Thesis (M.S.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (Feb. 23, 2007). Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Protection of potable water supplies demands a better understanding of the factors controlling migration of disease causing bacteria in subsurface environments. In this study, the migration behaviour of the waterborne pathogenic microorganisms Escherichia coli O157:H7 and Yersinia enterocolitica was investigated in water saturated granular systems. Both facultative bacteria were grown under aerobic and anaerobic conditions and further acclimatized to a microaerophilic or fully aerated environment for 21 h. Experiments were conducted using laboratory-scale packed columns over controlled extreme dissolved oxygen (DO) concentrations. The observed differences in the transport potential of these pathogens were found to depend strongly on the antecedent growth conditions under the tested environmental settings as well with the environmental DO in certain conditions. Further microbial characterization using cell titrations and FTIR spectroscopy gave a greater insight on the source of the surface charge that was found to dominate the attachment phenomena in sand packed columns. Techniques also revealed a probable role of other cell surface macromolecules (LPS) that could account for non-DLVO behaviour. The results illustrate the importance of considering physicochemical conditions relevant to the natural subsurface environment when designing laboratory transport experiments as evidenced by variations in microbe migration as a function of the DO under growth and acclimation. / Keywords: bacterial adhesion, bacterial transport, DLVO, physicochemical characterization, dissolved oxygen, porous media.

The contamination of drinking water by microbial pathogens is recognized as one of the most pressing water supply problems of our day. To minimize the impact of pathogens and parasites on the environment and public health, accurate methods are needed to evaluate their presence and concentration. Although various techniques exist to detect certain pathogens in water (e.g., immunofluorescence or PCR techniques), these are time- and labor-intensive. A direct, real-time method for detection and quantification of target organisms would thus be very useful for rapid diagnosis of water safety. A quartz crystal microbalance with dissipation monitoring (QCM-D) based biosensor for detection of waterborne pathogens (i.e., Escherichia coli O157:H7) was developed. The detection platform is based on the immobilization of affinity purified antibodies onto gold coated QCM-D quartz crystals via a cysteamine self-assembled monolayer. The results show that the optimal sensor response is the initial slope of the dissipation shift. A highly log-log linear response is obtained for detection of E. coli O157:H7 over a broad range of cell concentration from 3 x 105 to 1 x 109 cells/mL. The prepared biosensor also exhibits a log-log linear working range from 107 to 109 cells/mL for E. coli K12 D21, a non-pathogenic model organism. The biosensor also shows satisfactory selectivity using Bacillus subtilis . To our knowledge, this is the first study demonstrating the use of the slope of the dissipation shift as a sensor response when using QCM-D technology. / Keywords: Biosensor, QCM-D, E. coli O157:H7, polyc1onal antibodies, dissipation slope, cysteamine, self-assembled monolayer

The persistance of Escherichia coli in the larval, pupal and adult stages of both house flies, Musca domestica (L.), and stable flies, Stomoxys calcitrans (L). was examined. Abundance of E.coli declined over time in immature house flies, but remained constant in immature stable flies, suggesting house fly larvae digest E. coli but stable fly larvae do not. Survival of house fly and stable fly larvae averaged 62% and 25% respectively when reared on pure E. coli cultures. E. coli load in pupae decreased significantly one day before emergence of adult house flies, but remained constant until stable fly emergence. Nevertheless, E. coli was detected in 78% of emerging house flies and in 28% of emerging stable flies. House flies are more important E. coli vectors as adults, whereas stable flies may be overlooked vectors of E. coli during immature development. / ix, 89 leaves ; 28 cm.

Escherichia coli O157:H7 is an important human pathogen that resides primarily in cattle and feedlot environments. E. coli O157:H7 can be divided into phylogenetic groups termed lineages; lineage I strains are responsible for most human illnesses. An understanding of the etiology of these lineages within cattle and the feedlot environment could allow for more effective surveillance and mitigation strategies. There were no lineage associated differences in growth or survival of E. coli O157:H7 in bovine feces at 4°C, 12°C or 25°C. Lineage I strains more readily colonized cattle jejunum tissue and a bovine colonic cell line than lineage II and intermediate type strains. Enhanced colonization of cattle by lineage I strains may increase the persistence of these strains in feedlots via re-infection and increased shedding. This outcome could increase the transmission of lineage I strains to the food supply and increase the potential for these strains to cause human illness. / xiii, 101 leaves ; 29 cm

