New approach to identify colistin-resistant Pseudomonas aeruginosa by high-resolution melting curve analysis assay

Colistin-resistant Pseudomonas aeruginosa (CRPA), as a health care system threat, is increasing globally. This study aimed was to determine CRPA by high-resolution melting curve (HRM) analysis method. The HRM method was done on standard strains of P. aeruginosa and CRPA strains. Ninefold serial dilutions of known genomic DNA (gDNA) and plasmid DNA (pDNA) concentrations, extracted from P. aeruginosa NCTC13715 and P. aeruginosa NCTC10728 were prepared and tested by melting curve analysis and HRM assay. Data analysis was performed using the Step-One Plus Software v2.3 and hrm Software v3.0.1. The results of the melt curve analysis and HRM showed a very similar melt peak for all positive controls with a melt temperature that ranged from 88·1°C to 88·6°C for the 16srRNA, 90·0°C to 90·05°C for the mcr-1 and 91·2°C to 91·7°C for the pmrA gene. Furthermore, the data indicated that the HRM approach has the sensitivity to detect 10⁰  CFU per ml for the 16srRNA gene, 10¹  CFU per ml for the pmrA gene, and 10³  CFU per ml for the mcr-1 gene. According to our findings, it was concluded that the HRM method could detect 10⁰ to 10³  CFU per ml of P. aeruginosa gDNA and pDNA. Therefore, CRPA strains can be identified with high sensitivity and specificity by HRM assay. SIGNIFICANCE AND IMPACT OF THE STUDY: The highlight of our research is about the detection of bacteria resistance genes to antibiotics by advanced molecular methods, which means high-resolution melting curve (HRM) analysis. We determined that the HRM method in identifying colistin-resistant P. aeruginosa has high accuracy and speed, and with high sensitivity and specificity.