Microbiological Sciences Masters Theses

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lntraspecific Variation In Pathogenic Cryptosporidium parvum
(North Dakota State University, 2010) Herges, Grant Richard
Cryptosporidium causes cryptosporidiosis, an infectious diarrheal disease, which can become chronic and life-threatening in immunocompromised individuals. Cryptosporidium parvum and C. hominis are the primary causes of human cryptosporidiosis. Of these species, C. hominis only infects humans while C. parvum additionally infects ruminants, in particular neonatal calves. Therefore, understanding the transmission dynamics of C. parvum, particularly the specific contribution of zoonotic and anthroponotic transmission, is critical to the control of this pathogen. Cryptosporidium parvum genotypes have been identified which appear to be restricted to a single host, which suggests that this may be a heterogeneous species, with varying infection and transmission dynamics. The first objective to this thesis was to determine the population structure of pathogenic C. parvum in the upper Midwest United States. A total of 216 isolates from cases of human cryptosporidiosis in Minnesota and Wisconsin and 64 isolates from diarrheic calves in Minnesota, Wisconsin, and North Dakota were genotyped at 8 polymorphic loci. A total of 213 isolates, 167 from humans and 46 from calves, had complete multilocus types (MLT). There were 93 different Ml Ts and sixty of those Ml Ts were only represented by one isolate. Population analysis revealed a highly recombining, panmictic population that does not have any genetic, geographic, or host sub-structuring. The second objective to this thesis was to determine the variability in the in vitro infectivity of different C. parvum strains, IOWA II and Moredun. A quantitative RT-PCR approach was used to quantify expression of target genes during infection of HCT-8 cells. Fluorescence microscopy was used to quantify life cycle stages during infection. Our data showed that the IOWA II reached its sexual stages earlier and had a greater number of trophozoites/meronts at 24 h p.i. than Moredun. The host used for propagation of C. parvum also affected subsequent infectivity of HCT-8 cells.
Sexual Dimorphisms in the Adipose Biology of Vasoactive Intestinal Peptide Deficient Mice
(North Dakota State University, 2022) Hawley, Emma
Both VIP and PACAP deficient mice display impaired fat storage, but only the molecular adipose characteristics of PACAP KO mice are known. While mice deficient in these peptides are lean, supplementation could promote weight gain in livestock after establishing tissue distribution. We investigated both sides of this 'metabolic coin' by measuring serum metabolites, adipocyte area, and pscWAT gene expression in VIP deficient mice and screening 15 ruminant tissues for VIP, PACAP, and their receptors using RT-qPCR. We found male VIP deficient mice displayed hypertrophic adipocytes, low serum FFAs, and suppressed relative mRNA expression of TMEM26. In ruminants, VIP and PACAP showed similar expression patterns, whereas their receptors had divergent tissue distributions. We conclude that VIP deficiency affects the investigated adipose characteristics in male but not female mice. Additionally, we predict that VIP supplementation in ruminants would promote weight gain since its mRNA distribution is similar to mice.
Effects of Intensive Agricultural Management Practices on Soil Microbial Assembly and Recruitment
(North Dakota State University, 2022) Sciences, Microbiological; Nath, Debdutta
Soil microbial communities play an important role in ecosystems in various ways to promote healthy and fertile soil. However, intensive agricultural practices with excessive tillage and fertilizer applications can affect the abundance and community structure of microbial communities in soil as well as their assembly and recruitment by plant roots. Using amplicon sequencing and microscopy, we have examined bacterial and fungal communities under different tillage and fertilizer treatments in a 34-year-old field-trial at the Carrington Research Extension Center of NDSU. We observed that fertilizer application has a significantly stronger effect than tillage on soil properties, as well as the overall soil microbial abundance and community structure. Significantly higher mycorrhizal colonization was found under organic manure application. Overall, the results of this study can improve our understanding of the effects of fertilizer application on soil microbial communities and how management practices can be optimized to reduce the imprints of intensive agriculture.
