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Shiga toxin-producing Escherichia coli O157:H7 is a food borne pathogen responsible for bloody diarrhea, and potentially life-threatening hemolytic uremic syndrome. The study of O157:H7 has been ongoing since the first recorded outbreak in 1982. These studies have been limited by the lack of an adequate model system. Recent developments in stem cell technology have allowed for production of intestinal stem cell models that mimic the small intestine. Human intestinal organoids (HIOs), and enteroids (HIEs) have shown value for studying O157:H7 by studies in our lab. This work focuses on utilizing HIE monolayers (HIEMs), reverse polarity HIEs (RPHIEs), and HIOs to study O157:H7. It highlights the advantages of each potential model, addresses their disadvantages, and identifies methods to use these models for addressing key scientific gaps in O157:H7 literature. HIEMs have been used previously to study many enteric pathogens, including O157:H7. However, unlike previous HIEM studies, saline was added to the apical surface, while maintaining culture media in the basolateral well. The monolayers continued to grow and differentiate with apical saline. Apical infection with O157:H7 or commensal E. coli resulted in robust bacterial growth from 105 to over 108 over 24 hours. Despite this robust bacterial growth, commensal E. coli neither adhered to the cells nor damaged the epithelial barrier over 30 hours. However, O157:H7 was almost fully adhered (90%) by 18 h with epithelial damage observed by 30 hours. O157:H7 contains the locus of enterocyte effacement (LEE) pathogenicity island responsible for attachment and damage to the intestinal epithelium. Previous studies report the ability of nutrients such as biotin, D-serine, and L-fucose to downregulate LEE gene expression. O157:H7 treated with biotin or L-fucose, but not D-serine displayed both decreased attachment and reduced epithelial damage over 36 hours. While advantageous HIEMs have their limitations, we worked to address those by identifying other potential model systems in HIOs and RPHIEs. HIOs can recapitulate published data illustrating biotin treatment of O157:H7 reduces epithelial damage during infection. RPHIEs were identified as a feasible model for O157:H7 attachment studies.Identification of optimal methodology for HIEMs and HIOs allowed us to further investigate host-pathogen interactions in these models. We performed an in vitro analysis of intestinal pH in each model. HIEMs maintained a luminal pH of 6.7, which is decreased following inoculation with probiotic E. coli Nissle 1917, and pathogenic E. coli O157:H7. HIOs maintained a higher, more neutral pH of ~7.05. We additionally leveraged HIEMs to address the mechanisms of microvillus effacement and initial epithelial interactions, which are currently not well understood. We created a protocol to use lambda-red mutagenesis for removal of potential adhesion proteins with the goal to identify those involved in initial host-bacterial interaction prior to LEE activity. This work highlights the advantages of using stem cell technology for studying O157:H7 host-pathogen interactions, and lays groundwork for addressing key gaps in literature previously difficult due to the lack of adequate methodology.

The objective of this dissertation study was to elucidate how the level and composition of bacterial surface extracellular polymeric substances (EPS) contribute to indirect photochemical disinfection processes. This study was developed to generate mechanistic information on EPS function in bacterial die-off under natural occurring sensitizing environments to inform future process design. Specifically, this researchfocused on whether EPS promotes or prohibits disinfection, or be able to facilitate association with sensitizing surfaces. To accomplish this, the influence of bacterial EPS has been studied in batch (solar simulator and reactor) and flowing (parallel plate) systems simulating groundwater and agriculturally impacted surface water. Model strains of E. coli and Salmonella were utilized. In the initial work, the model Salmonella was incubated in different solution chemistries for varying times. Three EPS extraction methods (lyophilization, ethanol, and sonication) were applied and compared over the range of conditions for the sensitivity and extent of cell lysis after the extraction processes. The sonication method was deemed the best method for further use in subsequent disinfection kinetic studies inthe solar simulator with E. coli. This work was followed by E. coli transport and deposition experiments in the flow chamber. Throughout the research, a series of physical-chemical-biological characterization techniques have been applied on thebacterial strains including EPS compositional analysis, cell lysis testing, and the measurement of bacterial hydrophobicity, zeta potential, surface charge density, and size. The findings from this doctoral study provide the following fundamental insights. First, the composition and levels of EPS depends on the extraction protocol utilized, where no single method has the ability to separate all EPS from the cell surface without disturbing cell structure. Next, the presence of nitrate in photochemical disinfection was not sensitive to EPS level. Additionally, EPS did not hinder the disinfection by acting as a physical barrier or chemical quencher. Finally, this study has shown the ability of EPS to facilitate interactions between bacteria and sensitizers in photochemical disinfection processes.

Escherichia coli O157:H7 causes an estimated 73,000 of food borne illness annually. Varying levels of disease severity exist and include diarrhea, bloody diarrhea, and hemolytic uremic syndrome which can result in kidney damage or death. E. coli O157:H7 produces Shiga toxins 1 and/or 2, although it is Shiga toxin 2 that is linked to severe disease. Currently, a Shiga toxin-producing isolate will yield a positive diagnostic result, regardless of which Shiga toxin variant is produced. We have developed an ELISA that can differentiate between Shiga toxin 1 and Shiga toxin 2 in the presence of fecal material, and if used in the clinical setting, can increase the accuracy of prognosis for a patient infected with E. coli O157:H7. Epidemiological studies have suggested that antibiotics may be linked to HUS development. As such, we have completed an extensive study to determine which, if any, antibiotics are safe for the treatment of E. coli O157:H7. The data suggest that antibiotic-mediated Shiga toxin induction or reduction is based on the mechanism. Antibiotics that target DNA increase Shiga toxin production while protein synthesis inhibitors decrease Shiga toxin production. Human intestinal E. coli have been shown to amplify Shiga toxin. Our data suggest that protein synthesis inhibitors may be effective, even if the patient harbors a Shiga toxin-amplifying isolate, provided that the commensal isolate is susceptible to the antibiotic. Conversely, antibiotics would likely be of no benefit if the Shiga toxin-amplifier is resistant to the antibiotic. Future studies should focus on characterizing the flora of HUS patients, and examining antibiotic treatments within the context of these strains.