Synergistic antibacterial effects of berberine, baicalin, and plumbagin against carbapenem-resistant Acinetobacter baumannii: Mechanistic exploration

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a high-priority nosocomial pathogen with strong biofilm-forming capacity and limited treatment options. We evaluated three traditional Chinese medicine–derived compounds (berberine, baicalin, and plumbagin) against ten clinical CRAB isolates and investigated OmpA-linked mechanistic evidence. Antibacterial activity was assessed by broth microdilution minimum inhibitory concentration (MIC) testing; drug interactions were assessed using checkerboard assays, expressed as the fractional inhibitory concentration index (FICI) and summed FICI (∑FICI); and the triple combination was further evaluated by time–kill curve. Anti-biofilm activity was quantified using crystal violet staining supported by scanning electron microscopy (SEM) with image-based measurements, while molecular docking and molecular dynamics (MD) and RT-qPCR were used to examine OmpA binding and ompA transcriptional responses. Plumbagin showed the lowest MICs (16–128 µg/mL), whereas berberine and baicalin exhibited MICs ≥1024 µg/mL. Checkerboard assays indicated synergy for berberine + baicalin (FICI ≤ 0.5), predominantly additive effects for berberine + plumbagin (9/10 isolates; one isolate classified as indifferent), indifferent interactions for baicalin + plumbagin (1.0 < FICI ≤ 4.0), and robust synergy for the triple combination across all isolates (ΣFICI 0.188–0.406). In time–kill assays, ≥4×MIC triple therapy achieved rapid and sustained killing, reaching counts below the detection limit within 4 h in 9/10 isolates without regrowth. Biofilm biomass, adherent cell counts, and projected surface area were markedly reduced. Docking/MD suggested preferential baicalin binding to the OmpA N-terminal β-barrel, and RT-qPCR revealed compound-specific ompA responses, with the triple combination reducing ompA to 0.5-fold relative to the untreated control. Collectively, these findings support a multi-target, three-compound strategy against CRAB, with consistent synergy and OmpA-linked biofilm-associated mechanisms.