The Link Between Gut Microbiota Metabolism and Host Gluconeogenesis by Vasoactive Intestinal Peptide
(North Dakota State University, 2022) Dawlaty, Razia
The gut microbiota (GM) plays a beneficial role in host metabolism. In mammals, the GM ferments dietary fiber into short chain fatty acids (SCFA), like propionate, that improves glucose metabolism. Rats fed a propionate diet increased intestinal gluconeogenic (IGN) gene expression, which was blocked by treatment with the neurotoxin, capsaicin, suggesting a neuronal-dependent mechanism. We hypothesized that the gut neuropeptide, vasoactive intestinal peptide (VIP), links GM derived propionate to IGN expression. We fed VIP deficient mice a 5% propionate chow diet (n=60) for 2 weeks and measured mRNA levels for GN genes by dPCR. Basel intestinal and liver GN mRNA expression was dysregulated by the loss of VIP. GN mRNA levels in liver were differentially altered in males versus females fed a propionate diet in a VIP-dependent manner. We conclude that VIP regulates basal intestinal and hepatic GN mRNA expression and mediates propionate induced GN mRNA changes in liver.
In the Pursuit of Poultry: β-Phenylethylamine and Ethyl Acetoacetate as Antimicrobials on Chicken
(North Dakota State University, 2022) Bauer, Erika Shay Hedrick
This research examines the effect of β-phenylethylamine (PEA), a natural trace amine commonly found in food, and ethyl acetoacetate (EAA), an FDA approved flavoring agent and food additive, as novel antimicrobials on store-bought chicken thighs in a 5-minute immersion. In the first part of this experiment, 5%, 7.5%, and 10% treatments of β-phenylethylamine and ethyl acetoacetate were compared to control H2O treatments utilized on chicken thighs. 10% treatments of PEA and EAA had significant reductions in counts of total aerobic bacteria and Pseudomonas spp. grown at 20°C by >1 log10 CFU/g of chicken meat. In the next experiments regarded 10% EAA as an antimicrobial on potential pathogens on chicken meat. The treatments of 10% EAA only succeeded in partial efficacy in the reduction of inoculated Salmonella spp. and Campylobacter spp. on chicken thighs.
The Response of Salmonella enterica and Enterohemorrhagic Escherichia coli in Low-Moisture Environments
(North Dakota State University, 2021) Lauer, Jessica Rae
The recent increase in outbreaks of low-moisture foods due to contamination with foodborne pathogens has led to an increase in interest of the response of these pathogens to low-moisture environments. In addition, knowledge of the response of foodborne pathogens to immediate desiccation stress is sparce. The first objective was to evaluate the long-term survival of enteric pathogens on wheat grain over the course of a year. Hard red spring wheat was inoculated with strains of Salmonella enterica and enterohemorrhagic Escherichia coli (EHEC) and their survival was monitored for a year. Strains of Salmonella enterica were detected over the course of 52 weeks, while all strains of EHEC passed below the limit of detection by 44 weeks. The second objective was to evaluate the tolerance of various enteric pathogens to sudden desiccations stress and wide variation was seen between species and among strains and growth conditions.
Validation of Effects of SNV From GWAS on Biofilm Phenotype Using a CRISPR System With Universal Guide RNA Sequence
(North Dakota State University, 2021) Gramillo, Allan Kelvin
Extraintestinal E. coli have adapted to survive in secondary environments outside of the intestines of a host. The genetically diverse phylogroup D was use to validate two previous GWAS analysis that identified single nucleotide variants to have a positive or negative effect on biofilm formation. We selected ten variants from TreeWAS based on current literature to identify patterns among a new set of E. coli isolates, to identify SNV that can be used as genetic markers for biofilm formation. DBGWAS was used to predict and validate the predicted value of a SNV, by conducting an allelic exchange by using a CRISPR/Cas9 system, pCAGO, that allowed for scarless genome editing. This allelic exchange and deletion of the gene led to no statistical significance when biofilm formation and growth rate were tested. This led to variation in biofilm formation but didn’t affect the phenotype in a statistically significant manner.
