BACKGROUND AND OBJECTIVES: The present study aimed to evaluate the antimicrobial capabilities of biologically synthesized Fe3O4 nanoparticles. MATERIALS AND METHODS: The Fe3O4-NPs were generated using the culture supernatant from the Alcaligenes sp. strain CR8441, with Fe2O3 acting as the precursor. Following synthesis, the nanoparticles were subjected to an extensive analysis using a variety of spectroscopic and microscopic techniques. In the next step of the study the antibacterial activities of Fe3O4 nanoparticle were investigated using disc diffusion method and also determination of MIC and MBC values against four bacterial strains; two-gram positive bacteria, Staphylococcus aureus, Bacillus cereus and two gram negative bacteria, Escherichia coli and Pseudomonas aeruginosa. Then, the anti-virulence effects of nanoparticles such as inhibition of biofilm formation, also tested for synergistic effects with penicillin and Cefixime antibiotics, bacterial motility inhibition, cell membrane disruption and efflux pump inhibition were performed at sub-MIC concentrations. RESULTS AND DISCUSSION: Fe3O4-NPs were synthesized with the confirmation of strong absorption peaks at 235 and 293 nm. According to the analysis, the nanoparticles were found to be spherical or slightly elliptical and have an average size of 36 nm. The DLS results indicated an average size of 53.6 nm for the synthesized Fe3O4-NPs, with a dispersion index of 0.31. The zeta potential of the synthesized Fe3O4-NPs was measured as -2.3 mV. XRD analysis confirmed the formation of cubic spinel Fe3O4-NPs. FTIR analysis revealed that organic compounds containing O-H, C=O, C-O, and N-H groups acted as effective coating agents during the synthesis. Based on the results, the Fe3O4 nanoparticle has acceptable antibacterial activity against both Gram-positive and Gram-negative bacteria (MIC were from 2.5-50 µg.ml-1, and MBC were from 5- 100 µg.ml-1). Also, at sub-MIC concentration, the investigated Fe3O4 nanoparticle has effective anti-virulence factors potential, including: inhibition of biofilm formation at MIC/2 concentration,
