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.

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

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

E. coli O157:H7 is the most important serotype of enterohaemorrhagic E. coli (EHEC) which is of concern to public health worldwide. As a common cause of haemorrhagic colitis, EHEC infection can progress to life threatening sequelae including haemolytic uraemic syndrome (HUS) in humans. Human infection rates are higher in Scotland than found in the rest of the UK. Cattle are asymptomatic carriers of EHEC and are an important reservoir from which disease outbreaks can spread. The terminal rectum has been indicated as a site of E. coli O157:H7 colonisation in the bovine intestinal tract. This is the location of numerous lymphoid follicles which contain dendritic cells (DCs) which are professional antigen presenting cells and important directors of immune responses. DCs are likely to come into contact with EHEC and therefore could be key in this location for enabling EHEC to colonise the bovine host. The first aim of this project was to characterise dendritic cells within the bovine intestinal tract at various anatomical locations, including the terminal rectum, using immunohistochemistry techniques. Following this, work to extract and further phenotype dendritic cells from terminal rectal tissues was undertaken. Finally, a widely-used bovine dendritic cell model was employed to generate dendritic cells from circulating blood monocytes. This model was utilised to investigate the interactions of dendritic cells with EHEC strains compared with responses to bovine enterotoxigenic (ETEC) and bovine commensal E. coli strains. Early work identified that there are potentially numerous DCs within the bovine intestinal tissues and these cells were found in greater numbers at the terminal rectum. Protocols to extract and further characterise these cells were developed but proved inconsistent, with large variation between animals. Using the monocyte derived dendritic cells (moDCs), differences were observed between immunological responses to challenge with E. coli O157:H7 strains and bovine pathogenic or commensal E. coli strains. Cytokine production, cell surface molecule expression, cell phenotype and viability as well as intracellular bacterial counts were compared. The data presented here shows that the bovine moDCs respond differently to EHEC strains when compared with commensal or pathogenic E. coli in several key areas. This has important implications for the responses of the bovine host to various E. coli strains. This work also indicates that dendritic cells could be central to these responses and if studied further still, may hold the key to reducing the colonization and persistence of E. coli O157:H7 in cattle, and subsequent human disease outbreaks.

The performance of a novel, visible light-driven photoelectrocatalytic (PEC) batch reactor employing tungsten trioxide (WO₃) as a photocatalyst was assessed by studying the degradation of selected model pollutants (2,4-DCP, chloroform) and the disinfection of a human bacterial pathogen (<i>E. coli </i>O157:H7). Overall efficacy of the batch reactor was assessed by combining biological toxicity assessment (biosensing) with conventional analytical chemistry. Photoelectrocatalytic degradation of the organoxenobiotics (2,4-DCP, chloroform) was monitored toxicologically by applying bacterial <i>lux</i>-marked biosensors and analytically by HPLC. The bacterial biosensor traced the removal of the target, model pollutants during degradation experiments, and also monitored changes in toxicity in the analyte of the PEC batch reactor caused by the possible appearance/disappearance of toxic transient intermediates derived from the breakdown of the parent molecule. Chromosomally <i>lux</i>-marked, non-toxigenic <i>E. coli</i> O157:H7 was selected as a model human pathogenic bacterium to demonstrate the disinfection potential of the batch reactor. Results of disinfection experiments indicated that a substantial decline in the population density of culturable <i>E. coli </i> O157:H7 cells was achieved. Accurate differentiation between the effects of photoelectrocatalysis and photolysis on the cells of <i>E. coli</i> O157:H7 was not achieved. The observed rate of the degradation of the model chemical compounds and the disinfection of the model human pathogen, demonstrated that visible light-driven photoelectrocatalysis offers considerable potential for remediation of contaminated water. Furthermore, toxicological biosensing can bridge the gap between traditional chemical analysis and ecologically relevant sample evaluation and address suitability of reintroduction of treated solution back into mainstream wastewater treatment.

Enterohaemorrhagic Escherichia coli O157:H7 causes haemorrhagic diarrhoea and potentially fatal renal failure in humans. Ruminants are considered to be the primary reservoir for human infection. Vaccines that reduce shedding in cattle are only partially protective and their underlying protective mechanisms are unknown. Studies investigating the response of cattle to colonisation generally focus on humoral immunity, leaving the role of cellular immunity unclear. To inform future vaccine development, the cellular immune response of cattle during EHEC O157:H7 colonisation was examined. Calves were challenged with either a phage type (PT) 21/28 strain possessing the Shiga toxin (Stx) 2a and Stx2c genes or a PT32 strain possessing the Stx2c gene only. T-helper cell associated transcripts at the terminal rectum were analysed by reverse transcriptase quantitative PCR (RT-qPCR). Induction of interferon (IFN)γ and T-bet was observed, with peak expression of both genes at 7 days in PT32 challenged calves, whilst up regulation was delayed, peaking at 21 days in PT21/28 challenged calves. Cells isolated from gastro-intestinal lymph nodes demonstrated antigen-specific proliferation and IFNγ release in response to type III secreted proteins (T3SPs); however responsiveness was suppressed in cells isolated from PT32 challenged calves. Lymph node cells showed increased expression of the proliferation marker Ki67 in CD4+ T cells from PT21/28, NK cells from PT32 and CD8+ and γδ T cells from both PT21/28 and PT32 challenged calves following ex vivo stimulation with T3SPs. Epitope mapping of rectal lymph node CD4+ T cell responses to 16 EHEC O157:H7 proteins, identified 20 CD4+ T cell epitopes specific to E. coli. The highly conserved N-terminal region of Intimin, including the signal peptide, was consistently recognised by mucosal CD4+ T cell populations from multiple animals of different major histocompatibility complex (MHC) class II haplotypes. Studies investigating the impact of secreted bacterial proteins on bovine peripheral blood mononuclear cells (PBMC) identified the ability of these proteins to cleave the surface molecule CD8 and that this phenotype was dependent on the ler virulence regulator but not the type III secretory system (T3SS) machinery. This effect was also observed in murine and ovine, but not human lymphocytes. Preliminary in vitro experiments suggest that this activity may reduce the efficiency of CD8+ T cell killing. This study demonstrates that cattle mount cellular immune responses during colonisation with EHEC O157:H7, the temporality of which is strain dependent, with further evidence of strain-specific immunomodulation.

<p>The purpose of this study was to investigate the occurrence<strong> </strong>of Verotoxin-encoding bacteriophages in mussels, cultured downstream the sewage treatment plant<strong> </strong>in Lysekil.</p><p>Mussels were collected in three growing areas from April 2008 to March 2009. Real-time PCR was performed for detection of <em>vtx1</em> and <em>vtx2</em> genes and enrichment of bacteriophages on non Verotoxin-producing <em>Escherichia coli</em> O157: H7 was carried out. All samples in real-time PCR analysis were negative; no presence of Verotoxin-encoding phages was shown. No plaque was formed on blood agar base plates, indicating that no bacteriophages had been taken up by <em>E. coli</em> bacteria</p><p>The levels of Verotoxin-encoding phages and <em>E.coli</em> outside the sewage treatment plant in Lysekil were not high enough to be able to form VTEC in mussels, indicating that the faecal contamination was low. This does not exclude the presence of other more common pathogens such as norovirus and campylobacter.</p><p> </p><p> </p>