Performing Department
(N/A)
Non Technical Summary
The seafood consumption is the United States (US) is increasing. In 2018, for which the most recent data is available, the consumption of seafood was highest in the past decade. Seafood is an important source of long-chain omega-3 fatty acids and '2015-2020 Dietary Guidelines for Americans' recommend eating at least 8 ounces of seafood per week. However, this increased seafood consumption also enhances the health risk associated with seafood-borne infections. Vibriosis is a leading seafood-borne infection causing an estimated 80,000 illnesses and 100 deaths every year in the US. The economic burden of the Vibriosis, primarily stemming from seafood-borne infections is estimated to be around $300 million. Vibrio parahaemolyticus is the most common species associated with seafood-borne infections and accounted for approximately 34,664 foodborne infections in the US in 2016. The pandemic clone O3:K6 and its serovariants are widely distributed and cause the majority of infections worldwide.The shellfish industry relies primarily on processes such as rapid cooling of the harvest, restricted harvesting in warmer/summer and rainy months, relaying and depuration, high-pressure processing (HPP) and irradiation to control the microbial contamination. These approaches are not only inadequate but also restrictive for production (for example - restricted harvesting in summer) and may entail a high cost of operations e.g irradiation and HPP. Also, low consumer acceptance of irradiation has limited its usage and wide application by the producers. A few additional interventions such as organic acids and ozone treatment, are presented in literature but could potentially impact the organoleptic and textural properties of the product.The lytic bacteriophages are viruses that specifically lyse the target bacterial strains or a subgroup of bacterial strains. The use of lytic bacteriophages for food safety is gaining recognition recently, and emerging evidence strongly suggests that a direct application of lytic phages on various food surfaces significantly reduce or eliminate foodborne bacterial pathogen. The FDA and USDA have approved several commercial phage-products for biocontrol of foodborne bacterial pathogens in various foods. Bacteriophages are abundant in the marine environment and several studies have identified lytic bacteriophages against V. parahaemolyticus. For instance, the application of pVp-1, a lytic bacteriophage, on the surface of oysters reduced V. parahaemolyticus counts by approximately 6 log CFU/g. However, no commercial product is currently available to the industry that can help reduce the V. parahaemolyticus incidence in the seafood. A wide-scale availability of such a product will not only help improve the safety of seafood products but will also greatly benefit the industry by reducing monetary and reputational damages associated with recalls. Intralytix is uniquely positioned to develop a bacteriophage-based product and make it widely available. Noteworthy, Intralytix was the first company in the world to receive FDA/USDA clearance for its phage-based product ListShield™, designed as a food-additive to reduce Listeria monocytogenes in various foods (21 CFR§172.785; FSIS Directive 7120.1; GRN No. 528). Since then, Intralytix has obtained approvals for three more phage-products targeting Salmonella, STECs and Shigella spp. in a variety of food products (GRN No. 435, GRN No. 834, GRN No. 672, FSIS Directive 7120.1, FCN No. 1018) as well as several products for controlling bacterial pathogens in the pet-foods.Our overarching goal is to create a cocktail of monophages with optimal lytic efficiency against pathogenic strains of V. parahaemolyticus that will be made available to the seafood industry to help improve the seafood safety. The objective of this proposal is to develop a phage cocktail targeting V. parahaemolyticus, referred hereafter as "VibrioShield" and to determine the optimal treatment regimen for seafood such as oysters. This proposal addresses the critical need "topic area 8.5, Food Science, and Nutrition.1. Food Safety. Developing technologies for the detection or mitigation of foodborne hazards (microorganisms, chemicals, toxins) during pre- and post-harvest processing and distribution". To demonstrate the efficacy of phage-biocontrol on seafood and provide proof-of-concept, two specific aims are proposed:-Specific Aim #1. Formulation of a candidate phage cocktail with optimal lytic potency against Vibrio parahaemolyticusIntralytix has isolated and acquired several V. parahaemolyticus phages and their host strains for propagation as well as pathogenic strains for the development of the phage cocktail. The lytic efficiency of individual phages will be tested against all the V. parahaemolyticus strains in the Intralytix library using the spot test method. Additionally, an