In Situ Synthesis of Anisotropic Silver Nanoparticles on 2D Chitosan Nanosheets: A Polymer-Metal Nanocomposite with Multi-target Genetic Efficacy Against Multidrug Resistant Bacteria

Abstract

The escalating global crisis of antimicrobial resistance (AMR) necessitates the rapid development of non-traditional, materials-based strategies. Here, we report a simple, one-pot, in situ synthesis of anisotropic silver nanoparticles (AgNPs) directly and uniformly integrated onto the surface of two-dimensional (2D) chitosan (CS) nanosheets. This method exploits the intrinsic reducing and stabilizing capabilities of chitosan, enhanced by a modified synthetic approach using sodium borohydride (NaBH4) at low temperature, to create a robust, synergistic polymer-metal nanocomposite (CS-Ag). Comprehensive characterization (UV–Vis, FTIR, XRD, EDS, HRTEM, AFM) confirmed the successful immobilization and unique morphology, revealing anisotropic AgNPs with an average size of approximately 50 nm uniformly dispersed over the CS nanosheet. Moving significantly beyond standard inhibition assays, this work elucidates the multi-mechanistic and genetic-level action of the CS-Ag nanocomposite against a panel of multidrug resistant (MDR) clinical isolates. The material exhibited exceptional antibacterial potency, with maximum efficacy observed against Gram-negative bacteria, specifically Enterobacter ludwigii and Escherichia coli O157. To definitively uncover the underlying mechanism, we performed a novel gene expression analysis using a custom qPCR panel. This investigation provided molecular evidence of a multi-pronged attack that simultaneously targets cell structure and genetic defenses: a significant downregulation of key AMR genes (including the beta-lactamase gene (blaACT), the efflux pump gene (acrB), and the biofilm regulator (csgD)). Simultaneously, the bacteria reacted by upregulating genes indicative of cellular stress and repair, such as the heat shock protein gene (HSP60) and genes related to cell envelope damage (uge and nhaA). These compelling genetic findings were rigorously corroborated by molecular docking analysis, which mapped the high-affinity interaction of the nanocomposite with crucial bacterial membrane proteins (such as LolC). Collectively, these results establish the CS-Ag nanocomposite as a high-potential, next-generation hybrid material capable of circumventing and degrading AMR mechanisms by disarming MDR pathogens at the genetic level. This study offers a powerful, data-driven material-science approach for combating antibiotic resistance by simultaneously targeting both cell integrity and genetic defenses.