Corron, Jessica Lee, authorNightingale, Kendra, advisorBunning, Marisa, committee memberSofos, John, committee member2022-04-202022-04-202010https://hdl.handle.net/10217/234744Covers not scanned.Print version deaccessioned 2022.Listeriosis is a severe and often fatal disease caused by the foodborne pathogen Listeria monocytogenes. At least two distinct subpopulations of this pathogen have been observed, including (i.) Epidemic clone (EC) strains, which have been responsible for numerous outbreaks worldwide, as well as the majority of sporadic listeriosis cases in the U.S., and (ii.) strains carrying a premature stop codon mutation (PMSC) in a key virulence gene inlA, leading to natural virulence attenuation. The latter population is prevalent in foods, with strains harboring an inlA PMSC comprising approximately 50 percent of food isolates. We thus hypothesized that these two distinct subpopulations have undergone niche adaptation, where EC strains have become adapted to a human host niche while inlA PMSC strains have become adapted to a food niche and additionally alternative mammalian hosts (e.g. mice, rats, etc.). In order to test this hypothesis we assembled a strain set of eight strains, four EC strains and four in/A PMSC strains, and subjected these strains to various assays, which mimic a host or food environment. Specifically we performed intracellular growth assays and cytotoxicity assays in macrophage like-cells. Additionally we performed invasion assays in mouse L929 fibroblast cells to determine the ability of these cells to invade cells other than human cells. InlA, a surface protein, binds to the receptor E-cadherin and mice possess a different isoform which does not allow for efficient InlA binding; therefore, these assays should provide insight on invasion of cells with different non-human isoforms of E-cadherin. Lastly, we performed cold growth assays in BEII broth at 7°C as well as a growth study on deli turkey in order to evaluate growth in food-like or food conditions. Overall, we found that although all strains grew similarly in all broth cold growth conditions; EC and inlA PMSC strains showed differences in terms of virulence phenotype as well as the ability to grow in RTE deli turkey. Although all strains grew similarly at all time points during intracellular growth experiments, differences between EC and inlA PMSC strains were observed at later time points in the cytotoxicity assays. Specifically, the inlA PMSC strains exhibited higher cytotoxicity 9, and 12 hours following inoculation {P = 0.0028, and P < 0.0001 respectively). All inlA PMSC strains invaded mouse L929 cells better than a standard laboratory control strain, in comparison to only one EC strain which showed the same trend (P<0.05). Lastly, inlA PMSC strains had higher exponential phase growth rates (P = 0.0243) when compared to EC strains when grown on deli turkey for 28 days. These data support the conclusion that EC strains are better adapted for pathogenesis in a human host while inlA PMSC strains are better adapted to survival in foods as well as in non-human hosts where inlA mediated invasion is not necessary for internalization. Escherichia coli 0157:H7 causes an estimated 73,480 cases of illness each year, a small percentage of these cases progress to a potentially fatal disease called hemolytic uremic syndrome or HUS, sometimes leading to kidney failure or even death in serious cases. The majority of E. coli 0157;H7 cases are food related, and cattle are implicated as the major reservoir host for this pathogen. Antibiotics are not recommended for the treatment of E. coli 0157:H7 infections, thus treatment options are limited. Therefore, it is critical to minimize the load of E. coli 0157:H7 that reaches the consumer. In order to identify compounds to be used for the control and treatment of this deadly pathogen, we designed a high throughput, small molecule, turbidometric growth assay for the identification of bactericidal or bacteriostatic compounds. Through this screening we investigated the potential of nearly 65,000 compounds, which were assayed in duplicate, and identified 43 which inhibited E. coli 0157:H7 growth. Of the 43 compounds, 38 were known bioactive compounds, while the other five were from libraries of commercially available chemical compounds. Many of the known bioactive compounds were known antibiotics, specifically cephalosporins (n=13), fluorquinolones (n=12), and tetracyclines (n=9), as well as one carbapenem and two other antibiotics. Additionally, an antiviral agent, an inhibitor of the tricarboxylic acid cycle, as well as two structurally related disinfectants, which are used in a number of non-clinical applications, were also discovered. Specifically, the non-clinical disinfectants show promise for spray-wash and dip type treatments to be implemented in the food supply, due to their antimicrobial activities against E. coli 0157;H7, and their cytotoxicity, which is similar to other widely used disinfectants.masters thesesengCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.Listeria monocytogenesEscherichia coli O157:H7Text