Synergistic antibacterial action of AgNP-ampicillin conjugates: Evading β-lactamase degradation in ampicillin-resistant clinical isolates.

Abstract

Antibiotic resistance towards penicillin has been attempted to counter by chemically modifying ampicillin through the conjugation with silver nanoparticles (AgNPs). The current study optimizes the conditions for synthesizing and characterizing AgNP-ampicillin to quantify the conjugation extent, evaluate the antibacterial efficacy, and explore the underlying antibacterial mechanisms. AgNPs were synthesized from silver nitrate by chemical reduction method, silica-coated with tetraethyl orthosilicate (TEOS) and amine functionalized by (3-aminopropyl) triethoxysilane (APTES), which was then conjugated with ampicillin via the carbodiimide chemistry. UV-visible spectroscopy and DLS were employed to confirm the synthesis of AgNPs, while FT-IR and TGA were used to confirm ampicillin functionalization and conjugation, and SEM and EDX spectroscopy provided morphological insight. Microbial assays were conducted against Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa to determine the inhibition zones, MIC, MBC, MPC, MBIC, MBEC, FIC index, and time-dependent efficacy of AgNP-ampicillin. Cytotoxicity was assessed on Vero cells while molecular docking was performed using AutoDock Vina. The synthesized conjugates demonstrated an approximate conjugation efficiency of 57.7%, with four ampicillin molecules conjugated per AgNP. The AgNP-ampicillin conjugates exhibited enhanced antibacterial activity against the studied clinical isolates compared to AgNPs or ampicillin alone, as evidenced by significant differences in inhibition areas in One-way ANOVA (F=18-25.68, p<0.05), while Tukey's post-hoc analysis suggested synergistic effects. AgNP-ampicillin demonstrated enhanced bacteriostatic and bactericidal activity against both planktonic and biofilm-forming cells with mutant prevention ability, and upto 1.25 times faster bacterial elimination compared to ampicillin and AgNPs alone. Synergistic effects of AgNP-ampicillin were confirmed by an FIC index (≤0.5), and effective protection of ampicillin from β-lactamase degradation was established through molecular docking. Cytotoxicity testing confirmed >95% Vero cell viability, indicating minimal toxicity. The AgNP-ampicillin conjugates exhibited enhanced antibacterial efficacy, biofilm disruption, and protection against β-lactamase degradation while maintaining low toxicity.