Browsing by Subject "Microbiology"

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Androgen-Mediated Repression of the Maspin Tumor Suppressor Gene in Prostate Cancer
(2012-04-18) Hanson, Kevin
This year in the United States, cancer is projected to cause one out of every four deaths. Prostate cancer alone is estimated to take the life of over 30,000 men. One area of intense research as a potential therapy against cancerous growth is in tumor suppressing genes, which function in cell cycle checkpoint responses, detection and repair of damaged DNA, protein ubiquitination and degradation, mitogenic signaling, cell specification, differentiation and migration, and tumor angiogenesis. One particular gene, the mammary serine protease inhibitor (maspin), is critically important in both breast and prostate cancer, which in the latter the gene is repressed entirely. However, the exact mechanism that leads to the repression of the maspin gene is not entirely understood. In my thesis work, I first showed that the maspin tumor suppressor gene was under direct regulation by the androgen receptor protein. By analyzing the quality and quantity of the mRNA produced under various growth conditions, we confirmed that the activation of the androgen receptor was critical for the repression of maspin. To do this, cell cultures were selectively grown with or without androgens and the mRNA was extracted, converted to cDNA, and RT-PCR was performed to analyze relative levels of maspin expression. I next attempted to verify that the androgen receptor was physically binding the promoter region of maspin by performing chromatin immunoprecipitation (ChIP) assays. After immunoprecipitating any chromatin fragments with the androgen receptor bound, a PCR to amplify the maspin promoter was completed. Obtaining a PCR product confirmed that the AR binds the androgen response element (ARE), repressing its expression.
Celebrating a Century of Microbiology and Immunology
(Department of Microbiology and Immunology, 2019) University of Minnesota. Department of Microbiology and Immunology; Hasse, Ashley T.
Denitrification in Agricultural Surface Waters: Quantifying the Effect of Environmental Parameters and Hydrologic Connectivity on Nitrate Uptake and Microbial Communities
(2017-10) Tomasek, Abigail
The development of synthetic fertilizer has led to increases in crop yields and allowed for global population growth over the past century. However, this increase in available nitrogen has greatly altered the global nitrogen cycle, including increased nitrate loading to surface water and groundwater in the Midwestern United States, with negative effects on human health and aquatic ecosystems. Therefore, there is a need for effective management strategies and an understanding of the mechanisms for nitrate transport and uptake. Denitrification, the microbiological reduction of nitrate to nitrogen gas, can be viewed as a net sink for reactive nitrogen in aquatic systems. Small areas, termed hot spots, and short time periods, termed hot moments, frequently account for a large portion of denitrification. This research focuses on identifying the environmental parameters and hydrologic regimes that promote denitrification, along with determining how parameters, denitrification rates, and microbiological communities are related at multiple temporal and spatial scales. At the finest scale, a recirculating laboratory flume was used to determine the effect of turbulence and organic carbon on denitrification rates and the microbial community. An outdoor experimental stream and flow-through basin in the Outdoor StreamLab at the St. Anthony Falls Laboratory (SAFL) were used to determine the effect of short-term inundation and periodic inundation on denitrification. At the largest scale, water and sediment samples were collected over two years from a field site in an agricultural watershed in Southern Minnesota. The objectives of this research were to: (1) determine how turbulence and organic carbon affect denitrification, (2) investigate how inundation and hydrologic connectivity leads to the formation of denitrification hot spots and hot moments, (3) quantify and correlate the driving environmental parameters of microbial denitrification and the differences in these relationships for in-channel and riparian locations in an agricultural watershed, (4) develop and evaluate functional relationships between environmental parameters and denitrification rates, and (5) identify how denitrifying gene abundances, denitrification rates, and environmental parameters are related across a hydrologic gradient from channels to riparian areas.
