Survival against exogenous hydrogen peroxide of Photobacterium damselae subsp. piscicida under different culture conditions.

Photobacterium damselae subsp. piscicida is a fish pathogen responsible for important losses in aquaculture world-wide. Several studies on its virulence mechanisms have been carried out and outer membrane proteins involved in the acquisition of iron or production of extracellular products have been suggested as the main determinants of its virulence for fish (Magariños, Santos, Romalde, Rivas, Barja & Toranzo 1992; Magariños, Romalde, Lemos, Barja & Toranzo 1994). However, the actual methods of invasion and survival inside the host are still unknown and while some authors have reported the presence of intact bacteria inside fish cells, suggesting the ability of the bacterium to survive as an intracellular pathogen (Noya, Magariños, Toranzo & Lamas 1995; López-Dóriga, Barnes, dos Santos & Ellis 2000), others have observed that this pathogen is highly susceptible to oxidative radicals generated during the macrophage respiratory burst (Skarmeta, Bandı́n, Santos & Toranzo 1995; Barnes, Balebona, Horne & Ellis 1999a). Reactive oxygen species (ROS) such as hydrogen peroxide and superoxide are generated during the macrophage respiratory burst in response to microbial infection. Bacterial pathogens must overcome the toxic effects of ROS to establish infections. Production of superoxide dismutase and catalase enzymes, which decompose superoxide and peroxide radicals, respectively, have been reported to contribute to the virulence of a number of pathogens (Franzon, Arondel & Sansonetti 1990; Lefebre & Valvano 2001; Uzzau, Bossi & FigueroaBossi 2002). Thus, the ability of catalase to decompose peroxide radicals increases survival of bacteria in the presence of peroxide. In addition, increased levels of catalase activity when bacteria are cultured under certain conditions, such as the presence of peroxide radicals or until the stationary phase, have been reported (Stortz, Tartaglia & Ames 1990; Loewen 1997). Moreover, the fact that most catalases are iron-cofactored suggests that growth under different iron concentrations may have some effect on this enzyme activity. Catalase activity has been reported in P. damselae subsp. piscicida (Barnes et al. 1999a), however, the role of this enzyme in the protection against peroxide has not yet been determined. For this reason, the resistance to peroxide radicals of P. damselae subsp. piscicida cells grown under iron limited and replete conditions, and pulsed with hydrogen peroxide, has been evaluated in this study. Two strains of P. damselae subsp. piscicida have been included in this study. The virulent strain (Lg41/01) (LD50 1⁄4 2.2 · 10 CFU g) was isolated from diseased sole, Solea senegalensis Kaup, showing typical signs of pseudotuberculosis, and the non-virulent strain (Epoy) (LD50 > 1.0 · 10 CFU g; Magariños, Bonet, Romalde, Martı́nez, Congregado & Toranzo 1996) kindly supplied by Dr K. Muroga (Faculty of Applied Journal of Fish Diseases 2003, 26, 305–308