Progress 04/01/16 to 03/31/21
Outputs
PROGRESS REPORT 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. 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. 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 ~2ÿ104 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 01 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. 02 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.
Impacts
(N/A)
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(2020):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. Methods in Molecular Biology. New York, New York: 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.
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Progress 10/01/19 to 09/30/20
Outputs
Progress Report 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. 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. 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.
Impacts
(N/A)
Publications
- 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.
- 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.
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Progress 10/01/18 to 09/30/19
Outputs
Progress Report 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. 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. 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 9ÿG repeats: µ = 6.78, s = 2.86; Poly-C probability distribution of the number of 9ÿC 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. Accomplishments 01 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. 02 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. 03 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).
Impacts
(N/A)
Publications
- Armstrong, C.M., Ruth, L., Capobianco Jr, J.A., Strobaugh Jr, T.P., Fernando, R., Gehring, A.G. 2018. Detection of shiga toxin 2 produced by Escherichia coli in foods using AlphaLISA. Toxins. 10(11):422.
- Song, M., Li, Q., He, Y., Feng, Z., Lan, L., Fan, Y., Liu, H., Chen, D., Yang, M. 2019. A comprehensive multilocus sequence typing scheme for identification and genotyping of Staphylococcus strains. Foodborne Pathogens and Disease. 16(5):331-338.
- Xie, Y., He, Y., Ghatak, S., Irwin, P.L., Yan, X., Strobaugh Jr, T.P., Gehring, A.G. 2018. Whole genome sequencing and annotation of Staphylococcus aureus strain SJTUF_J27 isolated from seaweed. Data in Brief. 20:894-898.
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report 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. 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. Progress was made on all three objectives and their associated subobjectives which fall under National Program 108, Component I, Foodborne Contaminants by ARS researchers (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 and associated subobjective milestones were substantially met and include: 1A) processing of ground beef samples (250 g beef in 750 mL of buffer) with a benchtop flow-through centrifuge which was demonstrated to process the mixture (prefiltered with glass wool) at a rate of 50 mL/min, 1B) development of a novel antibody-based antigen separation device/method led to an invention disclosure that was approved for generation of a provisional U.S. patent application by ARS� National Life Sciences Patent Committee, and 1C) the InnovaPrep �concentration pipette� flow-through filtration unit was tasked for separation and concentration of background flora in frozen mixed vegetables and inoculated pathogens (Shiga-toxin producing E. coli or STEC) in ground beef with emphasis on particulate analysis (with a Malvern particle analyzer) and capture efficiency. Approx. 74-79% bacterial recovery was observed for the Innovaprep (30 vs. 37�C conditions and either Tris- or phosphate-buffered saline in-house diluent and wash preparations were assessed revealing no significant differences). Recovery of bacteria in ground beef was determined to be futile as the hollow fiber filtration membranes were readily clogged. Finally, regarding Subobjective 1D, the following scenarios were considered: if each food-based pellet (an emulsion of various micro-organisms, lipids, proteins, and assorted debris) after centrifugation is 1 mL and the number of organisms per pellet is 10000, then the volume per particle after re-suspension and break-up of the pellet needs to be = 0.1 �L which is not readily obtainable. On the other hand, if 10 pathogens (or colony forming units or CFUs), or fewer, are present in such a pellet, the suspended particle size needs only to be = 100 �L. These differences in particle size and the resultant non-random distribution of associated pathogen CFUs are the basis for non-stochastic sampling error. As for a more quantitative example of this problem: Assuming one could count the total number of all suspended particles (n) as well as the number of these with at least one bacterium, or CFU, attached (x) then if n = 1000 after the breakup of the concentrated sample (pellet) and if x = 900, with = 1 CFUs attached, the most probable number (MPN) of bio-attachments per particle = ln[n/(n-x)] is over 2. By increasing n 10-fold (by decreasing the volume per particle) the MPN of organisms per particle would drop to 0.0943. Under this experimental condition, the counting of positive occurrences of bacterial attachment was more equivalent to the number of CFUs present in the sample regardless of the food particle associations. For Objective 2, substantial progress was made for all subobjectives and includes: 2A) empirical demonstration of successful coating of a CRADA partner�s piezoelectric membrane sensors (PEMS) with insulative (and covalent cross-linkable) 3-mercaptopropyltrimethoxysilane via lack of electronic short-circuiting upon exposure to aqueous mixtures and fluorescence immunoassay-based results of binding of various analytes (Salmonella, STEC, and microparticles) to sensor cross-linked antibody. Currently, the PEMs were determined to be too thick (approx. 