We performed the first broad study aiming at the reconstruction of the evolutionary history of vibrios by means of multilocus sequence analysis of nine genes. Overall, 14 distinct clades were recognized using the SplitsTree decomposition method. Some of these clades may correspond to families, e.g., the clades Salinivibrio and Photobacteria, while other clades, e.g., Splendidus and Harveyi, correspond to genera. The common ancestor of all vibrios was estimated to have been present 600 million years ago. We can define species of vibrios as groups of strains that share >95% gene sequence similarity and >99.4% amino acid identity based on the eight protein-coding housekeeping genes. The gene sequence data were used to refine the standard online electronic taxonomic scheme for vibrios (http://www.taxvibrio.lncc.br).Vibrios are widespread in the aquatic environment, occupying a variety of ecological niches, such as the human and animal gut, the surface of chitinous organisms, most notably copepods, and the coral mucus layer. A better understanding of the ecology and the patterns of distribution of vibrios relies on the online electronic taxonomy. Polyphasic taxonomic studies of vibrios performed in recent years have underpinned this new paradigm in studies of the biodiversity and systematics of this group (16,17,19). Currently, we recognize 78 species of vibrios distributed into five phylogenetic robust clades corresponding to the genera Vibrio, Photobacterium, Salinivibrio, Enterovibrio, and Grimontia based on 16S rRNA gene sequences (16,17,19). Both genome content and architecture indicate that these genera share a common ancestor (12). In addition, the genera within vibrios are defined on the basis of their shared sequence similarities in different loci. Species within the genus Vibrio share at least 85% gene sequence similarity in recA, rpoA, and pyrH (18).Species of vibrios are defined as clusters of strains with high phenotypic and genotypic similarities. Clusters comprise strains with highly similar genomes as determined by multilocus sequence analysis (MLSA), amplified fragment length polymorphism analysis, and DNA-DNA hybridization (DDH) (16,17,19). Formal delineation of bacterial species still relies on DDH, with a cutoff level of Ͼ70% DDH similarity, but this technique is time-consuming and can be performed in relatively few laboratories and, more importantly, the DDH data are not cumulative in online databases. Clearly, a reliable and straightforward alternative is the use of MLSA. The usefulness of MLSA in the taxonomy of vibrios was described in previous papers (e.g., references 15 and 18). Overall, species form discrete clusters on the basis of recA, rpoA, and pyrH, with a species cutoff level of Ͼ94% gene sequence similarity (18). However, some groups of species, e.g., the Vibrio splendidus and Vibrio harveyi species groups, were somewhat fuzzy on the basis of recA, gyrB, and gapA (15, 18). Thus, it is very important to evaluate additional genetic markers that can distinguish closely related species o...

To date 142 species have been described in the Vibrionaceae family of bacteria, classified into seven genera; Aliivibrio, Echinimonas, Enterovibrio, Grimontia, Photobacterium, Salinivibrio and Vibrio. As vibrios are widespread in marine environments and show versatile metabolisms and ecologies, these bacteria are recognized as one of the most diverse and important marine heterotrophic bacterial groups for elucidating the correlation between genome evolution and ecological adaptation. However, on the basis of 16S rRNA gene phylogeny, we could not find any robust monophyletic lineages in any of the known genera. We needed further attempts to reconstruct their evolutionary history based on multilocus sequence analysis (MLSA) and/or genome wide taxonomy of all the recognized species groups. In our previous report in 2007, we conducted the first broad multilocus sequence analysis (MLSA) to infer the evolutionary history of vibrios using nine housekeeping genes (the 16S rRNA gene, gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA), and we proposed 14 distinct clades in 58 species of Vibrionaceae. Due to the difficulty of designing universal primers that can amplify the genes for MLSA in every Vibrionaceae species, some clades had yet to be defined. In this study, we present a better picture of an updated molecular phylogeny for 86 described vibrio species and 10 genome sequenced Vibrionaceae strains, using 8 housekeeping gene sequences. This new study places special emphasis on (1) eight newly identified clades (Damselae, Mediterranei, Pectenicida, Phosphoreum, Profundum, Porteresiae, Rosenbergii, and Rumoiensis); (2) clades amended since the 2007 proposal with recently described new species; (3) orphan clades of genomospecies F6 and F10; (4) phylogenetic positions defined in 3 genome-sequenced strains (N418, EX25, and EJY3); and (5) description of V. tritonius sp. nov., which is a member of the “Porteresiae” clade.

