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

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.

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.