1a. Objectives (from AD-416):
1: Develop rapid and efficient techniques that separate and concentrate and/or quantify targeted pathogens from food matrices. 1A. Apply rapid and high volume centrifugal flow concentration to the separation of bacteria from food matrices. 1B. Partition and concentrate bacteria using immunomagnetic separation with a new class of antibody-coated paramagnetic particles. 1C. Compare and contrast bacteria separation and concentration with flow-through filtration systems. 1D. Develop and validate procedures for the rapid and quantitative detection of multiple foodborne pathogens. 2: Develop and validate field testing kits that rapidly screen for the presence and quantification of pathogens and/or indicator microorganisms in foods at the initial processing level. 2A. Generate portable, label-free sensors (e.g., next generation cantilever microbalance) for rapid in-line or near-line screening of foods. 2B. Generate portable antibody and/or phage-based multiplex assays including integrated comprehensive droplet digital detection (IC 3D). 2C. Develop an AlphaLISA detection protocol for target pathogens. 2D. Develop a flow-through immunoelectrochemical detection device for field portable detection of target pathogens. 3: Develop and validate rapid methods for the identification of pathogens and/or indicator microorganisms in foods for application in either the field or testing laboratories. 3A. Generate phage and/or antibody typing arrays. 3B. Generate pathogen databases and improve the accuracy of the Beam (formerly BActerial Rapid Detection using Optical scattering Technology or BARDOT) system. 3C. Direct typing (colony isolates not required) of enriched samples using a targeted-sequencing method. 3D. Generate genome sequence-based typing and identification schemes using next-generation sequencing technology (e.g., MiSeq, Ion Torrent PGM, and MinION), and characterize virulence and antibiotic resistance of microbial pathogens.
1b. Approach (from AD-416):
The primary objective of the plan is to develop rapid screening and identification methods for top foodborne bacterial pathogens, including STECs, top Salmonella serotypes, and Listeria monocytogenes as well as those of intermittent concern. Novel or enhanced sample preparation techniques (e.g., flow-through centrifugation, hollow fiber filtration, immunomagnetic separation), most likely in conjunction with pre-filtration, will be key for rapid concentration of food-associated bacteria to readily de detectable levels by modern rapid methods. Subsequently, improved levels of detection sensitivity are expected, perhaps even to an extremely low goal of approximately 1 cell/100 mL of target pathogen as required for real-time testing in the field, processing plant, distribution center, or retail establishment. Total assay times are foreseen to be from a few minutes to = 2 hours. Also, enhanced detection systems will be needed in order to bypass growth enrichment and achieve the desired, quantifiable detection levels. Furthermore, numerous biomarkers and the potential for false positive results using cross-reacting biorecognition elements will require multiplex detection techniques (e.g., multiplex qPCR and microarrays) that may be employed to distinguish true positive results from interference by background matrix or flora. Methods will initially be developed with culture media or buffer as the sample matrix, and then extended to application with food (primarily ground meat) in multiple sample formats: N=60 samples, meat core samples, tissue homogenates, carcass rinses, etc. The efficacy of any newly developed methods will require comparison to current “gold standard” methods in order to validate assay performance. Initially, this will be accomplished by reliance on enumeration of known bacterial isolates, quantified in pure culture with total cell counting if a significant dead population is expected. For evaluation, artificially inoculated and unknown samples will be tested with new methods as assessed against selective enrichment followed by selective and differential plate agar analysis. Regulatory-based methods, such as biochemical testing, multiplex PCR, and serotyping, and possibly whole genome sequencing, may be invoked for additional comparison. Our sister agency, FSIS, will provide guidance as to the parameters and specifics regarding acceptable validation of desired rapid bacterial detection methods. We propose that our developed methods be initially tested at FSIS regional labs using inspector obtained samples, split/divided at the lab, and tested in parallel. Eventually, testing will move to the field- first off-line and near-line, then in-line for some analysis platforms (e.g., microcantilever balance biosensor) situated in the processing environment and/or retail establishments. It is expected that multitudes of tests will be conducted given that most samples will be negative. Regulatory, and perhaps legal guidance will be anticipated to be critical since validation testing may lead to recalls if “zero tolerance” organisms are detected or if threshold amounts of positive samples (e.g., for Salmonella) are discovered.
