Rapid ESBL-Carba Combo PN test for simultaneous detection of ESBL and carbapenemase activity in Enterobacterales.

UNLABELLED: A chromogenic biochemical assay (ESBL-Carba Combo PN test) was developed for the simultaneous detection of extended-spectrum β-lactamase (ESBL) and carbapenemase activity in Enterobacterales, as well as for discrimination of clinically relevant carbapenemase classes. The assay is based on a novel chromogenic substrate, B-Lac, and a low-cost Tris-based lysis buffer, providing a unified workflow for the detection of ESBLs and carbapenemases. The test was evaluated using cultures of well-characterized clinical isolates with resistance mechanisms previously defined by whole-genome sequencing. Results were obtained after a mean preparation time of approximately 3 min per isolate and were read after 20 min and 1 h of incubation. For ESBL detection (n = 67), sensitivity was 93.8% and 97.8% after 20 min and 1 h of incubation, respectively, with a specificity of 100%. Carbapenemase activity (n = 92) was detected with 100% sensitivity and specificity at both time points. Using the combined assay (n = 85), ESBL and/or carbapenemase activity was detected with 100% sensitivity and specificity. For Ambler classes A and B, 100% sensitivity and specificity were reached after 20 min of incubation. Regarding class D carbapenemases, 91.3% and 95.5% sensitivity after 20 min and 1 h of incubation, respectively, were observed, with 100% specificity at both times. The ESBL-Carba Combo PN test provides a simple, low-cost (<1 USD/sample), and functional method for the simultaneous detection of the major β-lactam resistance mechanisms in Enterobacterales and for carbapenemase class discrimination within one workflow, potentially supporting earlier optimization of antimicrobial therapy and antimicrobial stewardship in routine clinical microbiology laboratories. IMPORTANCE: Rapid identification of extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing Enterobacterales (CPE) is essential to optimize antimicrobial therapy and implement timely infection control measures. Most currently available rapid phenotypic assays detect these resistance mechanisms separately, increasing hands-on time, workload, reagent consumption, and cost, particularly in isolates co-producing multiple β-lactamases. In addition, discrimination between carbapenemase classes, particularly class A and class D carbapenemases, remains challenging for most biochemical assays. This study describes a simple chromogenic assay that allows the simultaneous functional detection of both enzyme types using a single substrate and a low-cost lysis buffer. By incorporating relebactam as a selective class A inhibitor, the assay improves phenotypic discrimination between class A and class D carbapenemases while preserving reliable ESBL detection. The rapid ESBL-Carba Combo PN test provides 1 h of phenotypic information on the most clinically relevant β-lactam resistance mechanisms and represents a useful tool to guide early antibiotic treatment.