effort will be made to isolate unique lytic phages from the Chesapeake Bay and seafood obtained from different sources. The isolated phages will be characterized for their lytic efficiency in a similar fashion described above. Intralytix has developed a unique proprietary tool PhageSelector™ program, which helps in identifying a combination of phages with the widest possible lytic spectrum. The resulting data from lytic efficiency tests will be fed to the PhageSelector™ program to identify phages for inclusion in the cocktail. Three different cocktails varying in their phage compositions and the three most wide lytic spectrum as predicted by PhageSelector™ will be tested further. The lytic spectrum of the three cocktails will be empirically determined and compared by testing the lytic activity of the cocktail against all the V. parahaemolyticus strains in the Intralytix library. The cocktail demonstrating the most comprehensive lytic spectrum will be named as VibrioShield and used for further studies. Since the phages isolated and screened against the most prevalent and pandemic serovars including O3:K6, we highly anticipate a successful formulation of the VibrioShield. Furthermore, the effort to isolate and identify new and unique phages will use a strain of O3:K6 serovar and other pathogenic strains which will further increase the likelihood of successful formulation of VibrioShield.Specific Aim #2. Perform pilot efficacy studies to determine the optimal treatment regimen for using VibrioShield to reduce V. parahaemolyticus loads on seafood products.Emerging evidence from our laboratory and other laboratories across the globe, as well as our experience with the food industry, clearly indicate that phages can efficiently reduce, and in certain cases completely eradicate, the levels of their targeted bacterial hosts contaminating various foods. Therefore, the goal of the studies proposed to meet Specific Aim #2 is to determine the optimal dosing regimen; i.e., the application time and dose (the volume and phage concentration of VibrioShield) that will result in a maximal reduction of V. parahaemolyticus in two types of seafood products: raw and ready-to-eat fish and shellfish. The efficacy of VibrioShield treatment will be determined by (i) the enumeration test (during which the levels of V. parahaemolyticus in foods, as expressed by the number of colony-forming units [CFU]/g of food, is determined), and (ii) the enrichment-detection test (during which the presence or absence of V. parahaemolyticus is determined). The enumeration test will be performed essentially as we and other investigators previously described for evaluating the ability of various phage preparations to reduce or eliminate specific bacterial pathogens from various foods. The enrichment-detection test will be conducted using the method described in the Bacteriological Analytical Manual (Revised May 2004; available at https://www.fda.gov/food/laboratory-methods-food/bam-vibrio), Chapter 9, Vibrio.
Animal Health Component
25%
Research Effort Categories
Basic
25%
Applied
25%
Developmental
50%
Goals / Objectives
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.
Project Methods
Specific Aim#11. We will begin our studies by (i) cultivating all of the anti-V. parahaemolyticus bacteriophages currently in our phage library, to obtain adequate sample size for each, and (ii) determining their lytic potency against all of our strains of pathogenic V. parahaemolyticus serotypes. The lytic potency will be determined by using a well-described and commonly used Spot Test method. The Spot Test method is commonly used to determine host-range and lytic efficiency and is now optimized to be performed by our high throughout robotic system Neptune™. The host range for each bacteriophage will be determined at two different concentrations of each phage against each strain: the commonly used phage concentration (ca. 1x109 plaque-forming units (PFU)/mL) followed by lower concentration (ca. 1x104 PFU/mL). We will take that approach because comparing the lytic potency of low (ca. 1x104 PFU/mL) and high (ca. 1x109-1010 PFU/mL) concentrations of a given phage against the targeted bacterial strains can be used (as described below) as a supplementary selection criterion when formulating VibrioShield™. As an additional measure, we will also determine the Virulence Index (VI) for all promising phages using the robotic system "Neptune". The VI data would be used as yet another selection criterion when formulating the VibrioShield ™ cocktail.2. The second step in the course of the project will be characterization of the bacteriophages. As part of our commitment to "safety first", the identity and purity of the selected bacteriophages with the strongest lytic activity in in-vitro sensitivity screening assays, will be confirmed. The purity/homogeneity will be verified by performing a series of studies to