Bacterial enzyme-mediated synthesis of silver nanoparticles and antimicrobial activity

Abstract

Extracellular synthesis of silver nanoparticles (AgNPs) using bacteria has been explored for their unique physicochemical properties. Studies have shown that nitrate reductase enzyme catalyzes the bioreduction of Ag+ to Ag0 and formation of AgNPs. The objective of this study was to detect the presence/absence of nitrate reductase enzyme in selected bacteria and to study the formation of AgNPs. The antimicrobial activity of the biosynthesized AgNPs was also examined. Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Acinetobacter baumannii (confirmed clinical strain) and Staphylococcus aureus ATCC 25923 were cultured in Nutrient broth. After 72 h of incubation, AgNO3 was added into the culture supernatant. AgNP formation was confirmed by Energy Dispersive X-ray analysis (EDX) and Transmission Electron Microscopy (TEM). For the nitrate reductase assay, heavy inocula of the above selected bacteria were inoculated in Nitrate broth and incubated at 37°C for 24 h. One dropper full of sulfanilic acid and α-naphthylamine were added to each tube and the colour change was observed. If no color change was observed a small amount of zinc (Zn) powder was added and the color change was observed. Well-diffusion method was performed to study the antimicrobial activity of the synthesized AgNPs against E. coli ATCC 25922, S. aureus ATCC 25923, P. aeruginosa ATCC 27853, Candida albicans ATCC 10231 and selected clinical isolates of P. aeruginosa, S. aureus and C. albicans. Positive controls were 0.5% AgNO3 and chemically synthesized AgNPs (0.436 mg/ml). All biosynthesized AgNPs were spherical in shape. The average sizes of the NPs were 11.14 ± 6.59 nm (S. aureus-NPs 0.435 mg/ml), 11.71 ± 2.73 nm (P. aeruginosa-NPs 0.45 mg/ml), 12.87 ± 2.95 nm (E. coli-NPs 0.99 mg/ml) and 12.22 ± 2.45 nm (A. baumannii-NPs 0.665 mg/ml). In general, zones of inhibition (ZOIs) given for chemically synthesized AgNPs, were higher than biosynthesized NPs. According to the well diffusion results, AgNPs produced by S. aureus resulted in the largest ZOI against the selected pathogens. Biosynthesized AgNPs were highly effective against Gram negative bacteria compared to Gram positive bacterial and fungal species, as well as Candida albicans, which were opportunistic pathogens. A. baumannii, E. coli and S.aureus, except P. aeruginosa, gave red colour after adding the two reagents and when Zn dust was added to P. aeruginosa, no colour change was observed. AgNP synthesis with a narrow size distribution was observed for all tested bacterial strains. AgNPs of S. aureus gave highest ZOI. Nitrate reduction was observed with all organisms. Further characterization of NPs is required to study the physical properties of silver NPs