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9 pages, 1 figure, 3 tables, Strain ISS155T, isolated from surface Mediterranean seawater, has cells that are Gram-reaction-negative, motile, strictly aerobic chemoorganotrophic, oxidase-positive, unable to reduce nitrate to nitrite, and able to grow with cellulose as the sole carbon and energy source. It is mesophilic, neutrophilic, slightly halophilic and has a requirement for sodium and magnesium ions. Its 16S rRNA gene sequence places the strain among members of Cellvibrionaceae, in the Gammaproteobacteria, with Agarilytica rhodophyticola 017T as closest relative (94.3¿% similarity). Its major cellular fatty acids are C18¿:¿1, C16¿:¿0 and C16¿:¿1; major phospholipids are phosphatidyl glycerol, phosphatidyl ethanolamine and an unidentified lipid, and the major respiratory quinone is Q8. The genome size is 6.09 Mbp and G+C content is 45.2¿mol%. A phylogenomic analysis using UBCG merges strain ISS155T in a clade with A. rhodophyticola, Teredinibacter turnerae, Saccharophagus degradans and Agaribacterium haliotis type strain genomes, all of them possessing a varied array of carbohydrate-active enzymes and the potential for polysaccharide degradation. Average amino acid identity indexes determined against available Cellvibrionaceae type strain genomes show that strain ISS155T is related to them by values lower than 60¿%, with a maximum of 58¿% to A. rhodophyticola 017T and 57¿% to T. turnerae T7902T and S. degradans 2-40T. These results, together with the low 16S rRNA gene sequence similarities and differences in phenotypic profiles, indicate that strain ISS155T represents a new genus and species in Cellvibrionaceae, for which we propose the name Thalassocella blandensis gen. nov., sp. nov., and strain ISS155T (=CECT 9533T=LMG 31237T) as the type strain, Sequencing was funded by grant CTM2016-80095-C2-1-R to C.P.A. from the Spanish Ministerio de Economía y Competitividad. Additional funding was obtained by grant CTM2017-87736-R to S.G.A. from the Spanish Ministerio de Economía y Competitividad, With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

21 pages, 7 figures, 6 tables, Two strains isolated from a sample of activated sludge that was obtained from a seawater-based wastewater treatment plant on the southeastern Mediterranean coast of Spain have been characterized to achieve their taxonomic classification, since preliminary data suggested they could represent novel taxa. Given the uniqueness of this habitat, as this sort of plants are rare in the world and this one used seawater to process an influent containing intermediate products from amoxicillin synthesis, we also explored their ecology and the annotations of their genomic sequences. Analysis of their 16S rRNA gene sequences revealed that one of them, which was orange-pigmented, was distantly related to Vicingus serpentipes (family Vicingaceae) and to other representatives of neighbouring families in the order Flavobacteriales (class Flavobacteriia) by 88–89 % similarities; while the other strain, which was yellow-pigmented, was a putative new species of Lysobacter (family Xanthomonadaceae, order Xanthomonadales, class Gammaproteobacteria) with Lysobacter arseniciresistens as closest relative (97.3 % 16S rRNA sequence similarity to its type strain). Following a polyphasic taxonomic approach, including a genome-based phylogenetic analysis and a thorough phenotypic characterization, we propose the following novel taxa: Parvicella tangerina gen. nov., sp. nov. (whose type strain is AS29M-1T=CECT 30217T=LMG 32344T), Parvicellaceae fam. nov. (whose type genus is Parvicella), and Lysobacter luteus sp. nov. (whose type strain is AS29MT=CECT 30171T=LMG 32343T), Financial support was received from grant AICO-2020-181 (Generalitat Valenciana) to M.J.P. S.G.A. was supported by Plan Nacional I+D+I 2020 (PID2020-116489RB-I00), With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

