Organic acids target key virulence factors of Aeromonas hydrophila to reduce infection in vitro

This study investigates the effects of the organic acid mixture AuraAqua (Aq) on the pathogenicity of Aeromonas hydrophila in primary gut cells of shrimp (SGP) and tilapia (TGP). A. hydrophila , an important pathogen in aquaculture, damages gut barrier integrity and promotes systemic infection with a significant economic impact. Organic acids are proposed as antibiotic alternatives, and we aimed to investigate their impact as a mixture AuraAqua (Aq—5% maltodextrin, 1% sodium chloride, 42% citric acid, 18% sodium citrate, 10% silica, 12% malic acid, 9% citrus extract, and 3% olive extract) on the growth, virulence traits, and host cell protection during A. hydrophila infection SGP and TGP cells. Aq exhibited an MIC of 1% and an MBC of 3%. Sub-inhibitory concentrations significantly reduced adhesion and invasion in SGP and TGP cells, preserved TEER, and decreased LDH release, indicating reduced cytotoxicity and barrier disruption. Aq impaired motility and biofilm formation in a dose-dependent manner. Transcriptional analysis showed downregulation of hcp1, act, fla, hlyA and acg , whereas TonB , aerA and alt were largely unaffected. Aq also strongly reduces capsular polysaccharide production both in vitro and during infection, which likely weakens immune evasion and persistence. In host cells, Aq significantly decreases oxidative stress markers (H 2 O 2 , SOD, CAT) during infection, indicating a beneficial effect on host responses. Overall, Aq acts as an anti-virulence agent, weakening A. hydrophila’s ability to colonize and damage gut epithelia while reducing host oxidative stress, supporting its use as a non-antibiotic preventive strategy in aquaculture, pending further in vivo studies.