We analyzed the usefulness of rpoA, recA, and pyrH gene sequences for the identification of vibrios. We sequenced fragments of these loci from a collection of 208 representative strains, including 192 well-documented Vibrionaceae strains and 16 presumptive Vibrio isolates associated with coral bleaching. In order to determine the intraspecies variation among the three loci, we included several representative strains per species. The phylogenetic trees constructed with the different genetic loci were roughly in agreement with former polyphasic taxonomic studies, including the 16S rRNA-based phylogeny of vibrios. The families Vibrionaceae, Photobacteriaceae, Enterovibrionaceae, and Salinivibrionaceae were all differentiated on the basis of each genetic locus. Each species clearly formed separated clusters with at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively. The genus Vibrio was heterogeneous and polyphyletic, with Vibrio fischeri, V. logei, and V. wodanis grouping closer to the Photobacterium genus. V. halioticoli-, V. harveyi-, V. splendidus-, and V. tubiashii-related species formed groups within the genus Vibrio. Overall, the three genetic loci were more discriminatory among species than were 16S rRNA sequences. In some cases, e.g., within the V. splendidus and V. tubiashii group, rpoA gene sequences were slightly less discriminatory than recA and pyrH sequences. In these cases, the combination of several loci will yield the most robust identification. We can conclude that strains of the same species will have at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively.Vibrios are gram-negative, usually motile rods, are mesophilic and chemoorganotrophic, and have a facultatively fermentative metabolism (5). They are generally able to grow on marine agar and on the selective medium thiosulfate-citratebile salt-sucrose agar and are mostly oxidase positive. Vibrios belong to the Gammaproteobacteria according to 16S rRNA gene sequence analysis. These bacteria are found abundantly in aquatic habitats and in association with eukaryotes. Associations established by vibrios range from mutualistic, e.g., Vibrio fischeri-bobtail squid (26), to pathogenic, e.g., V. cholerae-humans (45). Probiotic Vibrio strains for fish and shellfish have also been documented (44).The current family Vibrionaceae comprises the genera Enterovibrio (2 species), Grimontia (1 species), Photobacterium (7 species), Salinivibrio (1 species), and Vibrio (64 species). The novel species Photobacterium rosenbergii and Enterovibrio coralii have recently been proposed to encompass isolates associated with coral bleaching (41). Several new Vibrio species, mainly in the phylogenetic neighborhood of V. harveyi, V. halioticoli, V. splendidus, V. tubiashii, and V. fluvialis, have been described in the last few years, with V. neonatus, V. ezurae (28), and V. ponticus (22) being the most recent ones. V. harveyi, V. splendidus, and V. tubiashii are frequently associated with disease in different spe...

The microbial community associated with the reef building coral Pocillopora damicornis located on the Great Barrier Reef was investigated using culture-independent molecular microbial techniques. The microbial communities of three separate coral colonies were assessed using clone library construction alongside restriction fragment length polymorphism and phylogenetic analysis. Diversity was also investigated spatially across six replicate samples within each single coral colony using 16S rDNA and rpoB-DGGE analysis. Clone libraries demonstrated that the majority of retrieved sequences from coral tissue slurry libraries affiliated with gamma-Proteobacteria. This contrasted with clone libraries of seawater and coral mucus, which were dominated by alpha-Proteobacteria. A number of retrieved clone sequences were conserved between coral colonies; a result consistent with previous studies suggesting a specific microbe-coral association. rpoB-DGGE patterns of replicate tissue slurry samples underestimated microbial diversity, but demonstrated that fingerprints were identical within the same coral. These fingerprints were also conserved across coral colonies. The 16S rDNA-DGGE patterns of replicate tissue slurry samples were more complex, although non-metric multidimensional scaling (nMDS) analysis showed groupings of these banding patterns indicating that some bacterial diversity was uniform within a coral colony. Sequence data retrieved from DGGE analysis support clone library data in that the majority of affiliations were within the gamma-Proteobacteria. Many sequences retrieved also affiliated closely with sequences derived from previous studies of microbial diversity of healthy corals in the Caribbean. Clones showing high 16S rDNA sequence identity to both Vibrio shiloi and Vibrio coralliilyticus were retrieved, suggesting that these may be opportunist pathogens. Comparisons of retrieved microbial diversity between two different sampling methods, a syringe extracted coral mucus sample and an airbrushed coral tissue slurry sample were also investigated. Non-metric multidimensional scaling of clone library data highlighted that clone diversity retrieved from a coral mucus library more closely reflected the diversity of surrounding seawater than a corresponding coral tissue clone library.

The inhibitory properties of the microbial community of the coral mucus from the Mediterranean coral Oculina patagonica were examined. Out of 156 different colony morphotypes that were isolated from the coral mucus, nine inhibited the growth of Vibrio shiloi, a species previously shown to be a pathogen of this coral. An isolate identified as Pseudoalteromonas sp. was the strongest inhibitor of V. shiloi. Several isolates, especially one identified as Roseobacter sp., also showed a broad spectrum of action against the coral pathogens Vibrio coralliilyticus and Thallassomonas loyana, plus nine other selected Gram-positive and Gram-negative bacteria. Inoculation of a previously established biofilm of the Roseobacter strain with V. shiloi led to a 5-log reduction in the viable count of the pathogen within 3 h, while inoculation of a Pseudoalteromonas biofilm led to complete loss of viability of V. shiloi after 3 h. These results support the concept of a probiotic effect on microbial communities associated with the coral holobiont.

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Corals are inhabited by complex communities of microbes that affect their growth and survival. Several studies suggest that coral disease may be attributed to the success of vibrios in out-competing other bacteria in the mucus and tissues of corals. Vibrios utilize a variety of quorum sensing (QS) signal molecules to regulate processes that could be used to colonize corals during adverse environmental conditions. We therefore screened a range of Vibrios isolated from a variety of healthy and diseased corals, for the production of the QS signal molecules, N-acylhomoserine lactones (AHLs) and the AI-2 (autoinducer-2) small furanone signal molecule. All 29 strains examined activated the AI-2 biosensor, but only 17 activated an AHL biosensor. Using reverse phase thin-layer chromatography, we showed that the effect of temperature on AHL production varied considerably among the isolates. For the first time, the QS inhibition by Vibrio harveyi is reported. This only occurred at higher temperatures and does not appear to be due to degradation of AHLs. The large diversity of vibrios and the different effects of temperature on signal production may partly explain the complexity of coral-associated community changes in response to environmental factors.