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Item Brucellosis Epidemiology, Virulence Factors, Control and Molecular Targets to Prevent Bacterial Infectious Diseases(North Dakota State University, 2012) Mugabi, RobertBrucellosis is a bacterial zoonosis that infects both professional phagocytic and nonphagocytic cells in the hosts. Brucella intracellular survival is important for its virulence. In a study to establish the seroprevalence and risk factors of brucellosis in livestock in Kazo and Buremba sub-counties of Kiruhura district, Uganda, fifty goat and 112 bovine serum samples were tested for Brucella antibodies. The prevalence of Brucella antibodies in goats and cattle was 26.0% and 38.4% respectively, while individual seroprevalence rates by livestock breeds were 10.7% (cross-breed goats), 45.5% (local goat breeds), 49.1% (cross-breed cattle), 31.0% (local cattle breeds), and 17.4% (exotic cattle breeds) (p = 0.001). Sharing of watering points, using surface water for livestock, presence of wildlife on pasture, lack of vaccination was significantly correlated with Brucella seropositivity in livestock. The molecular study on biofilm in Escherichia coli included in this paper revealed that pflA knock out mutations had a significant effect on biofilm amounts when biofilms formed on D-serine and acetate. The ldhA formed generally high bacterial biofilm amounts on all carbon sources as compared to the wild type.Item Phenotypic and Genotypic Effects of FlhC Mediated Gene Regulation in Escherichia Coli O157:H7(North Dakota State University, 2011) Sule, PreetiEscherichia coli (E.coli) 0157:H7, a pathogen belonging to the enterohemorrhagic group of E.coli, has long been a concern to human health. The pathogen causes a myriad of symptoms in humans, ranging from diarrhea and malaise to renal failure. Human infection with the spread of the pathogen is mainly attributed to consumption of contaminated food material such as meat. Decontamination of meat via sprays have to date been the most commonly practiced method to reduce contamination, which now has little relevance in the face of developing resistance by the pathogen. In the following study we investigated FlhC mediated gene regulation in E. coli 0157:H7 on the surface of meat, in an attempt to recognize FlhC regulated targets, which may ultimately serve as targets for the development of novel decontaminating sprays. Microarray experiments were conducted to compare gene expression levels between a parental E. coli 0157:H7 strain and its isogenic flhC mutant, both grown on meat. Putative FlhC targets were then grouped based on their function. Realtime PCR experiment was done to confirm the regulation. Additionally, experiments were done to investigate the phenotypic effects of the regulation. To test the effect of FlhC on biofilm formation, an ATP based assay was first developed in E.coli K-12, which has been detailed in the following dissertation. This assay was used to quantify biofilm biomass in E. coli 0157. Microarray experiments revealed 287 genes as being down regulated by FlhC. These genes belonged to functions relating to cell division, metabolism, biofilm formation and pathogenicity. Real-time PCR confirmed the regulation of 87% of the tested genes. An additional 13 genes were tested with real-time PCR. These belonged to the same functional groups, but were either not spotted on the microarray chips or had missing data points and were hence not included in the analysis. All 13 of these genes appeared to be regulated by FlhC. The phenotypic experiments performed elucidated that the FlhC mutants divided to 20 times higher cell densities, formed five times more biofilm biomass and were twice as pathogenic in a chicken embryo lethality assay, when compared to the parental strain. The following dissertation also reports the development of a combination assay for the quantification of biofilm that takes advantage of the previously mentioned ATP assay and the PhenotypeMicroarray TM (PM) system. The assay was developed using the parental E. coli strain AJW678 and later applied to its isogenic flhD mutant to elaborate on the differences in nutritional requirements between the two strains during biofilm formation. Metabolic modeling and statistical testing was also applied to the data obtained. This assay will be used in the future to study biofilm formation by the parental strain E. coli 0157:H7 strain and its isogenic FlhC mutants on single carbon sources, hence identifying potential metabolites which differentially support biofilm formation in the parental and the mutant strain.Item 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.Item Gene Regulation in Biofilms(North Dakota State University, 2011) Samanta, PriyankarSessile 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.Item Attachment and Biofilm Formation of Foodborne Pathogens(North Dakota State University, 2017) Smith, Sara JeanOutbreaks 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.Item Role of ArcB/ArcA in the Regulation of Biofilm Formation in Escherichia coli in Conjunction with F1hD Expression(North Dakota State University, 2014) Nessa, Laura ChristineThe two goals of this study were to identify the affect that arcB has on biofilm formation and to determine whether this was dependent on AckA. Acetyl phosphate is formed from acetyl-CoA during acetate metabolism and is degraded to acetate through the enzyme AckA. ArcB is the sensor kinase of the ArcB/ArcA two-component system involved in anaerobic metabolism [1]. Another important factor is the FlhD/FlhC global transcriptional regulator complex involved in regulating motility. The overall conclusion indicates acetyl phosphate has an effect on arcB only when both genes are nonfunctional as demonstrated by a lack of biofilm, increased motility, and increased flhD expression. To test the hypothesis, several experiments were performed including scanning electron microscopy (SEM), growth curves, motility assays, and a ß-galactosidase assay measuring flhD expression. The tests were performed using a parent Escherichia coli K-12 strain (AJW678) and mutants in ackA, arcB, and an ackA arcB double mutant.