Rapid and sensitive detection of Shiga-toxin Escherichia coli (STEC) from food matrices using the CANARY biosensor assay

Authors: Tam, Christina; Du, Wen-Xian; WANG, YANGYANG - Smiths Detection; FLANNERY, ANDREW - Smiths Detection; He, Xiaohua

Submitted to: Toxins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/19/2024
Publication Date: 7/22/2024
Citation: Tam, C.C., Du, W.N., Wang, Y., Flannery, A.R., He, X. 2024. Rapid and sensitive detection of Shiga-toxin Escherichia coli (STEC) from food matrices using the CANARY biosensor assay. Toxins. 16,325. https://doi.org/10.3390/toxins16070325.
DOI: https://doi.org/10.3390/toxins16070325

Interpretive Summary: From 2009 to 2021, there were 1,019 reported Shiga-toxin producing Escherichia coli (STEC) outbreaks resulting in 14,010 illnesses, 2,218 hospitalizations, and 43 deaths in US alone. STEC has many different virulence factors, but Shiga toxins (Stxs) are the primary factor responsible for the development of severe complications like hemorrhagic colitis and hemolytic uremia syndrome. Early, rapid, and sensitive detection of STEC/Stx will greatly reduce morbidity and mortality since there are no therapeutics to treat STEC infections. In this study, we applied a new CANARY B-cell based biosensor assay that we developed to determine its translational applicability in the detection of Stx in common STEC-contaminated foods such as beef, lettuce, and milk. We found that the assay was able to sensitively detect STEC contamination in enriched cultures from the contaminated foods at the same time points as the commonly used FDA BAM method used by the USDA Food Safety Inspection Service. Additional assay optimization may improve the sensitivity of the assay to as early as 8 hours thus allowing for earlier diagnosis and product recall to protect the nation's food supply.

Technical Abstract: Shiga-toxin producing Escherichia coli (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Current FSIS testing methods for STEC use the FDA BAM protocol which includes enrichment, cell plating, and genomic sequencing which takes time to complete thus delaying diagnosis and treatment. Previously, we developed a rapid, sensitive, and portable assay that can identify STEC by detecting Shiga toxin (Stx) using the CANARY (Cellular Analysis and Notification of Antigen Risks and Yields) B-cell based biosensor technology with limits of detection (LOD) of 100- 200 pg/mL under optimal laboratory conditions. In this study, we applied this biosensor technology to detect Stx2 present in different food samples, including milk, lettuce, and beef. It was found that the STEC enrichment medium, mTSB, inhibited the biosensor assay, but the inhibition could be easily eliminated by dilution with assay buffer. Results with Stx2 toxoid-spiked food samples indicated an estimated LOD of 4 ng/mL When this assay was applied to food samples inoculated with STEC, it was able to detect 0.4 CFU/g or 0.4 CFU/mL of STEC at 16 hours post incubation (hpi) in enrichment medium containing mitomycin C. We also demonstrated that this assay was even able to detect STEC strains that were high expressors of Stx2 at 8 hpi. These results indicate that the STEC CANARY biosensor developed in this study is a rapid and sensitive assay applicable for detection of STEC contamination in food with minimal sample processing that can complement the current FSIS methodologies for STEC.