ß-phenylethylamine: A Novel Chemo-Attractant Agent
(North Dakota State University, 2021) Burg, August Henry
Bacterial metabolism and physiology are finely tuned mechanisms that maintain homeostasis for the bacterium and allow for responses to environmental signals. Responses could include anything from regulation of cell division to the expression of virulence factors leading to serious infection. This thesis explores the role of neurotransmitter molecules and ß-phenylethylamine (a structurally similar molecule to catecholamines) on the physiological characteristics of flhD expression, biofilm formation, and chemotactic behaviors in the E. coli organism. We observed changes in physiology leading to chemotactic changes in the presence of ß-phenylethylamine through the Plug Agarose Assay as well as the Microfluidic Assay. In a comparison with serine, the amino acid and documented chemoattractant agent, ß-phenylethylamine was revealed to be a novel chemoattractant agent with comparable influence on the bacterium.
Potential for Phenotypic Plasticity in the Hyperosmotic Stress Response of Diverse E. coli
(North Dakota State University, 2021) Schwartz, Madelyn Ashley
Escherichia coli inhabits multiple environments that contain varying physical stresses which emphasize the importance of adaptation. The large pangenome of E. coli can account for some strain-to-strain variances in phenotype, but phenotypic plasticity may be another key factor. The key behind phenotypic plasticity may be transcriptional regulators such as the sigma factor RpoS. The extent of phenotypic plasticity and possible mechanisms in stress conditions has not been heavily studied in bacteria. We looked at the presence of plasticity in the growth rate of E. coli under a hyperosmotic condition. RNAseq was used to explore the connection between RpoS and phenotypic plasticity. The conserved genes within all the isolates and their connection to hyperosmotic stress were also explored. Phenotypic plasticity of growth rate was observed among the strains on a phenotypic level. Genes associated with osmotic stress, fermentation, and cell envelope synthesis were found to be significantly expressed within all isolates.
Characterizing Humidity, Sex, and B-Cell Gene Regulation in Fungal Allergic Asthma
(North Dakota State University, 2020) Kusick, Emma Claire
Asthma is a debilitating lung disease that affects nearly 300 million people worldwide. Environments with high humidity and subsequent mold exposure often trigger allergic asthma. Sex differences have been reported in the incidence, prevalence, and severity of asthma. B-lymphocytes are recruited in high numbers to the allergic lung in response to the inhalation of Aspergillus fumigatus mold spores (conidia). In this work, we used a mouse model of allergic fungal asthma to assess environmental humidity, sex, and B-lymphocytes in an inhalational model of allergic fungal asthma. Our results showed that animals sensitized in low humidity conditions had no airway hyperresponsiveness (AHR), inflammation, but an increase in IgG3 antibody production. Males weighed more than females, female mice had more fibrosis and produced more IgG3 Ab, but sex showed no impact on low humidity. C19+ B-lymphocytes differentially downregulated multiple genes related to allergic asthma returning the body to homeostasis.
Efficacy of Chlorine Dioxide Fumigation on the Spores of Paenibacillus larvae, the Causative Agent of American Foulbrood Disease in Honeybees
(North Dakota State University, 2019) Mahdi, Osama Salih
Honeybees (Apis mellifera) play a critical role in agricultural pollination. However, their health and numbers are in decline. A major cause of this decline is bacterial diseases, of which American foulbrood disease (AFB) is particularly important and troubling. Since the causative agent, Paenibacillus larvae, is spore forming, it can resist antibiotics, many disinfectants, and environmental stresses. We provide protocols and methods for the growth, maintenance, sporulation, and germination of P. larvae. Also, this study investigates the sporicidal activity of ClO2 on P. larvae spores. The gas efficacy depends on treatment time and gas level. The effective level was 645-811 ng/ml ClO2 for 30 min, 191-198 ng/ml for 1 hour, 21-18 ng/ml for 2 h and 7-16 ng/ml ClO2 for 4 h. For decontamination of contaminated surfaces, 214- 245 ng/ml ClO2 for 1 h and 191- 200 ng/ml ClO2 for 2 h completely inactivate the spores.
