A New Strategy to Prevent Emerging Lactococcus garvieae Infections by Using Organic Acids as Antimicrobials In Vitro and Ex Vivo

The ever-growing global demand for animal protein forces the aquaculture industry to expand at a pace which imposes significant challenges in maintaining sustainable practices. This study aimed to investigate the efficacy of an organic acid mixture (Aq) in mitigating <i>Lactococcus garvieae</i> (<i>L. garvieae</i>) virulence through its effects on bacterial virulence (EPS production, biofilm, and haemolytic factors) and host pathogenicity, including its adherence to CHSE-214 cells, haemolysis, and proinflammatory responses. Our findings reveal that Aq significantly inhibits <i>L. garvieae</i> growth at a 0.125% concentration, suppresses EPS-related gene expression, reduces biofilm formation, and reduces cytotoxicity in fish epithelial cells (CHSE-214). Moreover, Aq decreased haemolysing gene expression (<i>hly1</i>, <i>hly2</i> and <i>hly3</i>) and attenuated red blood cell haemolysis, a hallmark of <i>L. garvieae</i> pathogenicity. Lastly, Aq was demonstrated to induce modulation in the host immune responses, lowering IL-1β and IL-8 expression, which are critical mediators of inflammation and pathogen recruitment. Therefore, we conclude that the main mechanism of action of Aq involves inhibiting <i>L. garvieae</i> adhesion to epithelial cells, reducing EPS production, and downregulating key virulence-associated genes (e.g., <i>hly1</i>, <i>hly2</i>, and <i>hly3</i>). After preventing <i>L. garvieae</i> adherence and suppressing proinflammatory cytokine expression (IL-1β and IL-8), Aq disrupts the pathogen's ability to breach epithelial barriers and induce red blood cell lysis, thereby mitigating its virulence and pathogenicity. Our results emphasised the potential of Aq as an alternative non-antibiotic intervention for controlling piscine lactococcosis, advancing our understanding of <i>L. garvieae</i> pathogenesis and providing the foundation for the future integration of environmentally friendly antimicrobials into aquaculture disease management.