1a. Objectives (from AD-416):
Objective 1: Develop rapid and effective means to separate and concentrate targeted pathogens from food matrices that can be coupled to very rapid detection methods such as real-time PCR. 1A. Develop filtration/centrifugation methods for separating and concentrating pathogenic Escherichia, Salmonella, Listeria, and Campylobacter spp. from a variety of food matrices. Optimize reagents, apparatus and conditions to achieve maximum speed and recovery with minimum detection limits. 1B. Develop DNA extraction methods providing rapid, efficient, unbiased recovery of inhibitor-free DNA from a variety of pathogens. Objective 2: Examine environmental factors and microbiological culture conditions affecting genotypes or phenotypes that are important for virulence, isolation, or detection of foodborne pathogens. 2A. Detection of foodborne threat agents (model system- pathogenic Yersinia spp.). 2B. Isolation and detection of foodborne pathogens maintaining mobile genetic elements. 2C. Enrichment of pathogens while maintaining mobile genetic elements. Objective 3: Develop protein- and nucleic acid-based methods for the multiplexed detection and characterization of food-borne pathogens. 3A. Protein-based microarray and other multiplexed methods for the analysis of foodborne pathogenic bacteria. 3B. Oligonucleotide-based microarray for multiple pathogen detection and characterization. 3C. Multiplex real-time PCR for multiple pathogen identification and quantification. Objective 4: Develop typing methods for pathogens of concern to associated food regulatory agencies. 4A. Develop Restriction Fragment Sequence Polymorphism method for typing. 4B. Fractionation of a naïve library of biorecognition elements for bacterial typing-An alternative to “molecular typing.”
1b. Approach (from AD-416):
This project plan has multiple goals that are distinct yet may be combined to generate improved, rapid techniques for the analysis of foodborne pathogenic bacteria (e.g., Campylobacter, E. coli, Listeria, Salmonella, and Yersinia spp.). Compared to traditional plate culture techniques, rapid methods for bacterial detection and typing [identification] primarily suffer from relatively poor sensitivity and/or selectivity. To improve detection limits for oligonucleotides using DNA microarray or multiplex RT-PCR, improved methods for DNA extraction, including an optimized alkaline/detergent reagent, will be developed for efficient extraction of nucleic acid from bacteria. Leukocyte removal filters will be used to separate bacteria from food matrices and concentrate the cells allowing for improved limits of detection by antibody microarray and/or time-resolved fluorescence. Culture enrichment conditions (e.g., slightly acidic pH, millimolar concentrations of calcium ion, with or without Irgasan) will be initially optimized for a model pathogenic bacterium (Yersinia) with the intent of concentrating the bacteria from the sample while maintaining mobile genetic elements [plasmids] required for expression of key genotypic and phenotypic markers. Prior to detection/typing with biosensor platforms, enriched Yersinia spp. will be carefully isolated and assessed for maintenance of virulence plasmids using organic dyes (crystal violet and/or Congo red) in conjunction with low calcium plating media. Novel biorecognition elements (initially, single chain variable fragment antibodies fractionated from naive phage display libraries) will be custom generated to improve accuracy of biosensor-based detection or phenotyping platforms (e.g., microarrays) for targeted pathogens. In addition, an abbreviated restriction fragment sequence polymorphism method will be developed and assessed as a novel genotyping method. Promising technologies will be directed towards usage by food producers and regulatory agencies for food safety monitoring and follow-up investigations.
