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:
Progress was made on all three objectives and their associated subobjectives which fall under National Program 108, Component I, Foodborne Contaminants by ARS researchers in Wyndmoor, Pennsylvania under Project Plan 8072-42000-084-00D, Development of Portable Detection and Quantification Technologies for Foodborne Pathogens. 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: 1) Rapid microbial sample preparation, 2) Rapid foodborne bacteria detection, and 3) Rapid bacterial identification. All Objective 1 subobjectives milestones were substantially met and include: 1A) Investigation of the ability of Scientific Methods high volume centrifugal flow concentrator (CFC) for the separation of bacteria from ground beef homogenate. From these studies, it was found that the CFC appeared to underperform the manufacture’s claims for the separation of bacteria from the matrix because a large number of bacteria were found to exist in the waste (eluate). Although it does appear to capture and concentrate certain components of the food matrix such as carbohydrates, protein and fats, which may be useful as a preparatory step for downstream detection techniques, it does not seem to have a large effect on the bacteria within the sample. Other filtration techniques tested, such as glass wool, appear to be better suited for separating the food matrix away from the bacteria, 1B) Assessments of the provisionally patented “Antibody Bar” magnetic capture device have continued. Although further improvements are underway, significant progress has been made towards increasing the capture efficiency of the newly developed device (USDA Docket No. 0068.18 US Provisional Patent Application No. 62/737,212). A Material Transfer Agreement was completed, allowing an interested existing CRADA partner in industry to evaluate the product, and 1C) the InnovaPrep “concentration pipette” flow-through filtration unit was further tasked for separation and concentration of background flora from select foods and inoculated pathogens (Shiga-toxin producing E. coli or STECas followed by particulate analysis (with a Malvern particle analyzer) and capture efficiency. For Objective 2, substantial progress was made for all subobjectives and includes: 2A) In collaboration with the Massachusetts Institute of Technology (Cambridge, Massachusetts), an interesting new technology has been devised which employed an optical biosensor that could focus or scatter light in response to external stimuli. This label-free sensor exploits a reversible reaction between boronic acid surfactants and carbohydrates at the hydrocarbon/water interface leading to a dynamic reconfiguration of the droplet morphology and subsequent angular distribution of the droplet’s fluorescent light emission. As proof-of-principle, Salmonella enterica was detected using this new technology, results of which are published in ACS Central Science, 2B) Significant progress was made in the development of a testing kit for the rapid screening of Shiga-toxin producing E. coli, which utilizes droplet digital PCR technology. This testing kit has the advantage compared to the older methodologies used by the Food Safety and Inspection Service of being able to determine if the two virulence genes utilized by the current screen (stx and eae) exist within the same organism or if several different bacterium are present within the sample, each expressing one of the two virulence genes. Testing performed on various food matrices including ground beef, skirt steak, chuck, brisket, stew beef, and round strips confirmed the robustness of the testing kit its applicability to different sample types, 2C) The evaluation of the bead-based AlphaLISA for the detection of Shiga toxin (Stx) in food matrices has been finalized in conjunction with a CRADA partner. A peer reviewed publication describing the AlphaLISA for the detection of Stx as tested in both Romaine lettuce and ground beef is currently available. This assay meets the current industrial standard for Stx detection (limits of 0.5 parts-per-billion) yet has several advantages over these approaches including a more rapid testing time, a larger dynamic range, and uses a method that is easily amendable to automation and high-throughput screening, and 2D) Experiments to determine the effectiveness of the flow-through immunoelectrochemical detection device for pathogen detection in buffer have been completed. The current design allowed liquid-based samples to flow through the electrode while simultaneously capturing target pathogens. Subsequent pathogen detection was performed via an oxidized substrate in a conventional sandwich immunoassay, yielding detection limits of ~1000 cells in a 60 mL volume. Results of the analysis were published in a peer-reviewed manuscript and are the subject to a U.S. Provisional Patent Application (No. 62/821,624; USDA Docket Number 0021.18). Significant progress has also been made on studies currently underway investigating its application to a large volume (=1L) of food matrix. For Objective 3, substantial progress was made on all subobjectives: 3A) A bacteriophage (fV10; a virus that may specifically infect bacterial serotypes) isolated in the laboratory of our collaborator with the Center for Food Safety and Engineering at Purdue University (West Lafayette, Indiana) was generated in E. coli O157:H7 and cross-linked to tosyl-activated superparamagnetic microparticles. The intent was to use the modified particles for both binding and transfection of the STEC to indicate both capture (screening/detection) and confirmation via identification through transfection, 3B) In conjunction with a collaborator at Lincoln University, Christchurch, New Zealand, BEAM/BARDOT scatter images were generated for Yersinia spp. on minced pork samples. On-going related work is intended to replace time-consuming (~10 day) detection/confirmation methods for the foodborne pathogen with the approx. 