Enhancing colistin efficacy against multidrug-resistant Klebsiella pneumoniae using farnesol-loaded lipid nanoparticles
Résumé
Background We previously demonstrated that farnesol-loaded lipid nanoparticles (F-LNPs) enhance colistin (CST) efficacy against Acinetobacter baumannii [1] and Escherichia coli [2]. In this study, we investigated the broader applicability of F-LNPs againstother Gram-negative bacteria, focusing on mechanisms of action and CST resistance prevention. Methods Checkerboard assays evaluated F-LNPs as a CST adjuvant against Pseudomonas aeruginosa (n=6) and Klebsiella pneumoniae (n=18) isolates. Effects on the bacterial inner (IM) and outer membrane (OM) were studied, followed by resistanceprevention via serial MICs and zeta potential (ZP) measurements. The efficacy of the combinations was evaluated in Galleriamellonella larvae infection models. Results F-LNPs had minimal impact on P. aeruginosa but significantly enhanced CST efficacy against all K. pneumoniae isolates in aconcentration- and isolate-dependent manner, achieving up to 1024-fold MIC reduction for an mcr1isolate (Fig.1). Mechanistic studies using nitrocefin, propidium iodide, and SYTOX™ blue showed that F-LNPs alone increased OM and IMpermeability, effects further amplified in combination with CST (Fig.2A-C). To further investigate OM destabilisation, thecombination was tested on CST-resistant K. pneumoniae strains with LPS modifications incorporating phosphoethanolamine (mcr-1), 4-amino-4-deoxy-L-arabinose (arnT), or both. F-LNPs enhanced CST efficacy by 4- to 1024-fold across all isolates,with no clear correlation between LPS modification type/extent and efficacy enhancement. This suggests that F-LNPs do notspecifically interact with lipid A. Additionally, electron microscopy revealed no visible OM alteration with F-LNPs alone,indicating minimal OM destabilisation by F-LNPs alone (Fig.2D). Resistance prevention studies demonstrated that in mcr1 K.pneumoniae , CST alone led to resistance after 11 passages (MIC≥1024 mg/L), while CST-F-LNP combination maintained alow MIC (0.03 mg/L). High CST resistance was associated with a significant ZP shift (-25.5±3.1 to -14.4±2.9 mV), whereasCST-F-LNP preserved a near-initial ZP (Fig.3). Similar trends were observed in arnT and mcr1-arnT bearing isolates. In vivo ,CST-F-LNP combination improved survival in larvae infected with 10^6 CFU/larva, achieving 43% survival on day 7, comparedto 0% with CST alone. Conclusions Our results highlight the potential of CST-F-LNP combinations to overcome K. pneumoniae resistance by enhancingmembrane permeabilisation and reducing resistance development.
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