Silver nanoparticles regulate antibiotic resistance genes by shifting bacterial community and generating anti-silver genes in estuarine biofilms

Biofilms are thought to be sinks for antibiotic resistance genes (ARGs) and nanoparticles (NPs), however, studies on the interactions between NPs and ARGs in biofilms are limited. This study focused on the occurrence and regulatory mechanisms of ARGs during the formation of biofilms with continuous treatment of zero-valent silver nanoparticles (Ag⁰-NPs) and Ag ions at an environmental concentration of 10 µg/L in the Yangtze Estuary. The biofilms could enrich large amounts of Ag, with the highest concentration of 97.60 mg/kg and 111.08 mg/kg in the Ag⁰-NPs and Ag ions group at 28 days. Compared to the blank at 28 days, the abundance of ARGs was reduced 2.2 times in the Ag⁰-NPs group, whereas it increased 1.3 times in the Ag ion group. Ag⁰-NPs and Ag ions induced the production of silver resistance genes (SRGs) or selected bacteria with SRGs in biofilms. Based on machine learning, the bacterial community, SRGs, and Ag concentration were the top three dominant regulators of ARGs, with 27.74 %, 25.57 %, and 17.93 % contributions, respectively. Structural equation modeling revealed that Ag could indirectly regulate ARGs by regulating the bacterial community in the Ag⁰-NPs group. Metagenomic sequencing further showed that most of the decreased ARGs were hosted by Betaproteobacteria in the Ag⁰-NPs groups. According to the KEGG pathway database, the possible molecular mechanism of Ag⁰-NPs/Ag ions regulating ARGs may be through the two-component system (arlS/silS-arlR) and beta-lactam resistance system (mexI-mexV-oprM/oprZ/smeF). Overall, this study provides new insights into the effects of Ag⁰-NPs at environmental concentrations on the ecological environment, especially regarding the mechanism of regulating ARGs in estuarine biofilms.