Location: Characterization and Interventions for Foodborne Pathogens
2021 Annual Report
Objectives
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.
Approach
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.
Progress Report
Progress was made on the two research objectives with associated 60 month subobjectives which fell under National Program 108, Component I, Foodborne Contaminants by ARS researchers at Wyndmoor, Pennsylvania under Project Plan 8072-42000-084-00D, Development of Portable Detection and Quantification Technologies for Foodborne Pathogens.
Objective 1: There were no milestones to be accounted for at the 60 month timeframe.
Objective 2: For Subobjective 2A “Generate portable, label-free sensors (e.g., next generation cantilever microbalance) for rapid in-line or near-line screening of foods,” pending grant proposals with Lenima Field Diagnostics LLC (Philadelphia, Pennsylvania) have been generated and include: “Demonstration of quantifying pathogenic Salmonella by its invA gene from sample to result in 30 minutes for rapid, on-site detection and control” submitted to Foundation for Meat and Poultry Research and Education (October 26, 2020) and "Handheld sensors designed to rapidly quantify pathogenic Salmonella contamination in foods” submitted to USDA SBIR Phase 1 (October 22, 2020). For Subobjective 2B “Generate portable antibody and/or phage-based multiplex assays including integrated comprehensive droplet digital detection (IC 3D),” this research was replaced with an analogous substitute of digital droplet PCR (ddPCR). A Materials Transfer Research Agreement to bring a ddPCR as well as all consumables required to complete the study to ERRC. A manuscript on application of ddPCR to detection of Shiga-toxin producing E. coli (STEC) was published. For Subobjective 2D, “Develop a flow-through immunoelectrochemical detection device for field portable detection of target pathogens,” studies demonstrated the application of the flow-through electrochemical to encompass genetic sequences that can distinguish Listeria monocytogenes from other Listeria species. The conditions necessary for genetic detection have been laid forth and the current results demonstrated the sensor’s ability to distinguish L. monocytogenes DNA from L. innocua with a limit of detection of ~2×104 cells per milliliter. Importantly, a timely culture enrichment period was not necessary and the assay may be performed with hand-held electronics, which would allow the platform to be adopted for near-line monitoring systems. Also, the incorporation of genetic-based capture probes helps overcome limitations based upon antibody availability and addresses specificity errors in phenotypic assays. This research resulted in a publication and submission of a US patent application. In addition, for the 60 month milestone for Subobjective 2D, a postdoctoral research associate was hired and a visiting student from a collaborating laboratory at Purdue University conducted relevant research in our ARS laboratories. Substantial progress was made on cross-linking transfecting phage to flow-through electrochemical platform transducer substrates.
Objective 3: For Subobjective 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,” the whole genome sequence of Campylobacter jejuni strain YH002, isolated from retail meat, was obtained using both Single Molecule Real-Time (SMRT; Pacific Bioscience) and MiSeq (Illumina) sequencing technologies. By comparative analyses of the genome sequences, annotated gene products, and phenotypic characteristics of these strains, we identified a number of important factors involved with the virulence and antimicrobial resistance of Campylobacter. In-depth methylome investigations into the genome seeking to identify areas unique to C. jejuni that could be utilized for pathogen characterization revealed a putative novel methylation motif (CGCGA) of a type II restriction-modification (RM) system in Campylobacter. Comparison of methylomes of this strain to well-characterized C. jejuni strains 81-176 and NCTC 11168 revealed non-uniform methylation patterns among the strains though the existence of the typical type I and type IV RM systems were also observed. Further investigations into the DNA methylation sites within promoter/regulatory sequences, which ultimately could alter the expression levels of transcription, revealed several virulence genes putatively regulated using this mode of action. Of those identified, a flagella gene (flhB), an RNA polymerase sigma factor (rpoN), a capsular polysaccharide export protein (kpsD), clustered regularly interspaced short palindromic repeats (CRISPR), and a multidrug efflux pump were highly notable. Together, the genome-wide studies combined with phenotypic characterization of C. jejuni isolates provide a better understanding of the genetic diversity and pathogenicity of this important foodborne pathogen. These investigations were detailed in a publication.
