Escherichia coli (E.coli) 0157:H7, a pathogen belonging to the enterohemorrhagic group of E.coli, has long been a concern to human health. The pathogen causes a myriad of symptoms in humans, ranging from diarrhea and malaise to renal failure. Human infection with the spread of the pathogen is mainly attributed to consumption of contaminated food material such as meat. Decontamination of meat via sprays have to date been the most commonly practiced method to reduce contamination, which now has little relevance in the face of developing resistance by the pathogen. In the following study we investigated FlhC mediated gene regulation in E. coli 0157:H7 on the surface of meat, in an attempt to recognize FlhC regulated targets, which may ultimately serve as targets for the development of novel decontaminating sprays. Microarray experiments were conducted to compare gene expression levels between a parental E. coli 0157:H7 strain and its isogenic flhC mutant, both grown on meat. Putative FlhC targets were then grouped based on their function. Realtime PCR experiment was done to confirm the regulation. Additionally, experiments were done to investigate the phenotypic effects of the regulation. To test the effect of FlhC on biofilm formation, an ATP based assay was first developed in E.coli K-12, which has been detailed in the following dissertation. This assay was used to quantify biofilm biomass in E. coli 0157. Microarray experiments revealed 287 genes as being down regulated by FlhC. These genes belonged to functions relating to cell division, metabolism, biofilm formation and pathogenicity. Real-time PCR confirmed the regulation of 87% of the tested genes. An additional 13 genes were tested with real-time PCR. These belonged to the same functional groups, but were either not spotted on the microarray chips or had missing data points and were hence not included in the analysis. All 13 of these genes appeared to be regulated by FlhC. The phenotypic experiments performed elucidated that the FlhC mutants divided to 20 times higher cell densities, formed five times more biofilm biomass and were twice as pathogenic in a chicken embryo lethality assay, when compared to the parental strain. The following dissertation also reports the development of a combination assay for the quantification of biofilm that takes advantage of the previously mentioned ATP assay and the PhenotypeMicroarray TM (PM) system. The assay was developed using the parental E. coli strain AJW678 and later applied to its isogenic flhD mutant to elaborate on the differences in nutritional requirements between the two strains during biofilm formation. Metabolic modeling and statistical testing was also applied to the data obtained. This assay will be used in the future to study biofilm formation by the parental strain E. coli 0157:H7 strain and its isogenic FlhC
mutants on single carbon sources, hence identifying potential metabolites which differentially support biofilm formation in the parental and the mutant strain.
| Identifer | oai:union.ndltd.org:ndsu.edu/oai:library.ndsu.edu:10365/29205 |
| Date | January 2011 |
| Creators | Sule, Preeti |
| Publisher | North Dakota State University |
| Source Sets | North Dakota State University |
| Detected Language | English |
| Type | text/dissertation |
| Format | application/pdf |
| Rights | NDSU policy 190.6.2, https://www.ndsu.edu/fileadmin/policy/190.pdf |
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