SBIR-STTR Award

Foodborne outbreaks of infectious disease have been associated with the contamination of fresh produce by infectious microorganisms such as hepatitis A virus. However, the indicator microorganisms currently used to assess the level of contamination in fresh produce are limited to bacterial microorganisms such as the total and fecal coliform groups that may have shorter half lives than the true enteric pathogens of interest. These bacterial indicators may not correlate with the presence/absence of enteric viruses effectively in fresh produce that may be consumed raw and sometimes fail to indicate contamination events. The project is to develop a simple and quantitative kit to detect coliphages as improved indicator microorganisms that more accurately index fecal contamination events. At the conclusion of the phase I study, the conceptual framework will be field-validated in a multi-state study to establish its robustness. OBJECTIVES: The proposed phase I study targets the development of a simple and economical product, C-phage+, that can be used to assess ready-to-eat food products for evidence of fecal contamination. Development of C-phage+ will take into account the lessons learned from previous efforts to validate fecal contamination indicators such as coliphages. The proposed research strategy will consider challenges such as ambiguities in scoring viral plaques caused by sample matrix effects and will also address limitations associated with extended assay turnaround times. The study will target the following objectives: validation of a simple, rapid and economical indicator system that can be used in modestly equipped laboratories; development and validation of an efficient extraction media for the recovery of coliphages from ready-to-eat produce; development of a simplified enumerative assay to quantify male-specific coliphages in the extraction buffer/eluate; validation of a pectin-based agar substitute that does not require steam sterilization and that eliminates the need for precise temperature monitoring during assay; and validation of a method to enhance visualization of viral plaques using a chromogenic substrate (e.g. X-GAL). APPROACH: An optimized elution buffer will be developed by evaluating a combination of base compounds, surfactants, and pH conditions to identify optimal recoveries of test microorganisms seeded onto model fruits and vegetables representing 'rough' and 'smooth' organic surfaces. The SMI research team will conduct carefully controlled experiments using food products seeded with known quantities of model indicators to characterize the recovery efficiency of each extraction buffer. A pectin-based agar substitute will be formulated and tested for comparison against traditional agar-based media. The utility of viral plaque enhancement techniques will be assessed by testing chromogenic substrates such as 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (X-GAL) to facilitate visualization of viral plaques within the C-phage+ pectin-based medium. During the final stage of phase I study, the research team will field-validate the optimized system by collecting a variety of fruit and vegetable samples from at least four midwestern U.S. states. PROGRESS: 2005/05 TO 2005/12 Several elution buffers were tested to optimize the recovery of male-specific coliphages as fecal indicators from a variety of vegetables and fruit. A number of surfactants and amino acids were investigated to determine their effectiveness in facilitating the release of male-specific coliphages from vegetables and strawberry. A beef extract based elution buffer supplemented with Tween80 and glycine has been developed and show high efficiency in recovering male-specific coliphages seeded on vegetables and subsequently assayed using agar based medium. Pectin-based media (Easyphage) has been developed as a viral plaque enhancement and tested for detecting coliphages from vegetables and strawberries during spiking experiments and from field samples. Blue plaques developed in Easyphage medium contrasted well against the red bacterial lawn and made them easy to observe and count. This pectin-based medium showed excellent recovery of wild-type male-specific coliphages in sewage. A unique elution buffer was created and evaluated to optimize phage recovery when pectin-based medium (Easyphage) was used. The traditional single agar layer (SAL) method was used to compare with Easyphage to determine the phage recovery by both methods. The results indicate that the Easyphage medium coupled with the unique elution buffer was equal to or better than SAL method. A feasibility study was conducted using ready-to-eat food samples collected from multiple states. Approximately 11% of samples, including several ready-to-eat vegetable were positive for male-specific coliphages using pectin-based Easyphages. The field study demonstrates the feasibility for using Easyphage to detect male-specific coliphages from fruits and vegetables. IMPACT: 2005/05 TO 2005/12 Food-borne outbreaks involving fruits and vegetables have been increased yearly and are responsible for more large-scale outbreaks than meat, poultry and eggs. Some of the causative agents have been identified as viruses including hepatitis A and Norwalk viruses. Detection of male-specific coliphages in vegetables and fruits may provide additional protection from viral contamination. One study indicates that male-specific coliphage could be useful conservative indicators of fecal contamination of produce and its associated virological risks (Allwood, et al 2004). The Easyphage detection kit developed under this grant is as easy to use as E. coli testing and can be performed in general food microbiology laboratories without additional equipment and special training

One of the most vulnerable sectors in the food industry includes "ready-to-eat" fruits and vegetables that may be consumed without peeling, pasteurization, or cooking. Increases in the year-round consumption of fresh fruits and vegetables that are out of season in the United States, coupled with the relaxation of international trade restrictions, have resulted in significant increases in the importation of produce from developing countries where oversight of irrigation, washing, and handling practices may not be as stringent as in the U.S. Because this produce is generally not cooked prior to consumption, foodborne outbreaks can result from microbiological contamination of crops irrigated using substandard water, reclaimed water or wastewater. Because it may never be possible to test ready-to-eat food products directly for all possible microbial contaminants, the development and commercialization of rapid, simple and economical indirect testing methods for assessing fecal contamination is highly desirable. This proposal addresses the need to develop a robust, rapid and economical method for the determination of fecal contamination of ready-to-eat produce. OBJECTIVES: This project continues the development of a rapid and simple fecal indicator detection system for ready-to-eat produce and will include beta-testing in food laboratories and a national-scale field survey of food markets. Coliphage detection kits will be produced according to several formats and validated using food samples from retail stores. The kits will permit testing for fecal contamination on ready-to-eat produce and the opportunity to diminish an important chain of transmission for foodborne infectious disease in the U.S. The product will also expand the tools available to those investigating the sources of infectious disease outbreaks. APPROACH: The phase II study continues the development of a simple and economical product, Easyphage, that can be used to assess ready-to-eat food products for evidence of fecal contamination. Development of Easyphage takes into account the lessons learned from previous efforts to validate fecal contamination indicators including coliphages. The research strategy considers challenges identified during the Phase I research efforts such as ambiguities in scoring viral plaques and limitations associated with extended assay turnaround times. The Phase II research study is divided into the following work assignments to achieve the project objectives: Validation of a simple, rapid and economical indicator system, Easyphage, that can be used by modestly equipped laboratories to assess ready-to-eat food products for evidence of fecal contamination. Development and validation of an efficient extraction media for the recovery of indicator microorganisms (coliphages) from ready-to-eat produce. Development of a simplified enumerative assay to quantify male-specific coliphages in the extraction buffer/eluate. Validation of a agar substitute that does not require steam sterilization and that eliminates the need for precise temperature monitoring agar during assay. Validation of a method to enhance visualization of viral plaques using a chromogenic substrate. Use of the Easyphage assay to perform a multi-state field study of ready-to-eat food purchased in US retail food stores