The misuse of antibiotics is causing widespread antibiotic resistance, creating an urgent need for new treatment options such as nanoparticle-based therapies. This study aimed to compare silver nanoparticles (AgNPs) produced via green synthesis methods with those made through traditional chemical processes. Furthermore, the study investigated and contrasted the bacterial responses to these two types of AgNPs over a 21-day period of selection pressure using experimental evolution techniques. Analysis using scanning electron microscopy and transmission electron microscopy revealed a consistent, uniform morphology among the AgNPs produced via chemical methods. In contrast, AgNPs synthesized through green methods displayed an irregular morphology. Despite these morphological differences, all nanoparticles from both synthesis approaches were under 100 nm in diameter. These findings were further supported by the absorption spectrum data, which showed a maximum absorption peak between the 400 and 500 nm wavelength range. E. coli exposed to green synthesized AgNPs for 21 days adapted to their presence, exhibiting both enhanced resistance to the green synthesized AgNPs themselves and the development of cross-resistance to ionic silver, a pattern not observed in chemically synthesized AgNP-selected populations. Populations selected using chemical synthesized AgNPs did not develop increased resistance to either chemically or green synthesized AgNPs; however, they showed a slight increase in resistance to ionic silver. Genomics analysis identified polymorphism in genes in a green synthesized AgNP-resistant line including but not limited to the multidrug efflux transporter system (EmrAB), DUF4756 family protein (D1792_RS05680), putative zinc-binding protein YnfU/cold shock-like protein (ynfU/cspB) and imcF-related family protein (D1792_RS10035). Bacterial resistance to chemical AgNPs involves specific polymorphisms in key bacterial components like the RNA polymerase sigma factor (RpoE) and the EmrAB efflux pump. Collectively, the method used to synthesize the AgNPs influences their antibacterial efficacy and the likelihood of bacteria developing resistance. Understanding this interaction is vital for developing effective and resistance-controlled applications of AgNPs across medicine, environmental science, and industry.

Rising antibiotic resistance necessitates alternatives such as silver nanoparticles (AgNPs), which exhibit bactericidal activity via multi-target mechanisms (e.g., membrane disruption, ROS production). While resistance to chemically synthesized AgNPs exists, the potential for resistance to green-synthesized AgNPs, such as those from reishi mushroom, is unknown. This study compared S. aureus resistance development against both AgNP types using experimental evolution by analyzing genomic and morphological changes. Additionally, this work evaluated potential cross-resistance responses to ionic silver and investigated how adaptation to green-synthesized AgNPs affects sensitivity to chemically synthesized AgNPs (and vice versa). Rapid resistance, along with cross-resistance to silver ions, emerged in bacteria following 14 days of sublethal exposure to silver nanoparticles, regardless of whether they were chemically or biologically synthesized. While green-synthesized AgNPs demonstrated a substantial resistance to chemical variants (p < 0.05), the reverse effect was not as strong, and resistant populations showed distinct morphological adaptations. Genomic analysis highlighted convergent hard selective sweeps, identifying common mutations across both chemical and green AgNP-treated populations, with limited unique mutations found for either. These findings enhance our understanding of bacterial resistance mechanisms to nanomaterials, contributing to the development of safer, eco-friendly, and high-efficacy treatments against multidrug-resistant infection.