Progress 07/01/23 to 02/29/24

Outputs
Target Audience:The target audience of our SalmonellaEnteritidis (SE) andE. coliO157:H7 (EC O157:H7)rapid test kits (RTKs) include food producers such as farmers and processing plants, food distributors, and regulatory authorities. Food producers, such as beef grinders (SE RTK) and/or poultry farm (EC O157:H7 RTK), would benefit from the rapid and on-site RTKs, which enable early diagnosis, medical intervention, and preventive measures. Although culturing method is the confirmatory method used by the Food Safety and Inspection Service(FSIS) Lab, the food industry has a need for early, rapid, on-site and accurate results. RTKs can provide rapid and convenient testing for surfaces, equipment and raw materials at different stages of production and processing (start, middle, and end). Samples can be sent for confirmatory tests if any RTK screening results indicate presumptive positive cases. Regulatory authorities provide food supply and safety regulatory oversight via testing/monitoring of food consignments to ensure food safety. If rapid and accurate test kits are available, it would help the authorities to avoid unnecessary consignment delays due to the conventional long culturing methods and any false-positive results. Our target audience will further benefit from our RTKs viademonstration of our RTKs, conducting on-site trials, and in-depth discussionsto determine if and how our RTKs can fit into their production/manufacturing and regulatory/inspection workflow. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Professional development (individual study) Through performing the research goals of this grant, the PI gained more skills and knowledge with regards to culturing different bacteria strains, safety handling, and their storage. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Conventional foodborne pathogen detection techniques, such as culturingmethods or other lababoratory techniques such as polymerase chain reaction (PCR), which take couple of days or require trained personnel and expensive instrumentation. Using our enzymatic-amplified rapid test kits (RTKs) immobilized with selected antibody pairs, foodborne pathogen detection can be greatly accelerated in a sensitive and specific manner. With our RTKs, the enrichment duration could be shortened to a few hours (vs. overnight culturing), and then followed by a 15-minute runtime on the RTK. This will greatly benefit food producers and farmers, enabling rapid on-site detection at various stages and early diagnosis, medical intervention, and preventive measures to prevent losses. Regulatory authorities can also clear food consignment quickly, and prevent delaying food items from getting onto market shelves. Achieving major goals of the project Heat-killedE. coliO157:H7 (EC) andSalmonellaEnteritidis were spiked into food matrices, i.e. beef, chicken, and pork meat, at different concentrations, and then tested on the RTKs to determine the limit of detection (LOD) of the respective bacteria targets, without culturing. The SE RTK was able to detect as low as103CFU/mL of SE in chicken and pork matrices (not tested in beef), while theEC RTK was also able to detect as low as 103CFU/mL of EC O157:H7 in chicken, pork, and beef matrices. As a comparison with existing commercial RTK, the same experiment was conducted on Neogen's "Reveal® for E. coli O157:H7" and Romer Lab's "RapidChek® SELECT™ Salmonella Enteritidis"RTK. Both the Neogen's EC RTK wasonly able to detect as low as104CFU/mL of spiked EC O157:H7, while Romer Lab's SE RTK was only able to detect as low as 106CFU/mL of spiked SE. This demonstrates that our EC and SE RTKs are 10 and 100 times more sensitive as compared to existing EC O157:H7 and SE commercial kits, respectively. This will enable our kit to work with less bacteria culture/enrichment time, leading to reduced turnaround time in detection workflows of food producers/manufacturers, farmers, and regulatory authorities. To determine the sensitivity and specificity of ourEC O157:H7 and SE RTKs, theEC O157:H7 RTK was challenged with the targetEC O157:H7 strain (20 samples) and 117 strains (148 samples) of non-target organisms; whereas the SE RTK was challenged with targetSalmonellaGroup D1 (18 strains, includingSalmonellaEnteritidis) from 29 samples, and53 strains(63 samples) of non-target organisms. Our RTKs achieved excellent sensitivity and specificity as summarized in the table below. SE EC O157:H7 Sensitivity 96.6% 28/29 100% 20/20 Specificity 100% 63/63 99.3% 147/148 RTKs should be stable at room temperature (RT, 30°C) for at least 2 years to enable easy storage for on-site applications. To determine the stability of our RTK, we conducted an accelerated stability test, in which we stored a sufficient amount of RTKs at 60°C for 31 days to simulate 2 years storage at RT. Acrosssix time points, the RTKs were retrieved from storage and challenged withheat-killed target bacteria to determine if the RTKs' detection capability is affected. The RTKs were challenged with low levels of target bacteria, i.e. at their limit of detection of 103CFU/mL, and 5 replicates were performed. At Day 31 of storage at 60°C, both RTKs were still able to detect their target organisms at their LOD level (results summarized below), indicating that our RTKs are stable for at least 2 years at RT. Days at 60°C Detected at 103 CFU/mL 0 Yes 5/5 4 Yes 5/5 7 Yes 5/5 15 Yes 5/5 21 Yes 5/5 31 Yes 5/5 Finally we determined if we are able to shorten the bacteria culturing time required for target detection (vs. overnight culturing of 16 to 20 hours). A low (10 CFU/mL) and medium (100 CFU/mL) load of bacteria (both EC O157:H7 and SE, 5 strains each) were cultured for 6 and 8 hours, and then tested on our RTKs for detection. Most (8/10) of the medium load targets can be detected after a 6-hour culture, while most (8/10) low load and all (10/10) medium load targets could be detected after an 8-hour culture. The reduced culturing time as compared to overnight culturing, willgreatly benefit food producers and farmers, enabling rapid on-site detection at various stages and early diagnosis, medical intervention, and preventive measures to prevent losses. Regulatory authorities can also clear food consignment quickly, and prevent delaying food items from getting onto market shelves. 6 hours 6 hours 8 hours 8 hours Organism ID No. 10 CFU/mL 100 CFU/mL 10 CFU/mL 100 CFU/mL 1 E. coli O157:H7 ATCC 35150 No Yes Yes Yes 2 E. coli O157:H7 EC1/14 No Yes Yes Yes 3 E. coli O157:H7 EC2/14 No Yes Yes Yes 4 E. coli O157:H7 EC3/14 No Yes Yes Yes 5 E. coli O157:H7 EC1/15 No Yes Yes Yes 6 S. Enteritidis ATCC 13076 No No No Yes 7 S. Enteritidis ATCC 4931 No Yes Yes Yes 8 S. Enteritidis ATCC 31194 No No No Yes 9 S. Enteritidis S53/20 No Yes Yes Yes 10 S. Enteritidis S44/20 No Yes Yes Yes

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