Mechanisms of protection of probiotics against bacterial pathogens in oyster aquaculture

Oysters are filter feeders and thus continuously exposed to large numbers of microbes present in the marine environment. The Eastern oyster, Crassostrea virginica is one of the most recognized aquatic species in the United States. Two major bacterial pathogens that have a serious impact on this species are Roseovarius crassostreae and Vibrio tubiashii. Probiotics are an environmental friendly method of disease management that potentially can control the proliferation of pathogens in the hatchery or farm. However, little is known about the actual mechanisms of action of probiotics. The goals of this study were to isolate potential local probiotic species for the control of infectious diseases in C. virginica culture and to investigate the mechanisms of probiotic action.;The present study successfully isolated two candidate probionts able to protect larval oysters C. virginica against V. tubiashii (Relative Percent Survival, Bacillus pumilus RI06-95: 29 ± 3 % and Phaeobacter sp. S4: 55 ± 2 %) and R. crassostreae (RPS, B. pumilus RI06-95: 42 ± 3 % and Phaeobacter sp. S4: 49 ± 3 %). Phaeobacter sp. S4 was isolated from the inner shell of healthy oysters and was identified to produce the antibiotic compound trophoditietic acid (TDA). This probiont was able to inhibit the growth of pathogens in an in vitro assay as well as significantly increase the survival of larval oysters in an in vivo bacterial challenge assay. Candidate probiont B. pumilus RI06-95, previously isolated from a marine sponge from Narrow River, RI, is known to produce the antibiotic amicoumacin. This candidate probiont showed antibiotic activity against R. crassostreae in an in vitro assay and protection of larval oysters to bacterial challenge.;In order to determine the relative roles of biofilm formation and antibiotic production on probiotic activity, several mutants of Phaeobacter sp. S4 were created. Mutants tdaA-, tdaB- and tdbD- no longer produce TDA and exhibit reduced biofilm formation; mutant clpX- forms normal biofilms without TDA production, mutant exoP- produces TDA but shows significantly decreased biofilm formation, and mutant rpoE- displays a similar phenotype to the wild type but shows a delay in TDA production. Our results shows the tdaA-, tdaB- and tdbD- mutants conferred significantly decreased protection to oysters challenged with V. tubiashii (tdaA- 24 ± 2 %, tdaB- 24 ± 4 %, tdbD- 23 ± 2 %). Meanwhile, mutant clpX- and exoP - conferred partial protection (clpX-: 33 ± 2 % and exoP-: 35 ± 8 %) and mutant rpoE - conferred protection just slightly lower than the wild type (rpoE- 54 ± 2 %). This study demonstrates that both TDA production and biofilm formation play a critical role in probiotic activity of Phaeobacter sp. S4. However, based on the fact that mutants with no ability to produce TDA and biofilm defective were still able to provide a small level of protection to bacterial challenge in larval oysters, we speculate that mechanisms other than antibiotic production and biofilm formation may be involved in the probiotic activity of Phaeobacter sp. S4.;In conclusion, probionts Phaeobacter sp. S4 and B. pumilus RI06-95 were able to protect oysters against bacterial challenge. The mechanism of actions of these probionts was proved to be complex, with production of the antibiotic TDA and an ability to form biofilms playing a major role in the probiotic activity of Phaeobacter sp. S4, and the ability to immune-modulate the responses of the host also providing a small contribution. Further research should be done in order to evaluate other potential mechanisms that may be involved in probiotic activity. (Abstract shortened by UMI.).