Performing Department
Food Science
Non Technical Summary
A pragmatic and efficient means of bacterial separation/concentration remains the bottleneck forrapid pathogen detection in food and agriculture. We are investigating a two-step process inisolating bacteria from a food and agricultural samples. The first step separates surface-adheredbacterial from a plant-based matrix. This is accomplished using a customized enzymaticdigestion of the plant material. As the plant material is rapidly broken down, the bacterial cells inand on the matrix surface are released into an aqueous mixture. While the digestion has beenshown to rapidly liquefy the plant matrix, there has been no effect on the viability of the bacterialcells. Our preliminary data demonstrate a significant increase in bacterial recovery followingenzymatic digestion.The second step involves the removal of the bacteria from the liquefied sample. This will beaccomplished using high affinity bacteriophage-tagged magnetic particles. The coliphage T7 andthe Listeria spp. specific phage A511 will be genetically modified to allow for biotin expressionon 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 havehigher 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 thenundergo a phage-based magnetic separation.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
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 separationPhage enabled separation of E. coli and Listeria spp. from liquid samples Validation of a digestion/phage-based bacterial separation method on food matrices
Project Methods
Enzymatic digestion of food and agricultural products for bacterial release and separation: We will build on the preliminary data obtained demonstrating a significant improvement in bacterial separation following enzymatic digestion. We will determine the enzyme concentrations and incubation conditions in order to maximize the digestion of food matrices (spinach, mung bean sprouts). The breakdown of the food components will allow the release of surface-adhered bacteria into an aqueous phase allowing a more efficient separation. We will use this method to separate inoculated E. coli and Listeria from spinach and mung bean sprouts, respectively. We determine the loss of mass from the plant material following digestion and screen enzymes/conditions to ensure bacterial viability is not compromised. The separation efficiency will be quantified for increasing bacterial concentrations by standard plating methods.Phage enabled separation of E. coli and Listeria spp. from liquid samples: We plan to build off our prior success with T7 bacteriophage to genetically engineer the Listeria specific phage A511 to express biotin on the major capsid protein. Both surface modified phages will be conjugated to streptavidin-coated magnetic particles (1µm) to allow for bacterial separation. We will take advantage of the high affinity between bacteria and their respective phages for the efficient separation of bacteria form a liquid sample (agricultural water). Following separation and bacterial lysis, qPCR will be conducted for the quantification of capture efficiency. Validation of a digestion/phage-based bacterial separation method on food matrices: A validation of the proposed bacterial separation will be conducted starting from the raw agricultural products. Extension staff will conduct the enzymatic digestion of spinach and bean sprouts inoculated with E. coli O157:H7 and Listeria monocytogenes, respectively. Following large-pore filtration, the bacteria will be separated from the liquefied matrix using phage-conjugated magnetic particles. The complete separation efficiency will be quantified with qPCR and compared to the inoculation concentrations.The novel bacterial separation method proposed herein has been designed to 1) release bacteria from a plant matrix into an aqueous mixture, and 2) separate the bacteria from the aqueous mixture into a clean buffer compatible with PCR. The methods can be used separately or in series and allows for the determination of viability through the use of bacteriophage-mediated lysis. By completing the objectives described herein, we support the long-term research goal: to improve food safety through the development to pragmatically separate bacteria from a complex sample.