Introduction: AmpC -lactamases are present in several species of Enterobacteriaceae and confer resistance to a wide range of -lactam antibiotics, including penicillins, 2 and 3 generation cephalosporins, and monobactams, although they generally do not hydrolyze cefepime or carbapenems.AmpC enzymes may be chromosomally encoded, with inducible expression after exposure to cephalosporins in some species, or plasmid-mediated (pAmpC), usually associated with high-level expression in different bacterial hosts.Therefore, monitoring AmpC -lactamases is essential to control the spread of resistance and to guide appropriate antimicrobial therapy.Objectives: This study aimed to detect plasmid-mediated AmpC (pAmpC) genes using qPCR-HRM, a method that combines the sensitivity of quantitative PCR with High-Resolution Melting analysis.This approach enables the differentiation of resistance genes based on their melting temperature (Tm) profiles, including the discrimination of mutant variants.Methodology: A total of 148 clinical Enterobacteriaceae isolates collected from hospitals and outpatient clinics in Rio de Janeiro between 2021 and 2024 were analyzed.Bacterial identification was performed using VITEK 2 and MALDI-TOF MS.Antimicrobial susceptibility testing was conducted by VITEK 2 and disk diffusion (BRCAST 2024).Among the isolates, 46 showed resistance to -lactam antibiotics and were subjected to conventional PCR to detect pAmpC genes.Positive results were confirmed by sequencing and analyzed using the PubMLST and Institut Pasteur MLST databases.Strains positive for pAmpC genes were then tested using primers specifically designed for qPCR-HRM, based on the previous analyses.Results: Three clinical strains producing pAmpC were confirmed: two Escherichia coli and one Klebsiella pneumoniae, harboring the CMY-44, CMY-69, and ACT-5 genes, respectively.In the qPCR-HRM analysis, distinct melting temperatures (Tm) were observed: 84.0 C (0.5) for CMY in E. coli and 86.0 C (0.5) for ACT in K. pneumoniae, allowing clear discrimination between the targets. Conclusion:The qPCR-HRM assay proved to be a rapid (approximately 2 hours), sensitive, specific, costeffective, and reliable method for the detection and differentiation of pAmpC genes, including variants harboring mutations.The observed prevalence of pAmpC was 6.52% among -lactam-resistant isolates.To date, there are no previous reports of these variants in Brazil.These findings highlight the need for expanded epidemiological studies to strengthen the surveillance of pAmpC dissemination in Brazil.
Mello et al. (Thu,) studied this question.