Thesis (Ph.D.)--Aberdeen University, 2009. / Title from web page (viewed on June 3, 2009). Includes bibliographical references.

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Cattle are naturally colonized by enterohemorrhagic Escherichia coli within the gastrointestinal tract. The most notorious of the enterohemorrhagic E. coli is E. coli O157:H7, which can cause serious illness to humans if ingested. To ensure that the United States has a safe food supply, research is ongoing in pre-harvest food safety and pathogen intervention strategies. While advances in pre-harvest intervention strategies are encouraging, no method has proven to completely eliminate and/or control O157:H7. A key limitation to successful pathogen intervention strategies is the inability to track and monitor pathogens in a real-time fashion. Through the use of bioluminescent plasmids harboring the luxCDABE cassette, pathogen tracking could be a viable solution. Bioluminescent plasmids are capable of facilitating the tracking, pathogenesis and physical locations of pathogens, thus enabling researchers to have a better understanding of the pathogenic process.

An increase in antimicrobial resistance bacteria has endangered our ability to treat infectious diseases. Lack of good in-vivo model has made it difficult to study antimicrobial resistance. In this study, we have used an inexpensive and short life span in-vivo model namely, Caenorhabditis elegans (C. elegans) to study antimicrobial treatment using pathogenic Escherichia coli O157:H7, a multidrug resistance bacterium that causes life threatening infection in humans. We have investigated the influence of live vs. heat killed non-pathogenic E. coli OP50 (OP50) as a food source on the growth and survival of infected C. elegans mutant AU37 with E. coli O157:H7 in the presence and absence of antibiotics. This is analyzed using a liquid-based C. elegans-E. coli O157:H7 infection assay. C. elegans was synchronized and grown on a lawn of live OP50 till they reached L4-young adult stage. L4-young adults were transferred to liquid medium where the C. elegans was infected with live E. coli O157:H7 or live non-pathogenic OP50 for 24 hours. After infection, C. elegans were fed live or heat killed OP50 depending on the experiment, and the life span and levels of E. coli O157:H7 were monitored, with and without ampicillin treatment in a 96 well transwell plate. Our results indicate that live OP50 is an ideal food source for C. elegans growth and survival to study antimicrobial treatment. C. elegans growth rate and survival decreased in presence of heat killed OP50, which makes heat killed OP50 as a non-ideal food source for antimicrobial assay. Moreover, using live OP50 we have discovered that the ampicillin dose 8mg/ml, 16mg/ml, and 32mg/ml are effective in increasing the survival of C. elegans infected with E. coli O157:H7. However, treatment on C. elegans infected with acid stressed E. coli O157:H7 is controversial.

Two studies investigating the use ultraviolet light at a wavelength of 253.7nm (UVC) into the inhibition of Salmonella spp. and Escherichia coli O157:H7 were conducted. The objectives of these studies were: to determine the rates for the destruction of Salmonella and Escherichia coli O157:H7 on the surface of agar and to investigate its effectiveness on the surface of fresh produce. Multiple replications of different doses and cocktail concentrations were performed and resulted in a 5 log reduction of Escherichia coli O157:H7 at doses exceeding 8.4 mW / cm2, while a 5 log reduction for Salmonella spp. was observed at doses exceeding 14.5 mW / cm². Samples of Red Delicious apples, green leaf lettuce and tomatoes were subjected to different doses ranging from 1.5 __ 24 mW / cm2 of UVC to determine effective log reductions of microbial populations. UVC applied to apples inoculated with E. coli O157:H7 resulted in the highest log reductions of approximately 3.3 logs at 24 mW/cm2. Lower log reductions (2.19 logs) were seen on tomatoes inoculated with Salmonella spp. and leaf lettuce (2.65 and 2.79) inoculated with both Salmonella spp. and E. coli O157:H7 respectfully. Due to the low capital involved in initiating a UVC system, the use of ultraviolet energy may prove to be a beneficial mechanism to decrease pathogens on fresh produce if used in conjunction with strict adherence to a sanitation program, Good Manufacturing Practices and Good Agricultural Practices in ensuring the safety of fresh produce. / Master of Science

