Date of Award

Spring 2010

Project Type

Dissertation

Program or Major

Microbiology

Degree Name

Doctor of Philosophy

First Advisor

Frank G Rodgers

Abstract

The severity of Escherichia coli O157:H7 disease is due in part to a major virulence factor produced by the microbe, the shiga-like toxin 2 (Stx-2). Antibiotic treatment to reduce pathogen numbers is controversial, as it is thought that antibiotics may increase the levels of Stx-2 released from the pathogen. Currently, recommended treatment for E. coli O157:H7 is palliative The purpose of this study was to examine three critical factors potentially important to disease outcomes, and to determine their effect on expression of the stx2 gene and on release of Stx-2 from the pathogen. Those factors selected for study were: (i) various classes of antibiotics; (ii) probiotic microorganisms; and (iii) carbon source variation together with cAMP. Stx-2 was assessed using MTT cytotoxicity assays and ELISA analysis, while the expression of stx2 was assessed using real time PCR. It was determined that antibiotics that affect microbial DNA increased stx2 expression and Stx-2 production, and this was linked to an upregulation in the SOS DNA repair response. A link was also observed between the upregulation of stx2 and those antibiotics that disrupt cell membrane integrity. However, these antibiotics did not increase the overall levels of Stx-2 released from E. coli O157:H7. The probiotic microorganisms Lactobacillus casei and L. plantarum were found to decrease both stx2 expression and Stx-2 release when grown in co-culture with E. coli O157:H7 at greater or equal numbers to the pathogen. This reduction in Stx-2 was at least in part attributable to organic acids produced by the probiotics, but other unknown factors produced by the lactobacilli cannot be excluded. Finally, it was determined that growth of the pathogen in glucose-supplemented media yielded significantly more stx2 expression and Stx-2 production than growth in glycerol-supplemented media. This observation was confirmed by a decrease in stx2 expression and Stx-2 production when exogenous cAMP was added to culture media. The examination of these three factors led to a clearer understanding of the intricacies involved in the regulation of stx2, and has demonstrated how such an apparently diverse group of external factors are interlinked through several complex mechanisms.

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