γ-Fe2O3@Prussian blue nanozyme-driven colorimetric detection of antimicrobial susceptibility in bacterial culture

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Antimicrobial resistance has emerged as a significant threat to global public health, necessitating accurate and rapid detection methods. To address this need, a colorimetric nanobiosensor was developed that exploits antibiotic-induced metabolic changes in bacteria, detected via the redox-dependent activity of a peroxidase-mimicking nanozyme (γ-Fe₂O₃@Prussian blue). This study aimed to differentiate resistant from susceptible E. coli strains and determine the minimum inhibitory concentration for susceptible ones using this novel principle. The experiments were conducted on two susceptible and two resistant strains of E. coli along with antibiotics, including ampicillin, cefazolin, ceftriaxone, and kanamycin. The γ-Fe 2 O 3 @PB NPs were characterized using UV-vis spectroscopy, dynamic light scattering (DLS), X-ray diffraction (XRD), transmission electron microscopy, and Fourier-transform infrared spectroscopy (FTIR). The synthesis of cubic crystalline nanoparticles with average crystallite size and hydrodynamic size of 31 nm and 219 nm, respectively, was confirmed. Upon adding H₂O₂ and the chromogenic substrate TMB to the bacterial culture supernatant, the intensity of the blue color (measured at 652 nm) in sensitive strains correlated with antibiotic concentrations. Compared to sub-MIC concentrations, a significant and sharp increase in absorbance at 652 nm was observed at the MIC, and similarly high absorbance levels were maintained at higher concentrations. By comparing the absorbance levels below and at the MIC, the MIC range can be determined. In contrast, for resistant strains, the intensity of the produced color remained nearly constant across different antibiotic concentrations. This innovative, label-free approach offers a simple and reliable method for antibiotic susceptibility testing. It achieves results in approximately 4 hours, with a detection limit (initial bacterial count) of 1.67×10 7 CFU/mL, without requiring expensive reagents or equipment.

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