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

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Shiga-toxin-producing Escherichia coli (STEC) are significant human pathogens.
• STEC infections can lead to severe conditions like hemorrhagic colitis and hemolytic uremic syndrome.
• Current STEC detection methods are often challenging and time-consuming.

Purpose of the Study:

• To develop a rapid, easy-to-perform assay for detecting Shiga toxins (Stx), the primary virulence factor of STEC.
• To utilize a novel FRET-based ssDNA substrate for real-time Stx activity detection.
• To evaluate the assay's efficacy in identifying diverse STEC strains and subtypes.

Main Methods:

• Design and synthesis of FRET-based single-stranded DNA (ssDNA) substrates targeting the sarcin ricin loop (SRL) of 28S rRNA.
• Incubation of bacterial culture supernatants or colonies with the substrate StxSense 4.
• Monitoring for fluorescence signal generation upon Stx-mediated cleavage of the substrate.
• Validation using a collection of 94 clinical bacterial strains, including STEC, Shigella, and other enteropathogens.

Main Results:

• The developed assay, using substrate StxSense 4, achieved optimal results within 30 to 60 minutes.
• The assay successfully detected Stx1 and Stx2 subtypes, various STEC serotypes, and Shigella species.
• Out of 94 clinical strains tested, 65 STEC and 11 Shigella strains were accurately identified based on Stx activity.

Conclusions:

• The assay provides a rapid and facile method for STEC detection based on real-time Stx activity readout.
• This approach has the potential to enhance STEC risk evaluation, guide therapy decisions, and aid in outbreak and source detection.
• The assay can also simplify research efforts for developing new antimicrobials against STEC.