Effects of Ancient Climate Change on Population Size of Thylamys Mouse Opossums
(2012-04-18) Comar, Courtney
Since the peak of the last ice age, approximately 23,000 years ago, climates have grown increasingly warmer on earth. This climate change has affected the habitats in the extremely biodiverse region of the Andes Mountains in South America. In order to determine the effect of the climate change on the populations of organisms inhabiting this region, I am exploring the changes in effective population sizes of the Thylamys mouse opossum species. Using multiple individual samples, I have focused specifically on the species Thylamys venustus, T. pallidior, and T. sponsorius. I hypothesize that the effective population sizes of Thylamys venustus and T. sponsorius have decreased since the last glacial maximum because their habitats have moved farther up the mountains and decreased in size. Multilocus nuclear genetic data was used to examine the genetic changes occurring in the Thylamys populations. Fifteen unlinked nuclear loci were amplified across multiple individuals for each species. PCR products were sequenced, aligned, and compared using phylogenetic software to examine phylogenetic relationships and effective population size over time. Data confirmed that my hypothesis is supported by a decrease in the effective population size of Thylamys sponsorius in the last 10,000 years. However, an increase in population size was observed for Thylamys pallidior over the last 25,000 years. Thylamys venustus population sizes seem to be relatively unchanged over the last 50,000 years. A possible reason for increased or unchanged population sizes could be expansion of territory of these Thylamys species and movement southward where the climate is colder. This project is significant because few studies have been conducted to examine the effect of past climate fluctuations on the evolutionary patterns of Thylamys. The Tropical Andes is considered a biodiversity hotspot and understanding evolutionary genetic patterns for the Thylamys species could be used to assist conservation tactics for these marsupials. Ultimately, this project arouses the question: what will happen in the future if the climate continues to grow warmer?
Gene expression control and antimicrobial peptide production by haute couture bacteria
(2013-05) Völzing, Katherine Giovanna
Over the last few decades, biology research has expanded from the study of naturally occurring systems to the engineering of new, synthetic functions of our choosing. This has been possible because of our detailed knowledge of biological systems, high resolution experimental biology tools, powerful computational resources and increasingly affordable de novo DNA synthesis technologies. For example, entire microbial genomes have chemically synthesized and interesting synthetic systems have been built with novel gene expression dynamics. Additionally, cells have been engineered to efficiently produce high value compounds, including a number of recombinant protein molecules. Now, wonderful opportunities abound for improving the human condition with synthetic biology. The work presented in this thesis introduces two new synthetic biological systems. One system focuses on gene expression control while the second is a new approach to producing and delivering antimicrobial molecules. The first project is a set of synthetic transcription activators called prokaryotic- TetOn and prokaryotic-TetOff, that upregulate gene expression in response to anhydrotetracycline. The molecular geometries of the prokaryotic-TetOn and prokaryotic-TetOff systems were first optimized using protein structure refinement and homology modeling. Next, the molecular devices were built and tested experimentally.Finally, both systems were characterized using stochastic models of gene expression. Prokaryotic-TetOn and prokaryotic-TetOff are the first prokaryotic devices of their kind, inducible synthetic activators of protein expression. Notably, they have also been designed to serve as firm stepping stones for developing a library of related synthetic transcription factors and networks. In a second, distinct project, we considered tacking the extensive use of antibiotics in agriculture. This overuse appears to have contributed to the emergence of antibiotic resistant pathogens that are critical to human health. This public health challenge motivated the development of antibiotic alternatives for agricultural applications. We engineered lactic acid bacteria to inducibly express and secrete antimicrobial peptides with activity against Escherichia coli and Salmonella typhimurium and infantis. This is the first demonstration of bacteria engineered to inducibly produce peptides with strong activity against Gram-negative pathogens. These systems may also be used as a foundation for developing a next generation of recombinant bacteria that produce and deliver antimicrobial peptides.
Global biogeography and local adaptation of Streptomyces
(2013-10) Schlatter, Daniel Cameron
Streptomyces play crucial roles in key ecosystem processes including nutrient and plant disease suppression in natural and agricultural systems. Moreover, Streptomyces are major producers of clinically relevant antibiotic compounds. Despite the importance of Streptomyces in natural, agricultural, and clinical settings, we have a limited understanding of Streptomyces ecology and evolutionary biology in natural habitats. Here we characterize the function diversity and biogeography of Streptomyces to shed light on the roles of local adaptation and coevolution in structuring soil Streptomyces communities. Specifically, this work focuses on patterns of antibiotic inhibition, antibiotic resistance, resource use, and phylogeny among sympatric and allopatric Streptomyces communities from across the globe. This work documents the extensive functional diversity of Streptomyces antibiotic inhibitory, resistance, and resource use phenotypes and provides strong evidence that local adaptation, coevolution, and resource competition are crucial drivers of antibiotic inhibition and resistance among Streptomyces.
