PFI:AIR - TT: Rapid, quantitative, molecular diagnostics for virulent Vibrio pathogens in water and shellfish

University Of North Carolina At Chapel Hill, Chapel Hill NC

Investigators

Abstract

This PFI: AIR Technology Translation project focuses on translating ecological research that has been previously conducted on a set of bacterial pathogens, Vibrios, found in estuaries and coastal systems. Vibrio cholerae is a well-known freshwater pathogen that is of concern in developing countries. Two lesser known, but important human pathogens in the USA are from the same bacterial group: Vibrio vulnificus and Vibrio parahaemolyticus. These are bacteria that are naturally found in estuarine systems, but they can also present a risk to human health when consumed in raw shellfish, or in rare cases, contacted in beach waters. This project will translate knowledge gained through prior ecological research into technology that permits the design of user-friendly, rapid, molecular testing kits. The existing ways to test for Vibrio species in water and shellfish samples are decades old, and they require 24-96 hours for results. Even new molecular approaches for testing of pathogenic forms of V. vulnificus and V. parahaemolyticus are compromised by a lack of specificity and/or a need for an enrichment step to improve sensitivity, making them require almost a day for completion. This project will permit generation of new, rapid molecular diagnostics for virulent forms of V. vulnificus and V. parahaemolyticus using a new approach for determining useful targets for the kits. It is envisioned that these kits could be used to 1) certify that shellfish is safe for raw consumption, 2) protect the health of at risk populations for water contact, 3) determine the patterns associated with dangerous forms of the bacteria for improved warning systems. The funded project will result in the design of commercially available molecular testing kits that will be easy to use, and will provide results in 2-3 hours. These features will permit accurate public notification and protection of public health. This project addresses a specific knowledge gap in the transition from research to commercial application. Vibrio sp. are complex bacterial organisms, that are naturally found in estuarine and coastal waters, but only a small subset of them are capable of causing disease. The assembled team has valuable expertise in next generation sequence and sequence data approaches, Vibrio sp. ecology, and Vibrio sp. pathogenicity. The combination of knowledge permits known virulent (pathogenic) Vibrio samples to be analyzed for specific sets of "DNA motifs" that are related to the capability of the bacteria to cause disease. From a repetitive process of analyzing known virulent and avirulent cells, the project team can identify the DNA motifs, or signatures that can be used to design a DNA test kit. Subsequently, through support from this project the molecular test kits will be developed, optimized, validated, and commercialized. In addition to the technical activities, undergraduates, senior graduate students, and post doctoral research associates as well as research assistant professors will be supported on this project. The will be exposed to a wide range of research and technology transfer activities, including market assessment, field based sampling and study, laboratory sample preparation, culture and molecular based analyses of samples, next generation sequence data annotation and analyses, and kit design and optimization. The project will be synergistic and contribute directly to the Molecular Training Facility run by PI Noble, where water quality professionals come to learn appropriate use of qPCR approaches and interpretation of qPCR data for water quality management. The project also engages the Marine Biotechnologies-Center of Innovation, a group dedicated to advancing the translation of marine science research to commercial application. In addition, Orion Integrated Biosciences is a project partner. Orion Integrated Biosciences is a company dedicated to the advancement of unique and disruptive computational approaches to survey "big data" for sequence and molecular application. This project will directly impact the advancement of marine metagenomics due to the fact that the project team is utilizing a holistic approach to target complex organisms. The project is novel in that the approach moves away from the "single gene target" qPCR design approach, permitting the generation of diagnostic kits that are rapid, and cost-effective that shellfish harvesting, and water quality managers can use to manage precious resources and protect public health.