1a. Objectives (from AD-416):
1: Development and evaluation of technologies for sample preparation, detection (label-based and label-free), and characterization of microbial, chemical, and biological contaminants of concern in foods that can be implemented for improved food safety and food defense. 1A. Spectroscopy-based identification of foodborne pathogens, toxins, and chemical contaminants. 1B. Antibody-based detection of foodborne pathogens and toxins. 1C. DNA - based detection of foodborne pathogens. 1D. Phage-based detection of foodborne pathogens. 1E. Cell-based detection of foodborne pathogens and toxins. 1F. Enabling technologies for pathogen detection. 2: Application of CFSE developed technologies either alone or in combination with existing methods to evaluate microbial populations and the microbial ecology of foods during production and processing. 2A. Expand the databases for BARDOT and HESPI techniques using pure cultures of known microorganisms (foodborne pathogens and indicator microorganisms) 2B. Apply BARDOT and HESPI techniques to analyze microbial populations in foods
1b. Approach (from AD-416):
The food supply must be protected from pathogens, toxins, and chemical contamination that cause disease or illness in humans. Detection technologies are a critical component for identifying and controlling the potentially harmful food contaminants. The overarching goal of the Center for Food Safety Engineering (CFSE), working in collaboration with USDA-ARS scientists, is to develop, validate, and implement new technologies and systematic approaches for improving food safety. We propose to develop a variety of timely, accurate, and cost-effective technologies for the pre-screening, detection, characterization, and classification of foodborne hazards. Our prototype pre-screening and detection technologies include hyperspectral light scattering, metal-enhanced plasma spectroscopy, phage-based detectors, cell-based assays, antibody- and DNA-probe inkjet-printed test strips, plasmonic ELISA, and enhanced lateral flow immunosensors. The accompanying algorithms and software for data processing, analysis, and interpretation of colorimetric, fluorometric, light-intensity, light-scattering, and spectroscopy-based assays, along with time-temperature tracking devices, will enable and enhance these technologies. These methods will detect Listeria monocytogenes, Shiga toxin-producing Escherichia coli (STEC), Campylobacter jejuni, and Salmonella enterica serovars, with demonstrated applications in meat, poultry, and produce, as well as detect toxins, metals, and chemicals of concern in foods. An experienced multidisciplinary team of investigators from Purdue University, the University of Illinois, and USDA will produce and evaluate operational technologies, and engage stakeholders and industry, in an integrated effort to validate and implement technologies for better detection of foodborne hazards along the food production continuum.
3. Progress Report:
The Center for Food Safety Engineering continues to develop novel methods for the sampling and detection of pathogens and other organisms that are indicators of contamination with pathogens, new methods to characterize microbial communities associated with food, and develop sensor technologies and bacterial growth models that will allow environmental data to be used to predict food safety risk. Despite the complications that arose from the Covid-19 pandemic, all of our projects continue to move forward with the majority of our 48-month milestones being fully or substantially met. Development of our elastic light scattering (ELS – formerly Bardot and BEAM) technology continues with improvements to both hardware and software, as well as the development of methodologies for using the technology to characterize entire microbial communities. Hardware enhancements involve production and testing of a hyperspectral instrument (HESPI). This has been tested on pure cultures and used to develop new theoretical models of the relationship between light scattering and colony morphology. Work also continues on improved classification algorithms, driven in part by our efforts to develop methodologies that will allow the classification of entire microbial communities. We have worked to define new classification models which work with our existing (ELS, LIBS) and future systems (Raman, FTIR, HESPI) using methodologies that are described below. As a part of this project we have also extensively characterized both the bacterial and fungal microbial communities associated with romaine lettuce using ELS and DNA sequencing and the results are being made available online. A similar analysis of poultry samples is in progress with a strain collection that has been subjected to DNA sequence analysis and will be characterized by ELS. We also continue to develop cell phone-based technologies to be used in portable detection systems. We have developed a smartphone-based spectrometer that was validated by detection of protein concentrations from liquid samples and milk. We have also developed a cell phone linked fluorometer that has been validated with FITC fluorochrome. We were successful in designing and building a gold-standard LIBS instrument that was capable of metal-based