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.

The metabolic activity of bacterial microorganisms may influence the growth and metabolic activities of other microbes that are present in any specific niche. Lactic acid bacteria (LAB) are antagonistic to some microbial pathogens by the metabolic production of compounds with antimicrobial activity. Consequently, investigators have measured the effects of those antimicrobials to inhibit specific pathogens. However, the mode(s) of action of LAB against foodborne pathogens on products and/or in broth is not completely understood. Therefore, the objectives of this research were to (i) determine the LAB dose required for inhibition of Escherichia coli O157:H7 and Salmonella enterica in vitro and on spinach, and (ii) identify and quantify the major antimicrobial substances synthesized by LAB as a function of postinoculation storage conditions. Assays were performed at 7 degrees C under aerobic conditions. The foodborne pathogens dose responses were assessed in a liquid microbiological medium (in vitro) and on spinach leaf surfaces. Different levels of foodborne pathogens and LAB cultures were used. The addition of LAB cultures did not reduce E. coli O157:H7 or Salmonella enterica populations when performed in vitro. However, when LAB cultures were sprayed on the surfaces of spinach leaves at 8.0 log10 CFU/g, there were significant reductions on E. coli O157:H7 of 1.62 and 0.73 log10 CFU/g (after 3 days) and on Salmonella enterica of 1.85 and 0.71 log10 CFU/g (after 6 days) for treatments inoculated with an initial level of 2.0 and 4.0 log10 CFU/g, respectively. After quantification of the antimicrobial compounds synthesized by LAB cultures, they were correlated against the population growth of targeted pathogens. The highest Llactic acid (3.71 plus/minus 0.14 micromoles/ml, day 12) and hydrogen peroxide (3.72 plus/minus 3.34 microM, day 6) production were obtained from the in vitro sample inoculated with 8.0 log10 CFU/ml of LAB and 0.0 log10 CFU/ml of pathogens. The highest bacteriocin production (0.1 plus/minus 0.01 mg/ml) was obtained from the in vitro sample with 8.0 log10 CFU/ml of LAB and 2.0 log10 CFU/ml of pathogens. In conclusion, the LAB cultures were able to produce detectable amounts of antimicrobials that may be used as intervention and/or sciencebased practice against foodborne pathogens by producers and the industry.

Ready-to-eat meat products such as dry-fermented sausages have been associated with foodborne outbreaks despite the multiple hurdles used in the manufacturing process to prevent growth of pathogens. As a result, new strategies such as natural products with antimicrobial activity are being used to control pathogens of importance like Escherichia coli O157:H7. This study investigated how different concentrations and sources of mustard can influence its antimicrobial activity against E. coli O157:H7 in dry-fermented sausage, as well as the contribution of residual myrosinase enzyme in mustard to this process. The genetic basis for the degradation of mustard glucosinolate by E. coli O157:H7, which is associated with the antimicrobial action of mustard, was also characterized. The ability of E. coli O157:H7 to withstand inhibitory allyl isothiocyanate (AITC) concentrations and the role of the two-component BaeSR system as a defense mechanism against AITC was also investigated. Results showed that 4% (w/w) deodorized yellow mustard powder was effective to control E. coli O157:H7 in dry-fermented sausage at 28 d. The presence of endogenous plant myrosinase in the mustard powder or meal enhanced E. coli O157:H7 reduction rates. Fully-deodorized, deoiled, yellow mustard meal as low as 2% (w/w) containing either 0.1% or 0.2% of residual plant myrosinase achieved the same results as 4% (w/w) mustard powder also containing similar residual myrosinase. Regardless of the type of mustard, the antimicrobial activity of yellow mustard derivatives were more pronounced than those of Oriental mustard. The initial genetic assessment through in silico analysis found similarity between plant myrosinase and enzymes encoded by genes (bglA, ascB, and chbF) from β-glucosidase families in E. coli O157:H7 strains. After disruption of these genes using lambda-red replacement, single (∆bglA, ∆ascB, ∆chbF) and double (∆bglAascB, ∆chbFascB, ∆chbFbglA) mutant strains were created and assessed for glucosinolate degradation. The comparison of the gene expression profiles and changes in the extent of sinigrin degradation by different mutants suggested that ascB have a prominent role in the degradation of this β-glucoside by E. coli O157:H7. E. coli O157:H7 did not develop resistance to AITC, the essential oil formed from sinigrin degradation that is responsible for the antimicrobial activity of Oriental mustard.

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.

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