Metallo-β-lactamase-producing Enterobacterales: cyanide-containing efflux pump inhibitors as potential dual-activity
Otávio Hallal Ferreira Raro1, Csaba Szabo2, Patrice Nordmann1,3
1Faculty of Science and Medicine, Medical and Molecular Microbiology Unit-Department of Oncology, Microbiology, Immunology (OMI), University of Fribourg, Fribourg CH-1700, Switzerland.
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.
Insights
Certain cyanide compounds like CCCP, FCCP, and KCN can restore carbapenem susceptibility in VIM-1-like metallo-β-lactamase producing bacteria. These compounds act as novel inhibitors of MBL enzymatic activity, offering potential as adjunct antibacterial agents.
Area of Science:
- Microbiology
- Biochemistry
- Pharmacology
Background:
- Metallo-β-lactamase (MBL)-producing Enterobacterales are a significant public health concern due to resistance to most β-lactams.
- Prevalent MBLs (VIM, IMP, NDM) are unaffected by current β-lactamase inhibitors.
Purpose of the Study:
- To investigate if cyanide-containing molecules can restore carbapenem susceptibility in MBL-producing bacteria.
- To explore the potential of efflux pump inhibitors (CCCP, FCCP) and potassium cyanide (KCN) as MBL inhibitors by targeting zinc-dependent activity.
Main Methods:
- Antimicrobial susceptibility testing (broth microdilution, checkerboard assays) was performed on E. coli and clinical strains.
- Enzymatic activity was assessed via IC50 determination and kinetic assays.
- Time-kill curve assays evaluated bacterial growth reduction.
Main Results:
- CCCP, FCCP, and KCN restored imipenem susceptibility in VIM-1-like MBL-producing E. coli strains.
- Synergistic effects and reduced bacterial growth were confirmed, with Zn2+ reversing the activity, suggesting a zinc-chelating mechanism.
- Inhibitory effects were selective for VIM-1-like MBLs, with no impact on VIM-2, IMP-1, or NDM-1.
Conclusions:
- CCCP, FCCP, and KCN demonstrate novel inhibitory activity against VIM-1-like MBLs at low concentrations.
- These compounds restore carbapenem susceptibility, indicating potential as adjunct antibacterial agents.
- Cyanide-containing molecules offer a new strategy for combating MBL-mediated antibiotic resistance.
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