detection and analysis of potential pathogens. The core of our progress was based on developing the assay component to target potential pathogens. This required the design and construction of a rapid assay platform and the design of the reagent complexes necessary for the successful detection of target molecules. We focused on lateral flow-based assays (LFA) because they are low cost, easy to transport and there is already a significant literature and commercial knowledge about the use of LFA tests. No previous work has been published using LFA testing with LIBS. We successfully designed and tested a LIBS-based LFA to detect the presence of E. coli from contaminated suspensions. In addition, we defined the limit of detection for four potential lanthanide elements using paper-based detection. We have also developed highly sensitive LFA sensors to detect pathogens in real food samples by just observing color changes with the naked eye. The technology is based on a lateral flow platform, but we utilize gold coated magnetic particles, so that the target pathogen captured by the particles can be concentrated at the signal generation zone of the sensor strip. The whole operation can be completed in 30-45 minutes and as low as 50-100 cfu/ml of pathogen can be detected. The sensor has been validated in fruit juices against E. coli O157:H7 and Salmonella spp. Recently we have extended our protocol to detect as few as 100 cfu/ml of pathogenic E. coli in blended lettuce samples in as much as 50 ml of sample volume in less than 60 minutes. Patterned microfluidic paper-based analytical devices (µ-PADS) have combined the well-known advantages of paper strips with the functionality and utility of microfluidics. This technology holds great potential for instrument-free, portable and multiplexed detection. To obtain quantitative measurements of food contaminant concentration, we developed an image analysis system to read the color response of the sensors by comparing different segmentation methods. We designed a pipeline for an image analysis system using a mobile phone camera to quantitatively correlate the response signal to target concentration, and also investigated variability of the response. Based on these results, we optimized our image analysis pipeline to produce a method which segments the test zones accurately and presents a quantitative estimate of color response in the test zones. Aptamers provide the high specificity for the target that antibodies offer, and at the same time are more robust towards environmental stressors and significantly cheaper. We were able to design aptasensors based on functionalization of gold nanoparticles with aptamers specific for mercury and arsenic and fabricated an effective multiplexed paper-based assay. We then tested the detection limit of µ-PADS, as well as reliability, repeatability, stability, and specificity. This work is, to the best of our knowledge, the first cell-phone integrated µ-PAD platform for multiplexed aptamer-based detection of analytical targets, which presents with a low standard deviation of analytical response and colorimetric signal quantification. We introduce the application of highly stable optical labels as a strategy for enhanced sensitivity of colorimetric detection. Finally, we present robust image analysis and processing, resulting in a limit of detection of 1 ppm for two targets, mercury and arsenic, and quantitative (analytical) response in an aqueous environment. Aptamers provide the high specificity for the target that antibodies offer, and at the same time are more robust towards environmental stressors and significantly cheaper. We were able to design aptasensors based on functionalization of gold nanoparticles with aptamers specific for mercury and arsenic and fabricated an effective multiplexed paper-based assay. We then tested the detection limit of µ-PADS, as well as reliability, repeatability, stability, and specificity. This work is, to the best of our knowledge, the first cell-phone integrated µ-PAD platform for multiplexed aptamer-based detection of analytical targets, which presents with a low standard deviation of analytical response and colorimetric signal quantification. We introduce the application of highly stable optical labels as a strategy for enhanced sensitivity of colorimetric detection. Finally, we present robust image analysis and processing, resulting in a limit of detection of 1 ppm for two targets, mercury and arsenic, and quantitative (analytical) response in an aqueous environment. We are also developing a plasmonic ELISA system (PES) that utilizes the growth of gold nanoparticles after the reduction of gold chloride solution with hydrogen peroxide to produce a visible color change that can be analyzed with the naked eye. This type of assay has been developed and tested for Salmonella enterica serovars and for Listeria monocytogenes. The PES assays developed have similar limits of detection to conventional ELISA assays, but are easier to score without the need for a spectrophotometer. We are also developing assays that are based on pathogen interactions with mammalian cell lines. We have developed Mammalian Cell-based ImmunoAssay (MaCIA) for the detection of viable Salmonella enterica serovar Enteritidis. In