3. Progress Report:
Progress was made on all four objectives and their subobjectives, all of which fall under National Program 108, Component I, [Microbial] Pathogens, Toxins and [non-biological-based] Chemical Contaminants: subdivided in Pre-harvest and Post-harvest. Progress on this project focuses on Problem Statement 1.C, Technologies for the Detection and Characterization of Microbial Contaminants. Substantial progress was made on every subobjective outlined in this project plan. The goal of this project is to develop accurate methods for the detection and identification (ID) of pathogenic, foodborne bacteria. Successful detection/ID typically necessitates concentration via physical means (e.g., filtration) or increasing the number of factors (or biomarkers) through non-deleterious cell growth or genetic material amplification (i.e., PCR). For Objective 1A, we have developed a fast method for separating and concentrating bacteria from foods using leukocyte reduction filters (commonly used in hospitals for blood component separation). For Objective 1B, we have optimized the extraction of unique DNA factors using in-house developed reagents. For Objective 2A, a simple, economical, and highly reliable test using select organic dyes was developed to promote the rapid detection and isolation of Yersinia spp. For Objective 3A, an antibody-based microarray platform was developed for the typing of “Big Six” Shigatoxin-producing E. coli. For Objective 3C, a database of scan patterns generated by the BARDOT (bacteria rapid detection using optical scattering technology) system was created for the rapid ID of Campylobacter spp., however, sporadic results for some Campylobacter spp. will improve with a newer version of BARDOT (recently acquired). Also, in collaboration with FDA, a multiplex qPCR method was developed for the detection of E. coli O157:H7, and Listeria monocytogenes in soft cheeses. When growing bacteria in a lab, it is impossible to use a rapid method (e.g., qPCR) to calculate the initial number of bacteria unless a Most Probable Number (MPN) technique is also used. qPCR-MPN was employed with Campylobacter spp., but we found that ca. ¼ of the results were problematic due to the non-random distribution of the bacteria in fat-laden chicken wash samples. For Objective 4A, in-silico studies were conducted to assess the feasibility of the proposed Restriction Fragment Sequence Polymorphism (RFSP) method for typing pathogens. Several hundred commercially available restriction enzymes were assessed with ca. 100 sequences of organisms currently in the CDC’s PulseNet PFGE database. Discrimination between bacteria was poorer using RFSP than by PFGE. Therefore, further pursuit of the RFSP approach is not recommended. In-silico studies were also conducted on Double Restriction Fragment Length Polymorphism methods. Preliminary results indicate that this is a promising approach and that a few combinations of restriction enzyme pairs could provide discriminatory power comparable to or better than PFGE.
1. Developed methods for separating and concentrating pathogenic bacteria from various food matrices. Foods and harmless bacteria in foods can interfere with accurate detection of harmful pathogens. ARS researchers at Wyndmoor, Pennsylvania have developed rapid filtration and centrifugation methods that not only separate, but to also concentrate target pathogens (Escherichia, Salmonella, Listeria, and Campylobacter spp.) from various food matrices including ground poultry and beef. Food regulators and producers will benefit immensely from applying these techniques since testing for harmful pathogens would be streamlined in terms of expense and time.
2. Ensuring the ability to detect harmful Yersina pestis (YP) in food. Accurate detection of virulent YP requires YP to have an intact virulence plasmid (small, circular strand of DNA). However, YP may lose this plasmid if it is mistreated during growth enrichment and/or isolation. ARS researchers at Wyndmoor, Pennsylvania have developed methods that were used to study the stability of the plasmid during growth of YP in raw ground meats (pork and beef). Producers and regulators may use this information to reduce the incidence of YP in foods.
3. Developed a typing assay for Shiga-toxin producing E. coli (STEC) using microarrays. Methods are necessary for the rapid identification of detection and identification (typing) of harmful bacteria in foods. Antibody and DNA typing (identification) microarray platforms have been developed on both glass and relatively inexpensive polystyrene plastic substrates. ARS researchers at Wyndmoor, Pennsylvania have produced systems that, using either antibodies or computer-designed nucleic acid probes/primers, interrogate large numbers of samples for E. coli O157:H7 and/or the “Big Six” non-O157 STECs. Total assay times were typically under 3 h with real-time (non-growth enriched samples) with relatively low detection limits. The published finding may arm bacterial typing labs and regulatory testing agencies with additional means for ensuring biosafety as well as biosecurity of foods.
4. Developed an identification method for foodborne pathogens using BARDOT and RT-PCR. There is a need to replace current methods for the identification of harmful bacteria in foods since such testing often takes long days to weeks. Faster identification reduces has benefits ranging from shorter food product holding times to accelerated epidemiological investigations. ARS researchers at Wyndmoor, Pennsylvania have combined BARDOT (bacteria rapid detection using optical scattering technology; technology developed by our collaborators in the Center for Food Safety Engineering at Purdue University, West Lafayette, Indiana) with multiplex RT-PCR (real time PCR) for the prescreening and identification of identification of pathogens (Campylobacter jejuni and C. coli) in foods. Food regulators and producers may apply this technique for the identification of harmful pathogens.
Gehring, A.G., Barnett, C., Chu, T., Debroy, C., D'Souza, D., Eaker, S., Fratamico, P.M., Gillespie, B., Hedge, N., Jones, K., Lin, J., Oliver, S., Paoli, G., Perera, A., Uknalis, J. 2013. A high-throughput antibody-based microarray typing platform. Sensors. DOI: 10.3390/s130505737.