Microbiological Sciences Masters Theses

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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.
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.
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.
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.
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.
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.
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.
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.
Characterization of a Novel Cryptosporidium Genotype in Red-Winged Black Birds
(North Dakota State University, 2014) Jesudoss Chelladurai, Jeba Rose Jennifer
Cryptosporidium species cause cryptosporidiosis, characterized by acute gastroenteritis in humans and animals worldwide. Knowledge of the diversity of Cryptosporidium among mammals and birds is incomplete, especially in North American passerines. In this first molecular study of Cryptosporidium in a North American passerine, C. parvum and a novel genotype, called the red-winged black bird genotype were isolated. Genetic characteristics and phylogenetic analyses of the red-winged black bird genotype were described at the 18S rRNA, actin and HSP70 loci, and it was distinct from previously described species and genotypes. The novelty of this genotype was also supported by propagation studies in chickens, zebra finches and cockatiels that failed to produce patent infections. The study adds to our understanding of the co-evolution of the parasite with its hosts and potential sources of C. parvum transmission to susceptible human and animal hosts.
Exploration of Laboratory Techniques Relating to Cryptosporidium Parvum Propagation, Life Cycle Observation, and Host Immune Responses to Infection
(North Dakota State University, 2014) Brown, Cheryl
Cryptosporidium causes cryptosporidiosis, a self-limiting diarrheal disease in healthy people, but causes serious health issues for immunocompromised individuals. Cryptosporidiosis has been observed in humans since the early 1970s and continues to cause public health concerns. Cryptosporidium has a complicated life cycle making laboratory study challenging. This project explores several ways of studying Cryptosporidium parvum, with a goal of applying existing techniques to further understand this life cycle. Utilization of a neonatal mouse model demonstrated laser microdissection as a tool for studying host immune response to infeciton. A cell culture technique developed on FrameSlides™ enables laser microdissection of individual infected cells for further analysis. Finally, the hypothesis that the availability of cells to infect drives the switch from asexual to sexual parasite reproduction was tested by time-series infection. The results suggest this isn’t accurate. These experiments open the door to several avenues of Cryptosporidium study and the host response to cryptosporidiosis.
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.
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.
Impact of Pre-Harvest Environmental Factors on the Survival of Enterohemorrhagic E. Coli and Salmonella on Lettuce
(North Dakota State University, 2015) Tyagi, Deepti
Enteric diseases linked to fresh produce consumption are on a rise. Pathogens can contaminate produce in the pre-harvest field and can survive for long time periods. Thus, this study quantified the survival of Enterohemorrhagic E.coli and Salmonella on pre-harvest lettuce under two relative humidity and seasonal conditions. The effect of relative humidity on pathogen survival depended on the seasonal conditions. The impact of chlorine stress on survival of the two pathogens after exposure to pre-harvest variables was also determined. A single EHEC strain developed resistance to chlorine after 3 days on lettuce plants. Gene expression analysis revealed the up-regulation of genes involved in osmotic and cell envelope stress. Up-regulation of a gene involved in oxidative stress was also observed which could possibly be responsible for imparting resistance to chlorine stress. Understanding these aspects will help develop effective post-harvest decontamination strategies to reduce consumer exposure to such pathogens on produce.
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.
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.
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.
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.
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.
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.

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