Phage biocontrol of Vibrio parahaemolyticus for enhancing seafood safety

The current interventions employed by the seafood industry such as rapid cooling of the harvest, restricted harvesting in warmer/summer and rainy months, relaying and depuration, high-hydrostatic pressure processing (HPP), mild heat processing, and irradiation are inadequate, restrictive, and in certain cases cost-prohibitive. The overarching goal of this project is to develop a commercial product (tentatively designated VibrioShield™) suited for direct application on food surfaces including raw or RTE fish and other seafood to eliminate or reduce their contamination with V. parahaemolyticus. Post successful commercialization, the product will be linked with a service component that will monitor product efficacy (including factors such as the development of resistance to specific phages), optimize and customize phage administration, and assist producers in their overall efforts to control V. parahaemolyticus contamination. Intralytix is the first company to have received an FDA/USDA approval for a phage-based product ListShield™ in 2006. The ListShield™ was designed for and is applied directly onto the surfaces of fish, shellfish, poultry, and other meat products (including RTEs), and significantly reduces the levels of Listeria monocytogenes in those foods. ListShield™ was also approved by the Environmental Protection Agency (EPA), as a "microbial pesticide" to decontaminate surfaces in food processing plants and other food establishments that may be contaminated with L. monocytogenes. Additionally, ListShield™ is also approved for food safety applications by Health Canada and the National Food Service of Israel. Following ListShield™, we have developed and successfully commercialized three additional phage products for food safety applications, including SalmoFresh™, EcoShield™ PX, and ShigaShield™ for controlling Salmonella spp., STECs, and Shigella spp., respectively. Noteworthy, the SalmoFresh™ preparation was developed under the auspices of USDA, utilizing SBIR Phase I and II grants. The product has been successfully commercialized and serves as an excellent example of how USDA SBIR funding can have a real and positive impact on the food industry in the United States. We believe that the VibrioShield™ product we propose to develop with support of this SBIR program will serve as another strong example of this impact.The etiology of V. parahaemolyticus contamination of fish and shellfish and subsequent transmission to humans is a complex phenomenon and involves various stages of harvesting and subsequent processing and packaging. While bacteriophage application can be effective/contemplated in various stages of that cycle, we believe 1-2 that the most effective and commercially viable approach is to use bacteriophages in the post-harvest environment, specifically by direct application of phages on (i) raw fish/shellfish carcasses after post-gutting/slicing and washing and before the carcasses are packaged, and (ii) on final food products, including RTEs. Correspondingly, our regulatory approval strategy would be to have VibrioShield™ approved as generally-recognized-as-safe (GRAS), for direct applications on post-harvest seafood products, including RTEs. Multiple bacteriophage preparations including ListShield™, SalmoFresh™, EcoShield™ PX, and ShigaShield™ have been already granted GRAS status 1, 3, so this type of regulatory approval strategy is clearly attainable. Our approach will be to undertake additional studies needed for assembling the GRAS application/notification package during our Phase II SBIR project after the studies suggested in this Phase I SBIR project are completed. The FDA typically issues a GRAS "no-objection" letter within 180 days after they receive a GRAS application. Thus, the overall timeframe for this project, from Phase I through Phase II and to commercialization is potentially very short - an excellent outcome for an SBIR application whose ultimate goal is to help rapidly commercialize the promising technologies. The studies proposed in this Phase I SBIR application are well focused and realistic, the necessary experience is in place, and we therefore, fully anticipate successfully completing them in the timeframe allocated to the project. We will proceed by addressing the following two specific aims:Specific Aim #1. Perform studies required to identify phages for inclusion in VibrioShield™ and formulate the candidate phage cocktail.Specific Aim #2. Perform pilot efficacy studies to determine the optimal treatment regimen for using VibrioShield™ to reduce V. parahaemolyticus loads on raw and RTE fish and shellfish.References1. Sulakvelidze, A.; Pasternack, G., Industrial and regulatory issues in bacteriophage applications in food production and processing. In Bacteriophages in the control of food- and waterborne pathogens, Sabour, P. M.; Griffiths, M. W., Eds. ASM Press: Washington, DC, 2010; pp 297 - 326.2. Sulakvelidze, A.; Barrow, P., Phage therapy in animals and agribusiness. In Bacteriophages: Biology and Applications, Kutter, E.; Sulakvelidze, A., Eds. CRC Press: Boca Raton, FL, 2005; pp 335-380.3. Vikram, A.; Woolston, J.; Sulakvelidze, A., Phage Biocontrol Applications in Food Production and Processing. Curr. Issues Mol. Biol. 2021, 40, 267-302.