determine their grossly observable plaque morphology and their electron microscopic (EM) appearance. Phages will also be characterized by (i) PFGE analysis of phage DNA, (ii) Sequencing followed by bioinformatic analysis and/or RFLP analysis of phage DNA (in case the phages are refractory to sequencing or restriction analyses).3. Following successful identification of effective bacteriophages and their characterization, candidate phages for preparation of a phage cocktail that lyses majority if not all of the targeted V. parahaemolyticus strains will be identified. The Intralytix has developed a proprietary software, the PhageSelector™ program, that facilitates the designing of optimally effective phage preparations. This tool will be employed to identify the bacteriophages for inclusion in candidate phage cocktail. After the phages for inclusion in the preliminary version of VibrioShield™ are identified, we will use a benchtop bioreactor to propagate small amounts of each component monophages. After confirming the identity, potency (lytic titer), and bacteriological sterility of each component phage preparation, we will blend them to form the VibrioShield™ cocktail to be used during the studies outlined in Specific Aim #2.Specific Aim#2The goal of the studies proposed to meet Specific Aim #2 is to determine preliminary optimal dosing regimen; i.e., the application time and dose (phage concentration of VibrioShield™) that will result in a maximal reduction of V. parahaemolyticus in two types of seafood products: raw fish/shellfish carcasses and cooked fish/shellfish. The efficacy of VibrioShield™ treatment will be determined by (i) the enumeration test (during which the levels of V. parahaemolyticus in foods, as expressed by the number of colony-forming units [CFU]/g of food, is determined), and (ii) the occurrence test (by enriching the contaminated food matric to determine the presence or absence of the V. parahaemolyticus).To achieve the objectives outlined in Specific Aim#2, We will generate an in house nalidixic acid-resistant mutant that (i) is fully susceptible to the candidate VibrioShield™ preparation, and (2) can grow on at least 25 µg/mL nalidixic acid without a growth defect. This strain will be used to experimentally contaminate the surfaces of raw and cooked fish/shellfish surfaces. The V. parahaemolyticus-selective V. parahaemolyticus sucrose agar (VPSA; recommended by the FDA) supplemented with nalidixic acid (25 µg/mL, to minimize the risk of nonspecific bacterial growth which could complicate our data analysis) will be used in all experiments. In tandem with experimentally contaminated specimens, aliquots from each food specimen will be analyzed for (i) naturally occurring V. parahaemolyticus and (ii) other bacteria capable of growing on VPSA medium supplemented with nalidixic acid to determine any potentially confounding bacterial species.We anticipate that a recovery of ca. 3 log CFU of V. parahaemolyticus from 25 g of the experimentally contaminated control (not treated with phage) food specimens will be needed to observe statistically significant reductions by phage treatment. Therefore, before initiating VibrioShield™ treatment, the challenge dose of V. parahaemolyticus to recover ca 3 log CFUs from 25 g of contaminated, untreated food will be empirically determined. After determining the appropriate challenge dose, the test specimens will becontaminated with that dose and treated with VibrioShield™ as briefly described below.The general design of the studies will be as follows: (i) contaminate the food specimens with the challenge dose of the V. parahaemolyticus test strain (as explained above, that dose will be different for the enumeration test than for the enrichment-detection test), (ii) allow the bacteria to adhere to the food surfaces for at least 30 minutes, (iii) apply VibrioShield™ to the contaminated specimens by spraying with a pre-calibrated spray gun (Basic Spray Gun, Model #250-2; Badger Air-Brush Co., Franklin Park, IL, or equivalent), and (iv) allow the contaminated specimens to remain in contact with VibrioShield™ for 15, 60, and 120 minutes before analyzing them by the enrichment-detection test (to determine the presence/absence of the V. parahaemolyticus test strain) and by the enumeration test (to determine the concentration [CFU/g of food] of the V. parahaemolyticus test strain). Food specimens experimentally contaminated with the same challenge dose of the V. parahaemolyticus test strain but treated with sterile saline or PBS will be used as positive controls.In addition to the time variable (i.e., the period for which VibrioShield™ is allowed to stay in contact with the food specimens before they are analyzed for residual V. parahaemolyticus contamination - item #iv above), we will examine the impact of phage concentration (PFU of VibrioShield™/g of food) on the efficacy of VibrioShield™ treatment.