Real-time monitoring and inhibition of the activity of carbapenemase in live bacterial cells: application to screening of β-lactamase inhibitors

Abstract

Antibiotic resistance mediated by β-lactamases including metallo-β-lactamases (MβLs) has become an emerging threat. In this study, we employed UV-vis to monitor the activity and inhibition of New Delhi MβL (NDM), K. pneumoniae carbapenemase (KPC) and MβL VIM-2 in live bacterial cells in real time. The monitoring showed that the faropenem was hydrolyzed by six strains which express β-lactamases, K. pneumoniae, P. aeruginosa, E. coli expressing extended-spectrum β-lactamase (ESBL), NDM-1 (EC07), VIM-2, and methicillin-resistant S. aureus (MRSA), but not by E. coli and S. aureus which do not express β-lactamases. Also, four β-lactams cefazolin, meropenem, faropenem and amoxicillin were hydrolyzed by the NDM and KPC expressed in K. pneumoniae. Cell-based studies revealed that the targets NDM and KPC expressed in K. pneumoniae cells were inhibited by three known inhibitors ethylene diamine tetraacetic acid (EDTA), ebselen and clavulanic acid, with a fifty percent inhibitory concentration (IC₅₀) of 1.2, 84.7 and 13.3 μM, and the VIM-2 expressed in E. coli cells was inhibited by EDTA, D-captopril and rhodanine with an IC₅₀ of 1.3, 510.8 and 9.3 μM, respectively. This study provides an approach that could be applied to screen and evaluate small molecule inhibitors of β-lactamases in whole cells or to identify drug resistant bacteria.