| Summary: | <i>Pseudomonas aeruginosa</i> is an opportunistic gram-negative human pathogen that causes a wide range of infections, including nosocomial infections. Aside from the intrinsic and acquired antimicrobial resistance against many classes of antibiotics, <i>P. aeruginosa</i> can produce an extracellular polymeric matrix called “biofilm” that protects bacteria from antibiotics and harmful factors. Biofilm enables <i>P. aeruginosa</i> to develop chronic infections. This study assessed the inhibitory action of ZnO-nanoparticles against biofilms formed by multidrug-resistant <i>P. aeruginosa</i> strains. A collection of 24 clinical strains of <i>P. aeruginosa</i> were tested for their antimicrobial resistance against different antibiotics using the disk diffusion method. The antibiofilm activity of ZnO-NPs was assessed using the microtiter plate biofilm assay. The application of ZnO-NPs dramatically modulated the resistance profile and biofilm activity of <i>P. aeruginosa</i>. The combination of ZnO-NPs and meropenem showed synergistic antipseudomonal activity with lower MICs. The scanning electron microscope (SEM) micrographs revealed complete inhibition of biofilms treated with the meropenem–ZnO-NPs combination. Reduced expression of biofilm regulating genes <i>lasR</i>, <i>pslA,</i> and <i>fliC</i> was detected, reflecting the enhanced antibiofilm effect of ZnO-NPs. In vivo application of this antimicrobial mixture completely cured <i>P. aeruginosa</i>-induced keratitis in rats. Our findings represent a dual enhancement of antibacterial and antibiofilm activity via the use of meropenem–ZnO-NPs combination against carbapenem-resistant <i>P. aeruginosa</i> infections.
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