Gene Regulation in Biofilms
(North Dakota State University, 2011) Samanta, Priyankar
Sessile bacterial communities which form on the solid surface or solid-liquid interface are known as biofilms. Both single species and multispecies biofilms are characterized by an extracellular matrix of polymeric substances which gives them several hundred times more antibiotic resistances than a planktonic bacterial culture. Though bacteria are the most common causative agent of various diseases, because of the high antibiotic resistance, biofilms cause complications of various diseases like cystic fibrosis, prosthetic valve endocarditis, chronic pulmonary diseases, catheter-associated urinary tract infections and several other diseases. From past studies, quorum sensing has been established as a novel target mechanism against biofilms; in this study, the two-component signal transduction systems (2CSTSs) have been focused. Once better understood, 2CSTSs can serve as a novel drug target and prevention mechanism for biofilm associated diseases. According to prior high-throughput experiments and phenotype microarray experiments by our lab, several 2CSTSs like OmpR-EnvZ, RcsCDB along with the global regulator FlhD/FlhC were hypothesized to have an important effect on various developmental stages of biofilm formation. From that past study, we postulated that acetate metabolism may be an important aspect for biofilm formation. In this study, we tested and confirmed this hypothesis. We observed biofilms formed by several mutants in 2CSTS, as well as mutants in acetate metabolism, using Scanning Electron Microscopy (SEM). We found quantitative and qualitative differences in the biofilm of the acetate mutants when compared to their isogenic parental Escherichia coli strain. An additional mutation in rcsB with acetate mutant strains forms less clumpy biofilms whereas an additional mutation in dcuR results in the formation of less biofilms. So the structural and the quantitative differences of acetate mutant biofilms depend on additional mutations in rcsB and dcuR. Though a number of studies have been done on the temporal gene expression within biofilms, spatial gene expression of the mature biofilm is a big gap of knowledge. The future aim of this study is to study the temporal as well as the spatial gene expression of different 2CSTSs in the biofilm. In my MS thesis, I have constructed selected promoter fused GFP /RFP plasmids and some other fusion plasmids were purchased from the promoter collections from Open Biosystems, lastly E. coli AJW678 bacterial strains were transformed with these GFP /RFP fused plasmids. A 96 well microtiter plate assay was performed to study the temporal expression from the promoters by quantifying the fluorescence intensity in the planktonic culture. According to this experiment, the highest expression of flhD was after 20 hours whereas, the expression of ompR increases up to 7 days, which indicates that the flhD expresses earlier than ompR. The decreasing phase of flhD expression was paralleled by the sharpest increase in ompR expression as phosphorylated OmpR is an inhibitor of flhD expression.
Biofilm Formation of Escherichia coli from Surface Soils is Influenced by Variation in Cell Envelope, Iron Metabolism, and Attachment Factor Genes
(North Dakota State University, 2018) Petersen, Morgan L.
Biofilm formation may increase survival and persistence of Escherichia coli in the highly variable conditions of soil environments, though it remains unknown the extent variation in biofilm formation affects survival. We asked what genetic traits influence biofilm formation in phylogroup D E. coli isolates from surface soils, and are they associated with the soil environment? Biofilm density was analyzed and compared with soil environment characteristics. Isolates produced more biofilm per unit growth at 15°C than 37°C. Biofilm formation was greater in soil isolates than fecal isolates and in soils with moisture and higher calcium and pH levels. A GWAS analysis found variants involved in cell envelope formation and structure were associated with biofilm formed at 37°C, and stress response and iron acquisition variants were associated with biofilm formed at 15°C. Motility variants were associated with a negative effect on biofilm formed and adhesion variants associated with a positive effect.
