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

Location: Characterization and Interventions for Foodborne Pathogens

2025 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
Development of an amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA)capable of differentiating members within the Campylobacter genus is underway. Using a variation of the AlphaLISA, known as the oligo-Alpha and devised by ARS researchers at Wyndmoor, Pennsylvania, nucleic acids from Campylobacter can now be detected with a simpler “no wash” alternative to the commonly employed enzyme-linked immunosorbent assay. This bead-based approach has been formulated to discriminate C. coli from C. jejuni, the two main species associated with human illness, in foods. and could be extended in the future to C. lari when AlphaPLEX 645 beads become commercially available. Experiments were conducted to determine the efficacy of long-read sequencing to detect Salmonella Typhimurium during the earliest stages of cell attachment to food-contact surfaces. Sequencing methods could detect S. Typhimurium after one hour of incubation on a surface as compared to three hours of incubation using culture- based methods. Salmonella are able to form biofilms that some sanitizers are not able to penetrate completely. Earlier detection of foodborne pathogen contamination on surfaces in food processing facilities will allow appropriate remediation methods to be applied sooner to kill the bacteria before biofilm formation occurs. Additional experiments were conducted based on the previously described study to further investigate biofilm formation in Salmonella on food-contact surfaces. The temperature, food-contact surface material, and serovar all influenced biofilm formation. Single nucleotide polymorphisms were identified in the curli genes that may have contributed to stronger biofilm formation by S. Montevideo and S. St. Paul and weaker biofilms in S. Senftenberg. Detection of these gene variants can indicate the presence of more virulent strains in a product or on equipment and guide proper remediation. A peer-reviewed journal article was published on experiments demonstrating that sequencing is capable of detecting only live bacteria. Treatment of live or dead Escherichia coli O157:H7 cultures with the intercalating dye PMAxx prior to DNA extraction prevented DNA from dead bacteria from being sequenced. Detection of only live bacteria overcomes a major disadvantage of sequencing as compared to culture-based methods.

Accomplishments
1. Detection of only live bacteria with long-read sequencing. The use of sequencing for foodborne pathogen testing is advantageous because bacteria can be identified at the strain level, virulence genes of interest can be detected, and antibiotic resistance genes monitored. However, a major disadvantage has been that DNA from both live and dead bacteria are sequenced, but only live bacteria are of concern because they can infect and cause disease. ARS researchers at Wyndmoor, Pennsylvania, pre-treated Escherichia coli O157:H7 with PMAxx, which resulted in the dye penetrating the permeable membrane of dead bacteria and intercalating the DNA, while live bacteria were unaffected. The DNA from dead E. coli were not sequenced because the intercalated DNA strands could not separate to go through the sequencing pores. Incorporating PMAxx into sequencing methods overcomes a major challenge to using sequencing for foodborne pathogen testing. When optimized, this method may be used by producers and regulators to directly sequence samples to test for live foodborne pathogens. Results will be obtained within 24 hours, drastically reducing the time needed for testing. This will allow food to be released to the market more quickly, reducing spoilage and financial loss, while protecting the consumer from illness.

2. Rapid, quantitative protocol for detecting Salmonella in raw and ready-to-cook poultry using a modified most probable number method coupled with multiplex real-time PCR. Traditional Food Safety and Inspection Service -approved protocols rely solely on qualitative screening for prevalence and offer no insight into contamination levels, limiting their utility for public health risk assessment. To address this, ARS researchers at Wyndmoor, Pennsylvania, integrated quantitative modified most probable number (MPN) enumeration with molecular detection to estimate viable Salmonella levels within the same 24-hour enrichment timeframe required by current regulatory methods. The protocol demonstrated strong correlation to theoretical inoculum levels and achieved reliable quantification down to approximately 30 cells/g. It also outperformed traditional plating in precision, with reduced variability and higher consistency across replicates. The method was validated on both fresh ground chicken and complex ready-to-cook products such as chicken cordon bleu, demonstrating adaptability across food matrices. To support technology transfer and broad adoption, the protocol was published in a peer-reviewed video format that provides step-by-step guidance for implementation by industry and regulatory laboratories. This work delivers a scalable, field-ready solution that closes the gap between qualitative screening and quantitative risk-based decision-making in poultry safety programs thus reducing hold times (for testing) and therefore shortening delivery time to market which positively impacts both poultry producers and associated regulators.

3. Enrichment-free protocol for rapid detection and quantification of foodborne pathogens in food matrices. Current methods used by regulatory and commercial laboratories require 15 to 24 hours of enrichment and separate polymerase chain reaction assays for each pathogen, extending time to results beyond 48 hours and introducing variability that complicates quantification. To address these limitations, a unified protocol was established by ARS researchers at Wyndmoor, Pennsylvania, to isolate, purify, and concentrate target bacteria in under 3 hours. The extraction is compatible with molecular and microbiological assays, enabling simultaneous detection of Salmonella, Listeria monocytogenes, Campylobacter, and E. coli in a single workflow. Quantification achieved a strong correlation to theoretical levels with a lower limit of quantification below 1 cell/g, meeting Food Safety and Inspection Service risk assessment criteria for regulatory use. Compared to the Most Probable Number method, this protocol more than doubles quantitative accuracy and reduces time-to-result by over 90 percent. Between reduced labor and reagent usage, a facility processing 1,000 tests per week, could save more than $500k/year while delivering same-shift, quantitative results that improve decision-making. These improvements promise to benefit food producers and regulators alike.

