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Submitted to: Meeting Proceedings
Publication Type: Proceedings Publication Acceptance Date: 10/6/2012 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: Shiga toxin-producing Escherichia coli (STEC) is an enteric pathogen known to cause human gastrointestinal illnesses with diverse clinical manifestations. The varying disease severity, such as the onset of the hemolytic uremic syndrome, has been associated with certain serotypes of STEC and with the production of Shiga toxins. In particular, serotype O157:H7 is considered the most-commonly reported serotype linked to outbreaks in North America. Epidemiological and genotyping studies have identified six non-O157 serotypes, O26, O103, O111, O121, and O145, linked to human illness worldwide. The rise in food-borne related outbreaks of STEC has heightened the importance of developing improved methods to rapidly detect and characterize virulent strains. Molecular-based technologies, such as DNA microarrays, offer a viable alternative to simultaneously screen multiple markers. To implement better methods to rapidly detect and genotype STEC strains, a novel colorimetric detection method, based on light-initiated signal amplification through polymerization (ampliPHOX technology), was evaluated for pathogen identification with DNA microarrays. Validation experiments have demonstrated that photopolymerization is a simple, rapid, and quantitative DNA microarray-based detection method for assessing the genetic composition of O157 and non-O157 STEC strains by examining genes encoding Shiga toxin (Stx) and selected virulence determinants that code for adhesins, proteases, cytotoxins, and effectors and that are considered to be necessary for E. coli strains to be pathogenic. Stxs are considered one of the best characterized virulence factors in STEC pathogenicity and a determinant for causing the life-threatening hemolytic-uremic syndrome. Based on sequence similarity and catalytic activity, Stxs have been divided into two major groups, Stx1 and Stx2. Epidemiological studies suggest that STEC strains expressing Stx2 may be more virulent than strains only expressing Stx1 or both Stx1 and Stx2. To examine the relative toxicities of Stx2 variants expressed by STEC strains, the Vero-d2EGFP fluorescent assay was employed. The assay uses the Vero-d2EGFP cell line, generated from Vero cells to constitutively express a destabilized variant of the enhanced green fluorescent protein (EGFP). The short, in vivo half-life (t1/2 = 2 hours) of this EGFP variant makes it a sensitive marker for measuring the inhibition of protein synthesis by Stx. Given that active Stxs inhibit protein synthesis, toxin-treated Vero-d2EGFP cells degrade the EGFP variant and do not produce more of the EGFP protein. By using the Vero-d2EGFP fluorescent assay, we were able to examine the relative toxicities of Stx2 variants expressed by STEC strains. Our results have demonstrated that the Stx2 variants are composed of a diverse and heterogeneous group of subtypes with differential cytotoxicities in mammalian cells. |
