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Post Harvest Treatment of Live Oysters and Investigation of Therapeutic Potential of Biological Controls

Gulig, Paul; Wright, Anita
University of Florida
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The specific goal of proposed research is to develop economical and logistically feasible practices to ensure microbiological safety of seafood. Interstate Shellfish Sanitation Conference (2003) mandated post harvest treatments (PHTs) of oysters, but PHTs kill oysters and generally are not used to produce a live product. Furthermore, effective PHTs (high hydrostatic pressure or irradiation) require expensive equipment, and other treatments may not completely eliminate pathogens.

Novel methods for processing shellfish and fish are needed to ensure safety of uncooked seafood and provide a viable oyster product for the half shell market. Proposed research will target a global regulatory system to effectively clear pathogens from live oysters. The rationale for this project is based on the discovery that GacS/GacA is central for the colonization of surfaces, cell-to-cell communication, motility, and virulence in all gamma-proteobacterial pathogens. We hypothesize that blocking this system will promote pathogen clearance from live oysters. Because GacS/GacA is conserved in gamma-proteobacteria and is absent from other organisms, the identification of GacS/GacA inhibitors offers an opportunity to develop safe PHT for simultaneous clearing of multiple pathogens from oysters.

The scientific objective of this research is to develop interventions that employ synthetic or natural inhibitors that disrupt GacS/GacA bacterial regulatory cascades that control colonization, infection, and survival of pathogens in oyster hosts. The specific objective of this proposed research is to test the efficacy and safety of GacS/GacA blockers for the PHT application in of live oysters, and develop a prototype model system for cost-effective and safe post-harvest treatment of live oysters with GacS/GacA blockers in a re-circulating depuration system. The overarching hypothesis is that blocking the GacS/GacA system will reliably clear human pathogens from live oysters by disrupting the ability of pathogens to attach to surfaces and colonize their hosts. Similar strategies to disrupt bacterial virulence gene expression and surface colonization by interfering with cell-to-cell communication are now a subject of intense academic and private research.

Research proposed here is distinct from these efforts because blocking the GacS/GacA cascade provides a more powerful approach due to its global regulatory role. Because the GacS/GacA system is highly conserved in vibrios and enteric pathogens, it is our expectation that this research will provide models that will be directly applicable to clearing of many different pathogenic bacteria from live oysters.

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NON-TECHNICAL SUMMARY: Novel methods are need to improve processing of shellfish and fish in order to ensure safety of seafood, especially with uncooked or live product that is frequently contaminated with indigenous bacteria that have the potential to cause human disease. The purpose of the project is to examine chemicals or natural products that block the ability of bacterial pathogens to attach and survive in fish and shellfish, using an oyster model of infection. The feasibility of these inhibitors as a post harvest processing will be tested for the production of safe, live oyster.

APPROACH: Our approach will accomplish the scientific, technical and extension components of this research by utilizing combined expertise in community microbiology (Teplitski), food microbiology (Wright), and molecular pathogenesis (Gulig). Over the last 25 years, Dr. Wright has investigated virulence, detection methods and PHT in V. vulnificus. Teplitski contributed to the characterization of GacS/GacA in Salmonella and constructed all the necessary reporter strains and purified proteins to characterize GacS/GacA blockers. Dr. Gulig has expertise in molecular pathogenesis includes both V. vulnificus and S. enterica. Dr. Wright has worked integrally with University of Florida extension faculty since 2002 to provide PHT validations required by the Florida oyster industry. Our approach to achieve the goals of this proposal will be accomplished by completing the following specific tasks: 1) Determine the efficacy of small molecule inhibitors of the GacS/GacA system in preventing bacterial colonization and infection of oysters by V. vulnificus, V. parahaemolyticus, and Salmonella enterica using an oyster model of infection, recombinase-based in vivo expression technology (RIVET), and signature-tagged mutagenesis. 2) Test therapeutic applications of GacS/GacA blockers in sub-cutaneous mouse models of V. vulnificus disease and evaluate their effects on bacterial virulence potential. 3) Evaluate the feasibility and environmental safety of the developing GacS/GacA blockers for large-scale oyster PHT. 4) Provide education and training to the oyster industry to promote acceptance and practical application of PHT in accordance with ISSC and HACCP regulations. As a result of proposed research, we expect to validate a novel PHT to safely remove human pathogens from live oysters. Existing extension programs will coordinate with this research to promote the application of these methods. The research will coordinate with the University of Florida Seafood Extension Program, Florida Department of Consumer and Agricultural Services (DACS), and with the Florida oyster industry. Findings will be integrated into our existing extension education and outreach programs on campus and at the Oyster Industry Laboratory in Apalachicola, FL.

