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

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 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.

This study was conducted to determine the effectiveness of trimming the original external carcass surfaces from subprimals during fabrication on the reduction of surrogates for Escherichia coli O157:H7. Carcass sides from five cattle (n = 10 sides) were inoculated along the pattern hide opening before entering the blast chill cooler with a gelatin slurry containing a bacterial cocktail of three rifampicin-resistant, nonpathogenic E. coli Biotype I strains. Following a 48 h chill, sides were fabricated to produce eight subprimals (brisket, chuck, clod, rib, bottom round, top sirloin, short loin, and inside round). Microbiological samples were taken from the original carcass fat surface area, initial lean surface area, trimmed fat surface area (where applicable), and trimmed lean surface area (where applicable). Trimming of the external fat surfaces reduced (P < 0.05) microbiological counts on the newly exposed lean surfaces of all eight subprimals during fabrication. However, these data also indicated that fat and lean surfaces that were not initially exposed to contamination became contaminated during the fabrication process. Trimming external surfaces reduces levels of pathogens, but under normal fabrication processes, pathogens may still be spread to the newly exposed surfaces.

Thesis (Phd- Agric) North-West University, Mafikeng Campus, 2011

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a human intestinal pathogen, which can cause severe disease. EHEC O157:H7 is responsible for outbreaks of diarrhea and hemorrhagic colitis. EHEC produces a potent cytotoxin known as Vero (Shiga-like) cytotoxin, which causes diarrhea-associated hemolytic uremic syndrome (HUS), the most common cause of acute renal failure in children. Current treatment remains predominantly supportive in nature because antibiotics and non-steroidal anti-inflammatory drugs exacerbate the condition. Therefore, alternative therapeutic approaches that will prevent the EHEC colonization without the release of toxins need to be delineated. Understanding the pathobiology of disease is likely to yield novel approaches to interrupt the infectious process. My hypothesis was that pathogen-derived effectors associate with lipid rafts and, thereby, promote the recruitment of host signal transduction proteins to lipid rafts in response to EHEC O157:H7 infection. In this thesis, specific host signalling pathways hijacked by EHEC O157:H7, through lipid raft signalling platforms, to elicit pathogenic effects are studied using complementary approaches, including epithelial model cell lines and an animal model of infection (Citrobacter rodentium challenge of mice). A lack of osteopontin resulted in decreased attaching effacing lesions and reduced colonic epithelial cell hyperplasia in response to C. rodentium infection. These findings suggest that C. rodentium, mimicking EHEC O157:H7 infection, is capable of utilizing host cell components to elicit its pathogenic effects. In vitro data showed that EHEC O157:H7 effector proteins manipulate cell signalling through lipid rafts employed as platforms to recruit and activate host second messengers. PKC and PI3K activation led to attaching and effacing lesions, disruption of tight junctions, and the initiation of both innate and adaptive host immune responses. The results pointed towards a role for atypical PKC in EHEC-induced attaching and effacing lesion formation. The role of lipid rafts in EHEC O157:H7 pathogenesis was also studied using Citrobacter rodentium-infected Niemann-pick type C (NPC) mice. Infection of NPC mice, which lack lipid rafts, with C. rodentium resulted in delayed colonization and delayed onset of attaching-effacing lesion formation, compared with infected wild type mice. C. rodentium-infected NPC mice also demonstrated reduced colonic epithelial hyperplasia and decreased secretion of the pro-inflammatory cytokine, interferon-γ. Taken together, the findings presented in this thesis highlight the importance of host cell signal transduction cascades in EHEC O157:H7 disease pathogenesis, and demonstrate a role for lipid rafts and OPN in mediating host cell signaling responses to non-invasive enteric microbial pathogens.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a human intestinal pathogen, which can cause severe disease. EHEC O157:H7 is responsible for outbreaks of diarrhea and hemorrhagic colitis. EHEC produces a potent cytotoxin known as Vero (Shiga-like) cytotoxin, which causes diarrhea-associated hemolytic uremic syndrome (HUS), the most common cause of acute renal failure in children. Current treatment remains predominantly supportive in nature because antibiotics and non-steroidal anti-inflammatory drugs exacerbate the condition. Therefore, alternative therapeutic approaches that will prevent the EHEC colonization without the release of toxins need to be delineated. Understanding the pathobiology of disease is likely to yield novel approaches to interrupt the infectious process. My hypothesis was that pathogen-derived effectors associate with lipid rafts and, thereby, promote the recruitment of host signal transduction proteins to lipid rafts in response to EHEC O157:H7 infection. In this thesis, specific host signalling pathways hijacked by EHEC O157:H7, through lipid raft signalling platforms, to elicit pathogenic effects are studied using complementary approaches, including epithelial model cell lines and an animal model of infection (Citrobacter rodentium challenge of mice). A lack of osteopontin resulted in decreased attaching effacing lesions and reduced colonic epithelial cell hyperplasia in response to C. rodentium infection. These findings suggest that C. rodentium, mimicking EHEC O157:H7 infection, is capable of utilizing host cell components to elicit its pathogenic effects. In vitro data showed that EHEC O157:H7 effector proteins manipulate cell signalling through lipid rafts employed as platforms to recruit and activate host second messengers. PKC and PI3K activation led to attaching and effacing lesions, disruption of tight junctions, and the initiation of both innate and adaptive host immune responses. The results pointed towards a role for atypical PKC in EHEC-induced attaching and effacing lesion formation. The role of lipid rafts in EHEC O157:H7 pathogenesis was also studied using Citrobacter rodentium-infected Niemann-pick type C (NPC) mice. Infection of NPC mice, which lack lipid rafts, with C. rodentium resulted in delayed colonization and delayed onset of attaching-effacing lesion formation, compared with infected wild type mice. C. rodentium-infected NPC mice also demonstrated reduced colonic epithelial hyperplasia and decreased secretion of the pro-inflammatory cytokine, interferon-γ. Taken together, the findings presented in this thesis highlight the importance of host cell signal transduction cascades in EHEC O157:H7 disease pathogenesis, and demonstrate a role for lipid rafts and OPN in mediating host cell signaling responses to non-invasive enteric microbial pathogens.