3. Progress Report:
This report documents progress for the parent project 8072-42000-084-00D Development of Portable Detection and Quantification Technologies for Foodborne Pathogens, which started April 1, 2016 and continues research from project 8072-42000-071-00D, Detection and Typing of Foodborne Pathogens in NP108. The Plan focuses on 3 major goals that address development of field-friendly methods for the rapid, real-time detection and identification of foodborne bacterial pathogens in the field: 1) Rapid microbial sample preparation, 2) Rapid foodborne bacteria detection, and 3) Rapid bacterial identification. Progress was made with all Objective 1 subobjectives and include: A) the purchase, setup, and testing of a benchtop flow-through centrifuge (demonstrated to process a prefiltered mix of 250 g of ground beef and 750 mL of buffer at a rate of 50 mL/min), B) pursuit of immunological-based sample concentration with a CRADA partner, and C) extensive testing of the InnovaPrep “concentration pipette” flow-through filtration unit. Regarding the latter, initial tests compared Purdue University’s Center for Food Safety Engineering’s (West Lafayette, Indiana) C3D flow-through filtration system with the InnovaPrep. Bacterial concentration with the C3D was very poor at best whereas the InnovaPrep worked well with ideal systems (i.e., dilute bacteria in buffer with over a 90% recovery) yet it failed with attempts to concentrate even simple, pre-filtered and clarified animal-based food washes. The InnovaPrep concentration process was efficacious with low concentration, plant (e.g., frozen vegetables)-borne contaminants. With the InnovaPrep, not only overall recovery rates were measured but also perturbations to the makeup of the microbiome (most probable composition using 16S rDNA sequencing) due to the concentration process itself. We found that the InnovaPrep device gives high recovery rates (80 +/- 18%; n = 5 samplings) with no significant alteration in the microbiological makeup of the samples. Finally, regarding Subobjective 1D, processing of samples contaminated with foodborne pathogens prior to quantitative analysis via PCR-MPN was conducted using centrifugation. Though efficient, centrifugation resulted in the bacteria being packed together with food particles, and it was impossible to evenly distribute the cells back into liquid without a large increase in non-stochastic sampling error (i.e., observed number of cells were less than the total since many food particles contained multiple cells) leading to inaccurate quantitation; Flow-through filtration is currently being assessed as an alternative, gentle approach. With Objective 2, significant progress was made with the latter 2 (of 4) subobjectives. Generation of an AlphaLISA for Shiga toxin 2 (Stx2) has proceeded as part of a CRADA with a collaborator. Ultimately, an optimized AlphaLISA (a homogeneous immunoassay) for the toxin is planned to be developed and marketed as a diagnostic kit. In addition, significant progress has seen the development of a working flow-through graphite-based immunoelectrochemical (IEC) system. The IEC system was recently demonstrated to detect 100 heat-killed Salmonella cells/mL in buffer. In addition for Objective 2, methods for the manufacturing of multilayer devices have been developed for the generation of biosensors capable of pathogen detection. Rapid, on-line, near real-time sensors are being developed to be used by regulatory agencies and the food industry to detect foodborne pathogens in an attempt to prevent contaminated food from reaching the marketplace. Sensor prototypes have demonstrated the potential to detect pathogens and their toxins in real world food processing conditions. However, the transition from prototype fabrication to larger scale manufacturing has presented a significant barrier to market entry for an engineering firm that is collaborating with ARS researchers at Wyndmoor, Pennsylvania. A process patent application was generated that describes engineering solutions to address obstacles associated with high volume manufacturing of quality sensors (piezoelectric membrane-based microcantilever balance transducers) in a manner that ensures high yields, and low costs. The combination of performance, cost, and manufacturability should help ensure that this sensor technology penetrates the market and addresses the unmet needs of both food regulators and producers. For Objective 3, numerous genomes of bacterial strains have either been characterized and/or detected via next generation DNA sequencing platforms with associated bioinformatic pipelines. In addition, we are continuing efforts on determining if the Oxford Nanopore Technologies (Oxford Science Park, United Kingdom) MinION sequencer can be used to rapidly detect pathogenic bacteria in food samples in the field. The MinIon has found application in the assistance of genome sequence closure for Campylobacter isolates described herein as well as Salmonella plasmids. Furthermore, genomic DNA from STEC “Super Seven” strains (obtained from the Center of Disease Control, Atlanta, Georgia) was analyzed and were identified to the serotype level using a custom-generated DNA sequence database and the MinIon. In addition for Objective 3, whole genome sequencing and analysis of Campylobacter coli YH502 from retail chicken has been investigated. Campylobacter infection, mainly caused by ingestion of undercooked poultry and meat products, is one of the most common bacterial-associated foodborne illnesses worldwide. Improving the accuracy of identifying this pathogen is required for faster analysis of potentially contaminated food samples. ARS researchers at Wyndmoor, Pennsylvania, in collaboration with other ARS researchers at Beltsville, Maryland and a visiting scientist from New Delhi, India applied next-generation DNA sequencing for detection, genotyping, and characterization of Campylobacter strains isolated from retail meat. This analysis, in conjunction with an advanced method for DNA sequence comparison known as “multilocus sequence typing” yielded very subtle differences (e.g., a change in a single DNA base of sequenced genomes) were exploited to delineate seemingly identical Campylobacter strains. Using bioinformatics to study the genes of this pathogen, including those associated with virulence (disease-causing potential) and antimicrobial resistance provides a better understanding of this important foodborne pathogen that will lead to improved control strategies.