Investigating the Effects of Antidepressants on Intestinal Bacteria
(2024-04-16) Lebakken, Sophia; Basting, Christopher M; Bailey, Melisa; Schroeder, Ty; Broedlow, Courtney A; Guerrero, Candace; Hemmila, Charlotte; Klatt, Nichole R
Introduction: The gut-brain axis (GBA) involves bidirectional communication between the gastrointestinal tract and brain, which contains many species of bacteria that play an important role in this communication. Major depressive disorder is often treated with antidepressant medications (ADMs) that pass through the gastrointestinal tract; however, the possible adverse effects of ADMs on the gut microbiome are not well characterized. Methods: This project investigates the impact of three selective serotonin reuptake inhibitors, sertraline, fluoxetine, citalopram; one norepinephrine and dopamine reuptake inhibitor, bupropion; and one tetracyclic antidepressant, mirtazapine, on the growth of eight species of gut bacteria, Bacteroides fragilis, Bifidobacterium longum, Bacteroides uniformis, Collinsella aerofaciens, Prevotella copri, Escherichia coli, Akkermansia muciniphila, and Lactobacillus plantarum. Bacteria were treated with various concentrations of each ADM to determine potential impact on growth. We calculated the concentration of drug needed to inhibit growth by 50% (IC50) using spectrophotometry. Results Several ADMs inhibited gut bacterial growth. At 50% bacterial growth inhibition, the most prominent was sertraline (28.742 μM), followed by bupropion (43.976 μM), then fluoxetine (76.449 μM). Citalopram (244.738 μM) and mirtazapine (294.316 μM) exhibited far less inhibition. Discussion These findings suggest ADMs have antibiotic effects that disturb the microbiome resulting in potential consequences for microbiota-GBA interactions. Building on these results, future experimentation will measure uptake and metabolism of ADMs by exposing bacteria to each drug longitudinally. Metabolites will be characterized using liquid chromatography-mass spectrometry. Conclusion Given the profound impact of the gut microbiome on the gut-brain axis, these data provide novel insights into potential mechanisms by which ADMs could have unintended consequences on the gut that may perpetuate, instead of treat, mood disorders thus the microbiome should be further investigated in relation to ADMs.
Investigating the Effects of NOM1 Deficiency on Human Cell Growth
(2009-04-08) Fares, Nancy
NOM1 (Nucleolarprotein with MIF4G domain 1) is a MIF4G/MA3 protein identified at the chromosome 7q36 breakpoint involved in 7;12 translocations associated with certain acute childhood leukemias. Hypothesis: Since NOM1 homologs are essential in both Saccharomyces cerevisiae and Caenorhabditis elegans, and since NOM1 interacts with a number of proteins involved in ribosome biogenesis, we predict that NOM1 is essential in human cells and that NOM1 deficiency will lead to decreased cell growth.
Investigating the Possible Role of a Glycosyl Transferase Protein in the Biosynthesis of Long-Chain Hydrocarbons in Shewanella oneidensis
(2012-04-18) Bergquist, Carl
In the search for alternative sources of energy, new organisms are being looked at as potential biofuel producers. It has been shown that the longchain hydrocarbons produced by the bacterium Shewanella oneidensis can be broken down into usable fuel. It is known that the enzyme OleA is largely responsible for hydrocarbon synthesis, but other proteins may also play a role. In my thesis work, I deleted the gene SO_3174, which encodes a glycosyl transferase protein and was found to be interrupted in a transposon screen for increased hydrocarbon production, from S. oneidensis MR-1. I then tried to show that deleting SO_3174 resulted in an increase in hydrocarbon production just as the interruption of the gene had. The deletion strain showed an increased fluorescence in the presence of Nile Red dye, a hydrophobic dye that can be used to indirectly detect hydrocarbon levels. However, the deletion strain did not exhibit increased hydrocarbons during direct analysis of nonpolar extractions. These same results were obtained from a strain containing both the SO_3174 deletion and Stenotrophomonas maltophilia OleA. Overall, I found that the protein encoded by SO_3174 most likely does not play a role in hydrocarbon biosynthesis in S. oneidensis.
