Biogenic spinel Fe₃O₄ nanoparticles from Alcaligenes sp. NV06 inhibit bacterial growth, biofilms, and swarming motility

The increasing prevalence of multidrug-resistant (MDR) bacteria has created an urgent need for antimicrobial agents with novel mechanisms of action. In this study, we report the extracellular biosynthesis of spinel Fe₃O₄ nanoparticles (NPs) using the cell-free supernatant (CFS) of Alcaligenes sp. strain NV06. To the best of our knowledge, this is the first report of Fe₃O₄ nanoparticle biosynthesis using the CFS of this strain. The NPs were synthesized from soluble Fe(III) and Fe(II) precursors, and their antibacterial and anti-virulence activities were subsequently evaluated. UV–visible spectroscopy revealed a distinct absorption band at 228–235 nm, confirming nanoparticle formation. FE-SEM analysis showed predominantly quasi-spherical NPs with some compact agglomerates. The mean diameter of the dried NPs was 36.00 ± 8.48 nm. DLS analysis showed a larger hydrodynamic diameter, with a Z-average hydrodynamic diameter of 53.6 nm and a PDI of 0.43. FTIR analysis identified biomolecule-associated functional groups on the nanoparticle surface, which may have contributed to surface capping and steric stabilization. However, the low zeta-potential value (− 3.2 mV) indicated limited electrostatic stabilization. The spinel IONPs exhibited antibacterial activity, with MIC values ranging from 6.25 to 100 µg/mL and MBC values from 12.5 to 400 µg/mL. At a sub-inhibitory concentration (½ MIC), the NPs compromised bacterial membrane integrity, as indicated by the crystal violet (CV) uptake assay. The NPs also markedly inhibited biofilm formation, with Gram-positive bacteria showing greater susceptibility than Gram-negative strains. Synergistic interactions were observed between the NPs and penicillin (against S. aureus and B. cereus ) as well as cefixime (against E. coli and P. aeruginosa ), with fractional inhibitory concentration index (FICI) values < 0.75. In the norA-overexpressing strain S. aureus SA-1199B, the NPs increased intracellular ethidium bromide accumulation, suggesting inhibition of efflux pump activity. At ½ MIC, they also significantly reduced the swarming motility of E. coli and P. aeruginosa . Overall, the biosynthesized spinel Fe₃O₄ NPs displayed antibacterial, anti-biofilm, and anti-virulence activities. Their synergistic interactions with selected antibiotics highlight their potential as adjunctive agents against MDR bacteria, although further in vivo studies are required to assess their therapeutic applicability. Clinical trial number Not applicable.