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.

<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>

The purpose of this study was to investigate the occurrence of Verotoxin-encoding bacteriophages in mussels, cultured downstream the sewage treatment plant in Lysekil. Mussels were collected in three growing areas from April 2008 to March 2009. Real-time PCR was performed for detection of vtx1 and vtx2 genes and enrichment of bacteriophages on non Verotoxin-producing Escherichia coli 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 E. coli bacteria The levels of Verotoxin-encoding phages and E.coli 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.

The Shiga-like toxins have DNA sequence homology to the toxins accountable for the dysentery brought about by the Shigella species. <i>Escherichia coli</i> which encode and produce shiga-like toxins are referred to as shiga toxin-producing E. coli (STEC). Upon infection with STEC, humans may develop a variety of clinical symptoms ranging in severity from bloody diarrhea to life threatening hemolytic uremic syndrome (HUS). Hemolytic uremic syndrome is the most fatal disease manifestation upon STEC infection for humans and has been documented to occur in up to 20% of patients upon STEC infection [29]. The Shiga toxins (Shiga toxin 1 and 2) are regarded as the principal virulence factor of STEC and are responsible for the clinical manifestations during HUS in humans [49].<p> Cattle are the primary non-human reservoir for STEC and therefore represent an attractive target for pre-slaughter intervention as a means to reduce human infections. To date, vaccination with secreted proteins including Shiga toxin 2 (Stx2), has reduced the numbers of bacteria shed in feces [3]. Even though published data exists supporting vaccination in cattle as a means to reduce STEC, commercially available vaccines are not being used by farms and STEC remain a significant zoonotic pathogen of humans causing disease and death. To further our knowledge about STEC pathogenesis in cattle, we examined the effect of Shiga toxin 2 on bovine immune responses. Bovine lymphocyte function was determined in the presence of Shiga toxin 2 and the magnitude of bovine immunological responses was measure after immunization with Shiga toxin 2. In general, results suggest that Shiga toxin 2 downregulates bovine immune responses suggesting vaccination with effector molecules that exclude Shiga toxin 2 may induce a better immunological response and improve vaccine efficacy.<p> To examine the possibility that Stx2 modulates bovine immune responses, we investigated lymphocyte function in the presence of Stx2. Menge et al [70] have reported that bovine lymphocytes express the Stx receptor and that Shiga toxin 1 inhibits lymphocyte proliferation in vitro. We isolated two populations of lymphocytes, peripheral blood mononuclear cells (PBMCs) and ileal Peyers patch lymphocytes (IPPL) and compared lymphocyte function in the presence and absence of Stx2. We found that Stx2 did not affect IPPL viability in vitro but did inhibit IPPL proliferation after 12 hours of incubation <i>in vitro</i>. In contrast, no altered PBMC function could be observed in the presence of Stx2. These results suggest that receptor-bound Stx2 may inhibit IPPL proliferation and that the two populations of lymphocytes isolated are unique and distinct from each other in their response to Stx2.<p> To determine the effect of Stx2 on bovine immune responses during STEC infection, a bovine ileal ligated loop model was employed. Ligated loops were inoculated with either a Stx2+ STEC strain or an isogenic Stx2- STEC strain. After 24 hours, IPPL populations were isolated from each ligated loop and immunophenotyped. The results indicated a significantly reduced CD4+ T cell population in the presence of Stx2. No differences in the levels of IFNá, TNFá, IL12 or IFNã could be detected between groups. These results suggest that Stx2 modulates bovine immune responses but not as a result of increased production of these cytokines. To extend this finding, we determined the effect of Stx2 on bovine immune responses during active immunization by using ELISA to measure serological responses in the presence and absence of Stx2. Serological responses to secreted proteins, as well as a co-administered antigen (hen egg lysozyme), were significantly reduced in the groups of cattle that were immunized with either purified Stx2 or secreted protein preparations isolated from STEC compared to groups vaccinated with antigens which did not contain the toxin. Bovine proliferative responses were also measured and the results indicated significantly reduced proliferation in the groups vaccinated with the formulations containing Stx2. Therefore, based on these results, we conclude that Stx2 downregulates bovine immune responses and thus may contribute to the colonization and persistence of cattle by STEC.

