Supplementary file 1_Evaluation of the efflux inhibitory potential of gallotannin to restore drug sensitivity in XDR Acinetobacter baumannii in vitro, and a zebrafish infection model.docx

IntroductionAcinetobacter baumannii is a highly problematic nosocomial pathogen, designated a “red alert” organism by the Infectious Diseases Society of America. Multidrug-resistant (MDR) strains are associated with mortality rates of 25–68%, particularly in critically ill patients. Efflux-mediated resistance, along with serum-induced upregulation of efflux pump genes, highlights the role of efflux systems in driving MDR phenotypes. Targeting these systems represents a promising therapeutic strategy.
MethodsThe efflux inhibitory potential of 13 plant-derived polyphenols was evaluated to restore antibiotic susceptibility in MDR/XDR clinical isolates of A. baumannii. Approaches included in silico molecular docking and molecular dynamics simulations, in vitro assays (MIC reversal, real-time efflux, checkerboard synergy, intracellular accumulation, membrane permeability, and ROS generation), and in vivo bioburden analysis using a zebrafish infection model.
Results and DiscussionGallotannin exhibited the strongest activity, achieving a 64-fold reduction in erythromycin MIC in an XDR strain. Efflux inhibition was confirmed through real-time efflux and accumulation assays, showing 32–64-fold MIC reductions across multiple antibiotic classes in five MDR/XDR isolates. Molecular simulations revealed stable binding of gallotannin to the AdeB efflux pump via persistent hydrogen bonding. Combination therapy resulted in a ∼5-log CFU reduction in vitro and ∼4-log reduction in zebrafish bioburden. Mechanistically, gallotannin enhanced membrane permeability and intracellular antibiotic accumulation, without affecting membrane potential. The combination also induced significantly elevated ROS levels.Gallotannin potentiates erythromycin activity by inhibiting efflux, increasing permeability, and promoting ROS-mediated killing, significantly reducing A. baumannii burden in vitro and in vivo.