Recipient Organization
UNIV OF TULSA
800 S TUCKER DRIVE
TULSA,OK 74104
Performing Department
(N/A)
Non Technical Summary
Foodborne illnesses impact the lives of millions worldwide. Retail meat is known to harbor several bacterial foodborne pathogens. The rapid detection of foodborne pathogens in meat and poultry products is vital to food safety. Developing an assay to detect multiple pathogens simultaneously in a food sample is needed. The primary objective of this two-year seed grant is to develop, optimize, and evaluate an advanced sequencing assay for the rapid detection of Campylobacter, Salmonella, Listeria, and STEC in retail meats using the pocket-sized Oxford Nanopore Technology platform. We believe that this is possibly the best solution for detecting microbes in the field due to the reduced times needed for assays, lower costs, and the convenient data analysis platform adapted for this portable device. This newly developed sequence-based method will accurately and simultaneously identify multiple foodborne pathogens in meat samples in a single sequencing run, which will shorten the length of time needed for detection. This grant addresses three priorities of the Food Safety and Defense program regarding the detection of foodborne pathogens in food. The long-term goal would be to eliminate the need for conventional culture methods and provide a rapid economical assay for detecting foodborne pathogens in various food products, which in turn will enhance food safety and reduce health risks for the consumers.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
The major goal of this project is to help develop a sequence-based rapid assay to simultaneously detect several foodborne pathogens if present in a retail meat sample. The development and validation of rapid detection assays using ONT sequencing will facilitate the rapid, cost-effective identification of foodborne pathogens without further culturing or serotyping. To achieve this major goal, this project proposal has one specific objective which is to develop, optimize, and evaluate ONT Flongle sequencing assays for the rapid detection of Campylobacter, Salmonella, Listeria, and STEC in retail meats. The long-term goal would be to eliminate the need for conventional culture methods and provide a rapid assay for detecting foodborne pathogens.
Project Methods
Briefly, ONT Flongle sequencing will be used to detect Campylobacter (5 species), E. coli O157:H7, Salmonella enteritidis, and L. monocytogenes in spiked, sterilized chicken rinsates after selective enrichment for 0, 6, 12, and 24 h. Validation of optimal enrichment times will be confirmed by repeating the optimization procedure with 10 additional chicken rinsates containing natural microbial flora. Bacteria will be isolated by enrichment and selective media as recommended in FSIS MLG protocols and confirmed by multiplex PCR. After optimization and validation of protocols for detection, 200 whole chicken carcasses retrieved from a poultry processing plant will be screened for foodborne pathogens using an optimized ONT Flongle sequencing assay. Approximately 250 samples will be used for ONT library preparation and analysis. Thirty-six samples will be used for optimization of the assay with spiked, sterilized chicken rinsate; these will include three dilutions (1000, 100 and 10 CFU per 30 ml), four incubation times (0, 6, 12, and 24 h) and three biological replicates (n=36 total). Ten samples of chicken rinsate with natural microbial flora will be used to validate the assay using spiked preparations. Naturally-contaminated chicken rinsates (n=200) will be used in screening assays, and six negative controls containing filter-sterilized chicken rinsates will be included.Experiments will be conducted to identify the minimal enrichment time and detection limits for selected foodborne pathogens by ONT Flongle sequencing. Sterile chicken rinsates will be spiked with bacterial pathogens at three different inoculum concentration, and ONT sequencing will be conducted with samples collected after enrichment for 0, 6, 12, and 24 h. C. jejuni ATCC 33560, C. coli ATCC 43478, C. lari RM 2100 (ATCC BAA-1060), C. upsaliensis ATCC 43954, C. fetus ATCC 19438, E. coli O157:H7 (ATCC 43895), S. enteritidis ATCC 13076, and L. monocytogenes ATCC 19115 will be used in these experiments.Sterile chicken rinsates will be spiked with dilutions of Campylobacter spp. (C. jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus), E. coli, Salmonella, and Listeria at concentrations of 1000, 100, and 10 CFU/30 ml for each strain. Three replicates will be included. Meanwhile, 100 ml of spiked chicken rinsate (0 h time point) will be inoculated to Campy-Cefex (Campylobacter, 42oC, microaerobic), modified Rainbow agar (E. coli, 