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Title: Interview of Lillian Kivela on her service in the U.S. Army Nurse Corps during WWII
Creator: Kivela, Lillian, 1920-2013
Date: 1986-01-22
Collection: Women's Overseas Service League Oral History Project
Description: Lillian Kivela talks about her service in the United States Army Nurse Corps during World War Two including, why she enlisted in June 1943, nurse's training, basic Army training, housing, uniforms, and her duties at the Schick General Hospital in Clinton, Iowa. She says that she was sent to New Jersey in preparation for being shipped to Europe and describes shipboard conditions and being seasick throughout the entire ten-day voyage. She talks about being housed in an unheated Welsh resort...
Show moreLillian Kivela talks about her service in the United States Army Nurse Corps during World War Two including, why she enlisted in June 1943, nurse's training, basic Army training, housing, uniforms, and her duties at the Schick General Hospital in Clinton, Iowa. She says that she was sent to New Jersey in preparation for being shipped to Europe and describes shipboard conditions and being seasick throughout the entire ten-day voyage. She talks about being housed in an unheated Welsh resort hotel, marching, walking a mile to the mess hall for meals, serving in the orthopedic ward at a hospital in Headington, a suburd of Oxford and experiencing an influx of patients following D-Day and the subsequent fighting, and the early use of penicillin to control infection. In her off-time, Kivela says that she often visited London for the theater, rode her bicycle around Oxford, became acquainted with British families and even met the Queen Mother and boxer Joe Louis when they visited the hospital. Back in the States, after the war, she says that she had a difficult time adjusting to civilian life and finally came to Michigan State College to finish her degree in microbiology. Kivela is interviewed by Elsie Hornbacher.
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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: 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: Identifying the underlying mechanisms of Marek's disease vaccine synergy
Creator: Umthong, Supawadee
Date: 2019
Collection: Electronic Theses & Dissertations
Description: Marek’s disease virus (MDV; Gallid herpesvirus 2, aka, serotype 1) is a ubiquitous and highly oncogenic α-herpesvirus that causes Marek’s disease (MD), a lymphoproliferative disorder affecting chickens with estimated annual costs to the poultry industry of ~$2 billion worldwide. Since 1970, MD has been largely controlled through widespread vaccination. While MD vaccines are very successful in preventing tumors, they do not prevent viral replication and spread. As a consequence, new and more...
Show moreMarek’s disease virus (MDV; Gallid herpesvirus 2, aka, serotype 1) is a ubiquitous and highly oncogenic α-herpesvirus that causes Marek’s disease (MD), a lymphoproliferative disorder affecting chickens with estimated annual costs to the poultry industry of ~$2 billion worldwide. Since 1970, MD has been largely controlled through widespread vaccination. While MD vaccines are very successful in preventing tumors, they do not prevent viral replication and spread. As a consequence, new and more virulent MDV strains have repeatedly emerged in vaccinated flocks. Thus, there is a need to understand how MD vaccines work in order to design future vaccines that are more protective, especially against more virulent MDVs. One promising insight for vaccine development is based upon protective synergism, a phenomenon where two vaccines when combined provide greater protection compared to either original vaccine when administered alone as a monovalent vaccine. The mechanism that underlines the synergistic effect between SB-1 (a Gallid herpesvirus 3, aka, serotype 2 strain) and HVT (herpesvirus of turkey, aka, Meleagrid herpesvirus 1 or serotype 3), two of the most widely used MD vaccines, has never been investigated, and thus, provides a highly relevant and useful model to explore. To investigate the mechanisms of protective synergy of SB-1 and HVT, we used three approaches. First, we investigated how monovalent SB-1 or HVT replicates when they were alone in the host or together as a bivalent vaccine. We observed that the replication patterns of SB-1 and HVT were different with respect to time after administration into the bird and the organs that they were found to replicate in regardless if the other vaccine were present. Based on the observation that HVT replicated primarily early in the bursa, we found that this organ was necessary for protection using both HVT and bivalent HVT + SB-1 vaccines. Second, we measured the effects of CD8 T cells in monovalent SB-1, HVT, and bivalent SB-1+HVT vaccine treatment. Specifically, we reduced CD8 T cells to see their effect of CD8 T cells on MD incidence and vaccinal protection by injecting the chickens with a monoclonal antibody directed against chicken CD8 T cells. In this study, we found that CD8 T cells were necessary for protection induced by vaccines. Third, we identified the cytokine profiles induced by SB-1, HVT, and the bivalent vaccine to see if cytokine synergy could be one of the mechanisms to explain protective synergy. We found that SB-1 induced an innate anti-viral response typified by IFN-α, IFN-β, IL-1β, T-cell proliferation cytokine IL-21, and Th2 cytokine IL-5, while HVT suppressed TGF-β3 and TGF-β4. The early stimulation of IL-1β and IL-21 (IFN-γ-promoting cytokines) at 4 days post vaccination (DPV) by SB-1 combined with the suppression of TGF-β (IFN-γ- suppressing cytokine) at 1 day post challenge (DPC) by HVT could result in the strong induction of IFN-γ found in the bivalent vaccine at 10 DPC. The induction of IFN-γ supports the synergistic effect of cytokines by a cooperative action mechanism where multiple cytokines work together to enhance the signal. Based on these findings, we propose a model to explain bivalent SB-1 and HVT vaccine synergy, which combines the replication of vaccines, T cell response to vaccinations, and cytokine synergy between SB-1 and HVT vaccine. Our proposed mechanism provides insights on how to generate rationally designed MD vaccines.
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Title: Transfer and inactivation of Salmonella during post-harvest processing of tomatoes
Creator: Wang, Haiqiang
Date: 2015
Collection: Electronic Theses & Dissertations
Description: Salmonella outbreaks have been historically linked to tomatoes, with cross-contamination during post-harvest processing having become a major public health concern. In response, a series of studies were developed to assess the extent to which dump tanks, conveyors, slicers, and dicers can spread Salmonella and other microorganisms. An evaluation of the microbiological quality of tomatoes and dump tank water was conducted during three visits to a local tomato packinghouse. At the beginning of...
Show moreSalmonella outbreaks have been historically linked to tomatoes, with cross-contamination during post-harvest processing having become a major public health concern. In response, a series of studies were developed to assess the extent to which dump tanks, conveyors, slicers, and dicers can spread Salmonella and other microorganisms. An evaluation of the microbiological quality of tomatoes and dump tank water was conducted during three visits to a local tomato packinghouse. At the beginning of whole-day processing and after 3 h of operation, bacteria and yeast/mold populations decreased < 2 logs on tomatoes, with these microbial counts greatly impacted by changes in organic load and sanitizer concentration during washing. When the spread of Salmonella was assessed during washing of tomatoes with various sanitizers and subsequent conveying in a pilot-scale packing line, ~ 90% of the original Salmonella inoculum transferred to sanitizer-free water. Acidified chlorine yielded the greatest Salmonella reduction on tomatoes (3.1 log CFU/g). After processing with sanitizers, Salmonella populations decreased to non-detectable levels (< 0.2 log CFU/100 cm2) on the equipment surfaces. Thereafter, Salmonella transfer was assessed during conveying of tomatoes with plastic, foam, or brush rollers. Overall, cross-contamination was greatest using foam, followed by plastic and brush rollers (P < 0.05). After 5 inoculated tomatoes were roller conveyed, 24 and 76% of all uninoculated subsequently conveyed tomatoes were cross-contaminated with Salmonella of 10 - 100 and 1 - 10 CFU/tomato, respectively, compared to 8% of 25 tomatoes using brush rollers.The next two studies focused on Salmonella transfer during slicing and dicing of tomatoes. For tomato slicing, one red round tomato was inoculated with Salmonella Typhimurium LT2 (~ 5 log CFU/g) and sliced using a manual or electric slicer, followed by 20 uninoculated tomatoes, all of which yielded quantifiable numbers of Salmonella after slicing. The quantitative data was fitted to a two-parameter exponential model (Y = A  e(B  X)). While significantly higher (P ≤ 0.05) percentages of Salmonella were transferred to wet (12.2%) as opposed to dry tomatoes (1.1%), with the variety of tomato also impacting transfer, post-contamination hold time, processing temperature and tomato slice thickness did not significantly impact the overall percentage of cells transferred. When one 0.9 kg batch of inoculated Roma tomatoes (~5 log CFU/g) was mechanically diced, followed by ten batches of uninoculated tomatoes, all uninoculated tomatoes yielded Salmonella, with populations exponentially decreasing from 3.3 to 1.1 log CFU/g. Flume tank washing in sanitizer-free water or water containing 80 ppm peroxyacetic acid, 80 ppm mixed peracid, or 80 ppm total chlorine decreased the Salmonella populations on diced tomatoes 1.3 ± 0.2, 2.3 ± 0.3, 2.4 ± 0.4, and 2.4 ± 0.1 log CFU/g, respectively. Spray sanitation on conveyor belts proved to be an effective way to enhance safety of diced tomatoes, with electrolyzed water being especially attractive due to its relatively low cost and ease of preparation.Finally, the impact of temperature, pH, and wash water organic load on Salmonella morphology and early-biofilm formation was assessed on different surfaces encountered in tomato packing houses. Both pH and temperature significantly affected the surface hydrophobicity of Salmonella. Early-biofilm formation on tomatoes was significantly affected by both time (P = 0.0004) and temperature (P < 0.0001). After 6 d, early-biofilms consistently developed on stainless steel and HDPE surface, with the former being more evenly distributed.
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Title: Quantitative detection of rotavirus and enterovirus in raw sewage using reverse transcription droplet digital PCR
Creator: Kiulia, Nicholas Mukaria
Date: 2017
Collection: Electronic Theses & Dissertations
Description: "The goal of this study was to survey the concentrations of rotaviruses (RVs) and enteroviruses (EVs) in raw sewage using reverse transcription droplet digital PCR (RT-ddPCR) and to compare the use of ViroCap filters, polyethylene glycol/sodium chloride (PEG/NaCl) precipitation and mixed cellulose ester (HA) filter methods for virus recovery." -- Abstract.

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: Genetic and chemical biology studies of Mycobacterium tuberculosis pH-driven adaptation
Creator: Dechow, Shelby J.
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Mycobacterium tuberculosis (Mtb) endures robust immune responses by sensing and adapting to its host environment. One of the first cues the bacterium encounters during infection is acidic pH, a characteristic of its host niche – the macrophage. Targeting the ability of Mtb to sense and adapt to acidic pH has the potential to reduce survival of Mtb in macrophages. A high throughput screen of a >220,000 compound small molecule library was conducted to discover chemical probes that inhibit Mtb...
Show moreMycobacterium tuberculosis (Mtb) endures robust immune responses by sensing and adapting to its host environment. One of the first cues the bacterium encounters during infection is acidic pH, a characteristic of its host niche – the macrophage. Targeting the ability of Mtb to sense and adapt to acidic pH has the potential to reduce survival of Mtb in macrophages. A high throughput screen of a >220,000 compound small molecule library was conducted to discover chemical probes that inhibit Mtb growth at acidic pH. From this screen, AC2P20 was identified as a chemical probes that kills Mtb at pH 5.7 but is inactive at pH 7.0. Through a combination of transcriptional profiling, mass spectrometry, and free thiol abundance and redox assays, I show that AC2P20 likely functions by depleting intracellular thiol pools and dysregulating redox homeostasis. Findings from this study have helped define new pathways involved in Mtb’s response to acidic pH using a chemical genetic approach.Upon sensing acid stress, Mtb can adapt accordingly by entering a nonreplicating persistent state, resulting in increased tolerance to host immune pressures and antibiotics. During growth in vitro, when given glycerol as a sole carbon source, Mtb responds to acidic pH by arresting its growth and entering a metabolically active state of nonreplicating persistence, a physiology known as acid growth arrest. To answer how Mtb regulates and responds to acidic pH, I performed genetic selections to identify Mtb mutants defective in acid growth arrest. These selections identified enhanced acidic growth (eag) mutants which all mapped to the proline-proline-glutamate ppe51 gene and resulted in distinct amino acid substitutions: S211R, E215K, and A228D. I demonstrated that expression of the PPE51 variants in Mtb promotes significantly enhanced growth at acidic pH showing that the mutant alleles are sufficient to cause the dominant gain-of-function, eag phenotype. Furthermore, I performed single carbon source experiments and radiolabeling experiments showing that PPE51 variants preferentially uptake glycerol at an enhanced rate, suggesting a role in glycerol acquisition. Notably, the eag phenotype is deleterious for growth in macrophages, where the mutants have selectively faster replication but reduced virulence in activated macrophages as compared to resting macrophages. This supports that acid growth arrest is a genetically controlled, adaptive process that could act as a potential targetable physiology in future TB therapeutics. My work with the carbonic anhydrase inhibitor, ethoxzolamide, sought to combine genetic and chemical biology to better understand pH-adaptation in Mtb. Ethoxzolamide is a potent inhibitor of Mtb carbonic anhydrase activity and the PhoPR regulon, suggesting a previously unknown link between carbon dioxide and pH-sensing. We hypothesized that the production of protons from carbonic anhydrase activity could be modulating PhoPR signaling. Mtb has three carbonic anhydrases (CanA, CanB, and CanC), and by using CRISPRi and gene knockout, I show that CanB is required for pathogenesis in macrophages, but I did not observe a function in controlling PhoPR signaling. However, transcriptional profiling at different pH and CO2 concentrations show that PhoPR is induced by high CO2 and also revealed a core subset of CO2 responsive genes independent of PhoPR or acidic pH regulation. Overall, these studies defined new functions for thiol- and redox-homeostasis, glycerol uptake, and CO2-concentration in regulating Mtb adaptation to acidic environments and provide new targets for the development of acidic pH-dependent therapeutics.
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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: 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: INSIGHTS INTO ORGANOSULFUR ASSIMILATION IN STAPHYLOCOCCUS AUREUS
Creator: Lensmire, Joshua Michael
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Bacterial pathogens deploy sophisticated strategies to acquire vital nutrients from the host during infection. Staphylococcus aureus is a considerable human pathogen due to its capacity to cause numerous life-threatening diseases. As such S. aureus has an intricate metabolism that promotes proliferation in distinct host environments. However, little is known regarding the sulfur sources the pathogen scavenges from host tissues. Sulfur is an essential nutrient due to its extensive redox...
Show moreBacterial pathogens deploy sophisticated strategies to acquire vital nutrients from the host during infection. Staphylococcus aureus is a considerable human pathogen due to its capacity to cause numerous life-threatening diseases. As such S. aureus has an intricate metabolism that promotes proliferation in distinct host environments. However, little is known regarding the sulfur sources the pathogen scavenges from host tissues. Sulfur is an essential nutrient due to its extensive redox capacity and consequently, it is a critical component of many cofactors. Prior studies started to define sulfur sources S. aureus can use including the inorganic sulfur sources, sulfide and thiosulfate, and organosulfur sources, cysteine, cystine, and glutathione. While we understand some of the sulfur sources S. aureus can use, we do not know the genetic determinants facilitating assimilation. The present studies sought to explain how S. aureus imports and catabolizes organic sources of sulfur. First, we wanted to uncover the proteins allowing S. aureus to utilize cystine and cysteine as sulfur sources. The S. aureus homologues of characterized cystine transporters, TcyP and TcyABC, were experimentally validated as cystine and cysteine transporters. We expanded the sulfur sources S. aureus can utilize to include homocystine and N-acetyl cysteine and show that both TcyABC and TcyP support growth on N-acetyl cysteine while only TcyABC supports growth on homocystine. Finally, a tcyP mutant is impaired in murine heart and liver when competing with WT S. aureus suggesting import of TcyP substrates is important for heart and liver colonization. While a tcyP mutant is reduced in competition with WT in murine heart and liver colonization is not fully ablated signifying more sulfur sources must be catabolized. We next examined how S. aureus imports and catabolizes GSSG. To identify S. aureus GSSG utilization strategies, we used a chemically defined medium containing GSSG as the sulfur source and isolated mutants harboring transposon insertions within a putative ABC-transporter and -glutamyl transpeptidase that fail to proliferate. The mutants also do not grow in medium supplemented with GSH. Consistent with these findings, we named the locus the glutathione import system (gisABCD-ggt). Biochemical analysis of recombinant Ggt confirms in silico functional predictions by demonstrating that Ggt cleaves both GSH and GSSG. Though Gis mutants display wildtype virulence, we find that Gis-Ggt promotes competition with Staphylococcus epidermidis when GSH or GSSG is supplied as the sole sulfur source in vitro. S. aureus resides as a nasal commensal in 30% of the population and once inside the body can infect nearly every organ. Throughout the changing host environments, S. aureus must sense and acclimate to nutrient availability. We sought to define how sulfur starvation and growth on different sulfur sources changes the transcriptional profile of S. aureus. We described the transcriptional changes when WT S. aureus or a CymR, the sulfur transcriptional regulator, mutant were grown in sulfur replete and deplete conditions. We show sulfur starvation leads to significant expression changes including upregulation of iron acquisition encoding genes and oxidative stress encoding genes. Furthermore, we provide evidence showing upregulation of CymR dependent sulfur transporters when S. aureus is grown on GSH and thiosulfate both of which are conditions in which CymR repression should be occurring. Finally, this dissertation ends with areas of future exploration of sulfur source utilization in S. aureus. These avenues include examining nutrient sulfur available in distinct infection sites and expansion of the sulfur sources S. aureus can use. Overall, the work presented here substantially contributes to our understanding of what sulfur sources S. aureus imports and catabolizes and how different sulfur sources change the transcriptional states of the cell.
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Title: UNDERSTANDING DRIVERS OF PLANT MICROBIOME IN MICHIGAN AGRICULTURE : STUDIES OF THE APPLE ROOT ZONE AND COMMON BEAN SEEDS
Creator: Bintarti, Ari Fina
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Plant-associated microbial communities are crucial for plant health and fitness, and may enhance plant tolerance to various environmental stresses. As global climate change threatens crop production and increases demands on sustainable agriculture, harnessing the plant microbiome has become one potential strategy to address these issues. Thus, it is fundamental to understand the relative contributions of both the host plant as well as the environment in shaping the plant microbiome. Moreover,...
Show morePlant-associated microbial communities are crucial for plant health and fitness, and may enhance plant tolerance to various environmental stresses. As global climate change threatens crop production and increases demands on sustainable agriculture, harnessing the plant microbiome has become one potential strategy to address these issues. Thus, it is fundamental to understand the relative contributions of both the host plant as well as the environment in shaping the plant microbiome. Moreover, the response of plant microbiomes to stress and any consequences of microbiome stress responses for the host plants are poorly understood, though this information is critical to achieve a basis of knowledge for plant microbiome engineering. My research aimed to contribute to this knowledge by investigating the factors that structure root- and seed-associated microbial communities of two valuable crops for Michigan’s agricultural economy: apple and common bean. The first chapter of my dissertation aimed to assess the biogeography of bacterial, archaeal, fungal, and nematode communities in the root zone of apple trees, and to determine their relationships with each other and their changes over natural abiotic gradients across orchards. I also assessed the influence of plant cultivar on microbiome structure in the root zone. I found that root zone microbiome community structure was strongly affected by geographic location and edaphic properties of soil. The next chapter of my dissertation investigated the variability of seed endophyte community of common bean (Phaseolus vulgaris L.). My results showed that plant-to-plant variability under controlled growth conditions exceeded within-plant variability among seeds from different pods. My study developed protocols and added insights to the growing toolkit of approaches to understand the plant-microbiome engagements that support the health of agricultural and environmental ecosystems. The last chapter assessed the responses of common bean seed endophytes to drought stress in the field across two growing locations and four genotypes of common bean. To summarize, this work advances foundational knowledge of the seed microbiome as a critical component of the plant microbiome, and in the context of two key crops for Michigan agriculture.
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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: DISRUPTIONS IN HEPATIC ONE CARBON METABOLISM AND THE GUT MICROBIOME DURING THE PROGRESSION OF NON-ALCOHOLIC FATTY LIVER DISEASE
Creator: Fling, Russell Ryan
Date: 2022
Collection: Electronic Theses & Dissertations
Description: The etiology of non-alcoholic fatty liver disease is complex, with multiple contributing factors including dietary, environmental, gut microbiome and genetic mechanisms. Accumulating evidence suggests exposure to polychlorinated dibenzo-p-dioxins and similar compounds may increases risk for NAFLD development. These environmentally persistent dioxin-like compounds bind and activate the aryl hydrocarbon receptor, a transcription factor that regulates intestinal homeostasis, xenobiotic and...
Show moreThe etiology of non-alcoholic fatty liver disease is complex, with multiple contributing factors including dietary, environmental, gut microbiome and genetic mechanisms. Accumulating evidence suggests exposure to polychlorinated dibenzo-p-dioxins and similar compounds may increases risk for NAFLD development. These environmentally persistent dioxin-like compounds bind and activate the aryl hydrocarbon receptor, a transcription factor that regulates intestinal homeostasis, xenobiotic and central metabolism. In a AhR-dependent manner, mice orally gavaged with 2,3,7,8-tetracholordibenzo-p-dioxin (TCDD) exhibit steatosis progressing to steatohepatitis with fibrosis akin to NAFLD progression. NAFLD and hepatocellular carcinoma (HCC) is also closely correlated with dysregulation of central metabolism e.g., hepatic one carbon metabolism (OCM), and gut dysbiosis contributing to NAFLD progression and worsening prognosis. This report investigates mechanisms involved in the dysregulation of the gut microbiome and OCM associated pathways relevant to NAFLD progression through comparisons of molecular analyses of TCDD-treated mice to human NAFLD and HCC. OCM describes the biosynthesis, homeostasis, and utilization of the cell’s main methyl donor, S-adenosylmethionine (SAM) including high flux anabolic biosynthesis of polyamines, phosphatidylcholine and creatine. In later stages of NAFLD, OCM is dysregulated with altered OCM gene expression as well as SAM and s-adenosylhomocysteine (SAH) levels. To assess TCDD-elicited effects on OCM, mice were orally gavaged with TCDD every 4 days for 28 days. Serum and livers collected at early (8 days) and late (28 days) time points were subjected to metabolomic analyses with integration of chromatin immunoprecipitation sequencing, transcriptomics and protein levels. Results from these studies suggest AhR-mediated repression of OCM required prolonged repeated TCDD-treatment and indirect effects elicited by AhR activation e.g., oxidative stress. Gut dysbiosis with disrupted enterohepatic bile acid metabolism is commonly associated with NAFLD and recapitulated in TCDD-treated mice. Similar to NAFLD, TCDD also increases systematic levels of secondary bile acids. These microbial transformed secondary bile acids are involved in modulation of host bile acid signaling pathways relevant to NAFLD. To investigate the effects of TCDD on the gut microbiota, the cecum contents of TCDD-treated mice were subjected to shotgun metagenomic sequencing. Taxonomic analysis identified dose-dependent increases in Lactobacillus species, notably Lactobacillus reuteri. Top enriched species were also associated with increased abundances of bile salt hydrolase sequences, responsible for the initial deconjugation reaction in secondary bile acid metabolism. L. reuteri levels were also attributed to enrichment of mevalonate-dependent isopentenyl diphosphate (IPP) biosynthesis genes, a pathway that was also elevated in cirrhosis patients. These results extend the role of Lactobacilli in the AhR/intestinal axis and NAFLD progression as well as highlight the similarities between TCDD-elicited phenotypes in mice to human NAFLD. Collectively, these studies evaluated TCDD-elicited mechanisms involved in disruptions in host and microbial metabolism, highlighting the AhR’s role in NAFLD progression.
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Title: Interactions Between Plasma and Material Surfaces for Sterilization and Impurity Adsorption
Creator: Mackinder, Madeline Ann
Date: 2022
Collection: Electronic Theses & Dissertations
Description: As the worldwide population increases, maintaining a standard of public health becomes more critical. Two major concerns in this area are nosocomial infections (NI) and water contamination. Current processes in sterilization and water treatment have limitations that could be overcome using plasma techniques. The unique characteristics of plasma make it a promising alternative for energy-intensive processes. This work investigated the characteristics of plasma that have the greatest impact on...
Show moreAs the worldwide population increases, maintaining a standard of public health becomes more critical. Two major concerns in this area are nosocomial infections (NI) and water contamination. Current processes in sterilization and water treatment have limitations that could be overcome using plasma techniques. The unique characteristics of plasma make it a promising alternative for energy-intensive processes. This work investigated the characteristics of plasma that have the greatest impact on sterilization and the reactivation of activated carbon. Previous studies have researched the physical and chemical surface properties of biochar but have not been able to establish an efficient process to activate biochar with desired characteristics. Plasma treatment would offer a way to etch the surface of biochar and specifically functionalize the surface. Successfully activating biochar would increase its adsorption ability and enable its use for water treatment. This project aims to harness these plasma properties and use plasmas to address three important topics related to public health: sterilization of surfaces, modulating commercial activated carbon (AC), and activations of biochar.Cold plasma sterilization offers an efficient way to sterilize medical components and instruments without the risk of deformation to heat-sensitive materials. This paper reports the use of magnetized plasma to realize low-temperature sterilization. A radio frequency dielectric barrier discharge was created in a quartz tube using a mixture of argon and oxygen gas. Glass slides inoculated with a uniform amount of Escherichia coli were exposed to the plasma afterglow at different pressures with and without a magnetic field. A global model was developed to evaluate the magnetically enhanced dielectric barrier discharges and predict species densities. Optical emission spectroscopy identified the plasma species present and validated the model. The magnetic field significantly promoted the intensity of the plasma and the sterilization efficiency. A process gas pressure of 100 mTorr presented the most effective treatment with a sterilization time less than one minute and sample temperature below 32 °C. The effects of O2 plasma on the adsorption capacity of activated carbon (AC) was investigated by varying treatment times. Transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), and Zeta potential were used to characterize the surface properties of the AC. The carbon was then applied to remove methylene blue (MB) from an aqueous solution. The adsorption kinetics and isotherm were also studied. Results showed that pseudo-second-order kinetics was the most suitable model for describing the adsorption of MB onto AC. Equilibrium data were well fitted to the Freundlich and Langmuir isotherm models. The highest adsorption capacity resulted from 4 minutes of O2 plasma treatment. This work shows that activation of AC by plasma can open the micropore and increase the effectiveness of chemical removal.Biochar was activated using a combination of O2 plasma and KOH. The adsorption capacity was investigated for different O2 plasma treatment times, KOH concentrations, and treatment temperatures. The adsorption capacity of methylene blue (MB) by the plasma activated biochar was evaluated. The adsorption kinetics and isotherm were also investigated. Results showed that pseudo-second-order kinetics was the most suitable model for describing the adsorption of MB onto biochar. Both the Freundlich and Langmuir isotherm models fit the equilibrium data. The highest adsorption capacity resulted from 10\% KOH + 300 °C for 5 minutes. This work shows that activation of biochar by plasma can improve adsorption capacity. The plasma treated AC and the plasma activated biochar were applied to the removal of PFOA. It was demonstrated that plasma treatment can improve PFOA adsorption. The negative surface charge was shown to negatively impact PFOA adsorption which aligns with the hypothesis that PFOA would preferentially adsorb onto more positive surfaces due to its anionic state in water.
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Title: MEMBRANE-LOCALIZED TRANSCRIPTION REGULATORS : UNDERSTANDING POST-TRANSLATIONAL REGULATION AND SINGLE-MOLECULE DYNAMICS OF TCPP IN VIBRIO CHOLERAE
Creator: Demey, Lucas Maurice
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Vibrio cholerae is a Gram-negative gastrointestinal pathogen that has evolved an elegant regulatory system to precisely time production of essential virulence factors. A key step in this regulatory system is the transcription of a soluble AraC-like transcription factor, ToxT. ToxR and TcpP, two membrane-localized transcription regulators (MLTRs), positively regulate toxT. Much work has contributed to our understanding of TcpP and ToxR regulation, yet major gaps remain in our knowledge of...
Show moreVibrio cholerae is a Gram-negative gastrointestinal pathogen that has evolved an elegant regulatory system to precisely time production of essential virulence factors. A key step in this regulatory system is the transcription of a soluble AraC-like transcription factor, ToxT. ToxR and TcpP, two membrane-localized transcription regulators (MLTRs), positively regulate toxT. Much work has contributed to our understanding of TcpP and ToxR regulation, yet major gaps remain in our knowledge of these MLTRs. MLTRs are unique one-component signal transduction systems because they respond to extracellular stimuli by influencing gene transcription from their location in the cytoplasmic membrane. In Chapter 2, I explore the prevalence and diversity of MLTRs within prokaryotes to enhance our understanding of TcpP and ToxR. I show that MLTRs are far more common among prokaryotes than previously anticipated and that MLTRs are an understudied class of transcription regulators. In Chapter 3, I describe the use of super-resolution single-molecule tracking to investigate how TcpP, a model MLTR, identifies the toxT promoter. I provide evidence that TcpP binds to the toxT promoter independent of ToxR, and TcpP transitions to a specific diffusion state. The data support the first biophysical model for how TcpP-like MLTRs locate their target promoters. TcpP is subject to a form of post-translational regulation known as regulated intramembrane proteolysis (RIP). RIP of TcpP results in its complete inactivation, resulting in loss of virulence factor production. TcpH inhibits RIP of TcpP under certain pH and temperature conditions. In Chapter 4, I describe the mechanism TcpH employs to inhibit TcpP RIP while V. cholerae is present in the mouse gastrointestinal tract. I demonstrate that the dietary fatty acid α-linolenic acid enhances inhibition. I also show that α-linolenic acid promotes TcpH-mediated inhibition of TcpP RIP by increasing association of both proteins with detergent-resistant membrane (DRM) domains. My work provides the first evidence that DRMs influence virulence factor transcription in V. cholerae and that a dietary fatty acid promotes V. cholerae pathogenesis.
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Title: Harnessing peat-based gnotobiotic plant growth to characterize microbiota-mediated immunocompetence in Arabidopsis
Creator: Paasch, Bradley Carlton
Date: 2021
Collection: Electronic Theses & Dissertations
Description: The increasingly evident involvement of microbes in basic host function has led to a more holistic perspective of plants to be considered. Here, plants and their associated microbiotas interact with each other as holobionts in performing various biological functions in natural ecosystems and crop fields as a single ecological unit. The extent to which microbial components of the holobiont contribute to host health, however, is not fully understood, especially in natural environments. Here, I...
Show moreThe increasingly evident involvement of microbes in basic host function has led to a more holistic perspective of plants to be considered. Here, plants and their associated microbiotas interact with each other as holobionts in performing various biological functions in natural ecosystems and crop fields as a single ecological unit. The extent to which microbial components of the holobiont contribute to host health, however, is not fully understood, especially in natural environments. Here, I applied the use of peat-based gnotobiotic growth systems to generate plants grown with and without exposure to microbiota to characterize the role of microbiota on the development of plant immunocompetence.In my first chapter, I review the current understanding on the interplay between microbiota and plant health and immunity. I begin with background on microbial detection and response in plants. I then discuss plant-associated microbiota and provide examples of how perturbation to microbiota homeostasis, such as during dysbiosis for example, can be associated with positive and negative impacts on host health. Host factors regulating microbiota homeostasis in plants are discussed. Lastly, I describe tools and approaches that can be used to the study of plant-microbiota interactions and highlight recent findings involving the modulation of plant immune responses by plant microbiotas. In my second chapter, I highlight the contributions I made to the improvement of two peat-based gnotobiotic plant growth systems for plant microbiome research recently developed in Dr. Sheng Yang He’s laboratory: the FlowPot and GnotoPot systems. I adapted the FlowPot system to use a field soil, highlighting its versatility, and optimized several abiotic conditions associated with plant growth in GnotoPots. Additionally, I used 16S rRNA gene amplicon sequencing to characterize the colonization of a natural, soil-derived microbial community and several preparations of a synthetic bacterial community, demonstrating the use of GnotoPots for colonization studies. We expect both systems to be useful tools for the research community to address a wide variety of questions related to plant-microbiota interactions. In my third chapter, I implement the optimizations made to peat-based gnotobiotic growth systems described in the previous chapter to characterize the role of basal microbiota colonization on plant immunocompetence. I found that compared to plants colonized by a soil-derived microbiota, axenic plants grown without exposure to a microbiota lacked robust age-dependent immunity. Axenic plants were defective in several aspects of pattern-triggered immunity including flg22-induced production of reactive oxygen species, signaling through MAPK pathways, and induction of defense-related genes and hypersusceptible to disease a bacterial foliar pathogen. Additionally, I found that a synthetic microbiota composed of culturable leaf endosphere bacteria was able to restore immunocompetence similar to plants inoculated with a soil-derived community in a growth substrate-dependent manner. These results demonstrate a role of microbiota in immunocompetence and age-dependent immunity, which was previously thought to be an intrinsic trait of plants.
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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: 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: IMPACT OF AGRICULTURAL MANAGEMENT AND MICROBIAL INOCULATION ON SOYBEAN (GLYCINE MAX) AND ITS ASSOCIATED MICROBIOME
Creator: Longley, Reid
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Soybean (Glycine max) is a globally important crop with uses as food, cooking oil livestock feed, and biodiesel. Soybean can be considered holobionts because they host diverse microbiomes which extend plant genotypes and phenotypes through various microbial functions such as nitrogen fixation and increased disease resistance. My research focused on assessing the impact of three agricultural management strategies on the soybean holobiont. Soybean cropping systems can be managed using various...
Show moreSoybean (Glycine max) is a globally important crop with uses as food, cooking oil livestock feed, and biodiesel. Soybean can be considered holobionts because they host diverse microbiomes which extend plant genotypes and phenotypes through various microbial functions such as nitrogen fixation and increased disease resistance. My research focused on assessing the impact of three agricultural management strategies on the soybean holobiont. Soybean cropping systems can be managed using various strategies, including conventional tillage, no-till, and organic management regimes. These management systems have been shown to impact the microbiomes of soybean-associated soils, however, their impacts on plant-associated microbiomes are still not well understood. In this study, I assessed the impact of conventional, no-till, and organic management treatments on soybean microbiomes at Michigan State’s Kellogg Biological Station Long-Term Ecological Research site (KBS LTER). I found that management impacted microbiome composition and diversity in soil, roots, stems, and leaves and that this impact persisted throughout the season. Additionally, when comparing the same soybean genotype grown in conventional and no-till management systems, tillage regime impacted the microbiome throughout the plant and the growing season. This effect impacted microbial taxa which are likely to be plant beneficial, including nitrogen fixing Bradyrhizobium. Another important management tool that is expected to impact plant-associated microbial communities is the application of foliar fungicides. While fungicides are known to protect plants from particular fungal pathogens, non-target impacts of fungicides on crop microbiomes, and the impact of management on microbiome recovery are not well understood. To address this knowledge gap, I assessed the impact of foliar fungicide application on the maize (Headline® fungicide, 2017) and soybean (Delaro® fungicide, 2018) microbiomes in conventional and no-till plots at the KBS LTER. I found that fungicide applications have a non-target impact on Tremellomycete yeasts in the phyllosphere and this impact was greater in soybean than maize. Co-occurrence network analysis and random forest modelling indicated that changes in fungal communities may lead to indirect impacts on prokaryotic communities in the phyllosphere. Importantly, this work demonstrated that phyllosphere communities of soybeans under no-till management had greater recovery from fungicide disturbance. This novel finding exemplifies how tillage regime can impact phyllosphere microbiomes and their responses to disturbance. Microbial inoculants in agriculture have long been used for biocontrol of pathogens, but there is also interest in their use to dampen the impacts of abiotic stress including drought. In this study, I tested whether inoculating soybeans with hub taxa identified through network analysis from no-till soybean root microbiome data from the KBS LTER could provide protection against water limitation. Soybean seedlings were enriched in consortia of hub bacteria and fungi and were grown in no-till field soil. Seedlings were then exposed to low-moisture stress, and plant phenotypes, plant gene expression, and amplicon sequencing of microbial DNA and cDNA were assessed throughout the stress period. Inoculation increased plant growth, nodule numbers, and led to increased expression of nodulation-associated genes. 16S sequencing of cDNA revealed higher levels Bradyrhizobium in inoculated samples. These results indicate that inoculation with hub microbes can benefit soybean plants, possibly through interaction with other microbes, interaction with the plant, or both. In summary, fungicide, tillage, and inoculation all impact the soybean microbiome, indicating that management choices impact the entire holobiont.
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Title: LISTERIA MONOCYTOGENES PLACENTAL COLONZATION AND CONSEQUENCES FOR PREGNANCY OUTCOME
Creator: Conner, Kayla Nicole
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Listeria monocytogenes (Lm) is a Gram-positive bacterium that causes the severe food-borne disease listeriosis. Listeriosis is particularly problematic in pregnant women as Lm colonizes the placenta, resulting in adverse pregnancy outcomes including stillbirth, miscarriage, and preterm labor. Despite numerous studies of placental listeriosis (PL) in various animal models, the mechanisms driving adverse outcomes following PL are largely uncharacterized. This dissertation addresses some of the...
Show moreListeria monocytogenes (Lm) is a Gram-positive bacterium that causes the severe food-borne disease listeriosis. Listeriosis is particularly problematic in pregnant women as Lm colonizes the placenta, resulting in adverse pregnancy outcomes including stillbirth, miscarriage, and preterm labor. Despite numerous studies of placental listeriosis (PL) in various animal models, the mechanisms driving adverse outcomes following PL are largely uncharacterized. This dissertation addresses some of the field’s knowledge gaps by analyzing the changes in placental gene expression and metabolism following infection with Lm and by characterizing a key Lm virulence factor, Internalin P (InlP), which plays a significant role in Lm placental colonization. Chapter 1 gives pertinent background information on the placenta, Lm, and PL and broadly addresses the knowledge gaps to be addressed by the rest of the dissertation. Chapter 2 describes an in vivo study of PL in mice. Infected and control placentas were analyzed for differences in gene expression profiles between the two groups. We identified an enrichment of genes associated with eicosanoid biosynthesis, suggesting perturbations in eicosanoid metabolism in infected tissues. By quantifying placental eicosanoid concentrations through mass spectrometry, we found a significant increase in the concentrations of several eicosanoids with known roles in inflammation and/or labor. This study provides a likely explanation for temporal disruptions of labor following placental infection. Chapters 3 and 4 discuss two studies of the Lm virulence factor InlP, a key player in placental colonization. InlP contributes to Lm’s placental pathogenesis likely by conferring the ability of Lm to transcytose through placental layers. Prior studies reported that no homologs of InlP exist in Listeria species other than Lm. Chapter 3 describes our discovery that at least two other Listeria species, L. ivanovii and L. seeligeri, encode InlP homologs. We characterized the domain architectures and genomic neighborhoods of these homologs and speculated on their implications for Listeria evolution. In chapter 4, I continue discussion of InlP and describe our identification and preliminary characterization of naturally occurring InlP variants. In this study, we used a bioinformatics approach to analyze Lm whole genome sequences (WGS) and identify InlP variants. We uncovered two InlP variants of interest in the Lm population. The first results from a start codon point mutation in the inlP gene, likely resulting in a truncated and potentially nonfunctional InlP protein product. The second is an InlP variant with a PRO to SER substitution in the InlP calcium binding loop, which is hypothesized to play a role in InlP activation or stabilization. These results provide two avenues for further investigation of InlP regulation and function and suggest the potential for InlP-dependent variation in placental colonization potential across Lm isolates. In chapter 5, I summarize this dissertation. This chapter reflects on the results, implications, and challenges of each study outlined in the prior chapters. I discuss the unique challenges faced due to the ongoing COVID-19 pandemic and its effects on my graduate training. Finally, I share concluding remarks and propose future directions for this project and the field of PL. Together, the chapters of this dissertation describe novel findings that contribute to the field by assessing genetic and metabolic changes to the placenta due to listeriosis and further characterizing a known key placental virulence factor.
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Title: The impact of lactobacillus and bacteriophage on group b streptococcus and the placental membranes
Creator: Shiroda, Megan
Date: 2019
Collection: Electronic Theses & Dissertations
Description: The human microbiota encompasses the microbes that live on and in the human body. While some body sites including the vaginal and intestinal tracts have been studied extensively for their role in human health, other body sites have been historically considered sterile and are less studied. One such site is the placental membranes that surround the fetus during pregnancy and serve as an important protective barrier during pregnancy. Several studies have established which bacteria are found in...
Show moreThe human microbiota encompasses the microbes that live on and in the human body. While some body sites including the vaginal and intestinal tracts have been studied extensively for their role in human health, other body sites have been historically considered sterile and are less studied. One such site is the placental membranes that surround the fetus during pregnancy and serve as an important protective barrier during pregnancy. Several studies have established which bacteria are found in this site, but few studies have been conducted to characterize their interactions in vitro or to understand their impact on the placental membranes. Further, our knowledge of the viral component of the microbiome in human health remains incomplete.In this dissertation, Lactobacillus, a well-studied probiotic in other body sites, was evaluated for its effect in the placental membranes. As these membranes were previously considered sterile, we sought to assess the ability of Lactobacillus to colonize these cells and evaluate its impact on them. A cell line model of the outermost layer of these membranes, the decidual cells, was used to establish that Lactobacillus can associate and impact a known cell signaling pathway, the Mitogen Activated Protein Kinase (MAPK) pathway, which is associated with inflammation and host cell death. Total protein level of p38, an important upstream protein in this pathway, was found to be significantly lower with Lactobacillus than in mock infection. These data suggest that Lactobacillus could maintain a commensal interaction in the placental membranes as described in other body sites. Lactobacillus is also known to inhibit pathogen invasion. Group B Streptococcus (GBS) can ascend from the vaginal tract to infect placental membranes, triggering premature birth or neonatal infection. Four Lactobacillus strains representing three species were characterized to assess their impact on two GBS strains (colonizing and invasive). We found live Lactobacillus does not affect GBS growth or biofilm production. L. gasseri increased association of both strains of GBS to the decidual cells but did not result in increased invasion of the cells. Instead, co-culture with Lactobacillus reduced host cell death. Secreted products of Lactobacillus drastically reduced growth in 35 GBS strains that broadly represent GBS diversity and could prevent biofilm formation; this inhibition was strain dependent. Unfortunately, increased GBS-induced host cell death with Lactobacillus supernatants was observed. Collectively, these data suggest that both live Lactobacillus and its supernatant could impact GBS interactions with the placental membranes.Bacteriophage are one of the most abundant members of the microbiome but their impact on opportunistic pathogens such as GBS remains unknown. As GBS can be isolated from gastrointestinal tract, we hypothesized fecal phage communities would inhibit the growth of GBS in vitro. Approximately 6% of the tested communities inhibited the growth of 35 GBS strains. To better understand differences in GBS strain inhibition, we examined capsule, sequence and clinical types of the strains. As no significant differences were found with these traits, we next examined Clustered Regularly Interspaced Palindromic Repeats (CRISPR), which serve as an adaptive immune system against invading foreign DNA by the acquisition of spacer sequences. GBS strains with fewer than nine spacer sequences were less likely to be lysed by a phage community than strains with more than sixteen spacers. We further hypothesized that presence of GBS in the corresponding bacterial component of each phage community would correlate to its ability to inhibit GBS growth. While this correlation did not exist across all GBS strains tested, sensitive strains of GBS were significantly more likely to be inhibited by phage communities with a lower abundance of GBS. Collectively, these data suggest that the phage component of the intestinal microbiome could impact GBS colonization. To further characterize these interactions, an individual bacteriophage should be isolated from these communities.
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Title: Postmortem microbiome computational methods and applications
Creator: Kaszubinski, Sierra Frances
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Microbial communities have potential evidential utility for forensic applications. However, bioinformatic analysis of high-throughput sequencing data varies widely among laboratories and can potentially affect downstream forensic analyses and data interpretations. To illustrate the importance of standardizing methodology, we compared analyses of postmortem microbiome samples using several bioinformatic pipelines, while varying minimum library size or the minimum number of sequences per sample...
Show moreMicrobial communities have potential evidential utility for forensic applications. However, bioinformatic analysis of high-throughput sequencing data varies widely among laboratories and can potentially affect downstream forensic analyses and data interpretations. To illustrate the importance of standardizing methodology, we compared analyses of postmortem microbiome samples using several bioinformatic pipelines, while varying minimum library size or the minimum number of sequences per sample, and sample size. Using the same input sequence data, we found that pipeline significantly affected the microbial communities. Increasing minimum library size and sample size increased the number of low abundant and infrequent taxa detected. Our results show that bioinformatic pipeline and parameter choice significantly affect the resulting microbial communities, which is important for forensic applications. One such forensic application is the potential postmortem reflection of manner of death (MOD) and cause of death (COD). Microbial community metrics have linked the postmortem microbiome with antemortem health status. To further explore this association, we demonstrated that postmortem microbiomes could differentiate beta-dispersion among M/COD, especially for cardiovascular disease and drug-related deaths. Beta-dispersion associated with M/COD has potential forensic utility to aid certifiers of death by providing additional evidence for death determination. Additional supplemental files including tables of raw data and additional statistical tests are available in supplemental files online, denoted in the text as table 'S'.
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