1. A novel method for rapid enrichment, amplification, and DNA sequence-based typing of foodborne pathogens. Methods currently used for detection of foodborne pathogens and for strain typing, necessary for epidemiological investigations, are not sufficiently rapid, are cumbersome, and can be inaccurate. To address these inadequacies, a novel enrichment, amplification, and DNA sequence-based typing (EAST) method was developed, by ARS researchers at Wyndmoor, Pennsylvania and a biotech company, that required 3 days or less to complete and provided strain resolution sufficient for source tracking and epidemiological investigation. EAST was applied to the detection of Salmonella-spiked ground turkey and Yersinia enterocolitica-spiked ground pork demonstrating a very high sensitivity and specificity for the target pathogens. Compared to existing typing technology (e.g., pulsed-field gel electrophoresis-based PulseNet coordinated by the CDC (Center of Disease Control), Atlanta, Georgia, EAST is more sensitive, specific, and simple as well as a relatively rapid, and less costly method. EAST can be used to both detect and type important foodborne pathogens directly from food enrichments containing background bacteria therefore assisting regulatory and public health agencies in epidemiological investigations during outbreaks of foodborne illness and food producers in source tracking of pathogen contamination during food processing.
2. Commercialization of two new antibody-based tests for Shiga toxin 1 and 2. Antibody-based detection methods referred to as enzyme-linked immunosorbent assays (ELISAs) have been developed to detect Shiga toxin producing E. coli (STEC) that may produce either one or both toxins: Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2). Stx2 is 400 times more toxic than Stx1, and the ability to differentiate STEC by their toxin production assists in epidemiological investigations. In collaboration with ARS researchers at Wyndmoor, Pennsylvania, a biotech company and CRADA partner developed and commercialized two new ELISAs that, unlike other similar rapid test products, has the ability to distinguish STEC that produce either Stx1 or Stx2 in food and environmental samples (beef, Romaine lettuce, recreational water, and pasteurized milk were tested). Each ELISA protocol incorporated antibodies (demonstrated to bind all known Stx1 or Stx2 variant subtypes) and an innovation of using an extraction reagent that proved to be effective for releasing and thus accurate detection of cell-bound Stx1. Sensitive, specific, and reproducible detection and differentiation of the Shiga toxin types was achieved and therefore the two ELISA kits should prove useful for application in food testing by producers and regulators alike.
3. The disease-causing potential of Campylobacter isolates from beef and poultry. Campylobacter is an important foodborne pathogen that causes gastrointestinal disease, and it is prevalent in poultry, as well as other meat products. ARS researchers at Wyndmoor, Pennsylvania used a novel method to isolate 27 Campylobacter strains from meat (chicken and beef), and the strains were identified as either C. jejuni or C. coli by molecular methods, including DNA next generation sequencing in collaboration with a Computational Biologist (ARS, Beltsville, Maryland). The whole genome sequences and encoded proteins of C. jejuni strain YH001 and C. coli strain YH501 were determined and have been deposited into the NCBI Genbank database under the accession numbers CP010058 and CP015528, respectively. Bioinformatics analysis revealed a significant genetic distance between the C. jejuni and C. coli species, and the isolates from the same food source appeared to be related more closely to each other than those from different sources. Comparative sequence analysis indicated potentially higher disease-causing potential and drug resistance for a strain (C. jejuni YH001) that was found to contain a key virulence gene cluster (cdtABC) and genes for a multidrug efflux pump (cmeABC). This study provided a better understanding of this important foodborne pathogen and will lead to development of better control strategies.
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