Bacterial pathogens, including several Vibrio spp. and Roseovarius crassostreae, cause severe mortality of larval and juvenile eastern oysters. The introduction of beneficial bacterial isolates in oyster hatcheries and nurseries for the biocontrol of bacterial diseases is a good alternative to the use of antibiotics. The goal of this study was to screen and characterize marine bacterial isolates as potential agents to prevent larval and juvenile mortality by the oyster pathogens Vibrio tubiashii and R. crassostreae. Screening of bacterial isolates from Rhode Island marine organisms and environment using agar-based assay methods for detection of antimicrobial activity against oyster pathogens led to the isolation of candidate probionts Phaeobacter sp. S4 and Bacillus pumilus RI06-95. Pretreatment of larval and juvenile oysters for 24 h with 102-106 cfu/mL Phaeobacter sp. S4 or B. pumilus RI06-95 protected larval oysters against mortality resulting from challenge with R. crassostreae and V. tubiashii (relative percent survival (RPS) range, 9%-56%). These probiotics also protected juvenile oysters against challenge with V. tubiashii (RPS, 37%-50%). Probiotic isolates had no negative impact on oyster survival. Protection conferred to larvae against bacterial challenge was short-lived, lasting for only 24 h after removal of the probiotics from the incubation water. These results suggest the potential of marine bacterial isolates Phaeobacter sp. S4 and B. pumilus RI06-95 to serve as biocontrol agents to reduce the impact of bacterial pathogens in the culture of Crassostrea virginica.

Members of the resistance nodulation cell division (RND) of efflux pumps play essential roles in multidrug resistance (MDR) in Gram-negative bacteria. Here, we describe the search for new small molecules from marine microbial extracts to block efflux and thus restore antibiotic susceptibility in MDR bacterial strains. We report the isolation of 3,4-dibromopyrrole-2,5-dione (1), an inhibitor of RND transporters, from Enterobacteriaceae and Pseudomonas aeruginosa, from the marine bacterium Pseudoalteromonas piscicida. 3,4-Dibromopyrrole-2,5-dione decreased the minimum inhibitory concentrations (MICs) of two fluoroquinolones, an aminoglycoside, a macrolide, a beta-lactam, tetracycline, and chloramphenicol between 2- and 16-fold in strains overexpressing three archetype RND transporters (AcrAB-TolC, MexAB-OprM, and MexXY-OprM). 3,4-Dibromopyrrole-2,5-dione also increased the intracellular accumulation of Hoechst 33342 in wild-type but not in transporter-deficient strains and prevented H33342 efflux (IC50 = 0.79 μg/mL or 3 μM), a hallmark of efflux pump inhibitor (EPI) functionality. A metabolomic survey of 36 Pseudoalteromonas isolates mapped the presence of primarily brominated metabolites only within the P. piscicida phylogenetic clade, where a majority of antibiotic activity was also observed, suggesting a link between halogenation and enhanced secondary metabolite biosynthetic potential. In sum, 3,4-dibromopyrrole-2,5-dione is a potent EPI and deserves further attention as an adjuvant to enhance the effectiveness of existing antibiotics.

At present no antiviral agents are available for treatment of infection by the pathogenic poxvirus molluscum contagiosum virus (MCV). Here we report the identification and characterization of an inhibitor active against the virus-encoded type-1 topoisomerase, an enzyme likely to be required for MCV replication. We screened a library of marine extracts and natural products from microorganisms using MCV topoisomerase assays in vitro. The cyclic depsipeptide sansalvamide A was found to inhibit topoisomerase-catalyzed DNA relaxation. Sansalvamide A was inactive against two other DNA-modifying enzymes tested as a counterscreen. Assays of discrete steps in the topoisomerase reaction cycle revealed that sansalvamide A inhibited DNA binding and thereby covalent complex formation, but not resealing of a DNA nick in a preformed covalent complex. Sansalvamide A also inhibits DNA binding by the isolated catalytic domain, thereby specifying the part of the protein sensitive to sansalvamide A. These data specify the mechanism by which sansalvamide A inhibits MCV topoisomerase. Cyclic depsipeptides related to sansalvamide A represent a potentially promising chemical family for development of anti-MCV agents.