Detection of Escherichia coli O157:H7 in Ground Beef Using Long-Read Sequencing.

Foodborne pathogens are a significant cause of illness, and infection with Shiga toxin-producing Escherichia coli (STEC) may lead to life-threatening complications. The current methods to identify STEC in meat involve culture-based, molecular, and proteomic assays and take at least four days to complete. This time could be reduced by using long-read whole-genome sequencing to identify foodborne pathogens. Therefore, the goal of this project was to evaluate the use of long-read sequencing to detect STEC in ground beef. The objectives of the project included establishing optimal sequencing parameters, determining the limit of detection of all STEC virulence genes of interest in pure cultures and spiked ground beef, and evaluating selective sequencing to enhance STEC detection in ground beef. Sequencing libraries were run on the Oxford Nanopore Technologies' MinION sequencer. Optimal sequencing output was obtained using the default parameters in MinKNOW, except for setting the minimum read length to 1 kb. All genes of interest (eae, stx1, stx2, fliC, wzx, wzy, and rrsC) were detected in DNA extracted from STEC pure cultures within 1 h of sequencing, and 30× coverage was obtained within 2 h. All virulence genes were confidently detected in STEC DNA quantities as low as 12.5 ng. In STEC-inoculated ground beef, software-controlled selective sequencing improved virulence gene detection; however, several virulence genes were not detected due to high bovine DNA concentrations in the samples. The growth enrichment of inoculated meat samples in mTSB resulted in a 100-fold increase in virulence gene detection as compared to the unenriched samples. The results of this project suggest that further development of long-read sequencing protocols may result in a faster, less labor-intensive method to detect STEC in ground beef.

Detection and Isolation of Campylobacter spp. from Raw Meat.

This article presents a rapid yet robust protocol for isolating Campylobacter spp. from raw meats, specifically focusing on Campylobacter jejuni and Campylobacter coli. The protocol builds upon established methods, ensuring compatibility with the prevailing techniques employed by regulatory bodies such as the Food and Drug Administration (FDA) and the U.S. Department of Agriculture (USDA) in the USA, as well as the International Organization for Standardization (ISO) in Europe. Central to this protocol is collecting a rinsate, which is concentrated and resuspended in Bolton Broth media containing horse blood. This medium has been proven to facilitate the recovery of stressed Campylobacter cells and reduce the required enrichment duration by 50%. The enriched samples are then transferred onto nitrocellulose membranes on brucella plates. To improve the sensitivity and specificity of the method, 0.45 µm and 0.65 µm pore-size filter membranes were evaluated. Data revealed a 29-fold increase in cell recovery with the 0.65 µm pore-size filter compared to the 0.45 µm pore-size without impacting specificity. The highly motile characteristics of Campylobacter allow cells to actively move through the membrane filters towards the agar medium, which enables effective isolation of pure Campylobacter colonies. The protocol incorporates multiplex quantitative real-time polymerase chain reaction (mqPCR) assay to identify the isolates at the species level. This molecular technique offers a reliable and efficient means of species identification. Investigations conducted over the past twelve years involving retail meats have demonstrated the ability of this method to enhance recovery of Campylobacter from naturally contaminated meat samples compared to current reference methods. Furthermore, this protocol boasts reduced preparation and processing time. As a result, it presents a promising alternative for the efficient recovery of Campylobacter from meat. Moreover, this procedure can be seamlessly integrated with DNA-based methods, facilitating rapid screening of positive samples alongside comprehensive whole-genome sequencing analysis.

Evaluation of Long-Read Sequencing Simulators to Assess Real-World Applications for Food Safety.

Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes are routinely responsible for severe foodborne illnesses in the United States. Current identification methods utilized by the U.S. Food Safety Inspection Service require at least four days to identify STEC and six days for L. monocytogenes. Adoption of long-read, whole genome sequencing for food safety testing could significantly reduce the time needed for identification, but method development costs are high. Therefore, the goal of this project was to use NanoSim-H software to simulate Oxford Nanopore sequencing reads to assess the feasibility of sequencing-based foodborne pathogen detection and guide experimental design. Sequencing reads were simulated for STEC, L. monocytogenes, and a 1:1 combination of STEC and Bos taurus genomes using NanoSim-H. At least 2500 simulated reads were needed to identify the seven genes of interest targeted in STEC, and at least 500 reads were needed to detect the gene targeted in L. monocytogenes. Genome coverage of 30x was estimated at 21,521, and 11,802 reads for STEC and L. monocytogenes, respectively. Approximately 5-6% of reads simulated from both bacteria did not align with their respective reference genomes due to the introduction of errors. For the STEC and B. taurus 1:1 genome mixture, all genes of interest were detected with 1,000,000 reads, but less than 1x coverage was obtained. The results suggested sample enrichment would be necessary to detect foodborne pathogens with long-read sequencing, but this would still decrease the time needed from current methods. Additionally, simulation data will be useful for reducing the time and expense associated with laboratory experimentation.

Draft Genomic Sequences of Nine Extraintestinal Pathogenic Escherichia coli Isolates from Retail Chicken Skin.

Extraintestinal pathogenic Escherichia coli strains were isolated from retail chicken skin. Here, we report the draft genomic sequences for these nine E. coli isolates, which are currently being used in agricultural and food safety research.