Most bacterial foodborne pathogens are shed intermittently from their animal hosts and are able to grow and persist in the environment. Cattle and pigs constitute the major animal reservoirs for these pathogens. The overall objective of this dissertation research was to improve understanding of the role of intermittent shedding and environmental persistence in the transmission and maintenance of Escherichia coli O157:H7 and Salmonella Typhimurium in their animal host populations. This objective was addressed through five interdepended studies. The study in Chapter II, describes the transmission of E. coli O157:H7 in a dairy herd using mathematical modeling that includes indirect transmission from the contaminated environment. The model predicts that the elevated ambient temperature during summer, together with the availability of large amount of drinking water per cattle, are the major factors for increased pathogen load in water and high prevalence of E. coli O157:H7 in cattle populations. The second study, in Chapter III, determined the variation in water-to-cattle ratios among feedlot pens and evaluated the association with the pen level management and environmental factors. Water-to-cattle ratio was found to vary greatly between feedlots and pens with lower water-to-cattle ratios on average had cooler drinking water. The study in Chapter IV, used a compartmental mathematical model of infection transmission, to evaluate the effect of cleaning on Salmonella Typhimurium control in a grower-finisher pig herd. Cleaning alone was not found to be an effective measure of control unless combined with other measures to reduce the level of bacterial shedding. The study in Chapter V, developed the multi-state Markov chain model to describe the fecal shedding pattern of three E. coli O157:H7 strains in cattle. One strain was not detected to shed, while the other two strains had on average different durations of host colonization, albeit not at the statistically significant level. The study in Chapter VI, used an experimental infection transmission approach to estimate and compare transmission rates for three different strains of E. coli O157:H7 in steers. The results revealed that the transmission rate of E. coli O157:H7 increases significantly with increasing levels of environmental contamination. Collectively, the five studies have highlighted the role of these pathogen characteristics in their transmission. The improved understanding of these characteristics will allow for better design of control measure to ensure food safety.