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Development of bacterial oxidative stress assays : towards using fluorescence methods

WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
Amanda Marie Schoonover (Creator)
Institution
Western Carolina University (WCU )
Web Site: http://www.wcu.edu/404.asp
Advisor
Lori Seischab

Abstract: Several classes of bactericidal antibiotics increase oxidative stress in bacteria by upregulating the production of reactive oxygen species. Reactive oxygen species are also produced by host immune cells as protection against infectious bacteria. Superoxide dismutase (SOD) is a defensive enzyme that protects bacteria from the damage caused by reactive oxygen species. It is hypothesized that the inhibition of SOD would increase bacterial cell death caused by oxidative stress. The goal of this study was to establish a fluorescence-based assay for the determination of the viability of Escherichia coli that have been treated with antibiotics and SOD inhibitors. The fluorescence-based assay was compared to traditional methods of assessing bacterial viability including spread plating and measuring growth by optical density. The concentration of E. coli cells and the type of 96-well plate used in the fluorescence microplate reader affected the ability of the fluorescence assay to accurately assess the bacterial viability. Quercetin, a Cu,Zn-SOD inhibitor, interfered with measurements due to fluorescence it emits when bound to targets in the cell. Diethyldithio carbamate (DDC), also a Cu,Zn-SOD inhibitor, caused a decrease of bacterial viability at high concentrations of inhibitor (100 µM and 1000 µM DDC) which was noted when using both fluorescence measurement methods and traditional optical density growth measurements. The effects of ampicillin, kanamycin and norfloxacin, representatives of three classes of bactericidal antibiotics, on cell viability were measured using fluorescence methods and spread plate methods. The affects of the antibiotics on the bacterial cells could not be accurately measured because using the fluorescence methods. E. coli incubated with DDC and antibiotic showed little to no difference from cells grown with DDC alone or antibiotic alone.

Additional Information

Publication
Thesis
Language: English
Date: 2009
Keywords
fluorescence, oxidative stress, Superoxide dismutase
Subjects
Antibiotics -- Analysis
Quercetin
Fluorimetry