Quantitating Shiga toxin production in environmental STEC isolated from a major produce production region in California

Authors: Silva, Christopher - Chris; Lee, Bertram; Yambao, Jaszemyn; Erickson-Beltran, Melissa; Quinones, Beatriz

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/27/2019
Publication Date: 6/21/2019
Citation: Silva, C.J., Lee, B.G., Yambao, J.C., Erickson-Beltran, M.L., Quinones, B. 2019. Quantitating Shiga toxin production in environmental STEC isolated from a major produce production region in California [abstract]. ASM Microbe 2019, June 20-24, 2019, San Francisco, CA. Poster AES-1194.

Interpretive Summary: N/A

Technical Abstract: Shiga toxin producing Escherichia coli (STEC) are responsible for approximately 260,000 infections, at least 3,600 hospitalizations, and direct costs exceeding 1 billion dollars per year in the United States alone. As dietary habits have changed, STEC infections, once more commonly transmitted by undercooked meat, are now associated with fresh produce. Shiga toxins are hexamers composed of a toxic catalytic A subunit and five identical non-toxic eukaryotic cell surface binding B subunits. We used a mass spectrometry-based method to quantitate the amount of Shiga toxins produced by a set of 45 environmental STEC strains. These strains were isolated from diverse environments in three of California’s agricultural counties. None of these strains have been associated with any STEC outbreak. The stx operon from each strain was sequenced and the serotype was determined by sequencing the O and flagellar antigen genes. Each strain belonged to either the O113:H21, O121:H19, O157:H7, O6:H34, O177:H25, or O185:H7 serotypes and contained either the stx2a or stx2c operon. Of the seven O113:H21, six contained two distinct stx2a operons. Eight of the O157:H7 strains possessed an A subunit (stx2c operon) gene that was interrupted by an insertion sequence (IS1203v). Production of Shiga toxins was induced by nutrient depletion and analyzed by mass spectrometry and a fluorescent Vero cell-based assay. The 37 strains produced Shiga toxins in a fifty-fold range (1.1 ng/mL to 49 ng/mL). Those strains possessing an IS-interrupted stx2c operon expressed low levels of B subunits (0.5 -1.6 ng/mL). By comparison, a Shiga toxin producing strain possessed an identical but intact stx2c operon and produced intact Stx2c (5.7 ng/mL). We identified a conserved peptide (FVTVTAEALR) that spans the active site of the A subunit. This peptide is suitable for our analysis and can be detected in the attomole range (10-18 mole). It provides an instrumental means of demonstrating the presence of both an intact A subunit and B subunits and quantitating the amount of Shiga toxin present in a sample.