For pathogenic microorganisms, heme uptake and degradation is a critical mechanism for iron acquisition that enables multiplication and survival within hosts they invade. While the bacterial proteins involved in heme transport had been identified at the initiation of our investigation, the fate of heme once it reached the cytoplasm was largely uncharacterized. Here we report the first crystal structures of two members of the heme utilization operon from the human pathogen Escherichia coli O157:H7. These are the heme oxygenase ChuS in its apo and heme-complexed forms, and the apo form of heme binding protein ChuX. Surprisingly, despite minimal sequence similarity between the N- and C-terminal halves, the structure of ChuS is a structural repeat. Furthermore, the ChuS monomer forms a topology that is similar to the homodimeric structure of ChuX. Based on spectral analysis and carbon monoxide measurement by gas chromatography, we demonstrated that ChuS is a heme oxygenase, the first to be identified in any E. coli strain. We also show that ChuS coordinates heme in a unique fashion relative to other heme oxygenases, potentially contributing to its enhanced activity. As ChuS and ChuX share structural homology, we extended the structural insight gained in our analysis of ChuS to purport a hypothesis of heme binding for ChuX. Furthermore, we demonstrated that ChuX may serve to modulate cytoplasmic stores of heme by binding heme and transferring it to other hemoproteins such as ChuS. Based on sequence and structural comparisons, we designed a number of site-directed mutations in ChuS and ChuX to probe heme binding sites and mechanisms in each. ChuS and ChuX mutants were analyzed through reconstitution experiments with heme and functional analyses, including enzyme catalysis by ChuS and mutants, and in culture development during heme challenge experiments by ChuX and mutants. Taken together, our results suggested that ChuX acts upstream of ChuS, and regulates heme uptake through ChuX-mediated heme binding and release. ChuS can degrade heme as a potential iron source or antioxidant, thereby contributing directly to E. coli O157:H7 pathogenesis. Functional implications that may be revealed from sequence and structure based information will be addressed as they pertained to our evaluation of ChuS and ChuX. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2007-04-27 11:34:50.272

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Refrigeration has been found to cause cell elongation in mesophilic enteric organisms like commensal Escherichia coli and E. coli O157:H7. As elongated cells may divide into multiple daughter cells, they may result in underestimation of pathogen numbers in foods when plate counts are used. When E. coli cultures were incubated at 6°C for ≤10 days, cells grew by elongation, did not divide, and lost viability (LIVE/DEAD vitality stain) at similar rates. Substantial fractions of cells in cultures elongating at 6°C were inactivated by an abrupt shift to 37°C. Direct microscopic observation of cells transferred to 37ºC after 5 days at 6°C showed that few or no cells of normal size (≤4µm) divided, while elongated cells (>4 µm) formed multiple daughter cells. Thus the threat from mesophilic pathogens with a low infective dose may be underestimated in refrigerated foods. It was also found that E. coli O157:H7 cultures containing elongated cells prepared at 6 or 15 °C have greater potential to attach to food contact surfaces than those grown at higher temperatures. Interestingly, at 6°C cell elongation was inhibited by ≥ 100 mg/l cinnamaldehyde and ≥ 200 mg/l cinnamaldehyde was lethal. In contrast, at 37°C 200 mg/l cinnamaldehyde initially delayed multiplication of E. coli cells by causing cell elongation, but from 2 to 4 h, growth resumed and cells reverted to normal length. To understand this transient behaviour, genome-wide transcriptional analysis of E. coli O157:H7 was performed at 2 and 4 h exposure to cinnamaldehyde in conjunction with reverse phase-high performance liquid chromatography analysis for cinnamaldehyde and other cinnamic compounds. At 2 h exposure, cinnamaldehyde induced expression of many oxidative stress-related genes, reduced expression of genes involved in DNA replication, synthesis of protein, O-antigen and fimbriae. At 4 h many repressive effects of cinnamaldehyde on E. coli O157:H7 gene expression were reversed. Data indicated that by 4 h, E. coli O157:H7 was able to convert cinnamaldehyde into the less toxic cinnamic alcohol using alcohol dehydrogenase or aldehyde reductase enzymes (YqhD and DkgA). The results also showed that the antimicrobial activity of cinnamaldehyde was likely attributable to its carbonyl aldehyde group.