Genistein Inhibits the Pathogenesis of Aeromonas hydrophila by Disrupting Quorum Sensing Mediated Biofilm Formation and Aerolysin Production

Abstract

Aeromonas hydrophila is an opportunistic pathogen that is responsible for a variety of infectious diseases both in human and animals, particularly aquatic animals. Moreover, the pathogen has become a foodborne pathogen by transmitting from seafood to human. The abuse of antibiotics in aquaculture results in the emergence of antibiotic resistance and treatment failure. Therefore, novel approaches are urgently needed for managing resistant A. hydrophila associated infections. Aerolysin, an essential virulence factor of pathogenic A. hydrophila strain, has been identified as target developing novel drugs against pathogenesis of A. hydrophila. In the present study, genistein, without anti-A. hydrophila activity, was identified that could decrease the production of aerolysin and biofilm formation at a dose-dependent manner. Transcription of aerolysin encoding gene aerA and quorum sensing related genes ahyI and ahyR was significantly down-regulated when co-cultured with genistein. Cell viability studies demonstrated that genistein could significantly improve aerolysin mediated A549 cell injury. Furthermore, genistein could provide a remarkable protection to channel catfish infected with A. hydrophila. These findings indicate that targeting quorum sensing and virulence can be a useful approach developing drugs against A. hydrophila infections in aquaculture. Moreover, genistein can be chosen as a promising candidate in developing drugs against A. hydrophila.

Keywords: Aeromonas hydrophila; anti-virulence; genistein; natural compound; quorum sensing.

FIGURE 1

Inhibitory effects of genistein on hemolytic activity of bacterial supernatants co-cultured with genistein. (A), chemical structure of genistein; (B), Growth curves of A. hydrophila XS-91-4-1 plus indicated concentrations of genistein; (C), genistein inhibits hemolytic activity of bacterial supernatants co-cultured with genistein, 1% Triton X-100 treated red blood cells represented 100% hemolytic activity, three independent hemolytic assays were performed and data in Figure 1C were mean value ±SD, *p < 0.05 and **p < 0.01 when compared with the genistein-free supernatants.

FIGURE 2

Detection of aerolysin production in bacterial supernatant with genistein by Western-blot.

FIGURE 3

Inhibitory effects of genistein on biofilm formation. (A), Determination of genistein against A. hydrophila biofilm formation, Data in Figure 3A represented mean value ±SD of three independent assays, *p < 0.05 and **p < 0.01 when compared with drug-free group; (B–F), inhibitory effects of genistein on biofilm formation by microscopic validation; (B), drug-free group; (C), co-cultured with 4 μg/ml genistein; (D), co-cultured with 8 μg/ml genistein; (E), co-cultured with 16 μg/ml genistein; (F), co-cultured with 32 μg/ml genistein.

FIGURE 4

Treatment with genistein decreased the transcription levels of aerA, ahyI and ahyR. Transcription levels of different genes were determined by qPCR assays in triplicate, data were mean value ±SD. *p < 0.05 and **p < 0.01.

FIGURE 5

Protective effects of genistein on aerolysin mediated A549 cell injury. Cell injury induced by aerolysin was evaluated by live/dead staining assay and LDH release assay. For live/dead staining assay, live cells were stained with green, while dead were red. (A), cells without any treatment; (B), cells treated with drug-free supernatant; (C), cells treated with supernatant obtained from bacterial culture plus 32 μg/ml genistein; (D), LDH release of A549 cells treated with bacterial supernatants plus indicated concentrations of genistein, LDH assay was performed in triplicate, data were mean value ±SD. *p < 0.05 and **p < 0.01.

FIGURE 6

Genistein treatment improved the survival rate of channel catfish infected with A. hydrophila. Fish infected with A. hydrophila were treated with 20 mg/kg genistein or sterile PBS every 12 h for 3 days, deaths were observed for 8 days after infection. Treatment with genistein could provide a significant protection to fish challenged with A. hydrophila when analyzed by log-rank test (p < 0.0001).