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ARS Home » Northeast Area » Wyndmoor, Pennsylvania » Eastern Regional Research Center » Characterization and Interventions for Foodborne Pathogens » Research » Publications at this Location » Publication #367750
Research Project: Advanced Development of Innovative Technologies and Systematic Approaches to Foodborne Hazard Detection and Characterization for Improving Food Safety

Location: Characterization and Interventions for Foodborne Pathogens

Title: Three dimensional vero cell-platform for rapid and sensitive screening of Shiga-Toxin producing Escherichia coli

Author
item TO, CELINA - Purdue University
item BHUNIA, ARUN - Purdue University

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/15/2019
Publication Date: 5/7/2019
Citation: To, C.Z., Bhunia, A.K. 2019. Three dimensional vero cell-platform for rapid and sensitive screening of Shiga-Toxin producing Escherichia coli. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2019.00949.
DOI: https://doi.org/10.3389/fmicb.2019.00949

Interpretive Summary: Pathogenic Escherichia coli is a foodborne pathogen of serious public health concern; causing 265,000 illnesses, 3,600 hospitalizations and 30 deaths in the United States each year. The current methods used for detection of Shiga toxin-producing E. coli (STEC) take long time to complete and may not accurately assess the pathogens virulence. One of the major factors that contributes to E. coli virulence is production of a potentially deadly toxin, Shiga toxin. We report here a simple color-changing method that detects the presence of STEC or the toxin in food samples in less than 24 hours and provides important information about potential pathogen virulence. The method can help food producers and manufacturers make science-based decisions to ensure the distribution of safe food to consumers and ultimately prevent foodborne illnesses.

Technical Abstract: Shiga-toxin producing Escherichia coli (STEC) is a serious public health concern. Current Vero cell assay, although sensitive, is lengthy and requires 48–72 h to assess STEC presence in a sample. In this study, we investigated if Vero cells in a three-dimensional (3D) platform would provide improved sensitivity for rapid screening of STEC. Vero cells (epithelial kidney cell line) were grown as a monolayer (2D) or in a collagen-matrix (3D) and exposed to Shiga-toxin (Stx) preparation or STEC cells that were pre-exposed to antibiotics (mitomycin C, ciprofloxacin, or polymyxin B) for toxin induction. Lactate dehydrogenase (LDH) release from Vero cells was used as a biomarker for cytotoxicity. Modified tryptic soy broth (mTSB) as enrichment broth containing mitomycin C (2 mg/ml) or ciprofloxacin (100 ng/ml) significantly induced Stx production, which was further confirmed by the dot-immunoblot assay. The 3D Vero platform detected STEC after 6 h post-infection with cytotoxicity values ranging from 33 to 79%, which is considerably faster than the traditional 2D platform, when tested with STEC. The cytotoxicity for non- Stx producing bacteria, Salmonella, Listeria, Citrobacter, Serratia and Hafnia was found to be below the cytotoxicity cutoff value of 15%. The detection limit for the 3D Vero cell assay was estimated to be 10^7 CFU/ml for bacteria and about 32 ng/ml for Stx in 6 h. STEC-inoculated ground beef samples (n=27) resulted in 38–46% cytotoxicity, and the bacterial isolates (n=42) from ground beef samples were further confirmed to be stx1 and stx2 positive in a multiplex PCR yielding a very low false-positive result. This 3D cell-based screening assay relies on mammalian cell pathogen interaction that can complement other molecular techniques for the detection of cell-free Stx or STEC cells from food samples for early detection and prevention.