Objective 1: Development and validation of sample preparation methods for the detection of foodborne bacterial pathogens and toxins. Subobjective 1A: Generate, evaluate, and transfer a new class of magnetic materials for the effective partitioning and concentration of bacteria from large volume samples. Subobjective 1B: Adaptation of surface chemistry for the effective separation and concentration of pathogens from foods. Objective 2: Development and validation of rapid screening methods for foodborne bacterial pathogens and toxins, and identification of biomarkers. Subobjective 2A: Transfer methods to quantify foodborne pathogens. Subobjective 2B: Application of droplet digital PCR (ddPCR) to pathogen detection and quantitation. Subobjective 2C: Improve and expand the utility to aid in the transfer of the immunoelectrochemical biosensor technology for the detection of toxins and pathogens in food. Objective 3: Rapid identification, genotyping, and sequence analysis of foodborne bacterial pathogens. Subobjective 3A: Generate, evaluate, and transfer a novel AlphaLISA to confirm the presence of select foodborne pathogens. Subobjective 3B: Generate pathogen databases and improve the accuracy of the BEAM (formerly BActerial Rapid Detection using Optical scattering Technology or BARDOT) system. Subobjective 3C: Rapid identification and enumeration of both E. coli O157:H7 and Salmonella by MPN combined with multiplex qPCR. Subobjective 3D: Rapid identification of Campylobacter and Salmonella by target amplification and next generation sequencing using portable MinION sequencer. Subobjective 3E: Whole genome sequencing analysis of the phylogenesis, virulence factors and antimicrobial resistance of Campylobacter spp. from meat samples.
The primary goal of this plan is to develop rapid screening and identification methods for top, foodborne bacterial pathogens (Shiga toxin producing E. coli or STEC, Salmonella serotypes, L. monocytogenes, etc.). Testing for specific pathogens in select foods is sometimes an intermittent demand as gaps in methodology and needs may arise. However, the technology to be generated in this plan will proactively be suited for quick adaption to these needs typically only requiring, for example, substitution of a recognition element (e.g., antibody or DNA primer) or bioinformatics-based mining for unique stretches of DNA sequences. The detection of low levels of pathogens is complicated due to a gap in screening platform sensitivity, therefore we will increase sample volumes in order to elevate the amount of pathogens per test, especially when culture enrichment is not suitable (e.g., for rapid, field-based testing for very low concentrations of bacterial adulterants). To achieve this, novel sample preparation techniques will be key for rapid concentration of bacteria typically from aqueous homogenates. Subsequently, higher levels of detection sensitivity are expected as well as quantitation of extremely low levels (~1 cell/100 mL) of pathogens as needed for real-time testing. Assay times should be a few minutes to = 2 hours. Also, enhanced detection systems will be needed to bypass growth enrichment and achieve the desired detection levels. Furthermore, numerous biomarkers and the potential for false positive results using cross-reacting biorecognition elements (such as antibodies) will require multiplex detection techniques. However, for food contaminated with very low levels of target pathogens, detection may benefit from enrichment for accuracy thus avoiding false negative results. Therefore, conditions warranting brief enrichment prior to detection will be addressed. Methods will initially be developed with culture media or buffer as the sample matrix, and then extended to application with food (primarily ground meats). Assay performance of developed methods will be compared against “gold standard” methods initially with reliance on bacterial enumeration. Evntually, developed methods will be tested using FSIS samples in comparison to state-of-the-art methods. Yet the 5-year time frame for this plan may not allow for full scale, multi-laboratory validation of methods. Hence optimization of robust and reproducible technologies may better merit the time and financial investment associated with such validation. Eventually, testing will move to the field first off-line, then in-line (for some methods) in regulated environments. It is expected that multitudes of tests will be conducted given that most samples are negative for contamination by pathogens. Regulatory and perhaps legal guidance will be anticipated to be critical since validation testing will lead to remediation or recall if zero-tolerance organisms are detected or if certain instances of positive samples are discovered.