Strain CECT 7735T, a marine Gram-reaction negative, aerobic, non-motile bacterium, was isolated from coastal seawater in Valencia, Spain. Strain CECT 7735T is chemoorganotrophic, mesophilic, slightly halophilic, grows at 15–28 °C but not at 4 or 37 °C, requires seawater for growth and grows up to 6 % salinity. The major cellular fatty acid is summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The G+C content of the genome is 55.7 mol%. Comparative analysis of the 16S rRNA gene sequence shows the strain is affiliated to the family Rhodobacteraceae , in the class Alphaproteobacteria , with highest similarities to Phaeobacter species (97.0–97.5 %), Shimia species (96.5–97.3 %) and Pseudopelagicola gijangensis (96.5 %). Further phylogenomic analysis through the up-to-date-bacterial core gene (UBCG) set showed P. gijangensis to be its closest relative. Average nucleotide identity and in silico DNA–DNA hybridization values are lower than 85 and 21 %, respectively, with its phylogenetic relatives, suggesting that strain CECT 7735T represents a new species. The average amino acid identity value was over 70 % with the genome of the type strain of P. gijangensis and with all those of Shimia species. These values, together with UBCG set trees, suggest that the new species and P. gijangensis belong to the same genus and that Pseudopelagicola should be reclassified as a Shimia species. We conclude that strain CECT 7735T represents a new species in the genus Shimia , for which we propose the name Shimia thalassica sp. nov. In addition, Pseudopelagicola gijangensis is reclassified as Shimia gijangensis comb. nov. From the same phylogenomic study, it can be concluded that Thalassobius activus should be reclassified in the genus Cognatishimia as Cognatishimia activa comb. nov.

A polyphasic taxonomic study was conducted on three strains isolated from drinking water systems that had previously been deposited as Chryseobacterium species at the Spanish Type Culture Collection in order to complete their classification. Strains CECT 9293T, CECT 9390T and CECT 9393T were isolated from sites in Barcelona, Spain, in the framework of a project aimed at generating the first MALDI-TOF database specific for bacteria present in water for human consumption. Their partial 16S rRNA sequences showed that their closest relatives among the type strains of Chryseobacterium exhibited 98 % similarity or less, supporting their taxonomic novelty. At the same time, comparison between them revealed that strains CECT 9293T and CECT 9393T could perhaps be related at the species level as they shared 99.5 % similarity. However, whole genome sequencing was performed and the subsequent calculation of relatedness indices, average nucleotide identity and estimated DNA–DNA hybridization, ruled out that possibility and confirmed instead that each of the strains should be considered a separate species in the genus Chryseobacterium . Having clarified their status, we also performed phylogenomic analyses and searched for possible environmental or non-type material sequences that could be related to any of them at the species level. In parallel, the strains were characterized phenotypically and compared to their closest relatives to determine diagnostic traits to support their formal proposal. The proposed species are Chryseobacterium potabilaquae sp. nov. with the type strain CECT 9293T (=LMG 32084T), Chryseobacterium aquaeductus sp. nov. with the type strain CECT 9390T (=LMG 32085T) and Chryseobacterium fistulae sp. nov. with the type strain CECT 9393T (=LMG 32086T).

16 pages, 5 figures, 3 tables, supplementary information https://doi.org/10.1186/s12866-020-01884-7, Isolation of marine microorganisms is fundamental to gather information about their physiology, ecology and genomic content. To date, most of the bacterial isolation efforts have focused on the photic ocean leaving the deep ocean less explored. We have created a marine culture collection of heterotrophic bacteria (MARINHET) using a standard marine medium comprising a total of 1561 bacterial strains, and covering a variety of oceanographic regions from different seasons and years, from 2009 to 2015. Specifically, our marine collection contains isolates from both photic (817) and aphotic layers (744), including the mesopelagic (362) and the bathypelagic (382), from the North Western Mediterranean Sea, the North and South Atlantic Ocean, the Indian, the Pacific, and the Arctic Oceans. We described the taxonomy, the phylogenetic diversity and the biogeography of a fraction of the marine culturable microorganisms to enhance our knowledge about which heterotrophic marine isolates are recurrently retrieved across oceans and along different depths, Projects Arctic Tipping Points (ATP, contract #226248), in the FP7 program of the European Union, and DOREMI (CTM2012–34294), from the Spanish Ministry of Economy and Competitiveness, allowed the collection of samples from the Arctic and NW Mediterranean Sea, respectively. Research, including laboratory experiments and analyses, was mainly funded by grant BIOSENSOMICS “Convocatoria 2015 de ayudas Fundación BBVA a investigadores y creadores culturales” and, MAGGY, Plan Nacional I + D + I 2017 (CTM2017–87736-R), awarded to S.G.A. Analyses were also possible thank to the King Abdullah University of Science and Technology (KAUST) through baseline funding to C.M.D. and a subaward agreement OSR-2014-CC-1973-02 between KAUST and Universitat Autònoma de Barcelona (UAB). Additional funding for analyses and interpretation of the data was provided by projects REMEI (ref. CTM2015–70340-R) and MIAU-S3 (ref. RTI2018-101025-B-I00) from the Spanish Ministry of Economy and Competitiveness, With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