Identification and classification of Vibrio species have relied upon band pattern methods (e.g., amplified fragment length polymorphism) and DNA-DNA hybridization. However, data generated by these methods cannot be used to build an online electronic taxonomy. In order to overcome these limitations, we developed the first standard multilocus sequence scheme focused on the ubiquitous and pathogenic Vibrio harveyi species group (i.e., V. harveyi, V. campbellii, V. rotiferianus, and a new as yet unnamed species). We examined a collection of 104 isolates from different geographical regions and hosts using segments of seven housekeeping genes. These two species formed separated clusters on the basis of topA, pyrH, ftsZ, and mreB gene sequences. The phylogenetic picture obtained by the other three loci, i.e., gyrB, recA, and gapA, was more complex though. V. campbellii appeared nested within V. harveyi in the recA trees, whereas V. harveyi formed a tight nested cluster within V. campbellii by gapA. The gyrB gene had no taxonomic resolution and grouped the two species together. The fuzziness observed in these three genes seems not be related to recombination but to low divergence due to the accumulation of only a few substitutions. In spite of this, the concatenated sequences provided evidence that the two species form two separated clusters. These clusters did not arise by recombination but by accumulation of point mutations. V. harveyi and V. campbellii isolates can be readily identified through the open database resource developed in this study (http://www.taxvibrio.lncc.br/). We argue that the species should be defined by evolutionary criteria. Strains of the same species will share at least 95% concatenated sequence similarity using the seven loci, and, most importantly, cospecific strains will form cohesive readily recognizable phylogenetic clades.The species Vibrio harveyi and V. campbellii are widespread in the marine environment and among the main species responsible for disease in many wild and reared aquatic organisms, most notably peneid shrimp, several fish species, and mollusks (2). Luminous vibrios related to V. harveyi have been implicated principally in disease outbreaks in shrimp larviculture facilities and in grow-out ponds worldwide. More recently V. harveyi has been associated with infections in corals (20). Indeed recent studies have shown that vibrios, including the V. harveyi group, are abundant in the mucus of corals and may cause infections during periods of environmental imbalances (15).In the past, the identification of V. harveyi and related species isolated from the marine environment has been imprecise and represents hard work as it involves performing many biochemical and/or physiological tests (14). Presumptive V. harveyi isolates grow on thiosulfate-citrate-bile saltssucrose agar are motile, ferment glucose, and are oxidase positive and sensitive to the vibriostatic agent 0/129 at 150 g. They are arginine dihydrolase negative and lysine and ornithine decarboxylase positive. Most presump...

Current growth in aquaculture production is parallel with the increasing number of disease outbreaks, which negatively affect the production, profitability, and sustainability of the global aquaculture industry. Vibriosis is among the most common diseases leading to massive mortality of cultured shrimp, fish, and shellfish in Asia. High incidence of vibriosis can occur in hatchery and grow-out facilities, but juveniles are more susceptible to the disease. Various factors, particularly the source of fish, environmental factors (including water quality and farm management), and the virulence factors of Vibrio, influence the occurrence of the disease . Affected fish show weariness, with necrosis of skin and appendages, leading to body malformation, slow growth, internal organ liquefaction, blindness, muscle opacity, and mortality. A combination of control measures, particularly a disease-free source of fish, biosecurity of the farm, improved water quality, and other preventive measures (e.g., vaccination) might be able to control the infection. Although some control measures are expensive and less practical, vaccination is effective, relatively cheap, and easily implemented. In this review, the latest knowledge on the pathogenesis and control of vibriosis, including vaccination, is discussed. 4INA-SALWANY ET AL.

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Vibriosis in cultured marine fishes: a review ABSTRCT For more than a century, vibriosis affects various species of economically important cultured marine fishes around the globe. The knowledge of this bacterial disease on many species of cultured fish is still lacking, but progressing. This review focuses on updated fundamental knowledge related to vibriosis including the history, taxonomy, and various epidemiological aspects such as socio-economy, clinical signs, pathological changes, diagnosis, pathogenesis, transmission, risk factors and control measures of vibriosis. This review revealed a rising prevalence of vibriosis in aquaculture, concomitant with the rapid development of this industry worldwide. Yet, information on Vibrio infection in cultured fish, particularly on the Vibrio of non-medical importance, the influence of their virulence toxins to host cells, effects of global warming and the socioeconomic impacts are still scarce, and need more profound studies. Moreover, comprehensive epidemiological information on vibriosis are quite limited in many Asian countries with tropical climate, limiting the progression in control and prevention aspects of the disease.