Titre de l'écran-titre (visionné le 25 septembre 2023) / Les bactéries telles qu'Escherichia coli O157 : H7, un pathogène pour l'humain, ont besoin de fer pour survivre et croitre. Puisque chez les mammifères ce nutriment est principalement contenu dans l'hème de l'hémoglobine, plusieurs bactéries ont évolué pour mener à l'obtention de voies métaboliques dédiées à l'acquisition et la dégradation de l'hème. Chez E. coli O157 : H7, un groupe de 8 gènes nommé chu est responsable de ces activités. L'un de ces gènes code pour la protéine ChuS pouvant dégrader l'hème d'une manière inédite en condition aérobie. La réaction effectuée par ChuS peut être alimentée par du peroxyde d'hydrogène et de l'oxygène moléculaire, et mène à la dégradation de l'hème en tripyrrole et acide hématique ce qui libère le fer et produit également du monoxyde de carbone. De plus, la réaction catalysée par ChuS est facilitée par la présence d'une seconde enzyme, ChuY, qui est une réductase de la famille des hydrogénases/réductases à courtes chaines. Les objectifs de ce projet étaient de mieux comprendre les réactions catalysées par ChuS et le duo ChuS/ChuY ainsi que leurs intermédiaires et produits de réaction. L'étude de la protéine ChuS in vitro montre que la réaction de dégradation de l'hème doit être préférablement effectuée dans le tampon phosphate de sodium ou le tampon citrate pour mener à une réaction complète qui libère le fer car d'autres tampons affectent différemment l'activité enzymatique. Au cours de la réaction de dégradation de l'hème, nous avons déterminé que ChuS génère deux isomères de l'intermédiaire verdohème, soit les isomères bêta et delta, contrairement à l'hème oxygénase humaine qui génère spécifiquement l'isomère alpha. Globalement, cette étude a permis une meilleure compréhension du mécanisme de dégradation de l'hème par la protéine ChuS in vitro et ouvre la voie à une meilleure compréhension de sa réaction en contexte biologique chez le pathogène.

This study explores relative levels of stxl and fur gene expression under antibiotic-stressed and control (non-stressed) Escherichia coli O157:H7 using real-time polymerase chain reaction (PCR) cycle threshold (CO value comparisons among replicates at designated time points of growth. Our data indicate that E. coli O157:H7 under the subinhibitory concentration(SIC) level of chloramphenicol decreases fur expression in early stationary phase cultures by 50% compared to non-stressed cells, but increases stxl expression by 35-50% during the log-to-stationary phase transition. Since the enterohemorrhagic E. coli stxl gene is negatively regulated by the fur gene product or results indicate that a separate fundamental transcriptional regulatory mechanism is functional in cultures grown under subinhibitory stress, such as antibiotic exposure. These data could support the clinical results obtained from treatment of EHEC-mediated toxicoinfections with antibiotics which have resulted inducing EHEC to prematurely produce cytotoxins within the host and speed the course of hemorrhagic colitis (HC) and/or hemolytic uremic syndrome (HUS). / Department of Biology

Thesis (Ph. D.)--Ohio State University, 2009. / Title from first page of PDF file. Includes vita. Includes bibliographical references (p. 138-157).

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-04-04T17:04:13Z No. of bitstreams: 1 Frozi, Jesieli Braz [Dissertação, 2012].pdf: 523167 bytes, checksum: 974a9ecfc8799188193c730cea58c7b9 (MD5) / Made available in DSpace on 2017-04-04T17:04:13Z (GMT). No. of bitstreams: 1 Frozi, Jesieli Braz [Dissertação, 2012].pdf: 523167 bytes, checksum: 974a9ecfc8799188193c730cea58c7b9 (MD5) / O objetivo deste estudo foi determinar a multiplicação e a sobrevivência de E. coli produtora de toxina Shiga (STEC) do sorotipo O157:H7 em queijo Minas frescal (QMF). Duas cepas de E. coli O157:H7 (RJ581 e EDL933) foram, experimentalmente, inoculadas no leite pasteurizado usado para a fabricação do QMF com inóculos de 102 e 103 células/mL. A avaliação da multiplicação e sobrevivência das cepas de E. coli, através da contagem de unidades formadoras de colônia (UFC) em ágar MacConkey Sorbitol suplementado com cefixime e telurito (CT-SMAC), foi realizado durante as diferentes etapas de processamento do queijo e durante os tempos de 0, 2, 4, 5, 7, 10 e 15 dias de armazenamento. Os queijos foram mantidos a 8ºC até o fim das análises. As colônias foram confirmadas como E. coli O157 através da pesquisa do gene rfb O157 por reação em cadeia da polimerase (PCR). Os padrões físico-químicos do leite utilizado como matéria prima e do QMF fabricado estavam de acordo com os padrões recomendados, com exceção de duas amostras que apresentaram o valor de pH levemente mais ácido que o encontrado na literatura. As análises microbiológicas do leite e do QMF apresentaram contagens de E. coli indicadora de contaminação fecal muito acima dos padrões recomendados pela legislação. Quando o QMF foi fabricado com leite contaminado experimentalmente com inóculos iniciais de 103 células/mL, ao final do processamento, o queijo apresentou contagem 10 vezes maior que a da matéria-prima para a cepa RJ581 e 100 vezes maior para a cepa EDL 933. Durante o armazenamento, os inóculos de 102 células/mL, de ambas as cepas, apresentaram o máximo crescimento no 4° dia de armazenamento, enquanto que os inóculos de 103 células/mL, apresentaram máximo de crescimento no 5° dia de armazenamento, a partir deste ponto observamos declínio na população, até ausência de detecção. Contudo, células viáveis foram encontradas até, pelo ao menos, o 7° e 10° dia de armazenamento dos QMFs fabricados com inóculos iniciais de 102 e 103 células/mL de leite, respectivamente. Estes resultados mostraram que E. coli O157:H7 foi capaz de se multiplicar e sobreviver no QMF, partindo de inóculos inicias com baixo número de células (102 e 103), encontramos na literatura que esta quantidade de células por grama ou ml de alimento é capaz de causar infecções em humanos e que o QMF, mesmo quando armazenado sob refrigeração é um veículo em potencial de doença provocada por STEC O157:H7. / The objective of this study was to determine the proliferation and survival of E. coli O157:H7 in Brazilian Minas Frescal Cheese (QMF). Two E. coli strains were experimentally inoculated into pasteurized milk used in Minas Frescal cheese production at level 102 and 103 cells/mL. The study of the proliferation and survival of E. coli strains, by counting the colony forming units (CFU) on MacConkey agar Sorbitol (CT-SMAC) was performed during various stages of processing of the cheese and during the times 0, 2, 4, 5, 7, 10 and 15 storage day. The cheeses were stored at 8ºC until the end of the analysis. The microbiological and physicochemical quality of pasteurized milk and cheese were also evaluated. The colonies on CTSMAC were confirmed as STEC O157: H7 through research of rfb O157 gene by polymerase chain reaction (PCR). The microbiological and physico-chemistry analysis of pasteurized milk and cheese were also evaluated, and were according to standards recommended. Microbiological analyzes of milk and QMF presented counts of E. coli far above the recommended legislation.When the QMF was made from milk inoculated with initial inoculum 103 cells/mL in the end of processing, the cheese had count 10-folds higher that of the raw material from the RJ581 strain and 100-folds higher from the EDL strain 933. During storage at 8° C, the inoculum of 102 cells/mL, both strains showed the most growth in the cheese on the 4th day of storage, whereas inoculum 103 cells/mL, grew up in 5th storage. Additionally, viable cells were found to at the least, the 7th and 10th day of storage in the QMF made from initial inoculum of 102 and 103 cells/mL of milk, respectively. These results showed that STEC O157:H7 was able to survive and multiply in counts able to cause infection in humans even when stored under refrigeration

