High-Resolution Melting Curve Analysis (HRMA) for the Identification of Class D β-Lactamases (CHDLs) in Pseudomonas aeruginosa Lung Infection

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Hamed Tahmasebi,1,2 Sanaz Dehbashi,3 Mohammad Reza Arabestani4,51School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran; 2Department of Bacteriology, Pasteur Institute of Iran, Tehran, Iran; 3Department of Laboratory Sciences, Varastegan Institute of Medical Sciences, Mashhad, Iran; 4Microbiology Department, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran; 5Nutrition Health Research Center, Institute of Health Sciences and Technology, Hamadan University of Medical Sciences, Hamadan, IranCorrespondence: Mohammad Reza Arabestani, Microbiology Department, Faculty of Medicine, Hamadan University of Medical Sciences, Pajoohesh Junction, Hamadan, Iran, Tel +989188662009, Email mohammad.arabestani@gmail.comObjective: Conventional approaches for laboratory detection of Carbapenem-hydrolyzing class D β-lactamases (CHDLs) from clinical isolates of Pseudomonas aeruginosa are laborious, slow, and have limited sensitivity. This study presents a novel high-resolution melting curve analysis (HRMA) assay for the rapid molecular characterization of CHDLs in P. aeruginosa lung infections.Methods: Detection of blaOXA genes in CHDLs was performed using an HRMA assay. ABI Step One-Plus Manager Software version 3.2 and Precision Melt Analysis Software version 3.02 (Applied Biosystems) were used to analyze a wide range of HRMA data.Results: Out of the 47 P. aeruginosa MBL-producing strains, 18 (38.2%) were blaOXA145 positive, 24 (51.0%) were blaOXA-161 positive, 20 (42.5%) were blaOXA-224 positive, 29 (61.7%) were blaOXA-539 positive, 7 (14.8%) were blaOXA-675 positive, and 19 (40.4%) were blaOXA848 positive. Distinct, non-overlapping melting peaks (eg, 81.70°C for blaOXA-145, 87.35°C for blaOXA-848) were achieved with an accuracy of ± 0.1– 0.5°C, enabling unambiguous genotype identification. Clinically, multi-locus sequence typing (MLST) of 100 isolates revealed that the dominant sequence types—ST09, ST15, ST111, and ST235—were significantly associated with MDR/XDR phenotypes, biofilm formation, and CHDL carriage (p < 0.05). ST235 and ST09 predominated among CHDL producers, and 57% of isolates were new or singleton sequence types, highlighting regional genetic diversity.Conclusion: Novelty lies in the precise differentiation of six clinically relevant OXA alleles exclusively through unique melting temperature (Tm) shifts and melt curve morphologies, eliminating the need for fluorescent probes. In practice, HRMA offers significant advantages over conventional methods, such as the modified Hodge test or Sanger sequencing. As a closed-tube, single-step assay, it eliminates post-amplification handling, reduces cross-contamination risks, and provides actionable results within two hours. This marked reduction in turnaround time and reagent costs makes HRMA a highly scalable, user-friendly tool, ideally suited for routine clinical microbiology laboratories to expedite antimicrobial stewardship and epidemiological surveillance in P. aeruginosa pulmonary infections.Keywords: Pseudomonas aeruginosa, high-resolution melting curve analysis, HRMA, New Delhi metallo-β-lactamase, NDM

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