Green Synthesis of CuFe2O4@Ag Nanocomposite Using the Chlorella vulgaris and Evaluation of its Effect on the Expression of norA Efflux Pump Gene Among Staphylococcus aureus Strains

Abstract

Increasing drug resistance among Staphylococcus aureus is a global health threat and finding alternative antimicrobial agents against it has been considered. Multidrug resistance efflux pumps, including NorA, are involved with resistance to different drugs, especially fluoroquinolones, in S. aureus. Using metal nanoparticles against pathogenic bacteria is a promising approach; however, physio-chemical synthesis of nanoparticles has limitations. Biosynthesis of metal nanoparticles with antibacterial activity has gained interest, recently. In this study, biosynthesis of CuFe₂O₄@Ag nanocomposite using aqueous extract from microalgae Chlorella vulgaris was performed, and its antibacterial property and effect on expression of norA efflux pump gene were investigated. Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), energy dispersive X-ray mapping analysis (EDX-map), differential reflectance spectroscopy (DRS), and dynamic light scattering (DLS) were used to characterize synthesized nanocomposite. Antibacterial activity of the prepared nanocomposite individually and combined with ciprofloxacin against S. aureus strains was evaluated using the disk assay method, and minimum inhibitory concentration (MIC) of each agent was determined using the broth dilution method. Anti-biofilm activity of this nanocomposite was checked. Finally, the effect of CuFe₂O₄@Ag nanocomposite alone and in combination with ciprofloxacin on the expression of norA was assessed by real-time PCR. The physical analysis revealed proper synthesis of spherical and well-dispersed CuFe₂O₄@Ag nanocomposite with an average diameter of 20 nm. Synthesized nanocomposite had synergistic antibacterial activity with ciprofloxacin. Moreover, expression of norA gene among clinical and standard strains treated with CuFe₂O₄@Ag nanocomposite combined with ciprofloxacin reduced by 59% and 65%, respectively. Thus, CuFe₂O₄@Ag nanocomposite synthesized in this study can be considered as a promising candidate to be used to inhibit staphylococcal efflux pump genes and increasing the antibiotic efficacy.

Keywords: Efflux pump; Ferrite; Nanocomposite; S. aureus; norA.