Amylase-silver nanocomposites to combat antibiotic resistance

In the present work, silver-based antimicrobial nanocomposites were prepared using alpha-amylase and silver nitrate as precursors. The enzyme acted as both a reducing and stabilizing agent, converting the metal salt into nanoparticles, as verified by a distinct absorption peak at 424 nm. In addition to the characteristic peak of silver nanoparticles, another peak around 260-280 nm confirms the presence of alpha-amylase. Fourier transform infrared (FTIR) spectroscopy also confirmed the association of alpha-amylase with the nanoparticles, validating the synthesis of amylase-silver nanocomposites. The resulting nanocomposites were analyzed through dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Microscopic examination revealed uniformly distributed, spherical nanoparticles with sizes ranging between 9 and 20 nm. The antibacterial efficacy of the amylase-silver nanocomposites was assessed by determining the minimum inhibitory concentration (MIC) against multidrug-resistant (MDR) isolates of Pseudomonas aeruginosa and Staphylococcus aureus, as well as against a dual-species co-culture comprising both organisms. The results demonstrated that the MIC value remained consistent at 15.625 µg/mL across all three experimental conditions, indicating comparable inhibitory effectiveness against individual as well as mixed bacterial populations. Additionally, the synergistic effects of the nanocomposites with antibiotics (kanamycin, gentamicin, and amoxicillin) were also investigated. The results demonstrate enhanced antibacterial efficacy in most combinations, suggesting a synergistic interaction. The nanocomposites also exhibit significant antibiofilm activity, confirmed by crystal violet (CV) staining and microscopic analysis. Furthermore, hemocompatibility evaluation using human red blood cells (RBCs) demonstrated ≤ 5% hemolysis even at concentrations up to four times the minimum inhibitory concentration (4 × MIC), indicating minimal membrane-disruptive effects. In parallel, cytotoxicity assessment via the MTT assay on the HaCaT cell line yielded an IC₅₀ value of 48.2 μg/mL, which is substantially higher than the MIC. This disparity between antimicrobial potency and cytotoxic threshold underscores the excellent biocompatibility and antimicrobial efficacy of the synthesized amylase-silver nanocomposites.