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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #361085

Research Project: Ecology and Detection of Human Pathogens in the Produce Production Continuum

Location: Produce Safety and Microbiology Research

Title: Quantitating and verifying Shiga toxin expression from Californian environmental STEC by mass spectrometry

item Silva, Christopher - Chris
item Lee, Bertram
item Yambao, Jaszemyn
item Erickson-Beltran, Melissa
item Quinones, Beatriz

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 2/19/2019
Publication Date: 2/20/2019
Citation: Silva, C.J., Lee, B.G., Yambao, J.C., Erickson-Beltran, M.L., Quinones, B. 2019. Quantitating and verifying Shiga toxin expression from Californian environmental STEC by mass spectrometry. Meeting Abstract. Poster. ARS-FSIS 2019 meeting.

Interpretive Summary: N/A

Technical Abstract: Shiga toxin producing Escherichia coli (STEC) have a disproportionately large impact on foodborne illness. Of the estimated 48 million cases of foodborne illness, they cause 260,000 infections and at least 3,600 hospitalizations. The direct costs of STEC infections exceeds 1 billion dollars per year in the United States. In the past STEC infections were associated with undercooked meat. As dietary habits have changed, STEC infections are now associated with fresh produce. Although Shiga toxin production is associated with specific E. coli strains, it is controlled by a lambdoid phage which infects the host E. coli. This means that the genes necessary to produce Shiga toxins (stx operon) may be present in the bacterial chromosome but may not be expressible if the stx phage is damaged or incapable of lytic replication. This can greatly complicate detection methods. We have developed a mass spectrometry-based method of rapidly and directly detecting the presence of Shiga toxins in complex matrices. Shiga toxins are hexamers composed of a toxic catalytic A subunit and five identical non-toxic eukaryotic cell surface binding B subunits. Conserved peptides in the B subunit were used to quantitate the Shiga toxins. These peptides were also used to determine the type of Shiga toxin. We identified a conserved peptide spanning the active site of the A subunit that can be used to demonstrate the presence of the A subunit of a Shiga toxin. 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. The Shiga toxins produced by a set of 45 environmental STEC strains (O113:H21, O121:H19, O157:H7, O6:H34, O177:H25, or O185:H7) were quantitated by this method. None of these strains have been associated with any STEC outbreak and all were isolated from various environments in three producing growing counties in California. In eight of the O157:H7 strains, the gene expressing the A subunit (stx2c operon) was interrupted by an insertion sequence (IS1203v). Shiga toxin production 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 nearly fifty-fold range. Those strains possessing an IS-interrupted stx2c operon expressed low levels of B subunits. By comparison, a Shiga toxin producing strain possessed an identical but intact stx2c operon and produced intact Stx2c.