolution of the communities, community B’s wastewater results were more closely tied to the onset of symptoms and not the case referral date. The ability to monitor indicators of pollution in watersheds and surveil etiological agents of disease in sewersheds provide non-intrusive methods for evaluating the potential risks and current burdens to community health. The first part of the work could be considered “downstream” monitoring identifying sources and potential exposures with the goal of reducing waterborne disease. While “upstream” monitoring was used for identifying the disease trends in the community and was focused on public health measures to prevent transmission. This project contributed novel methods, results and analysis providing valuable knowledge ultimately addressing the role of monitoring strategies to protect public health.
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- Title
- Environmental regulation of cyclic di-GMP turnover in vibrio cholerae
- Creator
- Koestler, Benjamin J.
- Date
- 2014
- Collection
- Electronic Theses & Dissertations
- Description
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3', 5'-cyclic diguanylic acid (c-di-GMP) is an intracellular bacterial second messenger that mediates the transition between a sessile, biofilm forming lifestyle to a motile, virulent lifestyle. Diguanylate cyclase (DGC) enzymes synthesize c-di-GMP, whereas c-di-GMP specific phosphodiesterase (PDE) enzymes hydrolyze the second messenger. Although numerous bacterial behaviors are regulated by c-di-GMP, the regulatory inputs of this system remain mostly undefined. Here, I examine how the marine...
Show more3', 5'-cyclic diguanylic acid (c-di-GMP) is an intracellular bacterial second messenger that mediates the transition between a sessile, biofilm forming lifestyle to a motile, virulent lifestyle. Diguanylate cyclase (DGC) enzymes synthesize c-di-GMP, whereas c-di-GMP specific phosphodiesterase (PDE) enzymes hydrolyze the second messenger. Although numerous bacterial behaviors are regulated by c-di-GMP, the regulatory inputs of this system remain mostly undefined. Here, I examine how the marine bacterium and human pathogen Vibrio cholerae utilizes c-di-GMP signaling to interpret and respond to environmental cues. The central hypothesis that underpins my research is that V. cholerae senses environmental signals with DGCs and PDEs to modulate c-di-GMP concentrations in different environments. As c-di-GMP is a widely conserved second messenger utilized by many different bacteria, the mechanisms by which V. cholerae utilizes c-di-GMP in different environments can be applied to other bacterial systems to further comprehend how they behave in and adapt to various surroundings. To examine the influence of environmental factors on c-di-GMP synthesis and hydrolysis, I have developed a novel method named The Ex vivo Lysate c-di-GMP Assay (TELCA) that systematically measures total DGC and PDE cellular activity. I have shown that V. cholerae grown in different environments exhibits distinct intracellular levels of c-di-GMP, and using TELCA have determined that these differences correspond to changes in both c-di-GMP synthesis and hydrolysis. These findings highlight that modulation of both total DGC and PDE activity alters the intracellular concentration c-di-GMP.While searching for specific environmental cues that regulate c-di-GMP, I have found that bile acids, a prevalent constituent of the human proximal small intestine, increase intracellular c-di-GMP in V. cholerae. This bile-mediated increase in c-di-GMP is quenched by bicarbonate, the intestinal pH buffer secreted by intestinal epithelial cells. These findings lead me to propose that V. cholerae senses distinct microenvironments within the small intestine, using bile and bicarbonate as chemical cues, and responds by modulating intracellular c-di-GMP.In addition to its function as a bacterial second messenger, c-di-GMP has potent immunostimulatory properties in eukaryotes; these properties make c-di-GMP a prime candidate for use as a vaccine adjuvant. Here, I present a novel method of delivering c-di-GMP into eukaryotic cells using adenovirus. I have demonstrated that c-di-GMP can be synthesized in vivo by delivering DGC DNA into the cell, and that this c-di-GMP increases the secretion of numerous cytokines and chemokines. This novel adenovirus c-di-GMP delivery system offers a more efficient and cost-effective method to administer c-di-GMP as an adjuvant to stimulate innate immunity.
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