A l'heure actuelle, les Escherichia coli producteurs de Shiga-toxines (STEC) sont considérés comme des pathogènes émergents importants en santé publique. Cependant, il n'existe aujourd'hui aucune réglementation officielle stipulant les procédures à suivre pour l'échantillonnage et la recherche des STEC dans les denrées alimentaires. <br />Ce travail a pour objectif d'étudier les différents protocoles utilisés pour la recherche des STEC, de manière à pouvoir proposer aux industriels des protocoles optimisés leur permettant une réelle maîtrise du « danger STEC » dans leur filière. Dans ce but, la cinétique de croissance de diverses souches de STEC a, dans différentes conditions d'enrichissement, été suivie simultanément à celle de la flore annexe de la matrice, puis modélisée.<br />Notre étude souligne qu'un enrichissement trop court, comme les 6 heures d'incubation dans le cas de l'IMS, peut conduire à l'obtention de résultats faussement négatifs. Il s'avère néanmoins inutile, dans certaines conditions, de prolonger l'étape d'enrichissement car une interaction de type compétition simple avec la flore annexe arrête la croissance des STEC. Cet arrêt est plus ou moins rapide selon la matrice analysée et sa densité en flore annexe naturelle (de 4 à 7 h pour les fèces et de 10 à 12 h pour le steak haché dans nos expérimentations). Dans le lait, des interactions plus complexes entraînent un arrêt de la croissance des STEC avant celui de la flore naturelle (8,5 à 11 h dans nos expérimentations).<br />L'utilisation d'agents sélectifs a pour but de freiner la croissance de la flore annexe, ce qui peut avoir pour impact de prolonger la croissance des STEC. L'ajout de sels biliaires dans le milieu d'enrichissement a un effet positif dans le cas de l'enrichissement d'échantillons de fèces de bovins et de lait cru mais n'a pas d'effet significatif pour la matrice « steak haché ». En revanche, l'addition de novobiocine dans le milieu peut inhiber certaines souches de STEC non-O157:H7 et ralentir la croissance de E. coli O157:H7. L'usage de cet antibiotique, potentiellement responsable de résultats faussement négatifs, devrait être abandonné.<br />Par ailleurs, cette étude a permis d'optimiser le protocole de recherche de E. coli O157:H7 dans le steak haché (ISO 16140) en validant, d'une part, l'analyse d'une plus grosse masse d'échantillon dans un même volume de milieu (ratio plus élevé) et en réduisant, d'autre part, le temps d'analyse grâce à l'utilisation d'une température d'incubation plus élevée de 41,5°C.

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

Shiga toxin-producing E. coli (STEC) cause diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). All STEC produce one or both of two Shiga toxins, Stx1 and Stx2. STEC strains that produce Stx2 are more strongly associated with HUS than strains that produce Stx1 or both Stx1 and Stx2. Epidemiologic evidence indicates a recent increase in the rate of HUS among STEC outbreaks. The increasing rate of HUS could be explained by a shift in the toxin profiles of STEC strains. The purpose of this study was to examine trends in toxin profiles of human STEC O157 isolates from 1996 to 2008 and to assess whether an increase in the number of Stx2-only-producing strains could be correlated with a recent increase in HUS cases. Data from three independent datasets, collected from PulseNet, eFORS and NARMS, were used. Additionally, trends such as seasonal variations, geographical variations, gender differences, and age differences were examined for each toxin profile. Results from this study show a shift in the toxin profile of human STEC O157 strains in the United States, in that the proportion of Stx2-only producing strains has increased dramatically since 1996.

Waterborne pathogen presence caused by fecal pollution is a leading cause of morbidity and mortality worldwide. In developed countries, this problem can result in waterborne outbreaks. Research suggests that there is a need for better fecal indicators because current methods (total coliforms and E. coli) are insufficient. This study investigated Bacteroidales 16S rRNA markers as a microbial source tracking tool in an agricultural watershed. Correlations between pathogens and markers were also investigated. Water quality monitoring was conducted following assay validation of ruminant-, bovine-, human-specific, and universal Bacteroidales markers. Results revealed a positive relationship between E. coli and the universal marker. Ruminant- and bovine-specific marker detection was associated with increased runoff due to precipitation; however, the human associated marker was not detected. Furthermore, no correlations between Campylobacter, Salmonella, or E. coli O157:H7 could be made. Consequently, these techniques have potential to become a powerful tool; however, further research is needed

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