Le travail de thèse a eu pour but de contribuer à une meilleure connaissance de la dangerosité pour l’homme des souches STEC d’origine bovine, en explorant la corrélation pouvant exister entre la présence du gène stx chez de telles souches et la réalité de son expression. La quantification de l’expression du gène stx2 présent chez 46 souches STEC bovines a été réalisée à l’aide d’un test ELISA commercial détectant spécifiquement les shiga toxines, le test ProSpecT® Shiga toxin (OXOID). L’ensemble des résultats de validation préalable obtenus pour ce test a permis de considérer qu’il pouvait être valablement appliqué à l’étude du panel de souches d’E. coli O157 :H7 bovines collectées au laboratoire, tout en déterminant les limites méthodologiques, et donc d’interprétation. Utilisé comme outil de quantification de la production de Stx2 par les souches du panel choisi, et dans deux conditions expérimentales différentes (présence ou absence d’induction par l’enrofloxacine), ce test a permis de mettre en évidence que seulement 15,2% des souches d’E.coli O157:H7/H- étudiés produisent des quantités significatives de Stx2 détectables sans induction, et ce à des niveaux variables. En revanche, la majorité de ces isolats, bien que n'exprimant pas la protéine Stx2 de manière constitutive, produit des quantités significatives de Stx2 en présence de concentrations subinhibitrices d'enrofloxacine, antibiotique de la famille des fluoroquinolones et utilisé en médecine vétérinaire. Enfin, des mutants résistants à l'enrofloxacine sélectionnés à partir de certaines souches d’E. coli O157:H7, produisent, après induction par l'enrofloxacine, 3 fois plus de toxine Stx2 que les souches sauvages. Les mutants sont également inductibles en utilisant des doses d'enrofloxacine 100 fois supérieures à celles utilisables pour les souches sauvages. L’ensemble de ces résultats montre (i) la corrélation, ou non, qui peut exister entre la présence du gène stx2 et son expression, (ii) que la proportion inductible des souches STEC bovines est potentiellement importante, (iii) que l’enrofloxacine induit fortement l’expression du gène stx2 chez les souches STEC bovines et que (iv) l’induction par l’enrofloxacine conduit à des taux d’expression du gène stx2 supérieurs chez des souches résistantes aux fluoroquinolones que chez les souches sensibles. Au final, cette étude contribue à documenter la variabilité des niveaux d’expression des gènes stx et illustre le risque que des STEC issus de bovins puissent devenir plus fréquemment pathogènes pour l'homme suite à l'usage croissant des fluoroquinolones vétérinaires. / The present study contributed to a better knowledge of the pathogenicity of STEC for humans by quantifying the expression of the stx2 gene from a panel of 46 cattle STEC isolates by ELISA. Succesful validation experiments of the ProSpecT® Shiga toxin ELISA (OXOID) first concluded to its capability to be used for a valuable quantification of the Stx2 protein. Stx2 expression was tested in presence and absence of subtherapeutic concentrations of enrofloxacin, an antibiotic of the fluoroquinolones family used in veterinary medicine. Whereas only 15.2% of the strains displayed significant amounts of detectable Stx2 in absence of induction, most of them were shown to be inducible, and at various levels, in presence of subtherapeutic concentrations of enrofloxacin. Also, enrofloxacin-resistant mutants of Stx2-producing E. coli O157:H7 were selected and produced 3-fold higher Stx2 levels than native strains after induction with enrofloxacin. Mutants were also inducible using hundred-fold higher enrofloxacin concentrations than the useful ones for native strains. At the end, these results show (i) the inconstant and variable expression of the stx2 gene from cattle STEC isolates in native conditions, (ii) the potentially high number of inducible STEC isolates in cattle, (iii) that enrofloxacin is a strong inducer of the stx2 expression in cattle STEC isolates and (iv) that the stx2 gene is stronger induced in isolates resistant to fluoroquinolones compared to susceptible ones. Finally, this all study documents the variable expression of the stx2 gene and also suggests that E. coli O157:H7 from cattle may become more frequently pathogenic to humans as a side-effect of the increasing use of veterinary fluoroquinolones.