PROGRESS: 2007/12 TO 2008/12
OUTPUTS: Rationale: Public health concern for the safety of raw shellfish, particularly oysters, is estimated to cost the seafood industry $3 to $14 billion per year (FDA). The goal of this research is to develop economically feasible practices to ensure the microbiological safety of seafood. The rationale for this research centers around the discovery of a global regulatory system that controls bacterial colonization, communication, motility, and virulence in all gamma-proteobacterial pathogens, specifically the GacS/GacA (also termed BarA/BarS, SirA/SirS, VarA/VarS), a two component regulatory system that promotes expression of small inhibitory RNA products.

Specific objectives for this reasearch include the following:
1. Determine the efficacy of small molecule inhibitors of the GacS/GacA in preventing colonization of oysters by V. vulnificus.
2. Test therapeutic applications
3. Evaluate the feasibility of developing large-scale oyster PHT.
4. Provide education and training to the oyster industry

Progress Report:
Objective 1 results: We identified GacS/GacA inhibitors in E. coli reporter system and have begun to genetically define the components of this system in V. vulnificus. We developed an oyster model of infection and demonstrated that motility, pili, CPS, and phase variation were important to the uptake of V. vulnificus in oysters. Candidate inhibitor molecules will be examined for their ability to decrease or eliminate survival in oysters. Inhibitors were identified by screening small chemical libraries for related activity using csrB-luxCDABE reporter constructs in E. coli. To date, we have independently screened two small chemical libraries. The csrB promoter from V. vulnificus was cloned into a luxCDBAE expression vector to determine its relationship to GacA. Results showed that expression from this promoter is GacA-dependent, as GacA deletion mutants did not show expression in S. enterica background, confirming that the GacS/GacA signal Transduction cascade is functional in V. vulnificus. A gacA deletion mutation was confirmed by PCR and DNA sequencing. Differences were observed between V. vulnificus wild type and gacA deletion mutant with respect to motility and rate of phase variation. Although log phase growth was essentially identical for both strains, growth yield during stationary phase was reduced in the gacA mutant.

Objective 2 results: Validation of virulence model. Prior studies showed that all strains of V. vulnificus were equally virulent in animal models; however, genetic profiling indicates that clinical strains of V. vulnificus are atypical and represent a relatively small proportion of the environmental population. We confirmed that these genetic distinctions were related to virulence potential in a mouse model developed by Dr. Gulig's group that discriminates the ability to cause systemic disease vs. localized infections.

IMPACT: 2007/12 TO 2008/12
These data provide the first investigations into the GacS/GacA pathway in V. vulnificus. We recently demonstrated that factors generally controlled by GacS/GacA also required for uptake and survival of V. vulnificus in oysters. Thus, blocking this system should promote pathogen clearance from live oysters. Signals that trigger GacS/GacA are unknown but are thought to involve small chemical molecules. Microarray libraries of potential chemical inhibitors were assayed as putative GacS/GacA blockers. Efficacy and safety of candidate blockers will be tested for PHT application in live oysters, using Vibrio vulnificus as the target pathogen. A prototype model delivery system for cost-effective and safe PHT will be developed in a re-circulating depuration system. The overarching hypothesis is that application of this PHT will disrupt of the ability of pathogens to attach to surfaces and colonize their host and/or facilitate host immune clearance. Because the GacS/GacA system is highly conserved in vibrios and other enteric pathogens, it is our expectation that this research will provide models that will be directly applicable to clearing of multiple pathogenic bacteria in a variety of raw food products.

Funding Source
Nat'l. Inst. of Food and Agriculture
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