200 �m as measured using a Keyence digital microscope) and therefore unlikely to exhibit any significant impedance-related response for binding by low concentrations of analyte [result empirically confirmed], 2B) As a draft MTRA with our potential IC 3D (integrated comprehensive droplet digital detection) collaborator (University of California, Irvine, California) is under review by their legal team, proof of concept of the key analytical aspect of IC 3D, i.e., ability to detect and characterize individual bacteria cells in complex food matrices was investigated with a comparable technology (droplet digital PCR or ddPCR) under an MTRA with the Food Science Division of Bio-Rad Laboratories (Marnes-la-Coquette, France). The objective of this collaboration is to determine if there are competitive advantages to ddPCR that can simplify and/or improve the detection of pathogens, specifically STEC in foods. Current screening PCR methods targeting stx and eae allow only independent detection of markers, while ddPCR enables the co-localization of STEC virulence genes which can help simplify the STEC identification process. Inherently quantitative, ddPCR does not rely on standard curves which can adversely affect the reliability and repeatability of the assay. The BioRad ddPCR system is in process of being benchmarked against the current FSIS MLG 5 standard, and Bio-Rad�s real time PCR assay, IQ Check for detection of STEC in ground beef. Preliminary results indicate that the assay is accurate and could serve as a more efficient screening protocol than the presently used BAX, real time PCR. Specifically, as the assay can determine if the target virulence factors are contained within one cell, as opposed to a mixture of cells, it will reduce the number of samples which need to be processed and further assessed. This could have a significant impact as FSIS relayed to ARS that 80% of the samples which test positive in the screening step of the MLG 5 are false positives. The device has also identified nonregulated serotypes which can cause foodborne illness that would not be identified by the present MLG protocol. The above data will be presented at the 6th qPCR & Digital PCR Congress at the 4Bio Summit (Fall 2018). For Subobjective 2C, in a collaboration with CRADA partner, AlphaLISAs were developed, optimized, and benchmarked (vs. a commercial ELISA) for STEC-generated Shiga toxin 2 (Stx2) in foods (Romaine lettuce and ground beef) allowing for more rapid analysis of Stx2 with less manual manipulation thus improving assay throughput and the ability to automate sample screening while maintaining detection limits of 0.5 parts-per-billion, and 2D) initial results focused on flow-through enzyme-linked immunoelectrochemical (FT-ELIEC) detection of Salmonella, and demonstrated that FT-ELIEC was two orders of magnitude more sensitive than ELISA, with a significantly larger dynamic range. FT-ELIEC was also demonstrated to accommodate sample volumes that are significantly larger than those used in conventional immunoassays; specifically the sensor can accommodate 60 mL sample volumes as opposed to 0.2 � 1 mL sample volumes used in ELISA platforms. For Objective 3, substantial progress was made on all subobjectives: 3A) bacteriophage (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) were tested for specificity to STEC (specifically E. coli serotype O157:H7 or STEC O157:H7) as it is the most common STEC causing foodborne illness. Previously an STEC O157:H7 detection system was developed employing the STEC-specific phage that was engineered to produce light when STEC O157:H7 is present. This year our collaboration further defined the binding requirements of the light-producing phage reporter to test if it is feasible to allow the detection method to be run while food samples are shipped from the producer to the testing laboratory. The success of these studies will minimize the likelihood of false-negative results and greatly improve the time-to-result for detection of STEC O157:H7, respectively. Specificity of the phage binding/infection was important for determining its effectiveness in array-based application. Currently we are working with our collaborator at Purdue University to fill a postdoctoral researcher vacancy with a predoctoral student who is also well-versed in the isolation, propagation, and manipulation of bacteriophage, 3B) a database for BARDOT scatter images was generated for Campylobacter species under various culture conditions that exploited the migratory/mobile-behavior of the bacteria, 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) generation of genomic sequence databases for important foodborne pathogens focused