MaCIA, instead of a capture antibody, which is generally used in conventional ELISA, a mammalian cell line was used to capture bacteria. In MaCIA, natural adhesion ability (achieved by using virulence factors) of a pathogen can be exploited for the detection of the viable pathogenic organism from food. In this assay, a human intestinal cell line, HCT-8 was grown in monolayers in 24-well tissue culture plates and Salmonella at variable concentrations was added and incubated for 30 minutes. Anti-Salmonella antibody was added to interact with the captured bacteria and an enzyme-conjugated second antibody was used as a reporter. MaCIA could detect viable S. Enteritidis (1-10 CFU/25g) in ground chicken, shelled eggs, whole milk, and cake mix, using a traditional enrichment set up; however, the detection time was shortened to 10-12 h using direct on-cell (MaCIA) enrichment. Formalin-fixed cells in the MaCIA platform permits a longer shelf life (at least 14-weeks at 4°C), minimum on-site maintenance care, and a stable cell monolayer for point-of-need deployment. Furthermore, the 24-well plate configuration is also convenient for testing multiple samples in a single run thus reducing cost per sample testing. We have also developed an assay using HEK293 reporter cell line expressing TLR-5. Interaction of flagellar antigen with TLR-5 leads to the activation of NF-¿B, IL-8 and alkaline phosphatase which can be detected with an appropriate substrate to generate a blue color. Our preliminary results indicate that the assay is highly sensitive (102-103CFU/ml) to Salmonella enterica (top-20 Salmonella serovars have been tested). We are now optimizing conditions to improve assay specificity by using Salmonella specific immunomagnetic beads from inoculated poultry meats. During this project period we were able to identify key genetic parameters for the optimization of production of the PhiV10 based reporter phage platform in a nonpathogenic E. coli host. The expression of these genes in the E. coli lysogen will counteract the lytic cycle repressor allowing release of progeny phage. During this project period we also developed a protocol for the freeze drying the reporter phage to ensure long-term storage with minimal loss during drying. The developed protocol will allow phage to be integrated into detection assays and inclusion in dry media formulations.
1. Identification of fungal indicator species from romaine lettuce. A better understanding of the interactions between the organisms that make up the microbial community found on lettuce will help to understand how human pathogens become part of that community and what we can do to prevent it. ARS-funded scientists at the Center for Food Safety Engineering in West Lafayette, Indiana, have identified a single, abundant and ubiquitous species of yeast present on healthy lettuce samples. The fact that this ubiquitous yeast is previously undescribed indicates how limited our knowledge of the fungal community on lettuce is. Characterization of this yeast and its interactions will help determine how its presence changes in response to invasion by human pathogens and/or food spoilage microbes.
2. Development of an enhanced elastic light scattering instrument for bacterial identification. Current practice for identification of bacterial species relies on microbiological methods to obtain individual bacterial colonies on an agar plate. Because visual examination of the isolated bacterial colony is not sufficient to confirm the identity of bacteria, complex and time-consuming assays are required following the isolation procedures. ARS-funded scientists at the Center for Food Safety Engineering in West Lafayette, Indiana, have previously developed a single-color laser optic method to directly identify bacterial isolates on plates without the need for additional complex and time-consuming assays for bacterial identification. While the method has proved successful for many bacterial species, we have improved this technology through the use of a multi-color laser. The system was tested for the identification of bacterial species from lettuce samples and shown to provide higher accuracy using newly designed analysis methods. This system will help laboratory personnel and food safety engineers in government and industry sectors with fast and accurate identification of bacterial species.
3. Cellphone-based spectrometer. A major limitation of many food safety assays is the requirement to use expensive equipment that is found only in centralized laboratories. This requires samples obtained in the field to be sent to those labs rather than being analyzed on site. ARS-funded scientists at the Center for Food Safety Engineering in West Lafayette, Indiana, have developed a smartphone-based spectrometer that can resolve the visible range of spectrum in transmission mode and can be used to analyze many types of food safety assays. The overall cost of the spectrometer is only $200 and functions with an app that can visualize, record, and analyze the visible spectrum. This device could be incorporated into many types of assays with visual readouts to allow them to be utilized at the point the sample is taken, simplifying the assay process and reducing the time required to get a result.