10 pages, 4 figures, 3 tables, Strain ISS653T, isolated from Atlantic seawater, is a yellow pigmented, non-motile, Gram-reaction-negative rod-shaped bacterium, strictly aerobic and chemoorganotrophic, slightly halophilic (1–15 % NaCl) and mesophilic (4–37 °C), oxidase- and catalase-positive and proteolytic. Its major cellular fatty acids are iso-C15 : 0, iso-C15 : 0 2-OH, and iso-C17 : 0 3-OH; the major identified phospholipid is phosphatidylethanolamine and the major respiratory quinone is MK6. Genome size is 4.28 Mbp and DNA G+C content is 34.9 mol%. 16S rRNA gene sequence similarity places the strain among members of the family Flavobacteriaceae, with the type strains of Mesonia phycicola (93.2 %), Salegentibacter mishustinae (93.1 %) and Mesonia mobilis (92.9 %) as closest relatives. Average amino acid identity (AAI) and average nucleotide identity (ANI) indices show highest values with M. mobilis (81 % AAI; 78.9 % ANI), M. phycicola (76 % AAI; 76.3 % ANI), Mesonia maritima (72 % AAI, 74.9 % ANI), Mesonia hippocampi (64 % AAI, 70.8 % ANI) and Mesonia algae (68 % AAI; 72.2 % ANI). Phylogenomic analysis using the Up-to-date-Bacterial Core Gene set (UBCG) merges strain ISS653T in a clade with species of the genus Mesonia. We conclude that strain ISS653T represents a novel species of the genus Mesonia for which we propose the name Mesonia oceanica sp. nov., and strain ISS653T (=CECT 9532T=LMG 31236T) as the type strain. A second strain of the species, ISS1889 (=CECT 30008) was isolated from Pacific Ocean seawater. Data obtained throughout the Tara oceans expedition indicate that the species is more abundant in the mesopelagic dark ocean than in the photic layer and it is more frequent in the South Pacific, Indian and North Atlantic oceans, Sequencing was funded by grant CTM2016-80095-C2-1-R to C.P.A. from the Spanish Ministerio de Economía y Competitividad. Additional funding was obtained by grant CTM2017-87736-R to S.G.A. from the Spanish Ministerio de Economía y Competitividad. Other parts of this work received no specific grant from any funding agency, With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

Two bacterial strains, EAod9T and SMJ21T, isolated from salt-marsh plants, were determined to be related to species of the genus Vibrio from from 16S rRNA sequence comparisons. Their closest phylogenetic relatives are members of the Gazogenes clade, Vibrio mangrovi and Vibrio rhizosphaerae , which show the greatest similarity to the SMJ21TrRNA sequence (97.3 and 97.1 %, respectively), while EAod9T had less than 97.0 % similarity to any other species of the genus Vibrio . Both strains share the basic characteristics of the genus Vibrio , as they are Gram-stain negative, motile, slightly halophilic, facultatively anaerobic bacteria. In addition, they are oxidase-negative and unable to grow on TCBS Agar; they grow between 15 to 26 °C, pH 6 to 8 and in up to 10 % (w/v) total salinity. They produce indol, are positive in the Voges-Proskauer test and are negative for arginine dihydrolase, lysine and ornithine decarboxylases. Strain SMJ21T is aerogenic and red-pigmented, due to prodigiosin production, while strain EAod9T ferments glucose without gas and is not pigmented. The major cellular fatty acids of both novel strains were C16 : 1ω7c/C16 : 1ω6c and C16 : 0. WGSobtained for both strains, along with the other five members of the clade, allowed the determination of ANI indexes and in silico estimations of DDH values, which confirmed that the two strains represent two novel species of the genus Vibrio: Vibrio palustris sp. nov. (with EAod9T=CECT 9027T=LMG 29724T as the proposed type strain) and Vibrio spartinae sp. nov. (with SMJ21T=CECT 9026T=LMG 29723T as the proposed type strain).

The genomes of the type strains of Roseivivax atlanticus and Roseivivax marinus ( Rhodobacteraceae , Alphaproteobacteria ), were analysed to determine their respective Average Nucleotide Identity (ANI) and in silico DNA–DNA hybridization (DDH) values. These species were proposed and effectively published relatively closely in time (February and August 2014, respectively) and so not taking account of the other. The intergenomic relatedness between both type strains, 97.0–97.4 % ANI and 82.8 % in silico DDH, confirm that they represent members of the same genomic species. This conclusion is also supported at the phenotypic level. Since the name Roseovarius marinus was validly published earlier, R. atlanticus (Validation List 161, IJSEM 65, 1–4. 2015) should be considered a later heterotypic synonym of R. marinus (Dai, Shi, Gao, Liu and Zhang, 2014), in application of the priority rule.

