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Objective I, PD, Birgit M. Pruess: A recent outbreak in Germany was caused by a new combination of an enteroaggregative Escherichia coli (EAEC) and an enterohemorrhagic one (EHEC) (Mellmann et al., 2011). The resulting strain constitutes a perfect example of a newly emerged pathogen, against which there is no current protection or treatment. This raises the intriguing question whether it is possible to develop novel prevention options for infectious diseases, without being able to characterize the pathogen ahead of time. Research by the PD of this proposal may be able to provide such a solution. The long term-goal is to develop novel prevention and treatment options against infection by E. coli. These techniques will be developed primarily with non-pathogenic E. coli K-12 and conventional E. coli O157:H7 strains. The molecular and physiological background of these techniques, however, is based on the central metabolism of the bacteria. Central metabolism is very similar across bacterial species. After the development of the prevention and treatment techniques, we will perform careful screening to determine against which additional bacterial species and strains they may be effective. Whether the technique will be effective against pathogens that have yet to emerge, will remain speculative. <P>Objective II, Co-PD, Penelope S. Gibbs: The microplate reader will minimize the amount of labor and supplies needed for certain virulence assays, such as complement resistance and biofilm formation (Lee et al., 1991, Sule et al., 2009). The long-term goal of this project includes seeking methods to prevent biofilm attachment to stainless steel (bulk milk tanks and utensils for reconstituting powdered infant formulat (PIF)), glass (blenders and bottles), and other surfaces that come in contact with the incoming milk supplies for manufacturing of PIF, and those surfaces involved in the reconstitution of PIF. In particular, the function of the zpx gene of C. sakazakii (Kothary et al., 2007) in forming biofilms (if any) and invasion of the blood-brain barrier will be a primary focus of this project. Future goals are to determine which nutrients are necessary to incite biofilm formation and finding ways to control C. sakazakii infections. <P>Objective III, Co-PD, Charlene Wolf-Hall: The microplate reader will be utilized for studies of the physiological responses of Fusarium spp. to changes in nutrient and environmental factors, as well as exposure of the fungi to interventions such as physical, chemical and biological treatments. The reader will assist with measuring growth, pigment and other measurable physiological responses for factorial studies. It is expected that the reader will also be utilized for various food microbiology projects in Wolf-Hall's research for which similar types of measurements may be needed.
Non-Technical Summary:<br/>
General Description of the Project: This equipment grant is intended to be used for the purchase of the GloMax Multi-Detection System from Promega that has capabilities for luminescence, fluorescence, and absorbance. Using a 96 well format, the PD and Co-PDs of this proposal plan to study the infectious disease molecular biology of several critical and emerging pathogens, with a long-term goal of developing novel intervention techniques. The reader will speed up this research. As one example, the PD is currently performing the luminescence assays in single tubes. This is neither time nor cost efficient. A 96 well format would allow us to perform these experiments in a more timely manner, while increasing reproducibility. In addition, we intend to move into new research areas by screening a library of promoter::fluorescence protein fusions, with the potential of discovering novel genes that may be used as drug targets to prevent and/or treat infectious disease. This will enable us to produce preliminary data that can then be used to be more competitive in applying for additional federal funds. The PD and Co-PDs have a history of successfully supervising students in their respective laboratories at both, graduate and undergraduate level. The Department of Veterinary and Microbiological Sciences just received three new research assistantships from the Experiment Station. Like this equipment grant, the assistantships will increase our competitiveness for future federal funding. They also indicate strong support for graduate research at NDSU from the State of North Dakota. Overall, being able to purchase the reader would be beneficial to our research, as well as our educational goals.
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Approach:<br/>
1.1 We will use the proposed microplate reader to screen hundreds of nutrients for their effect on biofilm formation in a selection of E. coli strains that include a biofilm forming parent strain, AJW678 (Kumari et al., 2000), and isogenic mutants in genes of two-component systems, as well as other global regulator genes. We will use the Phenotype MicroArrayTM (PM) system from BioLog (Hayward, CA) for the nutrient screen. The PM system consists of 96 well plates that have a different single nutrient dried to the bottom of each well. This permits the screening of approximately 2,000 growth phenotypes, when used with the redox dye that is provided by the manufacturer and measured as absorbance at 600 nm (Bochner et al., 2001). Previous research in the lab of the PD has modified this system to be used for the screening of biofilm amounts that form in the presence of the different nutrients (Sule et al., 2011b). Biofilm amounts will be determined with the established ATP assay, that converts ATP amounts into a luminescence signal (Sule et al., 2009).
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1.2 An additional screen will be performed in this context, taking advantage of the fluorescence option of the microplate reader. Not all of the regulators that the research proposed with this grant will identify as important in biofilm formation will be expressed at levels that permitt fluorescence microscopy. We have purchased the library of promoter::GFP fusion plasmids from Open Biosystems that contains 1,900 of the 2,400 E. coli promoters, fused to the GFP green fluorescence protein. We will use the 96 well microtiter plate format to determine the fluorescence levels from the promoters of interest. This will be more time and cost effective that attempting to perform fluorescence microscopy with poorly expressed genes.
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1.3 The final experiment that the microplate reader will be used for will advance the development of the spray that protects consumers from infection with E. coli from contaminated beef meat. We will use the same PM technology that was described as experiment 1.1 with the E. coli O157:H7 strain. Using the redox dye, we will determine growth at 10oC (refrigerator temperature) on individual carbon sources. Using the ATP assay (Sule et al., 2009, 2011b), we will determine biofilm amounts. Cell numbers will be determined by serially diluting planktonic bacteria from the PM plates. This will be indicative of cell division. The experiment will determine carbon sources that will increase the expression level of flhD, while decreasing the ability to form biofilm and divide effectively. The continuation of the project will develop such carbon sources into the protective spray. As explained for experiment 1.1, we will then test the effectiveness of the spray against additional bacterial pathogens. Altogether, the microplate reader will be used to determine nutrients that inhibit biofilm formation and can be used for the development of novel biofilm prevention and treatment options.