AmpC β-lactamase confers resistance to several antibiotics, including penicillins, second- and third-generation cephalosporins, and monobactams, but does not hydrolyze cefepime or carbapenems. Its origin may be chromosomal, inducible in some species after cephalosporin exposure, or plasmid-mediated (pAmpC), characterized by constitutive and abundant expression in different bacteria. This study aimed to develop specific primers for pAmpC gene identification using qPCR-HRM, a technique that combines the sensitivity of qPCR with High-Resolution Melting analysis, enabling differentiation of resistance genes based on melting temperature (Tm), including the detection of mutant variants.

A total of 148 clinical Enterobacteriaceae isolates from hospitals and clinics in Rio de Janeiro (2021–2024) were analyzed. Bacterial identification was performed using VITEK-2 and MALDI-TOF. Antimicrobial susceptibility was determined by VITEK-2 and disk diffusion according to BRCAST (2024). Forty-six β-lactam-resistant isolates were screened for pAmpC genes using conventional PCR, followed by sequencing and analysis in PubMLST and Institut Pasteur MLST databases.

Three pAmpC-producing strains were confirmed: two Escherichia coli and one Klebsiella pneumoniae, carrying CMY-44, CMY-69, and ACT-5 genes, respectively. These variants had not been previously reported in Brazil. qPCR-HRM showed Tm values of 82.0°C (± 0.5) for CMY in Salmonella enterica, 84.0°C (± 0.5) in E. coli, and 86.0°C (± 0.5) for ACT in K. pneumoniae, allowing clear distinction between CIT and EBC targets.

qPCR-HRM proved to be a rapid (2 h), sensitive, specific, cost-effective, and efficient tool for detecting and differentiating pAmpC genes, even in the presence of mutations. The observed prevalence was 6.52% among β-lactam-resistant strains. These findings underscore the importance of broader surveillance studies to monitor pAmpC epidemiology in Brazil.