Strain 96-237T, a Gram-reaction-negative, curved- to spiral-shaped motile bacterium, isolated from coastal marine water, was found to be related to species of the genus Grimontia by 16S rRNA gene sequence comparison, sharing 98.3 % similarity to Grimontia marina CECT 8713Tand 98.8 % to 'Grimontiaindica' AK16. Phenotypic analysis revealed that strain 96-237T is slightly halophilic, mesophilic and facultatively anaerobic, fermenting d-glucose, d-ribose, d-mannose, d-mannitol, maltose and sucrose. It was positive for oxidase and indole production and negative for arginine dihydrolase and lysine and ornithine decarboxylases. Its major fatty acids were C16 : 1ω7c/C16 : 1ω6c (SF3), C18 : 1ω7c and C16 : 0. Its DNA G+C content was 48.4 mol%. The strain was different at the species level from all other species of the genusGrimontia, with average nucleotide identity indices of 79.6 % to Grimontia. hollisae CECT 8713T, 87.8 % to G. marina CECT 5069T and 89.1 % to 'G. indica' AK16 genomes. Thus, the strain represents a novel species for which we propose the name Grimontia celer and 96-237T (=CECT 9029T =KCTC 42960T =LMG 29238T) as the type strain.

Strain CECT 5091T, an aerobic, marine, Gram-reaction- and Gram-stain-negative, chemoheterotrophic bacterium was isolated from oysters harvested off the Spanish Mediterranean coast. Analysis of the 16S rRNA gene sequence placed the strain within the genus Ruegeria , in the family Rhodobacteraceae , with 16S rRNA gene similarities of 98.7, 98.7 and 98.4 % to Ruegeria conchae , Ruegeria atlantica and Ruegeria arenilitoris , respectively. Average nucleotide identities (ANI) and in silico DNA–DNA hybridization (DDH) were determined, comparing the genome sequence of CECT 5091T with those of the type strains of 12 species of the genus Ruegeria : the values obtained were always below the thresholds (95–96 % ANI, 70 % in silico DDH) used to define genomic species, proving that CECT 5091T represents a novel species of the genus Ruegeria . The strain was slightly halophilic and mesophilic, with optimum growth at 26 °C, pH 7.0 and 3 % salinity, it required sodium and magnesium ions for growth and was able to reduce nitrate to dinitrogen. Carbon sources for growth include some carbohydrates (d-ribose, d-glucose, l-rhamnose, N-acetyl-d-glucosamine) and multiple organic acids and amino acids. The major cellular fatty acid was summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), representing 70 % of the total fatty acids. Carbon monoxide oxidation, cyanophycin synthetic ability and phosphatidylglycerol, diphosphatidylglycerol and phosphatidylcholine production are predicted from genome annotation, while bacteriochlorophyll a production was absent. The DNA G+C content of the genome was 56.7 mol%. We propose the name Ruegeria denitrificans sp. nov. and strain CECT 5091T (=5OM10T=LMG 29896T) as the type strain for the novel species.

Two strains of Gram-negative, facultatively anaerobic, slightly halophilic bacteria, isolated from healthy gilthead sea bream (Sparus aurata) cultured in Spanish Mediterranean fish farms, were different from their closest relatives,Vibrio scophthalmiandV. ichthyoenteri, by phenotypic, phylogenetic and genomic standards. The strains were negative for decarboxylase tests and lacked extracellular hydrolytic activities, but were able to fermentd-mannitol, sucrose, cellobiose andd-gluconate, among other carbohydrates. The major cellular fatty acids were C16:1and C16:0, in agreement with other species of the genusVibrio. Their 16S rRNA gene sequences were 98.4 and 97.2 % similar to those of the type strains ofV. scophthalmiandV. ichthyoenteri, and the similarities using other housekeeping genes (ftsZ,rpoD,recA,mreBandgyrB) and indices of genomic resemblance (average nucleotide identity and estimated DNA–DNA hybridization) between the isolates and those type strains were clearly below intraspecific levels, supporting the recognition of the strains as members of a separate novel species. Thus, we propose the nameVibrio renipiscariussp. nov., with DCR 1-4-2T( = CECT 8603T = KCTC 42287T) as the type strain.

