High-throughput real-time PCR approach using the LightCycler®1536 instrument for genotyping and high discrimination of E. coli pathogroups responsible for gastrointestinal diseases

Abstract
Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens responsible for a number of human gastrointestinal diseases, including watery or bloody diarrhea, and hemorrhagic colitis. Rapid and specific detection of STEC strains with high virulence for human has become a priority for public health authorities. The main virulence factor of STEC is the production of Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2) or its variants. STEC strains causing severe disease in humans mainly possess additional virulence factors such as the outer membrane protein intimin, essential for the intimate attachment and the formation of attaching and effacing (A/E) lesions on gastrointestinal epithelial cells whose genetic determinant is harboured by the LEE pathogenicity island. In addition to the LEE locus, several other putative pathogenicity islands (PAIs) have been identified in STEC strains which present a variable repertoire of effectors genes that encode potential virulence determinants (nle, as non-LEE encoded effector). Based on a high-throughput real-time PCR approach using the LightCycler®1536 instrument a selection of discriminative nle genetic markers mostly encoding type III secretion system effector proteins allowed to develop an assay for specific characterization of the most virulent strains of STEC. These nle genes were detected at different frequencies in pathogenic and apathogenic E. coli strains indicating their possible role in virulence. The presence of these genes may be used to make a “molecular risk assessment” to predict the virulence potential of STEC strains. For E. coli O26, these data were corroborated by a genotyping real-time PCR test based on allelic discrimination of the arcA (aerobic respiratory control protein A) gene. Results obtained with the LightCycler®1536 instrument indicate that a combination of molecular detection and genotyping tools are highly discriminative to clearly distinguish STEC strains constituting a severe risk for human health from STEC that are not associated with severe and epidemic disease. This simple diagnostic approach might be applicable in hospital service labs or public health laboratories to test strains isolated from foods or stools of patients suffering from diarrhea.