Attachment and Biofilm Formation of Foodborne Pathogens
(North Dakota State University, 2017) Smith, Sara Jean
Outbreaks of Listeria monocytogenes, Salmonella, and Escherichia coli are increasingly attributed to fresh produce. Current control measures have been assessed for decades, with no alternatives adopted. Sources were identified, reducing flhD transcription and biofilm amounts nearly 2-fold. β-phenylethylamine (PEA), reduced growth and biofilm 96% and 70%, respectively. Curli production was assessed and found to be microorganism-, strain-, and/or serotype-dependent. Reporter fusions were constructed, evaluating expression of Listeria cellulose protein (Lcp). Plcp was not impacted by conditions used. Conditions were then used in attachment of L. monocytogenes to stainless steel. Attachment was significantly reduced by 5 ppm chlorine and 2% lysate. Small molecules could be alternatives to current control measures. More research is needed on what induces curli production. Controls confirm that reporter fusions are an effective way to discover signals impacting gene expression. Attachment/expression assays, indicate that something other than Lcp are responsible for changes in attachment to stainless steel.
Glucose Uptake by the Cellulolytic Rumen Anaerobe Bacteroides Succinogenes
(North Dakota State University, 1986) Franklund, Clifton Victor
Glucose uptake by the cellulclytic rumen anaerobe, Bacteroides succinogenes S85, was measured under conditions that maintained anaerobiosis and osmotic stability. This organism was found to possess a highly specific, active transport mechanism for glucose. Evidence for a phosphoenol-pyruvate:g1ucose phosphotransferase system was not detected. Compounds that inhibit electron transport systems (non-heme iron chelators, and sulfhydryl reagents) were effective inhibitors of glucose uptake. The strongest inhibitors were compounds (proton and metal ionophores) that interfere with maintenance of the proton motive force. Compounds which interfere with ATP synthesis also inhibited glucose uptake, but a role for ATP in energizing uptake could not be inferred from these results. Oxygen prevented glucose uptake (75% inhibition), reflecting possible active sulfhydryl centers (above) or autooxidation of electron transport components. The results suggest the fumarate reductase-coupled electron transport system of B. succinogenes can generate a proton motive force that is used to energize glucose uptake. Na+ and Li+. but not K+, stimulated glucose uptake and may partly account for the growth requirement of B. succinogenes for Na+. However, the data were insufficient to conclude that glucose uptake occurs by a Na+ symport mechanism. Spheroplasts of B. succinogenes transported glucose as well as whole cells, indicating glucose uptake is not dependent on a periplasmic glucose binding protein. A variety of sugars including the nonmetabolizable analog, [inversely proportional symbol]-methylglucoside. did not inhibit glucose uptake. Only cellobiose and 2-deoxyglucose were active and neither behaved as a competitive inhibitor. Metabolism of both sugars was probably responsible for the inhibition. Cellobiose-grcwn B. succinogenes showed a reduced ability to transport glucose compared to glucose-grown cells. This may indicate regulation of synthesis of the glucose carrier protein by cellobiose through a mechanism other than catabolite repression. Differences in the ability to transport glucose were detected between transition cells (transition from lag to log phase of growth) and log-phase cells. However, the differences were not due to different glucose transport mechanisms. Alterations in the structural integrity of the cell envelope, as reflected by osmotic- and cold-sensitivity features of transition and log cells, may have affected the glucose uptake abilities in these cell types.
16S Ribosomal RNA and Phylograms: Characterizing Student Reasoning to Learning Outcomes from the American Society for Microbiology Curriculum
(North Dakota State University, 2017) Grassie, Chelsey Lee
The American Society for Microbiology (ASM) has established a suggested curriculum for introductory microbiology courses that includes a focus on evolution. However, no data is published to describe how proficiently students address the learning outcomes, in part because validated assessments do not exist. Thus, the goal of this project was to develop assessment prompts that capture student understanding about fundamental statement five under the core concept of evolution. In total, 167 written responses were collected from upper-division microbiology courses, with pre-pharmacy and microbiology majors comprising the majority of students (74.6%). Two coders coded all written responses, and five student interviews were conducted. Results indicate that students have not retained instruction on 16S rRNA, or have not been exposed to it in their classes. Additionally, most students have not been exposed to phylograms, and are unfamiliar with genetic distance being represented on a phylogenetic tree. Emergent reasoning techniques are described.
