The Novel CarbaLux Test for Carbapenemases and Carbapenem Deactivating AmpC Beta-Lactamases

Abstract

Objectives: To evaluate the rapid phenotypic CarbaLux test for routine diagnostics in the medical laboratory in a proof of concept study. Methods: isolates of Gram-negative bacteria suspicious for carbapenem resistance including Enterobacterales (67), Pseudomonas (10), Acinetobacter (5), and Stenotrophomonas (1) species, collected between 2016 and 2018 from in-patients, were tested for carbapenemase activity using a novel fluorescent carbapenem. When subjected to extracted bacterial carbapenemases its fluorescence disappears. All bacteria to be tested were cultured on Columbia blood agar and few on other commercial media. MALDI TOF MS, molecular assays, automated MIC testing, and in part, agar diffusion tests served to characterize the isolates. For comparison, few selected bacteria were also investigated by prior phenotypic tests for carbapenemase detection. Results: Under UV light, the CarbaLux test allowed a rapid detection of 39/39 carbapenemase-producing bacteria, including 15 isolates with OXA carbapenemases (e.g., OXA-23, OXA-24/40-like OXA-48-like or OXA-181). Several isolates had low MICs but still expressed carbapenemases. Among Enterobacter spp., it detected six strains with hyper-produced AmpC beta-lactamases, which deactivated carbapenems but were not detectable by prior rapid phenotypic assays. An unexpected high carbapenemase activity appeared with these enzymes. They were identified as AmpC variants by inhibition with cloxacillin. Conclusion: Other than prior rapid phenotypic assessments for carbapenemases, which use secondary effects such as a change of pH, the inactivation of the fluorescent carbapenem substrate can be visualized directly under UV light. The new test works at 100 to 200-fold lower, therapy-like substrate concentrations. It takes advantage of the high substrate affinity to carbapenemases allowing also the detection of less reactive resistance enzymes via a trapping mechanism, even from bacteria, which might appear unsuspicious from initial antibiograms. The novel fluorescence method allows simple and safe handling, reliable readings, and documentation and is suitable for primary testing in the clinical laboratory.