Rapid Enzymatic Detection of Shiga-Toxin-Producing <i>E. coli</i> Using Fluorescence-Labeled Oligonucleotide Substrates

Shiga-toxin-producing <i>Escherichia coli</i> (STEC) are important human pathogens causing diarrhea, hemorrhagic colitis, and severe hemolytic uremic syndrome. Timely detection of the multifaceted STEC is of high importance but is challenging and labor-intensive. An easy-to-perform rapid test would be a tremendous advance. Here, the major STEC virulence factor Shiga toxins (Stx), RNA-<i>N</i>-glycosidases targeting the sarcin ricin loop (SRL) of 28S rRNA, was used for detection. We designed synthetic FRET-based ssDNA SRL substrates, which conferred a fluorescence signal after cleavage by Stx. Optimal results using bacterial culture supernatants or single colonies were achieved for substrate <b>StxSense 4</b> following 30 to 60 min incubation. Stx1 and Stx2 subtypes, diverse STEC serotypes, and <i>Shigella</i> were detected. Within a proof-of-principle study, a total of 94 clinical strains were tested, comprising 65 STEC, 11 <i>Shigella</i> strains, and 18 strains of other enteropathogenic bacteria without Stx. In conclusion, the assay offers rapid and facile STEC detection based on a real-time readout for Stx activity. Therefore, it may improve STEC risk evaluation, therapy decisions, outbreak, and source detection and simplify research for antimicrobials.