Progress was made on all three research objectives as well as those associated with the 12 month subobjective milestones that fell under National Program 108, Component I, Foodborne Contaminants by ARS researchers in Wyndmoor, Pennsylvania, under Project Plan 8072-42000-093-000D Development of Improved Detection, Quantification, and Characterization Technologies for Foodborne Pathogens. Objective 1: There were no milestones to be accounted for at the 12-month timeframe. Regardless, for Subobjective 1A “Generate, validate, and transfer a new class of magnetic materials for the effective partitioning and concentration of bacteria from large volume samples", research was conducted to apply technology related to USDA filed patent application 62/737,212 (USDA Docket No. 0068.18) to expand the capabilities to also capture and concentrate proteins and oligonucleotides in complex mixtures. A peer reviewed manuscript is being drafted to summarize the development and results that address the associated 24-month milestone. In addition, research for Subobjective 1B, “Adaptation of surface chemistry for the effective separation and concentration of pathogens from foods” was conducted to assess the impact of enzymatic treatment to improve sample preparation/pathogen detection on meat. The impact of enzymatic treatment was assessed using 6x6 drop plate culture, qPCR, and LAMP within the confines of the Microbiology Laboratory Guidebook (MLG) 8th edition (USDA Food Safety and Inspection Service (FSIS)). A peer review manuscript and corresponding invention disclosure was drafted to address the 24-month milestone. ARS scientists are working with OTT to draft a CRADA to continue the collaboration. The parties plan to submit grant applications to support subsequent research. A NIFA-AFRI will be submitted in collaboration with University of Virginia (Charlottesville, Virginia) and Drexel University (Philadelphia, Pennsylvania). A USDA-SBIR will be submitted in collaboration with a small business that has an interest in commercializing the developed technologies. Objective 2: For Subobjective 2B, “Application of droplet digital PCR (ddPCR) to pathogen detection and quantitation, research was conducted to evaluate sample collection using the MicroTally cloth in conjunction with the dd-Check STEC protocols within the context of the established MLG-5 protocol utilized by USDA FSIS. Samples collected via the MicroTally cloth were analyzed using both qPCR and dd-Check Shiga-Toxin producing Escherichia coli (STEC). Data obtained was shared with researchers at ARS MARC (Clay Center, Nebraska). Though a peer-reviewed publication was anticipated at 12 months, the data was not fully sufficient to complete an associated manuscript. In 2020, the ddPCR instrument was claimed by our collaborator and research cannot continue until a replacement instrument is obtained. In addition, for Subobjective 2C, “Improve and expand the utility to aid in the transfer of the immunoelectrochemical biosensor technology for the detection of toxins and pathogens in food,” research was conducted to detect Listeria monocytogenes using the flow-through electrochemical biosensor. Due to a lack of commercially available biorecognition elements that can differentiate Listeria monocytogenes from species which are not pathogenic to humans, the technique was modified to use oligonucleotides for specific capture and subsequent detection. The research advanced the technology, contributed to a peer-reviewed publication, and a patent application which satisfied the anticipated 12-month milestone. Objective 3: For Subobjective 3A, “Generate, validate evaluate, and transfer a novel AlphaLISA to confirm the presence of select foodborne pathogens,” research was conducted to extend the utility of the amplified luminescent proximity homogeneous linked immunosorbent assay (AlphaLISA) to the detection of genetic material for the identification of Listeria monocytogenes. A peer-reviewed manuscript is being drafted to transfer the technology to satisfy the 12-month milestone. For Subobjective 3C, “Rapid identification and enumeration of both E. coli O157:H7 and Salmonella by MPN combined with multiplex qPCR”, research was conducted to determine multiple gene targets, qPCR primers/probes, and the specificity and efficiency of the multiplex qPCR assay to meet the 12-month milestone. Additional investigation was conducted to include Listeria as well within contaminated foods. Subobjective 3D, “Rapid identification of Campylobacter and Salmonella by target amplification and next generation sequencing using portable MinION sequencer” saw considerable progress with sequencing simulation used to select the initial experimental parameters for gene of interest identification using MinION sequencing. DNA from samples was extracted, prepared in a library, and run on a MinION sequencer using both MinION and Flongle flow cells. Optimal parameters for MinKNOW, the sequencing software, were established. The MinION flow cells were observed to provide better quality data than Flongle flow cells. A relatively short run time (1 hour) was sufficient to identify all genes of interest. However, the number of reads generated by the sequencing run was found to be a better predictor of data output than run time due to variations in pore availability on the flow cells. It was determined that 400k reads provided enough data to identify genes of interest. Finally, for Subobjective 3E, “Whole genome sequencing analysis of the phylogenesis, virulence factors and antimicrobial resistance of Campylobacter spp. from meat samples,” the whole genomes of three antibacterial-resistant Campylobacter species were sequenced and uploaded to GenBank. Additional work to study the antimicrobial nature of these species was used to prepare a manuscript.
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Elders, J., Liu, Y., Kanrar, S., Gehring, A.G., Sommers, C.H., Johnston, B.D., Johnson, J.R. 2021. Complete genome sequence of Escherichia coli strain FEX669, a ColV plasmid-containing isolate from retail chicken meat. Microbiology Resource Announcements. 10(8):e01340. https://doi.org/10.1128/MRA.01340-20.
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