Deep-sea vents harbor dense populations of various animals that have their specific symbiotic bacteria. Scaly-foot gastropods, which are snails with mineralized scales covering the sides of its foot, have a gammaproteobacterial endosymbiont in their enlarged esophageal glands and diverse epibionts on the surface of their scales. In this study, we report the complete genome sequencing of gammaproteobacterial endosymbiont. The endosymbiont genome displays features consistent with ongoing genome reduction such as large proportions of pseudogenes and insertion elements. The genome encodes functions commonly found in deep-sea vent chemoautotrophs such as sulfur oxidation and carbon fixation. Stable carbon isotope (13C)-labeling experiments confirmed the endosymbiont chemoautotrophy. The genome also includes an intact hydrogenase gene cluster that potentially has been horizontally transferred from phylogenetically distant bacteria. Notable findings include the presence and transcription of genes for flagellar assembly, through which proteins are potentially exported from bacterium to the host. Symbionts of snail individuals exhibited extreme genetic homogeneity, showing only two synonymous changes in 19 different genes (13 810 positions in total) determined for 32 individual gastropods collected from a single colony at one time. The extremely low genetic individuality in endosymbionts probably reflects that the stringent symbiont selection by host prevents the random genetic drift in the small population of horizontally transmitted symbiont. This study is the first complete genome analysis of gastropod endosymbiont and offers an opportunity to study genome evolution in a recently evolved endosymbiont.

Sulfurovum aggregans sp. nov., a hydrogenoxidizing, thiosulfate-reducing chemolithoautotroph within the Epsilonproteobacteria isolated from a deep-sea hydrothermal vent chimney, and an emended description of the genus Sulfurovum , was isolated from a deep-sea hydrothermal vent chimney at the Central Indian Ridge. The non-motile, rod-shaped cells were Gram-stain-negative and non-sporulating. Growth was observed between 15 and 37 6C (optimum 33 6C; 3.2 h doubling time) and between pH 5.4 and 8.6 (optimum pH 6.0). The isolate was a strictly anaerobic chemolithoautotroph capable of using molecular hydrogen as the sole energy source and carbon dioxide as the sole carbon source. The G+C content of the genomic DNA was 42.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the novel isolate belonged to the genus Sulfurovum and was closely related to Sulfurovum sp. NBC37-1 and Sulfurovum lithotrophicum 42BK T (95.6 and 95.4 % similarity, respectively). DNA-DNA hybridization demonstrated that the novel isolate could be differentiated genotypically from Sulfurovum sp. NBC37-1 and Sulfurovum lithotrophicum. On the basis of the molecular and physiological traits of the new isolate, the name Sulfurovum aggregans sp. nov. is proposed, with the type strain Monchim33 T (5JCM 19824Deep-sea hydrothermal vents represent one of the most productive marine ecosystems. The ecosystem is supported primarily by microbial chemosynthesis (Jannasch, 1985). Deep-sea vent chemolithoautotrophs are able to use a range of reductive substrates as energy sources (Campbell et al., 2006;Fisher et al., 2007;Sievert & Vetriani, 2012). Molecular hydrogen is one of the most abundant reducing gas components in vent fluids. A diversity of hydrogen-oxidizing, chemolithoautotrophic, thermophilic and hyperthermophilic genera have been isolated from deep-sea hydrothermal fields (Sievert & Vetriani, 2012). In addition, recent research indicated that H 2 could be an important energy source for symbiotic psychrophilic to mesophilic bacteria (Petersen et al., 2011). Various mesophilic, hydrogen-oxidizing Epsilonproteobacteria have been isolated from deep-sea vents, including Sulfurimonas paralvinellae GO25 T , Sulfurovum sp. NBC37-1 (Nakagawa et al., 2007), Thioreductor micantisoli BKB25Ts-Y T (Nakagawa et al., 2005a) and 'Thiofractor thiocaminus' 496Chim (Makita et al., 2012). In particular, members of the genus Sulfurovum are frequently found as endo-and/or epi-symbionts of various vent animals in global deep-sea hydrothermal fields (Haddad et al., 1995;Polz & Cavanaugh, 1995;Cary et al., 1997;Goffredi et al., 2004; Suzuki et al., 2005; Urakawa et al., 2005;Watsuji et al., 2010). However, the description of the genus Sulfurovum is based on just two strains originated from the 3Present address: