Molecular Mechanism of Artemisinin Against Aeromonas hydrophila LP-2 and the Combined Effect of Gallic Acid

Aeromonas hydrophila poses a growing threat in aquaculture due to high mortality and increasing antibiotic resistance, highlighting an urgent need for novel antimicrobial agents. Artemisinin (ART), a natural bioactive compound, exhibits antibacterial activity against A. hydrophila, yet its underlying mechanism remains poorly understood. In this study, quantitative proteomics was employed to elucidate the antibacterial mechanism of ART against A. hydrophila LP-2 and to evaluate its combined inhibitory effect with gallic acid (GA). The results showed that ART inhibited A. hydrophila in a concentration-dependent manner. At concentrations of 400 μg/mL and above, ART reduced biofilm mass independently of planktonic growth and also induced cell membrane damage and intracellular reactive ROS accumulation. Proteomic analysis indicated that ART significantly perturbed multiple metabolic pathways, including amino acid metabolism, redox metabolism, ABC transporters, and quorum sensing. Furthermore, the combination of ART and GA exhibited a pronounced antibacterial effect, which was closely associated with massive accumulation of intracellular ROS. Mechanistically, oxidative stress-related genes (orf00650, orf00652) were significantly upregulated, whereas the efflux transporter gene (orf00659) was markedly downregulated, suggesting that the combined effect is jointly driven by enhanced oxidative damage and impaired drug efflux. Hemocompatibility test showed that ART caused no detectable hemolysis. This study reveals the multifaceted antibacterial mechanism of artemisinin against A. hydrophila LP-2 and demonstrates that gallic acid significantly enhances its antibacterial activity in this strain, providing a theoretical basis for the further development of artemisinin as a potential antibacterial agent against A. hydrophila.