Detection of Escherichia coli O157:H7 in ground beef using long-read sequencing

Authors: Counihan, Katrina; Kanrar, Siddhartha; Tilman, Shannon; Capobianco, Joseph; Armstrong, Cheryl; Gehring, Andrew

Submitted to: Foods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/3/2024
Publication Date: 3/8/2024
Citation: Counihan, K.L., Kanrar, S., Tilman, S.M., Capobianco Jr, J.A., Armstrong, C.M., Gehring, A.G. 2024. Detection of Escherichia coli O157:H7 in ground beef using long-read sequencing. Foods. 13(6):828. https://doi.org/10.3390/foods13060828.
DOI: https://doi.org/10.3390/foods13060828

Interpretive Summary: Shiga toxin-producing Escherichia coli (STEC) is a bacterium that causes foodborne illness and has the potential to be life-threatening. The current methods to identify STEC in meat take at least four days to complete. However, newer technologies could reduce the time needed for testing. Therefore, the goal of this project was to evaluate using 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 STEC in pure cultures and spiked ground beef, and evaluating selective sequencing to enhance STEC detection in ground beef. Sequencing libraries were run on Oxford Nanopore Technologies’ MinION sequencer. Optimal sequencing output was obtained using the default parameters in the sequencing software, except for setting the minimum read length to 1 kb. There were seven genes (eae, stx1, stx2, fliC, wzx, wzy, rrsC) that we wanted to detect in the STEC DNA. They were detected in DNA extracted from STEC pure cultures within 1 hour of sequencing. All genes could be 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 the high concentration of bovine DNA in the samples. Growth enrichment of the inoculated meat samples 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.

Technical Abstract: Foodborne pathogens are a significant cause of illness in the United States, and infection with Shiga toxin-producing Escherichia coli (STEC) has the potential to produce severe, 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. The time needed for testing could be reduced by using long-read whole genome sequencing to identify foodborne pathogens. Therefore, the goal of this project was to evaluate using 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 STEC in pure cultures and inoculated ground beef, and evaluating selective sequencing to enhance STEC detection in ground beef. Sequencing libraries were run on 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, rrsC) were detected in DNA extracted from STEC pure cultures within 1 hour of sequencing, and 30X coverage was reliably obtained with 2 hours of sequencing. All virulence genes could be 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 the high concentration of bovine DNA in the samples. Growth enrichment of the 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.