Luteolin potentiates antibiotic activity against multidrug-resistant Pseudomonas aeruginosa and modulates efflux-associated resistance and biofilm-associated phenotypes

Abstract Multidrug-resistant Pseudomonas aeruginosa is a WHO critical-priority pathogen whose recalcitrance is driven largely by efflux pump overexpression and biofilm formation, two mechanisms rarely addressed by a single agent. Here we evaluated whether luteolin, a plant-derived flavone, could potentiate conventional antibiotics and suppress biofilm-associated phenotypes in multidrug-resistant P. aeruginosa . Checkerboard assays against PAO1 and 20 multidrug-resistant clinical isolates revealed predominantly additive interactions with no antagonism: luteolin–ceftazidime combinations were additive in 16 of 21 strains (indifferent in 5), and luteolin–ciprofloxacin combinations were additive in 18 of 21 strains (indifferent in 3); luteolin reduced the ceftazidime and ciprofloxacin MICs by 2- to 8-fold across the entire panel. At sub-inhibitory concentrations, luteolin inhibited biofilm formation by up to 71%, disrupted pre-established mature biofilms, reduced extracellular polysaccharide and DNA content, and suppressed swimming and swarming motility in a concentration-dependent manner; these anti-biofilm effects were further enhanced by combination with either antibiotic. Functional assays showed increased intracellular ethidium bromide retention, accompanied by transcriptional downregulation of efflux, biofilm-matrix, quorum-sensing, and flagellar genes. Luteolin also modestly increased outer membrane permeability while producing limited effects on inner membrane integrity, suggesting that both efflux-associated and membrane-related effects may contribute to its antibiotic-potentiating activity. Molecular docking suggested that luteolin may occupy a binding pocket within the MexB transporter, providing a plausible structural hypothesis pending experimental confirmation. In cell culture and an invertebrate infection model, luteolin exhibited low cytotoxicity, reduced macrophage injury during infection, and its combination with ceftazidime markedly improved larval survival in a Galleria mellonella infection model, suggesting that luteolin warrants further preclinical investigation as a multi-target antibiotic adjuvant candidate against multidrug-resistant P. aeruginosa .