Microbiological Sciences Masters Theses

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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.
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.
Cronobacter Sakazakii Characterization and Analysis of Cytotoxicity in Microvascular Endothelial Cells
(North Dakota State University, 2014) Hafner, Hilary Jayne
Contamination of powdered infant formulas by the bacteria Cronobacter sakazakii can pose serious risk to infants and neonates who consume the formula and subsequently develop C. sakazakii related illnesses such as sepsis and meningitis (1). The Gibbs’ lab assesses C. sakazakii isolates’ ability to cross the blood brain barrier and cause meningitis. This thesis research investigated C. sakazakkii cytotoxicity towards microvascular endothelial cells which comprise the first cell line encountered in the barrier. Understanding the mechanisms used to affect these cells will contribute to our understanding of early stages of invasion. Cytotoxicity assays performed for this research found that the cell line used could not sustain confluency when co-cultured with C. sakazakii isolates over periods beyond 24 hours of incubation. In addition, cell-free cytotoxicity assays found that live cells are not necessary to cause damage suggesting a toxin mediated effect.
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.
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.
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.
Happy Beef: The Development of ß-Phenylethylamine as a Novel Nutrient Treatment Reducing Bacterial Cell Count by Escherichia Coli O157H7 on Beef Meat
(North Dakota State University, 2013) Lynnes, Ty Cordell
Since its emergence in 1980's, Escherichia coli O157:H7 has often been associated with the consumption of contaminated meat. E. coli O157:H7 continues to persist as a food borne pathogen not only in beef but many other food products as well. One of the reasons for its persistence is its ability to overcome many of the current control effort including citric acid treatments. This research looks at the use of nutrients as a novel way to control E. coli O157:H7. In this research we used Phenotype MicroArray ™ technology from BioLog (Hayward, CA) technology to screen 95 carbon and 95 nitrogen nutrient sources for their ability to reduce respiration, biofilm amounts and cell number. The top eight performing nutrients were then screened a second time to look at the effects of concentration on their ability to reduce biomass, biofilm amounts and cell number in beef broth. The second screening allowed for the calculation of the concentrations needed to inhibit these factors by 50%. This screening reduced the number of chemicals from eight to two chemicals, acetoacetic acid and ß-phenylethylamine, both of which were characterized by low inhibitory concentrations (<10 mg/ml). In a final experiment, these two chemicals were used in various concentrations as treatment on beef, which was then inoculated with E. coli O157:H7. Only ß-phenylethylamine was able to reduce the bacterial cell counts of E. coli O157:H7 over the seven day incubation period. ß-phenylethylamine, a natural trace amine found in chocolate after fermentation, was able to reduce the recovered colony forming units by >74%. This shows that a nutrient can be used as a novel way to control phenotypic traits in E. coli O157:H7 in a preventative manner.
Investigations in Asthma Heterogeneity: The Roles of Aspergillus Fumigatus-Derived Eicosanoid Synthases and Occupational Exposures to Grain Dusts on the Development of Fungal Allergic Asthma
(North Dakota State University, 2013) Sharma, Akshat
Allergic asthma is an inflammatory syndrome of the respiratory system which changes the airway wall architecture. Using an aeroallergen, murine model of A. fumigatus-mediated asthma, the two studies herein examine the development of asthma in the contexts of host-allergen interactions via A. fumigatus knock-outs of eicosanoid synthases and occupational exposures to corn and soybean dusts. The lack of difference between control and treatment groups seen in post-methacholine airway responses, goblet cell metaplasia, peribronchial inflammation, and fibrosis in the first study show that fungus-derived eicosanoid synthases are dispensable in the development of fungal allergic asthma. However, the same set of respiratory parameters in the grain dust study reveals an increase in BAL neutrophilia and serum IgE titer. The study also underscores a need for modifications of dust exposure times and of time-points of data analysis. These two studies represent unique perspectives on asthma pathogenesis and emphasize the heterogeneity of the syndrome.
Physical and Chemical Treatments for Bacterial Biofilms
(North Dakota State University, 2014) Irsfeld, Meredith Lynn
Physical and chemical treatments have been investigated for the treatment to remove biofilms. This thesis examines the problem of the removal and prevention of biofilms by: (i) using a water jet to determine biofilm stability and (ii) testing the effect of β-phenylethylamine (PEA) on growth and biofilm amounts. Three dimensional structures of biofilms vary in different genetic backgrounds of E. coli, we wanted to see whether changes in structures were paralleled by differences in stability of the biofilm. The water jet apparatus was used to test biofilm stability of E. coli mutants. Alteration of the cell surface structures was detrimental to biofilm stability, while alterations in metabolism had less effect on stability. PEA (0 to 50 mg/mL) was applied to bacterial strains to see the effects on growth and biofilm amounts. PEA had an inhibitory effect on growth and biofilm amounts of some bacterial strains tested.
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.
Preliminary Investigation of Escherichia Coli K12 Biofilm Inhibition on an Antimicrobial Polysiloxane Coating using Whole Transcriptome Profiling
(North Dakota State University, 2012) Stafslien, Shane J.
Whole transcriptome profiling was examined in E. coli K12 when cultured on the surface of a pure polysiloxane coating (Sil) and a polysiloxane coating containing a tethered quaternary ammonium compound (QSil) shown to inhibit biofilm formation. An optimized protocol was developed for isolating high quality RNA from the surface of these coatings prepared in multi-well plates. DNA microarray data obtained from the Sil and QSil coatings revealed that 222 genes were differentially expressed between these two surfaces by a factor of at least 2-fold and with a 90% level of confidence. Several genes of the lsr operon, which encode the various components of the AI-2 based quorum sensing system, were repressed on the QSil coating surface. The QSil coatings ability to effectively interfere with the AI-2 based quorum sensing system was most likely the primary factor that contributed to the impairment of E. coli K12 biofilm formation on that surface.
Studies in Pathogenesis of a Novel Isolate of Cronobacter Sakazakii using an In Vitro Blood Brain Barrier Model
(North Dakota State University, 2013) Welker, Elliott Weston
Genus Cronobacter is a member of the family Enterobacteriaceae consisting of several opportunistic species. The primary focus of this study was to utilize an in vitro co-culture model of the blood brain barrier to investigate a bovine fecal strain of C. sakazakii to investigate pathogenicity. The strain was found to have the same effect on the barrier’s integrity as the positive Escherichia coli control. Additionally, C. sakazakii strain BAA-894 was found to have the same effect as the negative E. coli control. This study also focused on the development of a site-specific mutagenesis procedure for C. sakazakii. A procedure using linear transformation was able to replace the putative virulence gene zpx (zinc-containing metalloprotease) in C. sakazakii. A future virulence study would involve using this mutagenesis procedure to induce a mutation in genes of C. sakazakii speculated to play a role in BBB translocation followed by challenge in the BBB model.

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Browsing Microbiological Sciences Masters Theses by browse.metadata.program "Microbiology"