Synergistic Antimicrobial Activity of a Novel Synthetic Peptide and Silver Nanoparticles against Multidrug-Resistant E. coli

Abstract

The rising prevalence of multidrug-resistant bacterial infections poses a significant public health challenge globally. This study investigates the synergistic antimicrobial potential of a novel synthetic antimicrobial peptide, EcDBS1R4, in combination with biogenic silver nanoparticles (AgNPs) against multidrug-resistant Escherichia coli isolated from animal sources. The study analyzed 350 diarrheal samples from lambs, of which 91.14% yielded E. coli isolates. Molecular characterization revealed that 24.0% of the animal isolates harbored major diarrheagenic E. coli pathotypes, including EPEC (11.0%), ETEC (2.0%), EAEC (4.0%), and STEC (7.0%). Alarmingly, the animal ETEC isolates exhibited 100% multidrug resistance, while EPEC and EAEC also displayed high rates of multidrug resistance at 63.6% and 50%, respectively. In contrast, the STEC isolates were classified as non-MDR. To address this threat, the study synthesized AgNPs using cell-free extracts of pathogenic E. coli, which exhibited characteristic surface plasmon resonance at 423 nm and a narrow size distribution of 13.08-25.81 nm. The antimicrobial peptide EcDBS1R4 was designed and characterized, revealing a cationic, amphipathic structure capable of targeting bacterial membranes. Molecular docking studies suggested favorable binding interactions of the peptide with outer membrane protein A (OmpA) and inner membrane protein BamA of E. coli. The in vitro antimicrobial assays demonstrated the synergistic effects of the peptide-AgNPs combination. The disc well diffusion assay showed that the combination treatment produced significantly larger zones of inhibition compared to the individual components across all tested concentrations. At the highest dose (100 μg/mL AgNPs + 100 μM/mL peptide), the combination achieved a 24.8 ± 2.0 mm zone, representing a 71.0% enhancement over the expected additive effect. The time-killing assay revealed that the peptide-AgNPs combination exhibited rapid and sustained bactericidal activity, reducing E. coli counts by >6 log₁₀ CFU/mL within 24 hours, significantly outperforming the individual treatments. Similarly, the CFU count assay demonstrated that the combination reduced viable E. coli by >95% at the highest concentration, exhibiting a strong synergistic effect. In conclusion, these findings highlight the potential of this peptide-AgNPs approach as a promising alternative therapy against multidrug-resistant E. coli infections, particularly in animal populations. The synergistic antimicrobial mechanism likely involves the peptide's membrane-disrupting capabilities combined with the nanoparticles' multi-targeted effects, including oxidative stress and disruption of cellular processes.