Overall, the Project Plan saw significant impact in its 3 major goals that addressed 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. Accomplishments, nine in all, ranged from patent applications for a sample preparation magnetic capture device and a novel, flow-through electrochemical detection platform to new methylome-based techniques for analyzing bacterial genomes for virulence factors in addition to unique sequences for identification. Technology transfer has included development of 5 incoming agreements, 4 invention disclosures, and the filing of 3 patent applications. International collaborations were with research groups in 6 countries (China, France, Germany, India, Israel, and New Zealand). Finally, fifteen articles have been published in peer-reviewed journals to date.
Accomplishments
1. Improved performance of downstream detection platforms. Sensors and genetic sequencing technologies designed to detect foodborne pathogens are often hampered by components contained within food products. ARS researchers at Wyndmoor, Pennsylvania, employed a systematic approach to compare five different sample preparation techniques for their impact on pathogen retention, particle size recovery, and composition of the sample matrix post-processing. These studies demonstrate techniques that can be applied to a range of downstream detection sample preparation techniques for complex matrices such as foods.
2. Isolation and confirmation of the presence of the bacterial pathogens (Yersinia enterocolitica and Y. pseudotuberculosis) from ground pork. Isolation and confirmation of the presence of the bacterial pathogens (Yersinia enterocolitica and Y. pseudotuberculosis) from ground pork typically relies on a long (10-21 days), “cold growth” enrichment protocol prior to identification. In collaboration with researchers at Lincoln University (New Zealand) and Purdue University (West Lafayette, Indiana), ARS scientists at Wyndmoor, Pennsylvania, generated a significantly shorter approach that combined a novel growth method followed by identification of suspect bacterial colonies using Elastic Light Scatter (ELS) analysis that took less than 2 days (39 hours). In short, ELS involves shining a red laser light through a bacterial colony on a growth agar plate (Petri dish) and the resulting light refraction produces a unique pattern that can be used like a fingerprint to rapidly determine the specific bacteria that is under observation. The food industry and regulators of both countries, as well as the consumer, will benefit from the extension of the shelf life and reduced costs associated with the testing of this food product.
Review Publications
Capobianco Jr, J.A., Lee, J., Armstrong, C.M., Gehring, A.G. 2019. Rapid detection of Salmonella enterica serotype Typhimurium in large volume samples using porous electrodes in a flow through, enzyme-amplified immunoelectrochemical sensor. Analytical and Bioanalytical Chemistry. 411:5233-5242. https://doi.org/10.1007/s00216-019-01901-3.
Capobianco Jr, J.A., Armstrong, C.M., Clark, M., Cariou, A., Leveau, A., Pierre, S., Fratamico, P., Strobaugh Jr, T.P. 2019. Detection of Shiga toxin producing Escherichia coli (STEC) in beef products using droplet digital PCR. International Journal of Food Microbiology. 319:108499. https://doi.org/10.1016/j.ijfoodmicro.2019.108499.
Capobianco Jr, J.A., Armstrong, C.M., Lee, J., Gehring, A.G. 2020. Detection of pathogenic bacteria in large volume food samples using an enzyme-linked immunoelectrochemical biosensor. Food Control. https://doi.org/10.1016/j.foodcont.2020.107456.
He, Y., Reed, S.A. 2019. Pulsed-field gel electrophoresis typing of Staphylococcus aureus strains. In: Walker, J.M., editor. Methods in Molecular Biology. New York, NY: Springer. 2069:78-88. https://doi.org/10.1007/978-1-4939-9849-4_5.
He, Y., Reed, S.A., Strobaugh Jr, T.P. 2020. Complete genome sequence and annotation of Campylobacter jejuni YH003 isolated from retail chicken. Microbiology Resource Announcements. https://doi.org/10.1128/MRA.01307-19.
