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 most of their associated 48 month 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. For Objective 1, there were no milestones to be accounted for at the 48 month timeframe. However, several manuscripts concerning the previous milestones have now been accepted for publication and thus the following updates are appropriate: a) Subobjective 1A- evaluations of the HVCF regarding its efficiency to: 1) concentrate bacteria, and 2) concentrate other components of food matrices have been completed. Comparisons were made between the HVCF and other filtration devices such as glass wool, graphite felt, and 50 µm polypropylene filters. Details of the evaluations are presented in a manuscript (Armstrong et al., 2019, Impacts of clarification techniques on sample constituents and pathogen retention, “Foods” 8:636), b) Subobjective 1B- research was conducted related to a magnetic capture device, and a provisional patent application was filed; in the current FY, additional data was generated to convert the provisional application into a full patent application (Armstrong, et al., A. Magnetic Bar Capture Device. U.S. Patent Application 62/737,212, USDA Docket Number 68.18), and c) Subobjective 1C/1D- studies aimed at evaluating the flow through bacteria separation systems and quantitation of foodborne pathogens via MPN/PCR have been completed and a manuscript (Irwin, P. et al., Bacterial cell recovery after hollow fiber microfiltration sample concentration: most probable bacterial composition in frozen vegetables) was submitted to “Food Control” that summarizes the impact of the InnovaPrep “concentration pipette” flow-through filtration unit on the efficiency of separating pathogens from food matrices. For Objective 2: a) Subobjective 2B- the relevant milestone was to combine multiplex qPCR with an integrated comprehensive droplet digital detection PCR system. The anticipated product was a peer reviewed publication on foodborne pathogenic bacteria using the technology. An analogous multiplexed droplet digital PCR (ddPCR) system was evaluated as a screening assay for Shiga toxin-producing Escherichia coli (STEC) within the Food Safety and Inspection Service’s (FSIS) published protocol, Microbiology Laboratory Guidebook (MLG) 5C. The study benchmarked the response of ddPCR against the current real-time PCR assays which both used the pathotype-specific genetic markers stx and eae. In this comparative study the ddPCR assay demonstrated equivalent sensitivity to the established screening techniques. Further, due to the sample partitioning utilized by the ddPCR technology, the system was able to differentiate between the co-existence of both genes within the same cell from the co-existence of both genes within a mixed microbial population. This distinction from the conventional PCR assays could be used to reduce the number of false positives identified in the screening stage of the MLG 5C, which will alleviate some of the associated time and cost constraints associated with pathogen testing. In addition, Bio-Rad (Marnes-la-Coquette, France) is currently launching a product named dd-Check STEC Solution around the ddPCR technology, as reported on in “Food Safety” magazine (Oct. 16, 2019; https://www.foodsafetymagazine.com/products/first-commercially-available-droplet-digital-pcr-solution-for-detecting-pathogenic-stec/) and “Quality Assurance & Food Safety” (Oct. 24, 2019; https://www.qualityassurancemag.com/article/bio-rad-launches-droplet-digital-pcr-solution-for-pathogenic-stec/). On-going work is being conducted with some guidance from William Shaw to further investigate the potential value of the technology to FSIS testing laboratories, b) Subobjective 2C- the evaluation of the bead-based AlphaLISA for the detection of Shiga toxin (Stx) in food matrices was finished in conjunction with a CRADA partner (Abraxis, LLC; Warminster, Pennsylvania) ahead of schedule with the peer-reviewed publication describing the AlphaLISA for the detection of Stx published by the 36 month timeframe. Because of this, additional studies are underway to access the ability of the AlphaLISA to identify Listeria monocytogenes. These additional studies have been temporarily suspended due to the maximum telework schedule, c) Subobjective 2D- studies investigating the application of the flow-through immunoelectrochemical detection device for pathogen detection in a large volume food matrix have been completed. These studies were performed with live bacterial cultures and showed that significant pretreatment strategies were necessary in order to utilize the technology in conjunction with the current Food Safety Inspection Service (FSIS) standards for ground meat, which is defined as 325 g sample of the product stomached in a 1 L volume of buffer. Flow rates of 12.3 mL/min were achieved with this detection device for ground beef homogenate with overall detection limits of 400 cells/mL for E. coli O157. The associated peer-reviewed manuscript is in press to “Food Control.” For Objective 3: a) Subobjective 3A- in collaboration with Center for Food Safety Engineering (CFSE) at Purdue University, West Lafayette, Indiana), progress was made on detection of specific foodborne pathogenic bacteria using colorimetric and luminescence-based reporting with phage (peer-reviewed manuscripts are currently under preparation for these investigations), b) Subobjective 3B- research on rapid bacterial identification with the BEAM platform has progressed with collaborators at CFSE and Lincoln University (Christchurch, New Zealand). A manuscript on the detection of Yersinia enterocolitica in pork, which usually takes approx. 10 days has been reduced to 3 days, c) Subobjective 3C- no additional research has been carried out this period as this work was completed ahead of schedule, and d) Subobjective 3D- one of the leading causes of human gastrointestinal illnesses in the United States, Campylobacter has been isolated from retail meat including poultry and liver products (recognized as main sources for transmission of campylobacteriosis). Virulence and antimicrobial resistance of Campylobacter strains are also important risk factors for the illness. To identify the risk factors associated with the infection, we performed whole-genome sequencing and comparative analysis of the genomic and phenotypic characteristics of C. jejuni strain YH002 isolated from retail beef liver. By annotation of the complete genome sequence of the strain, we revealed several novel genetic features, including an integrated intact phage element, multiple antimicrobial resistance (AMR) genes, virulence factors, and a Phd-Doc type toxin-antitoxin (TA) system. Phenotypic tests of AMR found that C. jejuni YH002 was resistant to amoxicillin and tetracycline, which correlates with the AMR genes in the strain. Comparative analysis of cell motility at genotypic and phenotypic levels identified discernible patterns of amino acid changes, which could possibly be the genetic cause of the motility variations among C. jejuni strains. These results provided important clues to the genetic mechanisms of AMR and cell motility in C. jejuni. The finding of a Phd-Doc TA system in the genome of C. jejuni YH002 is the first description of this TA system in Campylobacter spp. Our genetic and phenotypic evidence consistently showed the multidrug resistance and high motility of the strain, suggesting it was a potentially disease-causing agent and therefore could greatly threaten food safety and public health. Expanded understanding of the genetic diversity and pathogenicity of this important foodborne pathogen will contribute to the control of this pathogen strain in consumer products by food producers and regulators alike.
5. Record of Any Impact of Maximized Teleworking Requirement:
Ghatak, S., He, Y., Reed, S.A., Irwin, P.L. 2020. Comparative analysis of genomic and functional characteristics of a multidrug resistant Campylobacter jejuni strain YH002 isolated from retail beef liver. Foodborne Pathogens and Disease. https://doi.org/10.1089/fpd.2019.2770.
Armstrong, C.M., Gehring, A.G., Paoli, G., Chen, C., He, Y., Capobianco Jr, J.A. 2019. Impacts of clarification techniques on sample constituents and pathogen retention. Foods. https://doi.org/10.3390/foods8120636.