The aim of this study was to determine whether the antibacterial activity of chitosan-modified Fe₃O₄ (CS@Fe₃O₄) nanomaterials against Acinetobacter baumannii (A. baumannii) is mediated through changes in biofilm formation and reactive oxygen species (ROS) production. For this purpose, the broth dilution method was used to examine the effect of CS@Fe₃O₄ nanoparticles on bacterial growth. The effects of CS@Fe₃O₄ nanoparticles on biofilm formation were measured using a semi-quantitative crystal violet staining assay. In addition, a bacterial ROS detection kit was used to detect the production of ROS in bacteria. The results showed that CS@Fe₃O₄ nanoparticles had a significant inhibitory effect on the colony growth and biofilm formation of drug-resistant A. baumannii (p < 0.05). The ROS stress assay revealed significantly higher ROS levels in A. baumannii subjected to CS@Fe₃O₄ nanoparticle treatment than the control group (p < 0.05). Thus, we demonstrated for the first time that CS@Fe₃O₄ nanoparticles had an inhibitory effect on A. baumannii in vitro, and that the antibacterial effect of CS@Fe₃O₄ nanoparticles on drug-resistant A. baumannii was more significant than on drug-sensitive bacteria. Our findings suggest that the antibacterial mechanism of CS@Fe₃O₄ nanoparticles is mediated through inhibition of biofilm formation in drug-resistant bacteria, as well as stimulation of A. baumannii to produce ROS. In summary, our data indicate that CS@Fe₃O₄ nanoparticles could be used to treat infections caused by drug-resistant A. baumannii.

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