Myco-synthesized silver nanoparticles from Aspergillus niger AH1 as multifunctional anti-Helicobacter pylori agents: Antibacterial, antibiofilm, anti-urease, and antibiotic-synergistic activities.

Helicobacter pylori remains a primary driver of chronic gastritis, peptic ulcers, and stomach cancer. However, surging antimicrobial resistance now causes treatment to fail in a significant number of patients. Because of their multi-target bactericidal processes, silver nanoparticles (AgNPs) have become a viable alternative antimicrobial therapy. In the current research, AgNPs were greenly synthesized employing Aspergillus niger AH1 (A. niger AH1) through an ecofriendly method. The biosynthesized AgNPs showed a characteristic surface plasmon resonance (SPR) peak at 420 nm, validating successful nanoparticles (NPs) development. Transmission electron microscope (TEM) analysis discovered primarily spherical and well-dispersed NPs with a size range of 8-38 nm, while dynamic light scattering (DLS) analysis exhibited an average hydrodynamic diameter of approximately 40 nm and a low polydispersity index (PDI = 0.122), indicating high colloidal stability. Furthermore, the X-ray diffraction (XRD) analysis illustrated the crystalline nature of the obtained AgNPs with characteristic face-centered cubic (FCC) diffraction peaks and 38.3 nm average crystallite size. The biosafety of the biosynthesized AgNPs was assessed toward normal cell lines; results revealed that the biosynthesized AgNPs are safe in use, where the IC50 was 306.4 and 336.9 µg/ml toward Wi-38 and Vero cell lines, respectively. The destructive ability of AgNPs against 17 clinical H. pylori isolates was thoroughly assessed using different microbiological assays. The minimum inhibitory concentration (MIC) values varied from 64 to 256 µg/mL, while minimum bactericidal concentration (MBC) values ranged from 64 to 1024 µg/mL. All isolates had MIC/MBC ratios of ≤ 4, indicating the bactericidal mode of action of AgNPs. In addition, AgNPs exhibited dose-dependent biofilm inhibition at sub-MIC concentrations, ranging from 1.31 to 60.42%. Mechanistic studies were subsequently performed using Hp13, the most susceptible isolate. In this representative isolate, AgNPs combined with amoxicillin and clarithromycin showed synergistic interactions in the checkerboard assay. AgNPs also suppressed urease activity in a concentration-dependent manner (IC₅₀ = 7.05 µg/mL), a concentration well below the isolate's MIC. Time-kill kinetics confirmed rapid bactericidal activity, while increased protein leakage indicated membrane disruption. These findings demonstrate that AgNPs possess potent antibacterial activity against clinical H. pylori isolates, whereas the mechanistic evidence was obtained using the representative isolate Hp13.