4. Development of a novel detection assay for Salmonella based on binding to mammalian cells. Rapid detection of major foodborne pathogens is of paramount importance to ensure food safety. At present, nucleic acid and antibody-based assays are the methods of choice for rapid detection, but these are prone to interference from inhibitors and resident microbes and the assay formats may limit multisample testing in a single run. ARS-funded scientists at the Center for Food Safety Engineering in West Lafayette, Indiana, have developed a mammalian cell-based assay that detects pathogen interaction with mammalian host cells and is responsive to only live bacterial pathogen cells. The assay has been formulated to use stabilized cell monolayers to provide longer shelf-life for possible point-of-need deployment and multi-sample testing in a single run. This assay provides a highly specific alternative to conventional assays that could be utilized either in testing labs or at remote locations.
5. Wireless, high-resolution, time-temperature measurement using low-cost tags. Continuous temperature monitoring is essential for the estimation of pathogen growth in ready-to-eat foods. ARS-funded scientists at the Center for Food Safety Engineering in West Lafayette, Indiana, have developed a system that can be integrated in deli-cases and is capable of acquiring temperature measurements using low cost tags that can be attached to food packages. The system provides high-resolution temperature measurement that can be integrated into the “Internet of Things” (IoT) through Bluetooth communication capabilities. Integrating the system in retail deli-cases can enable real-time risk assessment of stored products with direct notification of the management when irregular storage conditions occur.
6. Development of paper-based assays for use with laser induced breakdown spectroscopy (LIBS). A major goal of food safety research is to provide rapid, sensitive, low cost assays that reliably detect human pathogens. ARS-funded scientists at the Center for Food Safety Engineering in West Lafayette, Indiana, have developed new paper-based assays that take advantage of a previously developed LIBS instrument and metal tagged antibodies and have demonstrated that they can be used to detect pathogenic E. coli. The use of tagged antibodies provides the potential for detecting multiple pathogens at once in a single assay. This demonstration is a proof of concept that will facilitate the development of commercial assays based on this technology that could be used by the food industry and food safety regulatory agencies.
7. Affordable, reliable, sensitive, and repeatable multiplexed aptasensors for the detection of heavy metals. Mercury and Arsenic have been recognized as chemical threats for human health because of their atmospheric transport, environmental persistence, bioaccumulation in living tissue and detrimental effects for human health even at extremely low concentration. ARS-funded scientists at the Center for Food Safety Engineering in West Lafayette, Indiana, designed and demonstrated a novel cell-phone integrated µ-PAD platform for rapid and reliable multiplexed aptamer-based detection of mercury and arsenic in pristine and real environmental samples. This is a new technology that makes µ PAD sensitivity and specificity enhancement and colorimetric data quantification possible and opens the door to commercialization of this low cost and flexible assay technology.
8. Quantitative colorimetric detection of target molecules with low response variability. Quantitative detection of the level of a biological or chemical food contaminant is often limited by the reliability of the assay system. Response variations in test areas of lateral flow assay strips to the same concentrations of target prevent reliable quantitation. ARS- funded scientists at the Center for Food Safety Engineering in West Lafayette, Indiana, developed of a robust image analysis system that provides rapid quantitative measurement of the µ-PADs based on the analysis of color composition. This device is fully integrated into a mobile phone to capture and analyze the sensor images on-site and the method can be customized for a wide variety of analytes including whole cell foodborne pathogens. This technology provides the basis for a portable and inexpensive system that could be deployed widely in the food industry and has the flexibility to detect a wide variety of food contaminants.
