Strain-Specific Bacillus subtilis-Derived Silver Nanoparticles for Effective Antibacterial Activity Against Multidrug-Resistant Pathogens: In Vitro Model

Priyanka Singh,1 Ivan Mijakovic1,2 1The Novo Nordisk Foundation, Center for Biosustainability, Technical University of Denmark, Kogens Lyngby, DK-2800, Denmark; 2Systems and Synthetic Biology Division, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, SE-412 96, SwedenCorrespondence: Priyanka Singh, Email [email protected] Ivan Mijakovic, Email [email protected]: Nanoparticles, particularly silver and gold nanoparticles, have emerged as powerful tools for improving drug stability, bioavailability, and targeted delivery in medical applications. With the increasing global demand for nanoparticles (NPs), sustainable and biocompatible production methods are crucial. This study investigates the biosynthesis of silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) using two genetically related bacterial strains, Bacillus subtilis 168 and Bacillus subtilis 3610, and compares their synthesis efficiency and the antimicrobial potential of the generated NPs.Methods: AgNPs were synthesized extracellularly from bacterial cell supernatants, whereas AuNPs were generated intracellularly by incubating washed cell pellets with gold salt. The nanoparticles were characterized using UV–Vis spectroscopy, sp-ICP-MS, TGA, SEM, TEM, FTIR, MALDI-TOF, zeta potential, and AFM. Antibacterial activity against Pseudomonas aeruginosa and Escherichia coli was evaluated using minimum bactericidal concentration (MBC) assays, live/dead fluorescence staining, and SEM-based morphological analysis.Results: Both strains synthesized AgNPs and AuNPs, but Bacillus subtilis 3610 exhibited faster and more efficient production. AgNPs displayed uniform spherical morphology, whereas AuNPs were polydisperse and membrane-associated. AgNPs synthesis was completed within 2 days by strain 3610 compared with 3 days by strain 168. Antibacterial assays revealed markedly stronger efficacy of 3610-derived AgNPs, with MBC values of 2 μg/mL against P. aeruginosa and 8 μg/mL against E. coli, in contrast to 16 μg/mL and 32 μg/mL, respectively, for 168-derived AgNPs.Conclusion: This study highlights the superior NPs biosynthesis capacity and antibacterial potency of the wild-type strain B. subtilis 3610 compared to the domesticated laboratory strain 168. The findings emphasize the critical influence of strain-specific genetic and metabolic traits in optimizing microbial nanomaterial production and position 3610-derived AgNPs as promising candidates for biomedical applications. Keywords: B. subtilis, silver nanoparticles, gold nanoparticles, simultaneous production, antimicrobial resistance, multidrug-resistant pathogens, stability