on furthering investigations with Campylobacter spp., not only for its virulence, but also antimicrobial resistance properties. A total of 36 Campylobacter strains were isolated from retail chicken and beef liver samples by passive membrane filtration and confirmed to be either C. jejuni or C. coli by a multiplex real-time PCR assay previously developed in our laboratory. Genomic DNAs of all the strains were purified and sequenced using Illumina�s Miseq. Some of the genomes were also sequenced via PacBio�s Single Molecule Real-Time technology. Substantial progress was made on whole genome assembly of, annotation of, and determination of single nucleotide polymorphisms in the derived sequences. Multilocus sequence typing analysis of the strains, which provides genetic basis for the species identification and genotyping of the pathogen, was also performed. Comparative analyses at genomic and phenotypic levels have yielded a better understanding of the molecular mechanisms of virulence and antimicrobial resistance of the organism. Accomplishments 01 A novel test for detection of a bacterial toxin in food. Production of Shiga toxin (Stx) is both an important virulence factor for the pathogenic bacterium, Escherichia coli, and a distinguishing feature routinely screened for in meat samples by the USDA�s Food Safety and Inspection Service. Under a collaborative agreement between Abraxis, LLC (Warrington, Pennsylvania) and a team of ARS researchers in Wyndmoor, Pennsylvania, a novel antibody-based test (amplified luminescent proximity homogeneous assay or AlphaLISA) was developed for the detection of Shiga toxin 2 (Stx2) generated by Stx-producing E. coli (STEC) in foods (Romaine lettuce, ground beef). Efficacy and sensitivity trials showed that not only was AlphaLISA as sensitive as the industry standard test (enzyme-linked immunosorbent assay or ELISA), but it also demonstrated a superior signal-to-noise ratio with the ability to distinguish high concentrations of the toxin. These features in combination with the reduced hands-on workflow and amenability for automation, make the AlphaLisa method a more economically viable choice for Stx2 detection in foods by both regulatory agencies and food testing labs alike. 02 A new method for rapidly detecting harmful bacteria in processed food. The ability to quickly test food in a production facility for the presence of harmful bacteria like Salmonella requires a very sensitive, simple to use, and inexpensive method. This method must be very sensitive as it cannot rely on culture-based growth enrichment which takes too long and is too risky because there would be the potential to grow up high amounts of bad bacteria right where the food is being made or processed. ARS researchers in Wyndmoor, Pennsylvania have developed a new method that meets the needed criteria by combining existing electrochemical detection techniques with innovative sample concentration technology. This novel method was demonstrated to readily detect extremely low concentrations (approx. 10 cells/mL) of two major pathogens (E. coli O157:H7 and Salmonella) in up to 1 liter of ground beef samples in approx. 2 hours. An invention disclosure that describes this biosensor platform has been approved by the USDA�s National Mechanical & Measurements Patent Committee for submission as a patent application to the United States Patent and Trademark Office. This low cost and simple to use, disposable test may be applied for the rapid detection of foodborne pathogens in the field by production and regulatory personnel alike. With very minor changes, this method may also be applied to the rapid detection of other regulated substances including drug residues, chemical contaminants, toxins, and viruses as well as other bacterial pathogens and DNA.
Impacts
(N/A)
Publications
- Gurtler, J., Doyle, M.P., Kornacki, J.L., Fratamico, P.M., Gehring, A.G., Paoli, G. 2017. Advantages of virulotyping foodborne pathogens over traditional identification and characterization methods. Foodborne Pathogens Virulence Factors and Host Susceptability. New York, NY: Springer Publishing. p. 3-40.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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. 03 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.
Impacts
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Publications
- Edlind, T., Brewster, J.D., Paoli, G. 2017. Enrichment, amplification, and sequence-based typing of Salmonella enterica and other foodborne pathogens. Journal of Food Protection. 80(1):15-24.
- Gehring, A.G., Fratamico, P.M., Lee, J., Ruth, L., He, X., He, Y., Paoli, G., Stanker, L.H., Rubio, F.M. 2017. Evaluation of ELISA tests specific for Shiga toxin 1 and 2 in food and water samples. Food Control. 77:145- 149.
- Kong, Q., Patfield, S.A., Skinner, C.B., Stanker, L.H., Gehring, A.G., Fratamico, P.M., Rubio, F., Qi, W., He, X. 2016. Validation of two new immunoassays for sensative detection of a broad range of shiga toxins. Austin Immunology. 1(2):1007.
- Ghatak, S., He, Y., Reed, S.A., Strobaugh Jr, T.P., Irwin, P.L. 2017. Whole genome sequencing and analysis of Campylobacter coli YH502 from retail chicken reveals a plasmid-borne type VI secretion system. Genomics Data. 11:128-131.
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