The Rapid CarbaLux Combination Test to Uncover Bacterial Resistance and Heteroresistance Prior to Antibiotic Treatment

<b>Background/Objectives:</b> In this proof-of-concept study, the objective was to evaluate the phenotypic CarbaLux combination rapid test in terms of guiding the therapy of infections caused by multidrug-resistant Gram-negative bacteria with carbapenemase inhibitors and carbapenems, and to compare its results and practicability with standard diagnostic methods. <b>Methods:</b> In the classical CarbaLux test, a fluorescent carbapenem serves as a UV-visible diagnostic surrogate for clinically used carbapenem antibiotics. When exposed to extracted carbapenemases from bacterial colony growth on agar plates, fluorescence rapidly disappears, showing whether monotherapy with carbapenems is possible or must be rejected. It was expected that a specific inhibitor that protects imipenem or meropenem from enzymatic deactivation during antibacterial therapy would perform the same in vitro with fluorescent carbapenem and preserve its fluorescence. The new additional CarbaLux combination test is used if the classic test is positive for carbapenemases: a classic test tube pre-dosed with fluorescent carbapenem is spiked with cloxacillin; with recently launched carbapenemase inhibitors, e.g., avibactam, relebactam, zidebactam, nacubactam, or vaborbactam; or with picolinic acid. Fourteen Enterobacterales and six <i>Acinetobacter baumannii</i> isolates were analyzed. <b>Results:</b> At fixed concentrations, the new inhibitors protected fluorescent carbapenem from bacterial KPC-mediated inactivation and partially from AmpC beta-lactamase-mediated inactivation. In addition, avibactam also effectively inhibited OXA-48-like enzymes. Cloxacillin selectively inhibited AmpC beta-lactamases extracted from <i>Enterobacter</i> complex species. Non-therapeutic picolinic acid was specific for metallo-beta-lactamases and thus identified infections by pathogens that cannot be treated with carbapenems alone or in combination. <b>Conclusions:</b> Inhibitor/fluorescent carbapenem mixtures corresponding to therapeutic inhibitor/carbapenem combinations allow us to visualize the efficacy of carbapenemase inhibitors. The in vitro results are consistent with clinical experience regarding combination therapy. Enzymatic assays provide a rapid yes/no answer for carbapenem mono- or combination therapy and offer several advantages over current carbapenemase testing methods. In contrast to PCR and lateral flow tests, which only target a selection of carbapenemases, enzymatic assays work by employing a reproducible phenotypic mechanism. They are simpler, broader in scope, and more cost-effective; they can also detect antimicrobial heteroresistance or AmpC beta-lactamase hyperproduction, which is normally undetected when performing automated antibiotic susceptibility testing. The new tests are suitable for clinical diagnosis, public health purposes, and infection control.