Biochemical and Molecular Analysis of Bacteria Used in Protecting Oyster Larvae from Pathogenic Bacteria in Hatchery Culture

A major focus of the aquaculture industry involves the development of strategies for controlling diseases caused by bacterial pathogens.  One approach utilizes probiotic bacteria that may benefit the host and/or antagonize harmful competitors.  The Milford lab has identified several bacteriaÑstrains S1, S2, S7, and OY15Ñhaving probiotic activity in cultures of Crassostrea virginica larvae and providing resistance to challenge by a bacterial pathogen, strain B183.  To begin understanding the mechanisms involved in this activity, the present study examined several molecular and biochemical characteristics of these strains as well as strain B183.  Phylogenetic analyses based on both 16S and 23S rRNA genes indicated that strain S1 shared >98% similarity with Bacillus cereus; S1, S2, OY15 and B183 displayed >98% similarity to Vibrio parahemolyticus.  Extracellular amylase activities were detected for all strains but lipase activity could only be detected for OY15.  Protease activity was measured in culture supernatants from all strains with B183 having the greatest activity (~10X).  All activities were EGTA-sensitive (up to 90% inhibition) indicative of metalloprotease; some inhibition was found with PMSF signifying serine proteases as well.  S2, S7 and OY15 were b-hemolytic on blood agar media and trh (thermostable-related), tdh (thermostable-direct), and tlh (thermolabile) hemolysin genes could not be detected.  S1 and B183 were  a-hemolytic: a trh gene was found in S1 (98% similar to trh in B. cereus) and tlh and tdh were found for B183.  Except for B183, virulence regulator toxR was detected in all strainsÑS1 toxR was >99% similar to the B.  cereus orthologue whereas the others were identical to V. alginolyticus.   Production of hemolysin and highly-active protease by B183 is likely to play a role in pathogenesis; any role for the extracellular activities of S1, S2, S7 and, OY15 in interfering with B183 growth or stimulating oyster immunity remains to be determined.