Establishment of a method for the rapid detection of carbapenem-resistant Pseudomonas aeruginosa based on bla_NDM with one-tube RPA-CRISPR/Cas12a visualization.

Abstract

Pseudomonas aeruginosa (PA), which is a common Gram-negative bacterium, can become carbapenem-resistant Pseudomonas aeruginosa (CRPA) upon the development of antibiotic resistance, making clinical treatment difficult. CRPA with antibiotic resistance genes (ARGs), such as bla_NDM and bla_KPC, is common in China. The development of tests for ARGs would facilitate the more rapid identification of CRPA in China. Isothermal amplification research has improved, but limitations remain, including a lack of specialized equipment, the difficulty of developing sophisticated primers, and aerosol pollution. Thus, clinical examination methods must improve. We successfully integrated RPA with CRISPR/Cas12a, and we identified bla_NDM as our institution's primary CRPA. RPA-CRISPR/Cas12a could accurately detect target DNA within 40 min without cross-reacting with other bacteria. The results showed high concordance with conventional culture-based methods, including 100% agreement in clinical sample validation. The method reliably identified standard PA strains and 29 clinical isolates, detecting PA at concentrations as low as 10^-1 CFU. In addition, the closed-tube format effectively minimized the risk of aerosol contamination. This platform offers a rapid and user-friendly tool for identifying bla_NDM-positive CRPA, and this tool is particularly suitable for early screening and clinical management in resource-limited settings. The simplicity and accuracy of this approach make it a promising option for infection control and public health surveillance.