Antimicrobial activity and virulence attenuation of citral against the fish pathogen Vibrio alginolyticus

Vibrio alginolyticus is an important fish pathogen causing pandemic diseases in marine animals and brings about severe economic losses in aquaculture all over the world. The emergence of multi-drug resistant V. alginolyticus and the prohibition of antibiotics both require the development of new anti-virulence strategies and therapeutic agents with alternative modes of action. In this study, the effect of citral on V. alginolyticus growth and virulence was investigated to determine the basis of its use in disease prevention and treatment in aquaculture. The minimum inhibitory concentration (MIC) of citral against V. alginolyticus was determined to be 0.125 mg/mL. Citral caused membrane invaginations and damages as shown by field emission scanning electron microscopy (FESEM) observations. Besides, membrane damage was further verified through the increase of conductivity and extracellular malondialdehyde content. After treatment with sub-inhibitory concentrations (SICs) of citral, the virulence factors of V. alginolyticus, including motility, biofilm formation, extracellular polysaccharide, and alkaline serine protease production, were all significantly inhibited. Transcription analysis showed that citral repressed genes involved in flagella synthesis (fliS, flaK, lafA, lafK), biofilm regulation gene (sypG), and quorum sensing genes. Moreover, citral reduced the virulence of V. alginolyticus to zebrafish. These findings suggest that citral has the potential to be developed as an alternative or supplemental agent to mitigate the infections caused by V. alginolyticus in aquaculture.