Glutathione-linked antioxidant defence limits Edwardsiella tarda-induced cytotoxicity in organic acid-treated gill epithelial cells

Cytotoxic injury to gill epithelial cells is a major consequence of pathogen infection and can compromise barrier function in aquaculture species. Edwardsiella tarda is an important bacterial pathogen in aquaculture. Infection of gill epithelial cells can damage barrier integrity, induce cytotoxicity, and trigger oxidative and inflammatory stress responses. This study evaluated whether AuraAqua (Aq), an organic acid-based antimicrobial mixture, protects RTgill-W1 cells from E. tarda -induced cytotoxicity and the associated inflammatory and oxidative stress responses. Aq showed a minimum inhibitory concentration of 1% and a minimum bactericidal concentration of 2%. Aq treatment reduced E. tarda adhesion and intracellular bacterial recovery, indicating impaired early host-pathogen interactions. Importantly, Aq was not directly cytotoxic to RTgill-W1 cells at the tested concentrations (0, 0.1, 0.2, 0.5, 1 and 2%) and reduced infection-associated LDH release while preserving cell viability and transepithelial electrical resistance. These outcomes were accompanied by increased expression of tight-junction-associated genes, including cldn-10e , cldn-30 , and zo-1 . During infection, Aq enhanced intracellular glutathione (GSH) levels, reduced superoxide dismutase (SOD) and catalase (CAT) activity, decreased H 2 O 2 accumulation, and decreased NF-κB p65 activation. Aq also significantly downregulated IL-8, IL-1β, and TNF-α expression in infected cells. Together, these findings show that Aq can stimulate increased endogenous GSH production, which primarily protects RTgill-W1 cells and limits E. tarda -induced cytotoxic injury. This strengthens antioxidant defence, maintains epithelial barrier integrity, and suppresses NF-κB-associated inflammatory signalling.