Biogenic silver nanoparticles optimization using Plackett–Burman design and its synergistic effect with cefotaxime against multidrug resistant clinical isolates

The rise in antibiotic resistance has created an urgent need for alternative strategies to combat multidrug-resistant (MDR) bacterial infections. Silver nanoparticles (AgNPs) possess unique antibacterial properties, making them a promising option in biomedical applications. This study explores the green synthesis of silver nanoparticles (AgNPs) using Citrus sinensis peel extract and their synergistic potential with cefotaxime against multidrug-resistant (MDR) clinical isolates. For the optimization of AgNPs synthesis, Plackett-Burman experimental design (PBD) was implemented that demonstrated incubation time, temperature and extract: AgNO₃ ratio as significant factors. The UV-Vis spectroscopy analysis revealed a characteristic absorbance peak of CS-AgNPs at 470 nm. The size of biosynthesized AgNPs was analyzed using Scanning Electron Microscopy (SEM), that showed size range of 50-60 nm with spherical shaped morphology. Fourier Transform Infrared Spectroscopy (FTIR) analysis found different functional groups involved in the stabilization and capping of AgNPs, as indicated by the peaks at 2925 cm^-1, 1630 cm^-1, 1100 cm^-1 and 1016 cm^-1 revealing -CH stretching aliphatic carbon, the carboxyl group, OH group and C-O-C group, respectively. The cytotoxicity of the synthesized CS-AgNPs and its synergistic effect with cefotaxime (CTX) antibiotic was analyzed with MTT assay. The combination of CS-AgNPs and CTX showed significant decrease in cytotoxicity compared to CS-AgNPs alone. Antibacterial activity of CS-AgNPs against MDR clinical isolates was performed using minimum inhibitory concentration (MIC) method. The MIC of CS-AgNPs was observed within 3.125-12.5 µg/ml range. Synergism assay of CS-AgNPs with CTX was also evaluated to determine the fractional inhibitory concentration (FIC) index. Clinical isolates (E. coli), S-11, S-14, S-16, S-19, and S-20 showed FIC in the range of 0.162-0.402 indicating synergism whereas, S-04, S-06, S-10, S-15 and S-21 showed the FIC in the range of 0.644-0.804 indicating the additive effect. The MDR E. coli clinical isolates S-11, S-16, S14, S-19 and S-20 demonstrated 65-85% biofilm inhibition which was significantly (p ≤ 0.001) high in all tested isolates. Significant (p ≤ 0.001) eradication of preformed biofilm in the range of 60-78% was also observed in S-16 clinical isolate.