PMAxx dye-assisted colorimetric dual DNAzyme-LAMP (PD-cDDLAMP) for rapid phenotypic antimicrobial susceptibility testing of Escherichia coli.

UNLABELLED: Rapid antimicrobial susceptibility testing (AST) is crucial for combating antimicrobial resistance and guiding effective therapy. Conventional phenotypic AST methods are often time-consuming, requiring 18-72 h for results, while genotypic approaches often depend on pre-existing knowledge of resistance markers. Although nucleic acid-based phenotypic AST methods have reduced turnaround time, attempts to shorten antibiotic exposure often produce only small differences between susceptible and resistant bacteria. Consequently, most loop-mediated isothermal amplification (LAMP)-based phenotypic AST assays still require prolonged antibiotic exposure (up to 4 h) or rely on sophisticated high-resolution nucleic acid detection, such as digital LAMP, to accurately detect these subtle changes, limiting their simplicity. Therefore, there is a critical need to develop a rapid assay with both short antibiotic exposure times and a simple readout. To address this unmet need, we developed PMAxx dye-assisted colorimetric Dual DNAzyme-LAMP (PD-cDDLAMP), a rapid nucleic acid-based phenotypic AST method that enhances suppression of nucleic acid amplification from antibiotic-susceptible bacteria through the selective binding of photoreactive DNA-crosslinker dye (PMAxx) to the DNA of membrane-compromised cells. This strategy enables reliable discrimination between antibiotic-resistant bacteria ("signal-on") and antibiotic-susceptible bacteria ("signal-off") after only 30 min of antibiotic exposure using a user-friendly colorimetric DNAzyme-LAMP readout system. PD-cDDLAMP achieved 91.7% sensitivity, 83.3% specificity, and 87.5% accuracy in detecting Escherichia coli exposed to ampicillin and tetracycline, outperforming current methods by reducing antibiotic exposure time to just 30 min. The method was further validated in spiked milk samples, demonstrating its potential for on-site AST in dairy farming. PD-cDDLAMP's simplicity, speed, and reduced workflow complexity make it a promising tool for rapid AST. IMPORTANCE: Rapid, reliable antimicrobial susceptibility testing (AST) is essential for guiding targeted therapy and combating the escalating threat of antimicrobial resistance (AMR). However, current phenotypic ASTs are too slow for urgent decision-making, and genotypic assays cannot fully capture emerging or unknown resistance mechanisms. The PMAxx dye-assisted colorimetric dual DNAzyme-loop-mediated isothermal amplification (PD-cDDLAMP) assay introduced in this work provides a rapid nucleic acid-based phenotypic AST capable of distinguishing antibiotic-resistant from susceptible bacteria within 30 min of antibiotic exposure. By combining PMAxx-mediated suppression of nucleic acid amplification in susceptible cells with equipment-free colorimetric DNAzyme-LAMP readout, PD-cDDLAMP bridges the gap between speed and phenotypic accuracy. Its high diagnostic performance, compatibility with complex matrices such as milk, and minimal instrumentation requirements position this method as a promising point-of-care tool for clinical microbiology laboratories and on-site testing environments. PD-cDDLAMP has the potential to significantly shorten time-to-result while maintaining reliability, thereby supporting antimicrobial stewardship.