Phaeobacter gallaeciensis reduces Vibrio anguillarum in cultures of microalgae and rotifers, and prevents vibriosis in cod larvae

Abstract

Phaeobacter gallaeciensis can antagonize fish-pathogenic bacteria in vitro, and the purpose of this study was to evaluate the organism as a probiont for marine fish larvae and their feed cultures. An in vivo mechanism of action of the antagonistic probiotic bacterium is suggested using a non-antagonistic mutant. P. gallaeciensis was readily established in axenic cultures of the two microalgae Tetraselmis suecica and Nannochloropsis oculata, and of the rotifer Brachionus plicatilis. P. gallaeciensis reached densities of 10(7) cfu/ml and did not adversely affect growth of algae or rotifers. Vibrio anguillarum was significantly reduced by wild-type P. gallaeciensis, when introduced into these cultures. A P. gallaeciensis mutant that did not produce the antibacterial compound tropodithietic acid (TDA) did not reduce V. anguillarum numbers, suggesting that production of the antibacterial compound is important for the antagonistic properties of P. gallaeciensis. The ability of P. gallaeciensis to protect fish larvae from vibriosis was determined in a bath challenge experiment using a multidish system with 1 larva per well. Unchallenged larvae reached 40% accumulated mortality which increased to 100% when infected with V. anguillarum. P. gallaeciensis reduced the mortality of challenged cod larvae (Gadus morhua) to 10%, significantly below the levels of both the challenged and the unchallenged larvae. The TDA mutant reduced mortality of the cod larvae in some of the replicates, although to a much lesser extent than the wild type. It is concluded that P. gallaeciensis is a promising probiont in marine larviculture and that TDA production likely contributes to its probiotic effect.

Figure 1. Concentrations of Tetraselmis suecica and Phaeobacter gallaeciensis in the co-cultures.

Means and standard deviations of eight experiments: colony-forming units of P. gallaeciensis wild type (♦) and the TDA-negative mutant (•), and concentrations of T. suecica with V. anguillarum (▾), T. suecica with P. gallaeciensis wild type (▪), T. suecica with P. gallaeciensis TDA-negative mutant (▴), and axenic T. suecica (□). The P. gallaeciensis strains were inoculated at 10⁷ cfu/ml and remained as a steady population, while the algae went from late log into stationary phase.

Figure 2. Localization of bacteria in cultures of Tetraselmis suecica.

Phase-contrast (A,C) and fluorescence (B,D,E) micrographs. Co-culture of Tetraselmis suecica with Phaeobacter gallaeciensis dsRed (A,B), axenic T. suecica (C,D), co-culture of T. suecica with V. anguillarum gfp (E). Panel A and B show two single (left) and one dividing algal cell (right side), and a marine snow-like particle consisting of algae-debris which is colonized by red-fluorescent P. gallaeciensis. Red fluorescence of algae is due to chlorophyll. Panels C and D show an algal cell and particles from an axenic culture, recorded using the same settings as for the panels above. Panel E shows red-fluorescent algae cells and green-fluorescent V. anguillarum, which do not colonize particles, but remain in suspension as single, motile cells.

Figure 3. Reduction of V. anguillarum in cultures of Tetraselmis suecica by Phaeobacter gallaeciensis.

Colony-forming units of V. anguillarum inoculated at 10¹ cfu/ml (A) and at 10⁴ cfu/ml (B) in presence of P. gallaeciensis wild type (▪), in presence of the P. gallaeciensis TDA-negative mutant (▴), and in the monoxenic control (▾).

Figure 4. Expression of tdaC in co-culture with Tetraselmis suecica.

Phase contrast (A) and fluorescence (B) micrographs of P. gallaeciensis pPDA11 (tdaCp::gfp) in co-culture with T. suecica. The two panels show the same seven algal cells of which some are dividing, and a marine snow-like particle which is colonized by P. gallaeciensis carrying the promoter-fusion on a plasmid. The green fluorescence of P. gallaeciensis on the particle shows that the gfp gene is expressed from the tdaC promoter, indicating production of TDA.

Figure 5. Influence of bacterial strains on rotifer growth.

Rotifer numbers in co-culture with P. gallaeciensis wild type (▾), with the TDA-negative mutant of P. gallaeciensis (♦), with only V. anguillarum (▴), and axenic rotifers (▪), first experiment. All bacteria were inoculated at day 0. Both P. gallaeciensis strains promoted rotifer growth, whereas V. anguillarum had no influence.

Figure 6. Reduction of Vibrio anguillarum by Phaeobacter gallaeciensis in rotifer cultures.

Mean values of two duplicate experiments: colony-forming units of V. anguillarum in co-culture with P. gallaeciensis wild type (▴), with the TDA-negative mutant of P. gallaeciensis (▾), and in the monoxenic control (▪).

Figure 7. Mortality of cod larvae during the challenge trials.

Mean values of two independent triplicate experiments. The single-larvae cultures were simultaneously inoculated with P. gallaeciensis wild type and V. anguillarum (T5, •), or with the TDA-negative mutant of P. gallaeciensis and V. anguillarum (T6, □). Unexposed larvae and larvae exposed to single bacterial strains acted as controls: Negative Control (T1, ▪), only V. anguillarum (T2, ▴), only P. gallaeciensis wild type (T3, ▾), and only P. gallaeciensis TDA-negative mutant (T4, ♦).