Real-Time Monitoring of NDM‑1 Activity in Live Bacterial Cells by Isothermal Titration Calorimetry: A New Approach To Measure Inhibition of Antibiotic-Resistant Bacteria

The “superbug” infection caused by New Delhi metallo-β-lactamase (NDM-1) has become an emerging threat. Monitoring NDM-1 has proven challenging due to its shuttling between pathogenic bacteria. Here, we report an isothermal titration calorimetry (ITC) method that can monitor activity and inhibition of NDM-1 in live bacterial cells in real time. This method has been exemplified by monitoring of the activity and inhibition of the target enzyme and evaluating the breakdown of antibiotics by pathogenic bacteria expressing β-lactamases. Cell-based studies demonstrate that the NDM-1 expressed in bacterial cells was inhibited by four known inhibitors ethylene diamine tetraacetic acid (EDTA), d-captopril, ebselen and azolylthioacetamide with fifty percent inhibitory concentration (IC50) values of 3.8, 48, 0.55, and 17.5 μM, respectively, which are in good agreement with the data from inhibition kinetics using UV–vis and NMR spectroscopy in vivo. This approach could be applied to screen and evaluate small molecule inhibitors of metallo-β-lactamases (MβLs) in whole cells or to identify drug resistant bacteria.