A Porcine Circovirus Vaccine with Enhanced Capabilities
(North Dakota State University, 2016) Kolyvushko, Oleksandr Hryhorovych
Porcine circovirus type 2 (PCV2) is a pathogen of swine. Vaccines against PCV2 are available, although none are capable of differentiating infected from vaccinated animals (DIVA). Positive and negative DIVA markers were introduced in the vaccine constructs. Decoy epitopes were modified by site directed mutagenesis to avoid possible subversion of host immunity and achieve a rationalized vaccine design. Immunization of pigs with the modified vaccines, followed by challenge with a virulent field strain showed that the efficacy of the vaccine was comparable to a commercial vaccine. The average weight gain was significantly higher group vaccinated with experimental construct if compared to the group that received commercial vaccine. An appropriate response to the positive and negative DIVA tags was detected. Therefore, the strategy used in this study is the first to enable a DIVA capable vaccine and accompanying immuno-assay, while using an epitope based approach to target improved immunogenicity.
The Effects of Acetoacetic Acis on Bacterial Growth and Biofilm Formation of Cronobacter Sakazakii, Serratia Marcescens, and Yersinia Enterocolitica
(North Dakota State University, 2016) Murphy, Jennifer Marie
Prevention of bacterial biofilms is an area of research currently being investigated by many research teams. The ability of a chemical to be incorporated into a material that could be used in a medical or food production setting could be of a major value. In this study, we explored the ability of acetoacetic acid (AAA) to reduce biofilm amounts and bacterial growth. We also looked at the transcription of early and late expressed virulence genes in the presence of AAA. We conclude that AAA is a plausible candidate for preventing biofilm formation as we saw a reduction in of biofilm amounts and growth in C. sakazakii, S. marcescens and Y. enterocolitca. We also concluded that AAA was effecting the transcription of virulence genes.
Assessment of Vacuum Steam Pasteurization to Improve Safety and Quality of Low Moisture Foods
(North Dakota State University, 2016) Shah, Manoj Kumar
Low moisture foods such as grains, seeds, spices and flour are part of our daily diet. While they are rich in bioactive compounds, they can be minimally processed or often consumed raw. Several outbreaks due to Salmonella and E. coli O157:H7 have been attributed to low moisture foods. The efficacy of vacuum steam pasteurization in inactivation of Salmonella PT 30, E. coli O157:H7 and E. faecium on whole flaxseed, quinoa, sunflower kernels, milled flaxseed and whole black peppercorns was determined. The impact of pasteurization on microbial shelf life of whole and milled flaxseed was also monitored along with their water activity. Vacuum steam pasteurization was effective at inactivation of each bacteria, providing >5.0 log CFU/g reduction at temperatures as low as 75 and 85C. Also, there was no negative impact on microbial shelf life or water activity on pasteurized flaxseed as compared to unpasteurized products.
Variability in Plant Growth Promoting Properties Among Clinical and Environmental Isolates of Stenotrophomonas Maltophilia
(North Dakota State University, 2015) Domfeh, Yayra Ekui
Stenotrophomonas maltophilia has both negative and positive attributes by being a human pathogen and plant growth promoting rhizobacterium. This study sought to determine if environmental and clinical isolates of S. maltophilia are phenotypically distinct. A total of 18 S. maltophilia isolates from clinical and environmental sources were investigated. Under normal growing conditions, S. maltophila isolates did not enhance growth of canola seedlings. However, under sodium chloride stress (6 decisiemens per meter or 0.33% NaCl), canola seedlings inoculated with S. maltophilia isolates had significantly (P < 0.05) higher number of root branches (isolate D457), root length (D457, CDC 2004-33-01-01 and CDC 2007-23-08-03) and stem length (D457, CDC 2005-37-11-04 and CDC 2011-01-42) than the “no bacteria” control. A number of S. maltophilia isolates protected canola plants from the growth limiting effects of Leptosphaeria maculans and Burkholderia cenocepacia. No clear evidence was found between clinical and environmental isolates based on phenotypic data.

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