Evaluation of Efflux Inhibitory Potential of Gallotannin to restore drug sensitivity in XDR Acinetobacter baumannii in vitro & zebrafish infection model

Abstract

Abstract Acinetobacter baumannii is among the most dreaded nosocomial pathogens and has been designated a "red-alert" organism by the Infectious Diseases Society of America. Its remarkable ability to acquire and upregulate resistance determinants has propelled it to the forefront of pathogens threatening to undermine the antibiotic era. Multidrug-resistant (MDR) A. baumannii infections are associated with high mortality rates (25–68%), particularly among critically ill patients. Efflux-mediated resistance to commonly used antimicrobial agents, coupled with the upregulation of efflux pump genes upon exposure to serum, suggests that A. baumannii exploits efflux systems to achieve its MDR phenotype. Therefore, targeting efflux pumps offers a promising strategy to curb infections caused by this notorious pathogen. The present study aimed to evaluate the efflux inhibitory potential of 13 plant-derived polyphenolics to restore drug sensitivity in MDR/XDR clinical isolates of A. baumannii using in silico, in vitro, and in vivo (zebrafish infection model) approaches. Among the tested compounds, gallotannin demonstrated the most potent activity, causing a 64-fold reduction in the erythromycin MIC in an XDR A. baumannii strain. Real-time efflux and time-dependent accumulation assays confirmed that gallotannin inhibited efflux activity, leading to a 32–64-fold reduction in MICs of three different antimicrobial classes across five MDR/XDR isolates. Gallotannin also exhibited persistent hydrogen bonding with AdeB efflux pump throughout the molecular dynamic simulation studies. In time-kill assay, relative to the untreated control, gallotannin–erythromycin combination achieved a ~5-log reduction in CFU. Consistent with the in vitro results, the combination also produced a ~4-log reduction in bacterial bioburden in infected zebrafish. Mechanistic investigations revealed that gallotannin enhances bacterial membrane permeability, with the combination treatment increasing intracellular accumulation of dansyl chloride–tagged erythromycin more effectively than colistin. While gallotannin did not affect membrane potential, the combination induced significantly elevated reactive oxygen species (ROS) levels, exceeding those generated by hydrogen peroxide. In summary, gallotannin potentiates the antibacterial activity of erythromycin by enhancing its permeability, inhibiting efflux, and promoting ROS-mediated bacterial killing, resulting in a marked reduction of A. baumannii bioburden both in vitro and in vivo.