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.