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The food and agriculture industry would benefit greatly from a rapid screening method to determine the presence of potential pathogens in complex matrices. Unfortunately, a pragmatic and efficient means of bacterial separation remains the bottleneck for rapid pathogen detection. Without an effective separation method, the need to rapidly screen for pathogens on sensitive agricultural samples will go unmet. The current method of pre-enriching bacteria prior to separation and detection requires extended periods of time and may not be suitable to environments such as food production facilities. Additionally, there exists a need to separate bacteria in a manner which will facilitate both enumeration and identification. Once a product has been pre-enriched, the ability to enumerate the initial sample load becomes difficult. There is a fundamental need to develop next generation technologies which will enable the rapid and efficient separation and concentration of bacteria, thus improving abilities for early detection. The lack of reliable, robust and rapid separation methods is the current roadblock to rapid detection. Improvements in these methods would enable many rapid and sensitive detection technologies which require concentrated and clean samples. We propose a rapid bacterial separation method using a combination of enzymatic matrix digestion and phage-based separation which will allow the use of rapid detection methods on agricultural matrices. The overall goal of this hypothesis-driven research is to enable early detection methods by rapidly and efficiently separating and concentrating bacteria from agricultural matrices. We hypothesize that a bacterial separation method which 1) breaks down the sample matrix allowing removal internalized bacteria, and 2) magnetically concentrates bacteria by utilizing the irreversible binding bacteriophages will allow an improvement early detection and therefore food safety (Figure1). We further hypothesize that the use of phages for the separation and lysis of the bacteria will allow the determination of viability when followed with PCR. To accomplish this goal, we propose an interdisciplinary approach encompassing expertise in the fields of molecular biology, enzymology, materials engineering, and food microbiology, as outlined below. Specific Objectives: Enzymatic digestion of food and agricultural products for bacterial release and separation phage enabled separation of E. coli and Listeria spp. from liquid samples Validation of a digestion/phage-based bacterial separation method on food matrices.
A pragmatic and efficient means of bacterial separation/concentration remains the bottleneck for rapid pathogen detection in food and agriculture. We are investigating a two-step process in isolating bacteria from a food and agricultural samples. The first step separates surface-adhered bacterial from a plant-based matrix. This is accomplished using a customized enzymatic digestion of the plant material. As the plant material is rapidly broken down, the bacterial cells in and on the matrix surface are released into an aqueous mixture. While the digestion has been shown to rapidly liquefy the plant matrix, there has been no effect on the viability of the bacterial cells. Our preliminary data demonstrate a significant increase in bacterial recovery following enzymatic digestion. The second step involves the removal of the bacteria from the liquefied sample. This will be accomplished using high affinity bacteriophage-tagged magnetic particles. The coliphage T7 and the Listeria spp. specific phage A511 will be genetically modified to allow for biotin expression on the major capsid proteins. The result will be oriented conjugation of the phages ontostreptavidin-coated magnetic particles. This phage separation strategy has been shown to have higher capture efficiencies as compared to immuno-affinity based separation. Once separated, the bacteria will be quantified using QPCR.A validation of the proposed separation method will be performed by extension. E. coliinoculated spinach and Listeria inoculated bean sprouts will be enzymatically digested and then undergo a phage-based magnetic separation.