Investigating the Potential of Lemongrass Essential Oil Against Multidrug-Resistant Salmonella Heidelberg in Broiler Chickens
(2018-10) Dewi, Grace
Foodborne illness continues to persist as a global public health concern, despite technological advances. In particular, Salmonella has remained a major foodborne illness-causing pathogen in the United States. Epidemiological studies indicate that poultry are among the major source of Salmonella in developed nations, as food-producing animals like chickens often serve as natural hosts for the pathogen. Among the many serovars of Salmonella enterica, Salmonella enterica serovar Heidelberg has emerged as a pathogen of concern as it causes infections of greater severity in humans and possesses high potential for multidrug resistance. As Salmonella colonization in broiler chickens increases the risk of contamination during subsequent stages of processing, antimicrobial intervention strategies are warranted to ensure food safety and prevent outbreaks from occurring. Decades of efforts to control Salmonella in food systems have indicated that the problem must be approached holistically, with the best intervention strategy ideally applied at multiple stages of production. In that regard, the current study aimed to investigate the efficacy of lemongrass essential oil (LGEO), a generally recognized as safe (GRAS) compound that is obtained from plants of the Cymbopogon species, in reducing MDR S. Heidelberg in vitro, in broiler chicken, and on broiler chicken carcass. The direct effects of LGEO against MDR S. Heidelberg virulence factors, which are important for colonization, survival, and transmission, were first assessed through a series of in vitro assays. Additionally, the potential quorum sensing modulating properties of LGEO against S Heidelberg was indirectly assessed using the biosensor organism, Chromobacterium violaceum. C. violaceum produces violacein, a violet pigment under the vio operon regulation, which is easily observable and quantifiable. Effect of LGEO against S. Heidelberg multiplication was examined through macrodilution assays performed in either tryptic soy broth (TSB) or poultry cecal contents. The effect of LGEO on S. Heidelberg motility was assessed using a motility assay where S. Heidelberg was spot inoculated on the center of semi-solid agar plates supplemented with LGEO. Assays that investigated the effect of LGEO against S. Heidelberg biofilms formation and inactivation of mature biofilms were performed using 96-well microtiter plate assays and 24-well tissue culture plate assays, respectively. Finally, the quorum sensing modulation of LGEO was assessed based on violacein synthesis by C. violaceum. At concentrations of 0.5% (vol/vol) or higher, LGEO was effective in inhibiting multiplication S. Heidelberg by at least 4.0 log10 CFU/mL after 24 hours in TSB and poultry cecal contents (P ≤ 0.05). Addition of 0.15% LGEO in semi-solid motility agar yielded complete inhibition of S. Heidelberg motility (P ≤ 0.05). Concentrations of 0.15% and 1% LGEO were also found effective against MDR S. Heidelberg biofilm formation and inactivation of mature MDR S. Heidelberg biofilms (P ≤ 0.05). Additionally, a reduction in violacein production by C. violaceum was observed with 0.5% and 1% LGEO (P ≤ 0.05). Results of the in vitro assays indicate the potential of LGEO to mitigate the persistence and dissemination of MDR S. Heidelberg in poultry production systems. Subsequently, in vivo experiments were performed to determine the efficacy of LGEO as an antimicrobial supplement either through feed or drinking water to reduce S. Heidelberg colonization in broilers. The efficacy of in-feed supplementation of LGEO against MDR S. Heidelberg was evaluated in 3-week-old broiler chickens. Efficacy of LGEO supplementation through drinking water was first examined in 5-week-old broiler chickens and then in 7-week-old broiler chickens. All birds, except those in NC groups, in all studies, were challenged by crop gavage with 105, 104, and 108 CFU/mL MDR S. Heidelberg, at 2-, 4-, and 6-weeks of age, respectively. For both feed studies where 1% LGEO was supplemented through feed, no significant reduction in S. Heidelberg populations was observed (P > 0.05). Likewise, when supplemented through drinking water, 0.5% LGEO resulted in no significant reduction in both 5- and 7-week old broiler chickens (P > 0.05). However, a significant reduction of 2.4 log10 CFU/gram cecal S. Heidelberg was observed in one replicate involving 5-week old broilers (P < 0.05). In conclusion, the results of this study indicated that LGEO supplementation in either feed or water was not effective in reducing MDR S. Heidelberg colonization in broiler chickens at the current concentrations tested. Finally, the last set of experiments examined the effect of LGEO dip treatments on MDR S. Heidelberg attachment to broiler chicken skin and meat samples. Skin from drumsticks and breast meat samples were spot-inoculated with 4.0 log10 CFU MDR S. Heidelberg per sample and dipped in water containing 0.5, 1, or 2% LGEO for 2, 3, or 5 minutes at either 54°C or 4°C, individually, as well as at 54°C and 4°C in sequence. At 2%, LGEO consistently resulted in significant reduction of MDR S. Heidelberg on both skin and meat samples, though the magnitude of reduction was lesser on meat than on skin (P < 0.05). Sequential dip treatments performed on skin yielded comparable results to that observed in individual dip treatments at scalding temperatures. However, subsequent dip treatments were also not as effective on meat compared to skin samples. All concentrations of LGEO effectively inactivated S. Heidelberg from the scalding and chilling treatment waters for both skin and meat samples (P < 0.05). The results indicate that LGEO could be utilized as a natural antimicrobial intervention in scalding and chilling waters to reduce S. Heidelberg on poultry carcasses. In conclusion, the results are indicative of the direct antibacterial effects which LGEO exerts on S. Heidelberg in vitro and on broiler chicken skin samples. However, results of the in vivo study suggest that additional studies must examine the possible factors that may have impeded LGEO’s efficacy against foodborne pathogens in broiler chickens. Overall, the efficacy of LGEO observed on retail broiler chicken samples is indicative of its potential use to control MDR S. Heidelberg transmission in poultry production systems.
Mechanisms of Androgen-Mediated Repression of the Maspin Tumor Suppressor Gene in Prostate Cancer
(2009-04-08) Bader, David
It is estimated that one in six men in North America will be diagnosed with prostate cancer (PCa) during his lifetime. Localized PCa is often treated using surgery and radiation. Advanced and metastatic PCa can be treated by blocking the production or action of androgens, the male sex hormones. This androgen depletion therapy is only temporarily successful because PCa frequently returns in an androgen-refractory form that is resistant to hormonal manipulations and capable of growing in an androgen-depleted environment. Androgen receptor (AR) is a nuclear receptor transcription factor necessary for normal prostate cell growth and function as well as for growth of PCa. Androgens activate the AR, which translocates to the nucleus where it transcriptionally activates or represses target genes. One such gene is the Maspin tumor suppressor. Maspin is a proteinase inhibitor that serves to prevent proteinase degradation of the extracellular matrix, which is prerequisite to tumor invasion and metastasis. Androgens transcriptionally repress Maspin, but the mechanisms have not yet been fully characterized. To investigate the mechanisms of Maspin repression, a plasmid containing the luciferase reporter gene under the control of the Maspin promoter was constructed and transfected into VCaP and LNCaP PCa cell lines. Transfected cells were treated with dihydrotestosterone (a natural androgen) or mibolerone (a synthetic androgen) for 24 hours. Luciferase activity was subsequently measured by dual luciferase assay. These experiments have indicated that the AR may not directly repress Maspin transcription. Ongoing research will utilize real time PCR to determine whether AR inhibits Maspin transcription via a direct or indirect mechanism.
Mechanisms of Androgen-Mediated Repression of the Maspin Tumor Suppressor Gene in Prostate Cancer
(2010-04-21) Bader, David
Prostate Cancer (PCa) is the second leading cause of cancer death in American men; one in six will be diagnosed with the disease during his lifetime. The androgen receptor (AR) is a transcription factor necessary for normal prostate cell growth as well as for growth of PCa. When AR is activated by androgens, it translocates to the nucleus and exerts transcriptional activation and repression of target genes. Significant efforts have focused on characterizing genes that are activated by AR such as prostate specific antigen (PSA), a marker for PCa screening. Genes that are transcriptionally repressed by AR are also likely to play a role in the progression of prostate cancer, yet the mechanisms behind their repression have received less attention. One such gene is maspin, a tumor suppressor gene that is repressed by androgens. Maspin expression is associated with increased cellular adhesion, increased sensitivity to cellular apoptosis, and decreased angiogenesis in the tumor microenvironment. In this study, we demonstrated that (1) Maspin is repressed following androgen treatment, (2) the repression is mediated in a direct manner, and (3) Androgen antagonists such as bicalutamide do not affect the ability of AR to repress maspin. Ongoing research will continue to investigate AR’s role in the repression of maspin. Understanding the underlying mechanisms by which AR represses maspin and other target genes may reveal novel opportunities for developing new prostate cancer therapies.
Microbiome and Immune Response to Salmonella enterica serovar Typhimurium and Lawsonia intracellularis Infection in Swine
(2018-05) Leite, Fernando Lopes Leivas
Salmonella enterica is a leading cause of foodborne illness world-wide. In the US alone Salmonella is responsible for over 1 million cases of disease a year in humans and causes an estimated loss of more than 3.5 billion dollars annually. Pork is frequently associated with food borne illnesses caused by S. enterica in humans, many of which are attributed to Salmonella enterica serovar Typhimurium. Efforts to reduce the incidence of salmonellosis due to meat consumption have mainly remained ineffective. This study extends previous findings that pigs are more susceptible to colonization by Salmonella enterica when co-infected with the pathogen Lawsonia intracellularis. We determined the composition of the porcine gut microbiome in response to co-infection to determine how potential disturbances caused by L. intracellularis could favor S. Typhimurium. This analysis revealed that L. intracellularis led to a decreased abundance of Clostridium species and Clostridium butyricum in addition to other changes that may favor S. Typhimurium. We also investigated if vaccination against L. intracellularis could have an effect on the shedding S. Typhimurium and found that vaccination significantly reduced S. Typhimurium shedding in animals co-infected with L. intracellularis. To better understand the host response to L. intracellularis, we performed transcriptome analysis of infected mucosal tissue and found that infection induced a signature of genes associated with inflammation and proliferation in the gut. We then tested zinc supplementation, which is known to impact immune function, and found that zinc amino acid supplementation led to a significant reduction of lesions caused by L. intracellularis. Finally, we investigated whether co-infection of enterocytes in vitro caused increases in certain inflammatory cytokines. We found that L. intracellularis up regulated expression of IL-8 and TNFα, two pro-inflammatory cytokines crucial to the pathogenesis of S. Typhimurium infection. This research suggests that increased inflammation mediated by L. intracellularis along with changes in microbiome composition are likely responsible for enhancement of S. Typhimurium infection in swine. We have also identified that L. intracellularis vaccination and zinc amino acid complex supplementation are two promising alternatives to the use of antimicrobials in swine.
Non-Conventional Methods of Soil-borne Fungal Disease Management in Soybean and Pea
(2009-04-08) Testen, Anna L.
As demand increases for organic and sustainable methods of agricultural production, interest also increases in non-conventional methods of disease management. Non-conventional methods for managing fungal disease, such as biological control and biorational pesticides, are becoming more important as chemical inputs are scrutinized and fungicide resistance develops in pathogen populations. In this study, non-conventional methods of managing soybean sudden death syndrome, a fungal disease caused by Fusarium virguliforme, and Aphanomyces root rot of peas caused by A. euteiches, were compared to conventional (chemical) methods of control. A biological control agent Bacillus pumilus and rapeseed meal were compared to two chemical fungicides in greenhouse assays to determine which treatments could reduce disease severity and maintain plant biomass when plants were subjected to conditions suitable for disease development. B. pumilus was also tested for antagonism in vitro against these pathogens, and all fungi showed reduced growth in the presence of B. pumilus. The results of this study showed that conventional methods of fungal disease control were more effective than non-conventional methods in maintaining plant biomass under conditions conducive to disease. However, the non-conventional methods reduced disease severity and may possibly have value in organic production systems. In the United States, Minnesota is the third largest producer of soybeans and the largest producer of organic soybeans and a major producer of peas, so any treatment that reduces the impact of soil-borne pathogens has the potential to make a large economic impact.
Observing Bacterial Diversity in Glacial Till using 16S rRNA Sequencing
(2012-04-18) Burnes, Andrew
Recent surveys of domestic wells in west central Minnesota have shown that the levels of arsenic in 50% of wells are higher than standards set by the EPA, creating a serious health risk for the surrounding populations. Investigations show that the arsenic contamination originates from naturally occurring arsenic in the surrounding glacial sediments that are part of the Des Moines Lobe; However, wells present in the Des Moines lobe are not universally contaminated with high levels of arsenic, suggesting that the process of contamination is controlled by other factors such as the presence of bacteria. This experiment aimed to observe if there are bacteria present in these soil samples that could play a role in arsenic contamination. To do this, DNA was extracted from core soil samples and the 16S rRNA gene was amplified using PCR. The PCR product was then cleaned and sent to a private lab to be sequenced using 454 pyrosequencing. Results are expected to indicate the presence of bacterial genera that have the ability to trigger arsenic contamination. These findings could further show the importance and scope of microbial populations on a geological level and could lead to avenues of bioremediation.
Plastic Biotransformation Technologies: Development of a Novel Environmental QPCR Assay for Polyethylene Terephthalate Hydrolase, and Isolation/Characterization of Polyethylene Degrading Fungi and Bacteria from Environmental Samples
(2020-08) Wedin, Nelson
Plastic production, use, and accumulation in the environment—including in the bodies of humans and other animals—have been increasing for decades and are a cause of growing global concern. Common plastic waste is generally considered to be non-biodegradable. In recent years, though, a growing assortment of bacteria and fungi capable of degrading a variety of common recalcitrant plastics have been identified. In general, the enzymes capable of depolymerizing long-chain hydrophobic plastic polymers are not well studied. However, Poly(ethylene) Terephthalate (PET) Hydrolase is well described in the literature and is thus a suitable target for molecular identification and quantification technologies. PET is the plastic polymer used in most plastic water bottles and in polyester fabric. The discovery of PET-degrading organisms and PET hydrolases is leading to the generation of biochemical technologies for the recycling and upcycling of PET, as well as the search for PET hydrolases that have greater activity on commercially relevant PET polymers. The incidence of PET hydrolase in metagenomes appears rare, though the quantification of PET hydrolases in environmental samples is unknown. Because plastic-biotransforming organisms are considered rare and slow growing, the process of isolating and characterizing these organisms is long and involved. This thesis presents two distinct, but interrelated, experimental trajectories related to the advancement of the study of plastic biotransformation. The first study focused on the molecular level, and the second study focused on microorganisms. In the first study, novel Quantitative Polymerase Chain Reaction (QPCR) primers were developed and tested for the ability to selectively amplify PET hydrolase genes from environmental samples. The products from these primers, used on eight environmental DNA extracts, were subjected to amplicon sequencing. Multiple sequence analysis methods confirmed the successful amplification of published PET hydrolase sequences, as well as sequences that show a high potential for being PET hydrolases. The on-target hit percentage and on-target hits varied substantially across samples, and this assay will require further optimization for specificity and quantification efficacy before it can be used for absolute quantification (i.e., gene copies/ ng DNA). There is reason to suggest that this assay can measure relative abundance of PET hydrolases, and thus relative genetic PET bioconversion potential. By providing comparative analysis that is both faster and less expensive than traditional techniques, this tool enables the rapid determination of ideal conditions to find and cultivate PET hydrolytic organisms. The core results of this analysis are presented in Figures 26, 28, and 29. In the second study, the focus was to enrich for and isolate (as individual species or consortia), identify, and evaluate microorganisms capable of Polyethylene (PE) biodegradation and biomineralization by culturing microbes in media where PE is the sole carbon source. Although the impact on the environment of PET (the polymer studied in the first study) is substantial, it pales in comparison to the impact of PE, which is used primarily for single-use items and is the most abundant type of plastic manufactured on the planet. Currently, no enzymes capable of degrading PE are well described, though some fungi and bacteria have been shown to degrade and utilize PE as a carbon source. In this set of experiments, preferential focus was given to fungi. Microbes that degrade and live on LDPE powder were enriched from environmental sources. A cogent argument for the confirmation of Low-Density PE (LDPE)-biodegrading organisms is presented from the limited data available (see below for limitations resulting from COVID-19 lockdown). LDPE-biodegradation can be seen in the isolate “Ath” (flask 6, a filamentous fungi that macroscopically appears to be Trichoderma sp.). The macroscopic observation of PE biotransformation for culture “Ath” is documented in Figure 33, where Flask 6 clearly shows modification to the PE powder. Modification increases with longer incubation and is not observed in the otherwise-identical non-inoculated control (Flask 34, Figure 33). Similar results are observed for other cultures, along with the growth of biomass and spore production. Thus, LDPE-biodegradation is also the most likely explanation for at least nine other environmental isolates. And microscopic confirmation of growth in this culture as well as others is presented in conditions where the only carbon source is PE powder. Both bacteria and fungi were shown to degrade the low molecular weight PE powder, though quantitative analysis on commercially relevant PE films was not completed. Tentative taxonomic hypotheses and the exciting possible implications of PE degradation within these taxa are presented, though genetic identification was also unable to be performed due to lockdowns. This research project was cut short prematurely due to mandatory laboratory lockdown in response to the COVID-19 pandemic. While both prongs of the studies described in this thesis were affected, the isolation and PE biodegradation assay was more seriously limited in that all quantitative analyses were unable to be performed. The discussion section reflects the limitations that resulted, as well as the adjustments that were made to compensate for these limitations.
The relationship of gut microbiota in standard and overweight children, before and after probiotic administration
(2019) Linhardt, Carter A; Clayton, Jonathan B; Hoops, Suzie; Amin-Nordin, Syafinaz; Knights, Dan
The usage of probiotic foods and supplements has been widely considered part of a healthy diet by supplementing the gut microbiome with beneficial bacteria. Although the usage of probiotics is a common dietary accessory, there is limited reproducible evidence showing bacterial colonization, thus limiting long term effectiveness. We administered Yakult, a commercial probiotic composed of Lactobacillus paracasei strain Shirota, to overweight and standard weight school children in Malaysia. Using a crossover intervention study design, two groups of school children were administered the probiotic supplement or continued their typical diet in sequential 5-week intervention periods, separated by a 5-week washout period. Fecal samples were collected every five weeks over the course of the 15-week study period. The gut microbiome of each subject was analyzed using 16S rRNA gene sequencing. We observed significant differences in Lachnospiraceae, Coproccus, Roseburia, Pyramidobacter, and Bacteroides ovatus between weight classes. However, differences in overall microbiome diversity between weight classes were not found to be significant. Subjects clustered according to their relative abundance of well-known genera Bacteroides and Prevotella, regardless of age, gender, or weight class. Overall, individual-to-individual variation overshadowed trends in gut microbiome composition associated with probiotic administration.
Robert Koch – From Obscurity to Glory to Fiasco
(Journal of Opinions, Ideas & Essays (JOIE), 2011-10) Dworkin, Martin
Robert Koch went from an obscure country physician to be the discoverer of the etiology of anthrax, the inventor of the technique of pure culture bacteriology and with that to the isolation of the tubercle bacillus and its identification as the etiological agent of tuberculosis. These successes propelled him to world-wide glory. In his search for a cure for tuberculosis, he proposed that tuberculin was such a cure. Unfortunately this turned out to be false, and his continued advocacy was a fiasco. Nevertheless his formulation of the germ theory of disease transformed medicine and led to a remarkable series of successes that clarified the etiology of a large number of infectious diseases.
Seroprevalence of Powassan Virus in Healthy Central and Northeastern Minnesota Blood Donors
(2012-04-18) Braue, Krystina
Powassan virus is the only member of the tickborne encephalitis arboviral family present in North America. It is a flavivirus transmitted by Ixodes scapularis, the deer tick, and can cause severe, potentially fatal meningoencephalitis. Powassan has emerged as a national public health concern in the last sixty years, and as a local concern in Minnesota in the last five years. Here we show that Powassan exposure in 2009 had a much larger geographic and demographic range than indicated by clinical reporting of human cases. Potential exposures were identified in samples from northeastern Minnesota, particularly from St. Louis County. However, we also observed a number of exposures in the non-endemic metro area and surrounding suburbs, despite being early in the season. This suggests that “cabin culture,” the traffic of southern Minnesotans to cabins in north-central and northeast Minnesota, plays a role in non-endemic exposure rates. No significant risk was associated with a particular age group, although 66.7% (4 of 6 donors) of recent exposures were in donors younger than 25 years. More work needs to be done to determine the count of true Powassan infections, but the trends identified in the study appear to suggest that Powassan is positioned to become a significant public health threat similar to West Nile. Identification of the extent, direction, and speed of the emergence of Powassan will provide the framework for continued surveillance, educational research development, and analysis of potential economic impact.
Systems analysis of pheromone signaling and antibiotic resistance transfer in Enterococcus faecalis
(2018-01) Bandyopadhyay, Arpan Anup
Antibiotics have been an extremely important weapon in the fight against bacterial infections for over half a century. However, excessive use of antibiotics has led to increased frequencies of resistance among bacteria. Antibiotic resistance is an inevitable outcome of natural selection as organisms undergo random mutations to escape lethal selective pressure. Many of these resistant bacteria can also transfer their genetic material to other bacteria through direct cell-cell contact via conjugation, further facilitating the spread of resistance. The human gastrointestinal tract, replete with a high density of bacteria and often exposed to antibiotics, provides an ideal environment for antibiotic resistance genes to arise and propagate through bacterial populations. Enterococcus faecalis, a commensal bacterium of the human intestinal tract, has emerged as a major cause of healthcare-associated infections. Treatment of these infections has become increasingly difficult with the emergence of E. faecalis strains that are resistant to multiple major classes of antibiotics. The organism’s ability to acquire and transfer resistance genes and virulence determinants through conjugative plasmids poses a serious clinical concern. Here we present our study on conjugation of a tetracycline-resistance plasmid pCF10 which is regulated by intercellular communication using two antagonistic signaling peptides. An inducer peptide produced by the plasmid-free recipient cells functions as a “mate-sensing” signal and triggers the conjugative plasmid transfer in donors. The donors encode an inhibitor peptide on the plasmid which represses conjugation and functions as a "self-sensing" signal, reducing the response to the inducer in a density-dependent fashion. This form of dual signaling-controlled conjugation was also found to be prevalent across other pheromone-responsive plasmids, including pAD1 and pAM373. Though the donors calibrate their conjugation response in accordance with the relative abundance of donors and recipients, plasmid transfer can occur under otherwise unfavorable conditions, such as low inducing pheromone and high inhibitor concentrations. To better understand this apparent inconsistency, we formulated a stochastic mathematical model that integrates intracellular molecular regulation of conjugation and interactions between donors and recipients through the signaling peptides. Kinetic parameters for the model were estimated from literature and augmented by experimental RNA-Seq data and binding constant measurements. Simulations of the stochastic model and single-cell analysis using transcript quantification by HCR-FISH and GFP reporter fusions revealed distinct subpopulations of rapid responders under unfavorable conditions for plasmid transfer. We developed a series of fluorescent reporters to track the uninduced/induced donors, recipients, and uninduced/induced transconjugants in real-time using confocal microscopy and flow cytometry. We are further developing a microfluidic gut model which allow for co-culturing of human and bacteria cells in an in vivo-simulated microenvironment. This system will be used to model the in vivo biology of conjugation and gain a better mechanistic understanding of the community balance between the microbial inhabitants of the GI tract. A better understanding of the bacterial signaling mechanisms in vivo and the downstream effects on microbiome community balance may help us identify alternate strategies to prevent the spread of antibiotic resistance.

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