2-day BEAM technique. In part, this new method is accelerated by a proprietary-modified selective enrichment broth for Yersinia spp. generated by Lincoln University, 3C) All milestones for this subobjective were unexpectedly met way under schedule and a manuscript on this work has already been published and recorded, and 3D) Because of the high prevalence of Campylobacter spp. in chicken and other meat products, it is important to develop rapid and accurate techniques for typing and identifying Campylobacter spp. in food matrices. With increasing availability of whole genome sequences and various bioinformatic tools, we analyzed all complete genomes (n=199) of Campylobacter spp. in the NCBI database and revealed distinct trends of mononucleotide repeats in Campylobacter genomes. On a per-genome basis, the highest mean occurrence of poly-A repeats (1639) was found in C. sputorum. C. jejuni had the highest occurrences of poly-T and poly-C repeats while C. fetus harbored the largest number of poly-G repeats. In all the genomes, occurrences of poly-A and poly-T repeats were clearly reduced with the increase in length of repeats. On the contrary, occurrences of poly-G and poly-C repeats followed a Gaussian distribution with a peak at 9 nucleotides: Poly-G probability distribution of the number of 9×G repeats: µ = 6.78, s = 2.86; Poly-C probability distribution of the number of 9×C repeats: µ = 6.17, s = 2.67. Considering the diversity in occurrences of mononucleotide repeats, we have demonstrated the use of mononucleotide repeats as a putative typing scheme for Campylobacter spp. Three phylogenetic trees based on (i) occurrence of mononucleotide repeats; (ii) alignment of housekeeping gene sequences stipulated by MLST schemes; and (iii) whole genome sequences, indicated reasonably good conformity. While the tree based on whole genome distances was best in segregating various Campylobacter spp., trees based on mononucleotide repeats and MLST were similar. The typing scheme based on mononucleotide repeats may serve as a putative alternative to existing methods for Campylobacter typing.
1. Absolute quantification of shiga-toxin producing E. coli in beef with ddPCR. Because harmful bacteria often possess a combination of distinguishing traits/markers that allow them to cause disease, screening systems such as that utilized by the Food Safety and Inspection Service capitalize on the existence of these traits and can delineate potential disease-causing E. coli strains based on the presence of 3 such genetic markers. However, false positives result when a single sample contains more than one bacterium that possess 1 or 2 of the markers (but not all 3 of them) since the screening method does not define the specific organism from which each gene was derived. To overcome this shortfall, a new screening system known as Droplet Digital PCR (ddPCR) with the ability to determine when multiple genes are contained within a single source organism was developed and tested by the Food Science Division at Bio-Rad Laboratories, Inc. (Marnes-la-Coquette, France) in partnership with ARS researchers in Wyndmoor, Pennsylvania. Ultimately, this system results in cost-savings by reducing both wasted man-hours and expenses associated with subsequent evaluation of false-positive samples and testing kits are expected to be released for purchase by Bio-Rad Laboratories, Inc. in the fall of 2019.
2. Rapid flow-through immunoelectrochemical detection of low numbers of bacteria in large volumes. To address the safety of the food supply chain, the size of raw meat samples collected for testing was increased from ~0.9 ounces to ~11.5 ounces. Although testing involving larger samples sizes can increase the likelihood of detecting pathogens present at low levels, it also creates an unmet need for rapid detection methods that can be used on large volume food samples. In response to this need, an electrochemical sensor was engineered in order to provide a testing method that can accommodate the larger sample size. The newly designed sensor consisted of a porous transducer (sensing element) that allowed for 1 L of sample to be filtered through within one hour. This sensor demonstrated the ability to detect different common foodborne pathogens in food samples that are aligned with protocols currently employed by regulatory agency’s (e.g., Food Safety and Inspection Service) and has resulted in a peer reviewed publication and the filing of a provisional patent application on the technology.
3. Rapid detection of Salmonella via emission from dynamic double emulsion droplets. The testing of samples for the presence of pathogens is an important method of ensuring the safety of our food supply chains. Because adoption of a testing method is dependent upon several factors including, the number of samples that can be processed, the need for specialized equipment, the overall accuracy of the test, the time to process the samples, and the costs associated with the testing method; there is a constant need for more rapid, portable, and low-cost detection methods for pathogens in food. Therefore, a new sensing model for the early-stage detection of foodborne pathogens that is based on the unique chemical-structural-optical coupling in chemical (boronic acid)-functionalized fluorescent double emulsions was developed. This novel sensor demonstrated the ability to detect Salmonella in both enrichment media, and chicken rinse samples and the results of this testing were published in a high impact factor, peer-reviewed, scientific journal. (ACS Central Science; Impact Factor = 12.8).
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