Synergistic Effect and Mechanism of Plumbagin with Gentamicin Against Carbapenem-Resistant Klebsiella pneumoniae

Background: Aminoglycosides are one of a few susceptible antimicrobials available for carbapenem-resistant Enterobacteriaceae (CRE). However, the altered pharmacokinetics and increasing drug resistance of aminoglycosides will make them hardly effective if used in monotherapy. The purpose of this study was to identify herbal compounds that potentiate the antibacterial effect of gentamicin against carbapenem-resistant Klebsiella pneumoniae (CRKp) with gentamicin resistance and explore the action mechanisms. Methods: A collection of 280 Chinese herbal compounds was screened for synergistic effect with gentamicin against CRKp by broth microdilution method according to the standard of the Clinical and Laboratory Standards Institute (CLSI). Intracellular gentamicin was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The membrane potential was evaluated by BacLight TM Bacterial Membrane Potential Kit. Plumbagin-induced metabolite changes of vital metabolic pathways were measured by an optimized untargeted metabolomics method based on gas chromatography-mass spectrometer (GC/MS). Intracellular nicotinamide adenine dinucleotide (NADH) was detected via EnzyChrom NAD/NADH assay kit. Results: We identified plumbagin to remarkably potentiate the antimicrobial activity of gentamicin against the CRKp with gentamicin resistance. Plumbagin at 100 mu M could bring the MIC of gentamicin from >16 mu g/mL to similar to 4 mu g/mL despite its minimal inhibitory effect on the CRKp. A similar synergistic effect with gentamicin was also observed in an antibiotics-susceptible strain of Klebsiella pneumoniae. Compared with gentamicin monotreatment, the combination group showed a higher intracellular concentration of gentamicin and increased membrane potential in CRKp. Metabolomics analysis indicated remarkable increases of malate and alpha-ketoglutarate in the tricarboxylic acid (TCA) cycle in the CRKp upon plumbagin treatment. Further analysis revealed higher intracellular NADH concentration in plumbagin-treated CRKp, supporting increased proton-motive force (PMF) that facilitates aminoglycosides uptake. Conclusion: Herbal compound plumbagin was identified to stimulate gentamicin uptake by CRKp via enhancing TCA efflux and PMF to achieve a synergistic antibacterial effect. Plumbagin may be used in combination with aminoglycosides for severe CRKp infection by potentiating their therapeutic efficacy and lowering dosage.