4. Characterization of 34 new plasmids from 43 Campylobacter jeuni and C. coli strains isolated from retail meat using long-read and short-read genome sequencing and hybrid assembling. ARS researchers at Wyndmoor, Pennsylvania, conducted phylogenetic analysis of the relatedness and diversity among isolated Campylobacter plasmids that revealed five distinct groups, including “pTet” providing tetracycline resistance and “pVir”, a group associated with virulence. These plasmids, particularly the large plasmids, carry multiple genes important for plasmid transfer virulence, antibiotic resistance, and/or the persistence of Campylobacter. They serve as the main vectors for transferring genetic material and spreading resistance and virulence between bacteria. Pan-genomic analysis identified the core and accessory genes in each group, indicating a high degree of genetic similarity within groups and substantial diversity between the groups. The identification and comprehensive genetic characterization of new plasmids from Campylobacter food isolates contributes to understanding the mechanisms of gene transfer, particularly the spread of genetic determinants of virulence and antibiotic resistance by this important pathogen. The advancement of knowledge by this research will benefit the poultry production industry as well as associated regulators.

Review Publications
Harper, S.A., Counihan, K.L., Kanrar, S., Paoli, G., Tilman, S.M., Gehring, A.G. 2024. Investigating the quantification capabilities of a nanopore-based sequencing platform for food safety application via external standards of lambda DNA and lambda spiked beef. Foods. https://doi.org/10.3390/foods13203304.
Rao, P., Huang, S., Armstrong, C.M., Capobianco Jr, J.A., Duan, Y., Shih, W., Shih, W. 2025. Rapid and accurate detection of huanglongbing in citrus by elasticity testing using a piezoelectric finger. Analytical Methods. https://doi.org/10.1039/d4ay01952k.
Bermudez-Aguirre, L.D., Tilman, S.M., Niemira, B.A., Counihan, K.L., Uknalis, J. 2025. Rapid detection of Salmonella Typhimurium in egg biofilms on three different surfaces using long-read sequencing. Microorganisms. https://doi.org/10.3390/.
He, Y., Dykes, G.E., Kanrar, S., Liu, Y., Gunther, N.W., Counihan, K.L., Lee, J., Capobianco Jr, J.A. 2025. Comparative genomic analysis of food-originated plasmids identified in Campylobacter jejuni and C. coli. Microorganisms. https://doi.org/10.3390/microorganisms13010206.
Armstrong, C.M., Chen, C., Xie, Y., Atencia, J., Pierre, S., He, Y., Lee, J., Dykes, G.E., Johnson, K.M., Froment, B., Martinos, S., Capobianco Jr, J.A. 2025. Quantification of Salmonella in raw poultry using droplet digital PCR with a whole cell, enrichment-free approach. Journal of Food Protection. https://doi.org/10.1016/j.jfp.2025.100498.
Capobianco Jr, J.A., Armstrong, C.M., Chen, C., He, Y., Counihan, K.L., Lee, J., Dykes, G.E., Johnson, K.M., Tilman, S.M., Koppenhofer, H.L., Atencia, J., Martinos, S. 2025. Multiplexed detection of Salmonella, Escherichia coli, Campylobacter and Listeria in raw poultry. Foods. https://doi.org/10.3390/foods14071137.
Counihan, K.L., Tilman, S.M., Chen, C., He, Y. 2025. Detection of live Escherichia coli with long-read sequencing. International Journal of Molecular Sciences. https://doi.org/10.3390/ijms26052228.
He, Y., Capobianco Jr, J.A., Dykes, G.E., Armstrong, C.M., Chen, C., Counihan, K.L., Lee, J., Reed, S.A., Tilman, S.M. 2025. A modified most probable number (MPN) assay to quantify Salmonella in raw and ready to cook chicken products. Journal of Visualized Experiments. Article e67910. https://doi.org/10.3791/67910.
Dykes, G., He, Y., Jin, Z.T., Fan, X., Lee, J., Reed, S., Capobianco Jr, J.A. 2025. Transcriptomic analysis of Campylobacter jejuni following exposure to gaseous chlorine dioxide reveals an oxidative stress response. International Journal of Molecular Sciences. https://doi.org/10.3390/ijms26073254.
Jin, Z.T., He, Y., Fan, X., Capobianco Jr, J.A. 2025. In-package release of gaseous chlorine dioxide for improving the safety and shelf-life of grape tomatoes. Food Control. https://doi.org/10.1016/j.foodcont.2025.111384.
Renye Jr, J.A., Chen, C., Miller, A.L., Lee, J., Oest, A.M., Lynn, K., Felton, S., Guragain, M., Tomasula, M.M., Berger, B., Capobianco Jr, J.A. 2025. Integrating bacteriocins and biofilm-degrading enzymes to eliminate L. monocytogenes persistence. International Journal of Molecular Sciences. https://doi.org/10.3390/ijms26010399.
Patch, C., Larsen, K., Armstrong, C.M., Kanrar, S., Michaelides, A., Chakraborty, P., Harper, K., Devlin, V., Martin, L., Lunna, A., Blackwell, H., Nguyen, S.C., Penny, A., Etter, A. 2025. Prevalence, risk factors, and human health implications of Salmonella enterica and Campylobacter spp. in Vermont backyard poultry. Zoonoses and Public Health. http://doi.org/10.1111/zph.70004.