<OL> <LI> Develop a multiplex PCR assay and real-time PCR assay to detect and quantify Pseudomonad species significant in biofilm formation, spoilage, and disease. <LI>Characterize biofilms that contain foodborne pathogens including Campylobacter, Salmonella, E. coli, and Listeria. <LI>Determine the virulence of foodborne bacterial pathogens, including Campylobacter, Salmonella, E. coli, and Listeria, associated with stressed phenotypes.</ol>
NON-TECHNICAL SUMMARY: Economic loss due to food spoilage has been estimated at somewhere between 5 and 17 billion dollars annually. To the poultry industry, the Pseudomonads are responsible for a majority of this spoilage cost, primarily as spoiled meat and egg products. While the ability of bacteria to form a biofilm varies greatly among the genera, one consistent finding is that all Pseudomonas species are excellent biofilm formers and these biofilms can promote the survival of the foodborne pathogens. Characterizing biofilms in terms of exoploymer production, diversity, mechanisms of resistance, survival, virulence, and cell signaling cascades will give information that can be used to target the bacteria responsible for biofilm formation, and which may help to control foodborne pathogens. Furthermore, determining the mechanisms by which bacteria resist stress or enhance virulence could provide novel targets for antibiotic therapy. <P>APPROACH: <br>Objective 1: A molecular approach will be used to create a multiplex PCR assay. In the first phase, individual primers will be optimized in separate reactions to detect and distinguish multiple Pseudomonad species. The assay then will be combined into a mulitplex to reduce reagents and costs which will improve efficiency. Chicken wash samples will be evaluated primarily and the ability of the multiplex to be used with other food products will be performed at a later time. The multiplex assay will be paired with a real-time PCR assay to quantify the amount of Pseudomonas present, which can be used to determine the expected shelf life. <P>Objective 2: In order to obtain bacteria within a biofilm community, bacteria will be isolated and identified from poultry environments using standard microbiological procedures. Samples will be taken at points typically thought to harbor biofilms or difficult to clean, including but not limited to poultry scald tanks, poultry chill tanks, corners of stainless steel tanks, rubber gaskets, rubber nipple drinkers, hauling pens, and drains. The members of the microbial community will be identified by traditional culturing, selective plating, and biochemical methods as well as using density gradient gel electrophoresis (DGGE) for whole genome profiling. The individual members will also be characterized in terms of biofilm formation, glycocaylx production, and cell signaling as described below. Pathogens will be further characterized by comparing biofilm and planktonic phenotype virulence abilities. <P>Objective 3: To identify stress response and the adaptive ability of pathogens, bacteria will be stressed and allowed to recover, then given further homologous and heterogeneous stresses using the following method. Stresses will include acid exposure, aerobic atmosphere, non-optimal temperatures and starvation conditions. All stressed cells will be inoculated onto appropriate medium and allowed to recover for 24h. Stressed cells then will be challenged with acid, aerobic, starvation, or temperature stresses, and samples will be taken at different times during the experiment to construct a survivor plot. By comparing the survival plot of stressed and non-stressed cells, the adaptation of pathogens to homologous and heterogeneous stresses will be observed.