In Vitro antibacterial, anti-biofilm, and virulence-modulating activity of Cinnamomum verum-mediated iron oxide nanoparticles against carbapenem-resistant uropathogenic Escherichia coli.

Abstract

Background: Carbapenem-resistant Enterobacteriaceae (CRE) represent a critical antimicrobial resistance threat globally, with uropathogenic Escherichia coli (UPEC) CRE isolates increasingly documented in both community and healthcare settings across the Middle East and worldwide. Conventional antibiotic options for CRE UPEC infections are severely constrained, with susceptibility typically retained only to aminoglycosides and fosfomycin, necessitating urgent development of alternative therapeutic strategies.

Methods: Iron oxide nanoparticles (Fe₂O₃-NPs) were synthesised using aqueous bark extract of Cinnamomum verum, rich in trans-cinnamaldehyde and eugenol. The synthesised Fe₂O₃-NPs were characterised by UV-Visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), field-emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (EDX), atomic force microscopy (AFM), and X-ray powder diffraction (XRD). Antibacterial and anti-biofilm activities were evaluated against two CRE UPEC clinical isolates confirmed by VITEK 2 (carbapenem MIC values: ertapenem ≥ 8 mg/L, imipenem ≥ 16 mg/L, meropenem 8 mg/L). Virulence gene expression was quantified by RT-qPCR using the 2⁻ᴰᴰᶜᵗ method, targeting csgD and fimH with 16 S rRNA as the reference gene.

Results: The C. verum extract displayed a UV absorption maximum at 300 nm, which shifted to 290 nm in the Fe₂O₃-NP preparation, consistent with nanoparticle formation. FTIR confirmed Fe-O stretching at 621.08 cm⁻¹ with retained phytochemical surface-capping bands. FE-SEM revealed a mean particle diameter of 65.63 nm; AFM confirmed a mean of 49.23 nm (n = 905 particles). Elemental analysis confirmed the formation of iron oxide nanoparticles with phytochemical capping, while XRD indicated a predominantly amorphous phase consistent with room-temperature biogenic synthesis. The nanoparticle preparation exhibited an MIC of 0.19 mg/mL against the tested CRE UPEC culture, compared with 25 mg/mL for the crude extract. Anti-biofilm activity reached 93.4% (blank-corrected). RT-qPCR indicated reduced csgD and fimH expression in the two evaluable treated samples; these preliminary findings require confirmation using additional biological replicates.

Conclusion: These proof-of-concept findings indicate that C. verum-mediated Fe₂O₃-NPs show potent in vitro multi-mechanism antibacterial activity against two clinically confirmed CRE UPEC isolates, combining promising antibacterial potency with transcriptional suppression of key virulence determinants; validation in a larger isolate panel is required to confirm generalisability. These findings identify the nanoparticle preparation as a promising in vitro candidate for further antibacterial and anti-virulence investigation. However, cytotoxicity, biocompatibility, validation using a larger collection of clinical isolates, and in vivo studies are required before any therapeutic application can be considered.