Nonculturability of the pathogenic Vibrio parahaemolyticus in live culture of Grateloupia turuturu is associated with bacterial attachment to the algal thalli

The invasive red alga Grateloupia turuturu Yamada could turn Vibrio parahaemolyticus into nonculturable state in live algal culture. In order to elucidate the mechanism of such an effect, a series of culture experiments were performed in this investigation based on three hypothesized causes, namely bacterial attachment, production of reactive oxygen species (ROS) and the discharge of water soluble secondary metabolic compounds. The results reveal that attachment to the thallus surface of G. turuturu was the major reason for the decrease of V. parahaemolyticus in seawater. Further investigations show that V. parahaemolyticus attachment to the surface of algal thallus in live cultures of seaweeds was a common phenomenon. However, the disappearance of the culturability of V. parahaemolyticus occurred only on the thallus of G. turuturu over 72 h among all six algal species tested. Electron microscopic scanning shows that most of V. parahaemolyticus attached to G. turuturu changed from the initial normal bacilli to coccoid-shape after 72 h. The enclosure experiments by enclosing the algal thallus in tubes demonstrate that the nonculturability of V. parahaemolyticus in the water of live culture of G. turuturu occurred after the physical contact of the V. parahaemolyticus to the alga. The capacity of G. turuturu in affecting the culturability of V. parahaemolyticus was not influenced after inhibition of photosynthesis by treatment of 3''-(3,4-dichlorophenyl)-1'',1''-dimethyl urea (DCMU) at non-lethal levels. Production of reactive oxygen species after addition of live culture of bacteria was excluded by on-line analyzing the oxidation of dichlorohydrofluorescein (DCFH) to dichlorofluorescein (DCF) in the presence of peroxidase on a VersaFluor fluorometer.