The Shiga-like toxins have DNA sequence homology to the toxins accountable for the dysentery brought about by the Shigella species. <i>Escherichia coli</i> which encode and produce shiga-like toxins are referred to as shiga toxin-producing E. coli (STEC). Upon infection with STEC, humans may develop a variety of clinical symptoms ranging in severity from bloody diarrhea to life threatening hemolytic uremic syndrome (HUS). Hemolytic uremic syndrome is the most fatal disease manifestation upon STEC infection for humans and has been documented to occur in up to 20% of patients upon STEC infection [29]. The Shiga toxins (Shiga toxin 1 and 2) are regarded as the principal virulence factor of STEC and are responsible for the clinical manifestations during HUS in humans [49].<p> Cattle are the primary non-human reservoir for STEC and therefore represent an attractive target for pre-slaughter intervention as a means to reduce human infections. To date, vaccination with secreted proteins including Shiga toxin 2 (Stx2), has reduced the numbers of bacteria shed in feces [3]. Even though published data exists supporting vaccination in cattle as a means to reduce STEC, commercially available vaccines are not being used by farms and STEC remain a significant zoonotic pathogen of humans causing disease and death. To further our knowledge about STEC pathogenesis in cattle, we examined the effect of Shiga toxin 2 on bovine immune responses. Bovine lymphocyte function was determined in the presence of Shiga toxin 2 and the magnitude of bovine immunological responses was measure after immunization with Shiga toxin 2. In general, results suggest that Shiga toxin 2 downregulates bovine immune responses suggesting vaccination with effector molecules that exclude Shiga toxin 2 may induce a better immunological response and improve vaccine efficacy.<p> To examine the possibility that Stx2 modulates bovine immune responses, we investigated lymphocyte function in the presence of Stx2. Menge et al [70] have reported that bovine lymphocytes express the Stx receptor and that Shiga toxin 1 inhibits lymphocyte proliferation in vitro. We isolated two populations of lymphocytes, peripheral blood mononuclear cells (PBMCs) and ileal Peyers patch lymphocytes (IPPL) and compared lymphocyte function in the presence and absence of Stx2. We found that Stx2 did not affect IPPL viability in vitro but did inhibit IPPL proliferation after 12 hours of incubation <i>in vitro</i>. In contrast, no altered PBMC function could be observed in the presence of Stx2. These results suggest that receptor-bound Stx2 may inhibit IPPL proliferation and that the two populations of lymphocytes isolated are unique and distinct from each other in their response to Stx2.<p> To determine the effect of Stx2 on bovine immune responses during STEC infection, a bovine ileal ligated loop model was employed. Ligated loops were inoculated with either a Stx2+ STEC strain or an isogenic Stx2- STEC strain. After 24 hours, IPPL populations were isolated from each ligated loop and immunophenotyped. The results indicated a significantly reduced CD4+ T cell population in the presence of Stx2. No differences in the levels of IFNá, TNFá, IL12 or IFNã could be detected between groups. These results suggest that Stx2 modulates bovine immune responses but not as a result of increased production of these cytokines. To extend this finding, we determined the effect of Stx2 on bovine immune responses during active immunization by using ELISA to measure serological responses in the presence and absence of Stx2. Serological responses to secreted proteins, as well as a co-administered antigen (hen egg lysozyme), were significantly reduced in the groups of cattle that were immunized with either purified Stx2 or secreted protein preparations isolated from STEC compared to groups vaccinated with antigens which did not contain the toxin. Bovine proliferative responses were also measured and the results indicated significantly reduced proliferation in the groups vaccinated with the formulations containing Stx2. Therefore, based on these results, we conclude that Stx2 downregulates bovine immune responses and thus may contribute to the colonization and persistence of cattle by STEC.

The beef industry has made tremendous strides in reducing pathogen contamination on carcasses. Multiple antimicrobial interventions have been validated for their use during harvesting. Information in regards to cross-contamination with pathogens in the post-harvest environment is limited. Surrogate microorganisms for enteric pathogens are commonly used to validate antimicrobial interventions and might allow for the simulation of cross-contamination through the post-harvest environment. The purpose of this study was to determine how the post-harvest environment impacts the direct and indirect transmission of pathogens. This was achieved by using fluorescent protein-marked surrogate strains of Escherichia coli O157:H7 and Salmonella spp. from inoculated carcasses to the adjacent ones and to the equipment and facility in three different abattoirs. Thirteen hide-on carcasses were inoculated using a gelatin-based slurry containing three nonpathogenic fluorescent protein-marked strains of E. coli biotype I. In order to determine direct and indirect cross-contamination, inoculated and adjacent carcasses were sampled (300 cm2) during the harvesting process at different stages: after hide opening (AHO), prior to evisceration (PE), after evisceration (AE), after splitting (AS), and after final intervention (AFI). Environmental samples consisting of the floor, walls, and air were tested as well as personal equipment including gloves, boots, and aprons. Equipment including hand knives, air knives, meat hooks, hide puller and split saw were also sampled. Results showed evidence of cross-contamination between inoculated carcasses and the adjacent non-inoculated ones for all abattoirs. Although this occurred in all abattoirs, surrogate counts on carcasses were below detectable levels (<1.4 log CFU/cm2) after antimicrobial interventions. Surrogates were found in low levels for all environmental samples. However surrogate counts from equipment such as knives, split saws, meat hooks, and hide puller were more frequently detected (15 percent) than those found on the floor, air and walls samples (10 percent). In the case of aprons, boots, and gloves, the prevalence of countable surrogate samples was 7 percent.