Metallo-β-lactamase-producing Enterobacterales: cyanide-containing efflux pump inhibitors as potential dual-activity inhibitors.

BACKGROUND AND

OBJECTIVES

Metallo-β-lactamase (MBL)-producing Enterobacterales represent a major public health threat because they confer resistance to all β-lactams except monobactams. The most prevalent MBLs include VIM, IMP, and NDM types, which render all clinically approved β-lactamase inhibitors ineffective. This study evaluates whether cyanide-containing molecules such as the efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP), carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP) or the cytochrome C oxidase inhibitor potassium cyanide (KCN) can restore carbapenem susceptibility in MBL-producing strains by disrupting zinc-dependent enzymatic activity.

METHODS

A series of isogenic Escherichia coli TOP10 and MG1655 strains carrying the shuttle vector pUCP24 harbouring various MBLs and clinical strains were included in this study. The strains were subjected to antimicrobial susceptibility testing by broth microdilution, checkerboard assays, determination of the 50% inhibitory concentration (IC50) of carbapenemase activity, kinetic assays and time-kill curve assays.

RESULTS

CCCP, FCCP and KCN restored carbapenem (imipenem) susceptibility in E. coli strains producing VIM-1-like enzymes. Synergy was observed in checkerboard assays, and reduction of bacterial growth was confirmed by time-kill experiments. The addition of Zn2+ reversed this effect, supporting a Zn2+-chelating or Zn2+-displacing mechanism. Non-VIM-1-like MBLs, including VIM-2, IMP-1 and NDM-1, were not affected by the addition of cyanide-containing compounds, indicating strong selectivity of these inhibitory effects. IC50 measurements confirmed the selective activity of CCCP, FCCP and KCN against VIM-1-like MBLs.

CONCLUSIONS

CCCP, FCCP and KCN exhibited inhibitory activity against VIM-1-like MBLs, restoring imipenem susceptibility in E. coli at very low concentrations (0.04-19.55 µM). Thus, certain efflux pump inhibitors, as well as the gaseous biological mediator cyanide, have a novel action as inhibitors of MBL enzymatic activity, which, in turn, renders them potentially useful as adjunct antibacterial agents.