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Infections caused by Vibrio species in the U.S. have greatly impacted the shellfish industry. However, despite multi-faceted efforts to address this problem, the incidence of Vibrio disease in the U.S. increased 78% in 2005-2006. V. vulnificus (Vv), V. parahaemolyticus (Vp), and V. cholerae (Vc) are the primary human pathogen in the Vibrio genus and are responsible for approximately 75% of bacterial seafood-borne disease and 95% of fatalities related to bacterial infections in the U.S.. Increasing global temperatures have led to an increased incidence of outbreaks as well as an increase in the global distribution of these pathogens. These changes in disease pattern will likely warrant boarder application of PHP of seafoods raised in the US and those imported from international markets. Several current PHP methods are able to effectively reduce the level of Vibrios in seafood, but do so at the expense of the raw shellfish product. The long-term goal of the proposed study is to provide a greater understanding as to the feasibility of using an alternate PHP method involving application of antimicrobial peptides (AMPs) to raw oysters. One of the more immediate goals of this research is to evaluate the susceptibility of the three primary Vibrio pathogens to various AMPs and determine AMP concentrations that induce growth inhibition and/or death in these bacteria. Therefore, the specific objectives proposed to meet these goals are as follows: Objective #1: To investigate the effects of hepcidin on the survival and persistence of V. vulnificus under environmentally and commercially relevant in vitro conditions and determine the range of efficacy for this compound; Objective #2: To evaluate the effects of additional AMPs against Vibrio spp. of significant importance to the seafood industry by expanding the scope of the previous objective to include other AMPs, such as nisin, on growth inhibition and killing of Vv, Vp, and Vc, and Objective #3: To investigate the ability of AMPs to reduce or eliminate V. vulnificus in live oysters by evaluating potential in vivo applications of hepcidin and/or other AMPs to the raw seafood industry. These results will provide strong evidence for the feasibility of using AMPs in post-harvest applications. During Year 1 of the proposed project, all in vitro work evaluating AMPs and as well as chemical and physical synergistic factors that inhibit growth and survival of Vv, Vc and Vp is anticipated to be completed. During Year 2, in vivo applications will be tested to determined concentrations needed for killing Vv populations in oysters. A significant outcome from this research will be knowledge of conditions under which the antimicrobial compounds hepcidin and nisin are effective in inhibiting growth and killing for the pathogens Vv, Vp, and Vc. These studies will also reveal compounds or physical stressors that may increase the efficacy of the above AMPs against these bacteria. The primary deliverable of this project will be the development of an oyster model will which reveal the feasibility and efficacy of using such treatments in oysters and potentially serve as a model for AMP use in other seafoods.
Non-Technical Summary: Infections resulting from consumption of seafoods have recently been on the rise and are primarily caused by human pathogens in the Vibrio genus. These bacteria are responsible for approximately 75% of bacterial seafood-borne disease and 95% of seafood related fatalities in the U.S. Increasing global temperatures have led to an increased incidence of outbreaks as well as an increase in the global distribution of these pathogens. Current post-harvest processing methods of seafoods are either inadequate for removal of these bacteria or are detrimental to the animal and render it unsuitable for the raw seafood market. In recent years consumer demand for natural foods that contain little or no chemical additives has greatly increased as has the demand for raw, minimally processed, and ready-to-eat foods. This poses a significant challenge for the food industry to meet these needs while still providing a safe product. One answer to these demands is the use of antimicrobial peptides (AMPs), which are produced by bacteria and kill closely related species. Currently, Nisin is the only AMP generally recognized as safe by the FDA and approved for use as a food additive. However, Nisin has limited effectiveness against the human pathogens found in seafoods. Fortunately, recent research has discovered conditions that make similar pathogens susceptible to Nisin. Therefore, the purpose of this project is to 1) identify conditions in which Nisin is effective against Vibrio spp and 2) apply these conditions to raw oysters and examine their ability to remove indigenous Vibrio populations. <P> Approach: Growth inhibition and killing of V. vulnificus (Vv) by hepcidin, Nisin and other AMPs will be evaluated using a microtiter format through optical density measurement. Assays will also be conducted using larger culture volumes. For these, as well as all killing (or survival) and survival studies, standard plating methods will be employed. Initial studies will examine the effectiveness of AMPs against Vv. MIC and killing concentrations of these antimicrobials will also be evaluated under in vitro conditions typical of estuarine environments with at least the following variations: 1) incubation medium, 2) incubation temperature, and 3) incubation time. Studies examining the effectiveness of AMPs against gram-negative bacteria have uncovered exogenous factors and physical stressors which enhance AMP bactericidal activity. Therefore, the addition of food grade chelators and permeabilizers as well as exposure to physical stressors will be examined for their influence on the killing capacity of bacteriocins against Vv. These studies will look at the inhibitory and/or killing ability of these conditions against Vv independently and also in conjunction with Nisin or other AMPs. AMPs, synergistic compounds, and physical stressors shown to be effective against Vv will then be tested against V. parahaemolyticus and V. cholerae. Studies evaluating growth inhibition and killing will be conducted in a similar manner as those assays described above, with modified culture conditions as necessary for each specific pathogen. Experiments investigating the ability of AMPs to reduce or eliminate Vv in live oysters will use a recently published oyster infection model. Freshly harvested oysters from Apalachicola Bay, FL will be treated to remove indigenous Vibrios and then inoculated with V. vulnificus. Inoculated oysters will then be transferred to experimental tanks and exposed to: 1) AMP only, 2) synergistic compound only, 3) AMP and synergistic compound added simultaneously, 4) synergistic compound added prior to AMP addition, 5) non-treated controls. After treatment, oysters will be processed by standard methods for enumeration of Vv. Conditions shown to remove Vv from artificially inoculated oysters will also be applied to oysters containing natural populations of the bacterium. Freshly harvested oysters will be exposed to AMPs and/or synergistic compounds and tested for ability of these treatments to remove indigenous Vv. A student's T-test or Analyses of variance (where applicable) will be performed to determine significant differences in culture density between treated and untreated samples as well as among Vibrios. Results from this research will be integrated into testing facilities and state programs through relationships with coordinators of the Seafood Extension Program at the Univ. of FL, the Oyster Industry Lab at Apalachicola, FL, and the FL Dept. of Agriculture and Consumers Services and the Dept. of Health. Additionally, Dr. Anita Wright (mentor) is currently working with collaborators to develop an international forum centered on seafood pathogens, and results from this research will be incorporated into this forum.