The Effects of Antibiotics, Metals, and Biotic Interactions on the Assembly of Taxonomically Diverse Single and Mixed Species Biofilms

To better understand the assembly of the sturgeon egg microbiome, we purified six bacterial isolates from eggs and characterized their ability to form biofilms under the stress of tobramycin, with and without exogenous protein. In experiments with single species biofilms, tobramycin reduced the metabolic activity of all isolates and increased biofilm biomass of three. The addition of exogenous protein to the assay countered the inhibition of biofilm and metabolic activity by tobramycin of Pseudomonas sp., Brevundimonas sp., Flavobacterium columnare and mixed biofilms of Pseudomonas-F. columnare and Brevundimonas-Hydrogenophaga. Two of the isolates (Pseudomonas spp.) that produced antimicrobial activity, were effective at reducing biofilm formation by Brevundimonas, but enhanced biofilm formation in other isolates. Increasing concentrations of Mg2+ had no effect on biofilm formation but Ca2+ enhanced biofilm formation of Pseudomonas aeruginosa PA01 (positive control) and Brevundimonas. Biofilm assembly by these two bacteria was inhibited by low concentrations of Ni2+. Mixed biofilms of Brevundimonas and Hydrogenophage consistently produced more robust biofilm than the strains in isolation, suggesting synergism. Established Brevundimonas biofilm appeared adept at recruiting pelagic Acidovorax and Hydrogenophaga into biofilm, suggesting that it plays an important role in the selection of species into the microbiome.