37oC, aerobic), Brilliant Green Sulfa agar (Salmonella, 37oC, aerobic), and Modified Oxford agar (Listeria, 35oC, aerobic) to determine initial inoculum concentrations. Individual, spiked rinsates will be equally distributed in three replicate sets (30 ml each) for enrichment, which will be conducted using FSIS MLG 41.07 (Campylobacter), FSIS MLG 4.11 (Salmonella), FSIS MLG 5C.02 (STEC), and FSIS MLG 8.13 (Listeria).DNA will be extracted with the HostZERO Microbial DNA Kit (HZMD) (Zymo Research) according to manufacturer's instructions. The HZMD kit depletes contaminating host DNA in samples and enriches DNA from viable microbial cells. Extracted DNA will be immediately used for ONT library preparation and any remaining DNA will be stored at -70oC until needed.After DNA extraction of each sample, library preparation will be carried out with the Native Barcoding Kit 96 (Q20+; SQK-NBD112.96) as recommended (Oxford Nanopore). A maximum of four barcoded samples will be included together in one library preparation. Libraries will be quantified and then loaded to Flongle flow cells (R9.4.1, FLO-FLG001) for sequencing with the MinION platform. Flow cell priming will be executed with priming mix via the priming port and library samples (75 ml) will be added in a dropwise manner to the flow cell via the SpotON sample port. Sequencing will be initiated with MinKNOWN software, and real-time base calling and data analysis will be conducted with the EPI2ME 'What's in my pot' (WIMP) pipeline. The minimum abundance cutoff for species-level identification will be set to 3% with default settings for other parameters. Further analysis of sequences will be conducted with the latest versions of Bonito, Guppy, MG-RAST, MEGAN and Kraken2.For molecular confirmation of bacteria isolated from selective media, colonies will be suspended in 40 µL of single cell lysing buffer as described previously. Cells will be lysed by heating at 80oC for 10 min followed by cooling at 55oC for 10 min. Solutions will be diluted (1:2) in nuclease-free water, centrifuged for 30 s at 5000 X g to remove cellular debris, and supernatants will be used in PCR assays for identification. Molecular confirmation will be done for each pathogen as described previously.The detection limit (10, 100, or 1000 CFU/30 ml) will be determined for each pathogen. The minimal enrichment time (6, 12, or 24 h) needed to detect all pathogens at the highest dilution after enrichment will be identified. For example, if 6 h of enrichment is sufficient for Salmonella but 12 h is needed for Campylobacter, the minimal common enrichment time would be 12 h.Ten whole chicken carcasses will be collected and transferred to the lab aseptically on ice. Chicken rinsates from individual whole chickens will be prepared as described above but the filter sterilization step will be excluded. The minimal bacterial concentration will be used to spike chicken rinsates, which will be inoculated to the selective media for incubation. At the minimal, common enrichment time, samples (2 ml) from enrichment media will be collected for DNA extraction and ONT Flongle sequencing as described above. Samples will also be inoculated to selective media for total counts and confirmed by multiplex PCR. The minimal, common enrichment time will be increased if the time determined before is not sufficient to detect the spiked bacterial pathogens in chicken rinsates containing native microflora. For example, if a 6 h enrichment time before is not adequate, 12 h will be considered; if 12 h is not sufficient, 24 h will be used.Whole chicken carcasses (n=200) will be collected from a local poultry processing plant and transferred to our laboratory aseptically on ice. Over a period of six months, eight chicken carcasses will be collected weekly and processed for identification of foodborne pathogens. Chicken rinsates will be prepared immediately as described above except that filter sterilization will be omitted. Chicken rinsate will be mixed with enrichment media as described above. Samples (2 ml) will be collected from enrichment media at the minimal, common enrichment time, and DNA will be isolated with the Host ZERO Microbial Kit (Zymo Research) as described above. Library preparation, loading protocols for Flongle flow cells, sequencing, and real-time data analysis will be executed as described above. The isolation and identification of bacterial pathogens after a 24 h enrichment will be conducted with selective media as described previously. The identify of bacterial isolates from selective media will be confirmed by multiplex PCR, and taxonomic assignments for ONT Flongle sequencing will be compared with the culture method. The sensitivity, specificity, and accuracy of assays will be calculated as described in MLG 1.01.