Examining the Fish Microbiome: Vertebrate-Derived Bacteria as an Environmental Niche for the Discovery of Unique Marine Natural Products

Historically, marine invertebrates have been a prolific source of unique natural products, with a diverse array of biological activities. Recent studies of invertebrate-associated microbial communities are revealing microorganisms as the true producers of many of these compounds. Inspired by the human microbiome project, which has highlighted the human intestine as a unique microenvironment in terms of microbial diversity, we elected to examine the bacterial communities of fish intestines (which we have termed the fish microbiome) as a new source of microbial and biosynthetic diversity for natural products discovery. To test the hypothesis that the fish microbiome contains microorganisms with unique capacity for biosynthesizing natural products, we examined six species of fish through a combination of dissection and culturedependent evaluation of intestinal microbial communities. Using isolation media designed to enrich for marine Actinobacteria, we have found three main clades that show taxonomic divergence from known strains, several of which are previously uncultured. Extracts from these strains exhibit a wide range of activities against both Gram-positive and Gramnegative human pathogens, as well as several fish pathogens. Exploration of one of these extracts has identified the novel bioactive lipid sebastenoic acid as an anti-microbial agent, with activity against Staphylococcus aureus, Bacillus subtilis

Phylogenetic Relationships of Taxa Related to FI-1004.

<p>The evolutionary history was inferred using the Neighbor-Joining method. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Saitou1" target="_blank">[50]</a> The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Felsenstein1" target="_blank">[49]</a> Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Felsenstein1" target="_blank">[49]</a> The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Tamura2" target="_blank">[51]</a> and are in the units of the number of base substitutions per site. The analysis involved 24 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1353 positions in the final dataset. Evolutionary analyses were conducted in MEGA5. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Tamura1" target="_blank">[30]</a> The sequence of <i>Brevibacterium linens</i> DSM 20425^T was used as an outgroup.</p

Chemical Structure for Sebastenoic Acid.

<p>a, b and c are subunits found using 2D NMR methods. HMBC correlations depicted by solid arrows, COSY correlations depicted by bold lines, NOESY correlations depicted by dashed arrows.</p

Fish Origins and Taxonomy, Microbial Isolates, and NCBI Closest Relatives.

<p>Fish Origins and Taxonomy, Microbial Isolates, and NCBI Closest Relatives.</p

Bioactivities of Fish Microbiome Isolates.

<p>A = Actinobacteria, F = Firmicutes, α = Alphaproteobacteria, γ = Gammaproteobacteria, (−) = Gram-negative, (+) = Gram-positive. Check mark indicates activity in growth inhibition assay.</p

Molecular Phylogenetic Analysis by Maximum Likelihood for all Isolated, Culturable Strains of Bacteria Isolated from Fish Intestines.

<p>The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2-parameter model. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Kimura1" target="_blank">[48]</a> The bootstrap consensus tree inferred from 2000 replicates is taken to represent the evolutionary history of the taxa analyzed. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Felsenstein1" target="_blank">[49]</a> Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (2000 replicates) are shown next to the branches. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Felsenstein1" target="_blank">[49]</a> Initial tree(s) for the heuristic search were obtained automatically as follows. When the number of common sites was <100 or less than one fourth of the total number of sites, the maximum parsimony method was used; otherwise BIONJ method with MCL distance matrix was used. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+<i>G</i>, parameter = 0.4869)). The rate variation model allowed for some sites to be evolutionarily invariable ([+<i>I</i>], 38.9340% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 53 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1244 positions in the final dataset. Evolutionary analyses were conducted in MEGA5. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Tamura1" target="_blank">[30]</a> Strains identified as psychrophilic bacteria in NCBI denoted with blue diamonds. Strains whose closest published NCBI relatives are uncultured clones denoted with open triangles.</p

Sebastenoic Acid (1) MICs Against Bacterial Panel.

<p>Sebastenoic Acid (1) MICs Against Bacterial Panel.</p

Biofilm Formation and Detachment in Gram-Negative Pathogens Is Modulated by Select Bile Acids.

Biofilms are a ubiquitous feature of microbial community structure in both natural and host environments; they enhance transmission and infectivity of pathogens and provide protection from human defense mechanisms and antibiotics. However, few natural products are known that impact biofilm formation or persistence for either environmental or pathogenic bacteria. Using the combination of a novel natural products library from the fish microbiome and an image-based screen for biofilm inhibition, we describe the identification of taurine-conjugated bile acids as inhibitors of biofilm formation against both Vibrio cholerae and Pseudomonas aeruginosa. Taurocholic acid (1) was isolated from the fermentation broth of the fish microbiome-derived strain of Rhodococcus erythropolis and identified using standard NMR and MS methods. Screening of the twelve predominant human steroidal bile acid components revealed that a subset of these compounds can inhibit biofilm formation, induce detachment of preformed biofilms under static conditions, and that these compounds display distinct structure-activity relationships against V. cholerae and P. aeruginosa. Our findings highlight the significance of distinct bile acid components in the regulation of biofilm formation and dispersion in two different clinically relevant bacterial pathogens, and suggest that the bile acids, which are endogenous mammalian metabolites used to solubilize dietary fats, may also play a role in maintaining host health against bacterial infection

Bile acid components induce detachment of <i>V</i>. <i>cholerae</i> biofilms.

<p>(A) CSLM images of horizontal (xy) and vertical (xz and yz) projections of biofilm structures formed by the wild-type strain (wt). Biofilms were formed at 30°C for 5 hours. These biofilms were either untreated or exposed to DMSO, 0.4% Ox bile, 150 μM of TCA or TCDCA. CSLM images of biofilms were taken at 5, 7, and 24 hours. (B) Detachment from biofilms was evaluated by quantification of the planktonic population. CFU/mL of cells released from biofilms to the planktonic population was quantified for each condition at 7 h (left) and 24 h (right). Error bars indicate standard deviations of three biological replicates. *<i>P</i> < 0.05, **<i>P</i> < 0.005, n.s., <i>P</i> > 0.05.</p

Bile acid components effect biofilm formation in <i>P</i>. <i>aeruginosa</i>.

<p>(A) CLSM images of horizontal (xy) and vertical (xz and yz) projections of biofilm structures formed by <i>P</i>. <i>aeruginosa</i> Δ<i>wspF</i> mutant in the presence of DMSO control or 50 μM of TLCA. Biofilms were incubated at 37°C and images were taken at 24 hours. (B) CSLM images of preformed biofilms after treatment with either DMSO control or 50 μM of TLCA for 19 hours at 37°C.</p