Diagnostic algorithm for the detection of carbapenemases and extended-spectrum β-lactamases in carbapenem-resistant .

can acquire carbapenem resistance through various mechanisms, including genomic mutations leading to the overexpression of efflux pumps, intrinsic AmpC-β-lactamase, and/or reduced permeability, and/or through the acquisition of plasmid-mediated carbapenemases and/or extended-spectrum-β-lactamases (ESBLs). Unfortunately, carbapenemase/ESBL-producing-carbapenem-resistant- (CP/ESBL-CRPA) cannot be differentiated from non-CP/ESBL-CRPA based solely on susceptibility testing results of conventional β-lactam (BL)-antibiotics. Knowing that these two groups display different activity profiles toward novel BL/β-lactamase-inhibitor (BLI) combinations, we developed and verified a cost-effective and easy-to-use diagnostic algorithm for screening and differentiation of carbapenemase and ESBL production in CRPA. We determined disc diffusion inhibition zones and gradient strip minimal inhibitory concentration values of 136 whole-genome-sequenced CRPA (70 metallo-β-lactamase-[MBL-], 1 GES-5-, 1 KPC-2-, 12 ESBL-, and 53 AmpC-hyper-producing isolates). We used the following BL-BLI combinations: ceftolozane-tazobactam (C-T), ceftazidime-avibactam, imipenem-relebactam (I-R), meropenem-vaborbactam, cefepime-enmetazobactam (C-E), and aztreonam-avibactam. We also included a lateral flow immunoassay (Carba-5, NG-Biotech) for confirmation of MBL production and double disc synergy testing (DDST) to improve ESBL detection. C-T was the most effective screening antibiotic for distinguishing MBL and ESBL producers from AmpC-hyperproducing CRPA, achieving a sensitivity of 100% for both MBL and ESBL producers. I-R reliably confirmed MBL production in C-T positive screened CRPAs, with a sensitivity of 92.8% and specificity of 100%. Incorporating Carba-5 into the phenotypic algorithm improved sensitivity for confirming MBL production to 100%. For the remaining C-T positive but I-R negative isolates, C-E showed 75% sensitivity and 78.6% specificity in detecting ESBL production. The DDST further confirmed ESBL production in six out of nine ESBL producers (66.6%). In conclusion, we established a simple and cost-effective diagnostic algorithm, enabling screening and confirmation of carbapenemase and ESBL production in CRPA.IMPORTANCECarbapenem-resistant (CRPA) is a major global health threat, and rapid identification of its resistance mechanisms is crucial for effective treatment and infection control. Differentiating between carbapenemase-producing (CP), extended-spectrum β-lactamase-producing (ESBL), and AmpC-hyperproducing CRPA is challenging, as conventional susceptibility testing cannot reliably distinguish these resistance mechanisms. Our study presents a simple, cost-effective, and easy-to-implement phenotypic diagnostic algorithm that enables accurate screening and confirmation of CP and ESBL production in CRPA. This method is particularly valuable for laboratories lacking access to molecular diagnostics, as it provides a practical alternative for routine testing. By facilitating the early detection of resistant strains, this approach has the potential to improve patient outcomes, optimize antimicrobial therapy, and enhance global surveillance efforts against multidrug-resistant pathogens.