Introduction
Lactococcus garvieae ( L. garvieae), a Gram-positive lactic acid bacterium belonging to the Streptococcaceae family, has gained notoriety as a significant pathogen in aquaculture, particularly in freshwater and marine fish, including high-value species like rainbow trout and European Sea Bass —causing piscine lactococcosis. The disease is marked by high morbidity, haemorrhagic symptoms, and systemic infections. Its adaptability and capacity for biofilm formation complicate eradication and contribute to persistent outbreaks. Conventional antibiotic treatments are losing effectiveness due to rising antimicrobial resistance, necessitating the exploration of safer, more sustainable alternatives. Plant extracts and o rganic acids (OAs) have emerged as promising candidates due to their natural antimicrobial, anti-inflammatory, and immunomodulatory effects. This study evaluates the efficacy of a plant extract and organic acid mixture AuraAqua (Aq), against L. garvieae in vitro (CHSE-214 fish epithelial cell line) and ex vivo (fish red blood cells), focusing on its impact on bacterial growth, virulence gene expression, host immune responses, and cytotoxicity.
Materials and Methods
This study employed CHSE-214 epithelial cells and rainbow trout red blood cells to model infection. The MIC and MBC of Aq were determined using standard dilution techniques. The anti-virulence activity of Aq at the established MIC (0.125%) was assessed by, measuring bacterial growth, EPS gene expression , biofilm formation , cytotoxicity via LDH release , adhesion of L. garvieae to epithelial cells and gene expression of haemolysins (hly1, hly2, hly3). Immunomodulatory and inti-inflammatory effect of Aq was assessed by measuring host inflammatory cytokine responses (IL-1β and IL-8). Finally, the protective effect of Aq was measured by challenging trout red blood cells with L. garvieae and measuring haemolysis in an ex vivo assay. Gene expression was analys ed using quantitative RT-PCR, and statistical analysis was performed using t-tests and ANOVA.
Results
Aq showed inhibitory and bactericidal activity at 0.125% (MIC) and 0.75% (MBC), respectively.
At a concentration of 0.125% Aq significantly reduced number of L. garvieae vilulence factors including growth, EPS gene expression (epsA , epsB , epsC, etc.), and biofilm formation. In addition, Aq inhibited bacterial adhesion to CHSE-214 cells and reduced cytotoxic effects (Fig. 1).
In ex vivo experiments a significant reduction in haemolysis was observed when Aq was co-administered with L. garvieae in red blood cell assays (p = 0.02). Expression of L. garviae virulence genes hly1 and hly3 was significantly downregulated, while hly2 showed no significant change. IL-1β and IL-8 expression was significantly reduced in infected CHSE-214 cells treated with Aq, highlighting its anti-inflammatory potential.
These effects collectively indicate Aq’s ability to suppress bacterial virulence and mitigate host inflammatory responses without harming host cells.
Conclusion
This study demonstrates that a natural blend of plant extracts and organic acids (Aq ) can significantly reduce the pathogenic potential of L. garvieae by inhibiting key virulence factors such as exopolysaccharide production, haemolysin gene expression, and host cell adhesion. Additionally, Aq reduces inflammation and cytotoxicity in host cells, suggesting its dual benefit as both an antimicrobial and immunomodulator. These findings position Aq as viable non-antibiotic alternatives for controlling L. garvieae infections in aquaculture, contributing to sustainable fish health management strategies and addressing the growing need for antibiotic-free disease control.