Studies on Phenylalanine Metabolism in Enhancing the ε-Polylysine-Mediated Killing Effect of Oxytetracycline-resistant Aeromonas hydrophila

Aims: Aeromonas hydrophila is a zoonotic pathogen, and its prevalence of drug resistance underscores the urgency of elucidating alternative bactericidal strategies. This study employed the oxytetracycline-resistant strain OTC-A01 to investigate the bactericidal mechanism of ε-PL. Methods and Results: With increasing ε-PL concentration, the survival rate of OTC-A01 decreased to 0.101% at 100 μg/mL. SEM and membrane integrity assays confirmed that ε-PL disrupted the membrane structure, leading to leakage of nucleic acids and proteins, increased AKP activity, and decreased relative conductivity, ATPase and NADH activity. Non-targeted metabolomics identified 142 differentially expressed metabolites, with S-plot analysis revealing 74 downregulated and 68 upregulated metabolites. KEGG enrichment indicated that the phenylalanine metabolism pathway was the most significantly affected, which was validated by RT-qPCR showing suppressed expression of key enzyme-encoding genes (DAADH, CAT, HPPD, AST). Notably, supplementation with downregulated exogenous metabolites significantly enhanced the bactericidal effect of ε-PL, with 2-phenylacetamide exhibiting the strongest synergistic effect (up to 436-fold), at which point intracellular ε-PL concentration reached 50.26 μg/mg protein. Conclusion: These results demonstrated the phenylalanine metabolic pathway is the key pathway through which ε-PL exerts its bactericidal effect against OTC-A01. This study innovatively applied non-targeted metabolomics to identify this pathway as a critical metabolic target, and was the first to demonstrate that exogenous metabolite supplementation enhances ε-PL efficiency via metabolic reprogramming. This work uncovered a novel molecular mechanism of ε-PL’s antibacterial activity and provides theoretical support for developing antimicrobial strategies targeting bacterial metabolism, with significance for addressing A. hydrophila antibiotic resistance in aquaculture and public health.