5. Record of Any Impact of Maximized Teleworking Requirement:
Many of our research laboratories were affected by COVID-19 related policies in effect at the universities that house the Center for Food Safety Engineering. The magnitude of these effects varied greatly from one laboratory to another since to what degree research was allowed to continue varied depending on whether the research was classified as “essential” or not. Rather than trying to generalize about how big the impact was, we have included individual statements from the faculty that felt their work was impacted by requirements to work remotely to the greatest degree possible. Applegate: The restrictions placed on research due to the Maximized Teleworking Requirement have thwarted research to some degree although our group was allowed to continue longitudinal studies. However, we have used this time to focus on preparing manuscripts on the completed work. Manuscripts on the freeze-drying protocol (submitted), H7 antigen specificity, and application of the reporter phage for detection of O157:H7 on lettuce are being prepared. Irudayaraj: We were not able to do any experiments. However, we focused our efforts on reviewing current literature in food safety and in methods for optimizing our technology. A possible extension of the technology to detect virus is also in development. Oliver, Peroulis, Mousoulis: The maximized teleworking requirement and research lab closures due to the Covid-19 pandemic have had a negative impact on the progress of improving our developed technology. Since the experiments required instrumentation and tools located at research labs, the focus of our efforts shifted to the development of analytical models, embedded systems programming, and the preparation of manuscripts. Planned experiments for the acquisition of performance metrics and system optimization, as well as the fabrication of tags using low-cost manufacturing, have been postponed until the reopening of the research labs. Similarly, growth curves with new sensors has been delayed due to lab closure. Pruitt: The maximized telework had both positive and negative impact. On one side, it slowed down the research activity which requires physical presence in the lab, access to buildings, facilities and equipment that were closed during the stay-at-home order, as well as the delay of processing purchase orders to get reagents and laboratory supplies. On the other hand, that research slowdown helped to finish some writing activities (papers, reports, data organization and analysis) which always are put aside due to the time being mainly consumed by the research activity. Singh: Planned experiments for the confirmation of Salmonella and validation of the BARDOT have been postponed due to an early spring shutdown at UGA. This has delayed our plans for the next steps of shipping isolates to Purdue University for validation of the rapid detection technologies. Stanciu, Chiu, Allebach, Deering: The abrupt closure of the laboratories affected the transition between one graduating student (Susana Diaz Amaya) and the new student assigned to the project. At the same time, in the Spring Semester, we had planned to implement strategies to move from the multiplexed detection of heavy metals on paper platforms to foodborne pathogens (E. Coli O157:H7 and Listeria Monocytogenes). The laboratory work has been halted in this response and progress has been slowed. However, the time has been used to prepare and submit a manuscript on the results on multiplexed detection of heavy metals. The manuscript has been submitted to ACS Materials and Interfaces in May 2020 and it is currently under revisions. A second manuscript is in preparation, on the electrochemical detection of heavy metals.
Bhunia, A.K. 2019. Microbes as a tool to defend against antibiotic resistance in food animal production. Indian Journal of Animal Sciences. 58(2). https://doi.org/10.36062/ijah.58.2SPL.2019.01-18.
Doh, J., Gondhalekar, C., Patsekin, V., Rajwa, B., Hernandez, K., Bae, E., Robinson, J.P. 2019. A portable spark-induced breakdown spectroscopic (SIBS) instrument and its analytical performance. Applied Spectroscopy. 73(6):698-708. https://doi.org/10.1177/0003702819844792.
Gondhalekar, C., Biela, E., Rajwa, B.B., Patsekin, V., Sturgis, J., Reynolds, C., Doh, I.J., Diwakar, P., Stanker, L., Zorba, V., Mao, X., Russo, R., Robinson, J.P. 2020. Detection of E. coli labeled with metal-conjugated antibodies using lateral-flow assay and laser-induced breakdown spectroscopy. Analytical and Bioanalytical Chemistry. 412:1291-1301. https://doi.org/10.1007/s00216-019-02347-3.
Mathipa, M.G., Thantsha, M.S., Bhunia, A.K. 2019. Lactobacillus casei expressing internalins A and B reduces Listeria monocytogenes interaction with Caco-2 cells in vitro. Microbial Biotechnology. 12(4):715-729.
Rodriquez-Lorenzo, L., Garrido-Maestu, A., Bhunia, J.K., Espina, B., Prado, M., Diéguez, L., Abaldecela, S. 2019. Gold nanostars for the detection of foodborne pathogens via surface-enhanced raman scattering combined with microfluidics. ACS Nano. 2(10):6081-6086. https://doi.org/10.1021/acsanm.9b01223.