Research Project: Detection, Quantification and Characterization Technologies for Foodborne Pathogens

Location: Characterization and Interventions for Foodborne Pathogens

2023 Annual Report

Objectives
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.

Approach
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 Report
Progress was made on all three research objectives as well as those associated with the 24 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 Detection Quantification and Characerization Technologies for Foodborne Pathogens. Objective 1: 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,” a peer reviewed manuscript is currently under review which compares the capture rates of Escherichia coli by the newly developed magnetic particles (USDA filed patent application 62/737,212) and the commercially available superparamagnetic particles. It also summarizes the compatibility of the new magnetic materials to be used in conjunction with a non-PCR based approach for detection, namely the detection of the enhanced green fluorescent protein (EGFP) through fluorescent measurements. 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 and an Accomplishment statement was generated. Objective 2: For Subobjective 2A, “Transfer methods to quantify foodborne pathogens,” research is underway for the development of a method to quantify Salmonella in contaminated raw frozen, breaded chicken products at low (0.1-1.0 CFU/g) levels. No product was anticipated for 24 month milestone but a manuscript is being drafted in FY24. There was no milestone for Subobjective 2B, “Application of droplet digital PCR (ddPCR) to pathogen detection and quantitation,” however research conducted with the ddPCR demonstrated the ability to detect Salmonella from a poultry matrix. It was demonstrated that use of ddPCR with whole cells was more sensitive than via the use of extracted DNA. 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,” a prototype housing device was constructed to allow temperate regulation of the flow-through electrochemical-based biosensor. A flow-through electrochemical-based biosensor composed of alternative transducer materials was also produced and an invention disclosure was submitted concerning the technology (Invention disclosure 38.23). Objective 3: For Subobjective 3A, “Generate, validate evaluate, and transfer a novel AlphaLISA to confirm the presence of select foodborne pathogens,” both internal and external databases were mined and 17 putative targets were identified for the differentiation of Campylobacter (C.) coli, C. jejuni and C. lari using a newly developed bioinformatic pipeline. Testing is beginning to determine the validity of the targets. A manuscript is expected to be submitted in FY23 concerning the detection of genetic material for the identification of Listeria monocytogenes. In addition, a CRADA was initiated (agreement No. 58-8072-2-006) for the use of AI driven optical sensors for the detection of foodborne pathogens with engineered nanoparticles. For Subobjective 3B, “Generate pathogen databases and improve the accuracy of the BEAM (formerly BActerial Rapid Detection using Optical scattering Technology or BARDOT) system”, considerable progress was made towards the identification of Salmonella. Twelve serotypes of Salmonella, including Typhimurium, Infantis, Newport, and Enteritidis (per high priority need of FSIS) were included in new optical scatter database generation. In addition to improving robotic interface between culture chamber/incubator and the BEAM platform, a grid arraying flow system was introduced to afford better bacterial CFU separation hence greatly improved discrimination of as well as minimization of “cross-talk” between bacterial colonies. Finally, enhancements to analysis software were made for improved identification. For Subjective 3.C, “Rapid identification and enumeration of both Escherichia coli (E. coli) O157:H7 and Salmonella by most probable number (MPN) combined with multiplex qPCR”, multiple detection technologies were evaluated in an MPN assay, and it was identified that a LAMP-based protocol was the most time efficient and sensitive approach for Salmonella. Currently efforts are being directed toward applying the technique to E. coli so that multiplexed detection with Salmonella may be achieved. For Subobjective 3D, “Rapid identification of Campylobacter and Salmonella by target amplification and next generation sequencing using portable MinION sequencer,” gene targets were selected that indicate the presence of specific pathogens down to the serotype level. Library preparation and sequencing on the Mk1b and Mk1c MinION platforms were optimized. A 3 hour run time is sufficient to reliably produce data for 30x genome coverage. Finally, excellent progress was made towards Subobjective 3.E, “Whole genome sequencing analysis of the phylogenesis, virulence factors and antimicrobial resistance of Campylobacter spp. from meat samples.” Campylobacter is one of the most common foodborne pathogens often found in poultry meat and livers with most cases associated with C. jejuni and fewer cases by C. coli. So, it is important to develop methods for detecting the pathogen at the species-level; this sequencing-based research revealed antibiotic resistance and virulence determinants for the organism which will assist regulators or epidemiologists with tracing its transmission source.

Accomplishments
1. Sample preparation for improved pathogen detection capabilities. Improvements in pathogen detection not only empower food producers to conduct more comprehensive assessments of their processing facilities, but also provide much needed support to regulatory agencies for the implementation of evidence-based policies concerning food safety standards. Therefore, ARS researchers in Wyndmoor, Pennsylvania, and their collaborators have advanced food safety by developing an approach that utilizes an innovative enzymatic treatment to dislodge and disperse pathogens resulting in improved detection capabilities within current pathogen detection protocols. To facilitate the adoption of this method, a patent application has been filed in collaboration with a commercial partner, in an effort to scale up production, distribute the enzyme, and facilitate its practical implementation across the food industry. This platform technology has the potential to revolutionize pathogen detection, strengthen food safety protocols, and ultimately ensure the protection of public health.

Review Publications
Felton, S., Armstrong, C.M., Chen, C., He, Y., Lee, J., Reed, S.A., Akula, N., Walker, S., Berger, B., Capobianco Jr, J.A. 2022. Enhancing detection of Listeria monocytogenes in food products using an enzyme. Food Control. https://doi.org/10.1016/j.foodcont.2022.109445.
He, Y., Reed, S.A., Gunther, N.W., Armstrong, C.M., Capobianco Jr, J.A. 2023. Complete genome sequence of Campylobacter jejuni BSD5, a multidrug-resistant isolate from a poultry processing facility in the United States. Microbiology Resource Announcements. 12(6). https://doi.org/10.1128/mra.00284-23.
Dong, R., Liang, Y., He, S., Cui, Y., Shi, C., He, Y., Shi, X. 2022. DsrA modulates central carbon metabolism and redox balance by directly repressing pflB expression in Salmonella Typhimurium. Microbiology Spectrum. 10(1):1. Article e01522-21. https://doi.org/10.1128%2Fspectrum.01522-21.