Pubescine as a Novel Antibacterial Agent Against Vancomycin-Resistant Enterococcus: Growth Inhibition, Antibiotic Synergy, and Anti-Biofilm Activity.

Introduction: The rise of Vancomycin-Resistant Enterococcus (VRE) has become a major public health concern due to its resistance to conventional antibiotics and ability to form biofilms. The urgent need for novel therapeutic strategies has led to increased interest in natural compounds with antimicrobial potential. Pubescine (PBN), a steroidal alkaloid isolated from Holarrhena pubescens, has demonstrated antimicrobial properties, but its efficacy against VRE remains unexplored.

Methods: PBN was isolated and purified from Holarrhena pubescens using chromatographic techniques and identified through spectroscopic analysis. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were determined via broth microdilution assays. Time-kill assays assessed the bacteriostatic or bactericidal nature of PBN. Resistance development was evaluated through prolonged bacterial exposure to subinhibitory concentrations. Synergistic interactions with vancomycin and cefoxitin were analyzed using checkerboard microdilution assays. Biofilm formation and eradication were assessed via crystal violet staining and fluorescence imaging. Metabolic activity and oxidative stress induction were measured using the Alamar Blue assay and Reactive Oxygen Species (ROS) quantification, respectively.

Results: PBN exhibited concentration-dependent inhibition of VRE growth, primarily exerting a bacteriostatic effect without promoting the development of resistance. Checkerboard assays revealed strong synergy between PBN and vancomycin (FICI = 0.1875) and cefoxitin (FICI = 0.3125), suggesting that PBN enhances the efficacy of these antibiotics.

Discussion: PBN significantly reduced biofilm formation and facilitated biofilm disruption at concentrations as low as 4 μg/mL. Metabolic assays demonstrated that PBN suppresses bacterial metabolic activity, while ROS quantification indicated a substantial increase in oxidative stress, suggesting a multi-targeted mechanism of action.

Conclusion: These findings establish PBN as a promising antimicrobial agent with potent activity against vancomycin-resistant Enterococcus faecalis. Its ability to enhance antibiotic efficacy, inhibit biofilm formation, and induce oxidative stress underscores its potential as a novel therapeutic strategy against multidrug-resistant infections. Further in vivo studies and pharmacokinetic evaluations are warranted to assess its clinical applicability.