The synonymy between Vibrio hemicentroti Kim et al. 2013 and Vibrio splendidus (Beijerinck 1900) Baumann et al. 1981 was suggested after a recent multilocus sequence analysis of the Splendidus clade, which included the type strains of both species. To clarify their status, we have determined genomic indexes from whole genome sequences of strains V. hemicentroti CECT 8714T and V. splendidus NCCB 53037T. Average Nucleotide Identities of 96.0–96.7 % and an in silico DNA–DNA hybridization value of 70.2 %, as well as similarity levels of selected housekeeping gene sequences support the consideration of V. hemicentroti as a later heterotypic synonym of V. splendidus .

Strain R46T, a marine alphaproteobacterium, was isolated from Mediterranean seawater at Malvarrosa beach, Valencia, Spain. It is an aerobic chemo-organotrophic, mesophilic and slightly halophilic organism, with complex ionic requirements. Phylogenetic analyses based on the 16S rRNA and gyrB gene sequences showed that strain R46T formed a separate branch within the family Rhodobacteraceae , bearing similarities below 94.7 and 80.3 %, respectively, to any other recognized species. It contained Q10 as the predominant isoprenoid quinone and C18 : 1ω7c/C18 : 1ω6c as the major cellular fatty acid. Phosphatidylglycerol was the only identified polar lipid, although other lipids were also detected. The DNA G+C content of the genomic DNA was 61.3 mol%. On the basis of extensive phenotypic and phylogenetic comparative analysis, it is concluded that the strain represents a novel genus and species, for which the name Actibacterium mucosum gen. nov., sp. nov. is proposed. The type strain of the type species is Actibacterium mucosum R46T ( = CECT 7668T = KCTC 23349T).

Nereida ignava strain 2SM4T (= CECT 5292T = DSM 16309T = CIP 108404T = CCUG 49433T) is a marine bacterium belonging to the Roseobacter group of the family Rhodobacteraceae within the class Alphaproteobacteria. The strain was isolated from sea water surrounding cultivated oysters 2–3 miles off the Mediterranean coast near Valencia (Spain) and was phylogenetically related to uncultured clones of gall symbiont bacteria of some species of Prionitis alga. Here we describe the genome sequence and annotation of this organism, the type strain of the single species of this genus. The genome comprised 2,888,349 bp, 2,872 protein-coding genes and 52 RNA genes. The annotation revealed the capacity to produce bacteriocins, vitamins and auxins. Besides, it contained sulfur cycling related genes.

Two strains of Gram-stain-negative, chemo-organotrophic, aerobic and halophilic gammaproteobacteria, isolated from within the stem and roots of Spartina maritima in salt marshes from the south Atlantic Spanish coast, were found to represent a novel species in the genus Marinomonas through phylogenetic analysis of their 16S rRNA genes and phenotypic characterization. 16S rRNA gene sequences of the two strains shared

A facultatively anaerobic marine gammaproteobacterium, designated strain M46T, was isolated from Mediterranean seawater at Malvarrosa beach, Valencia, Spain. The strain was characterized by using a polyphasic approach and was found to be situated within the genus Photobacterium in the family Vibrionaceae. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain M46T was closely related to P. rosenbergii CECT 7644T, P. halotolerans CECT 5860T and P. ganghwense CECT 7641T, showing sequence similarities of 96.8, 96.4 and 96.2 %, respectively. According to the results of phylogenetic analyses based on recA and gyrB gene sequences, the most closely related taxon was P. ganghwense CECT 7641T with 87.4 and 85.0 % sequence similarity, respectively. Regardless of the gene used in phylogenetic analysis, strain M46T always formed a separate and stable clade containing these three species of the genus Photobacterium. Strain M46T was not luminescent and produced a diffusible brown pigment. It required NaCl to grow, reduced nitrate to nitrite and oxidized a small number of substrates in Biolog GN plates. Strain M46T was positive for arginine dihydrolase (ADH), β-galactosidase, aesculin hydrolysis and DNase activity. In API ZYM tests, the novel strain was positive for alkaline phosphatase, leucine arylamidase and acidic phosphatase activities. The major cellular fatty acids were unsaturated C18 and C16, as in other members of the genus Photobacterium, but their relative amounts and the presence or absence of other fatty acids differentiated strain M46T from its closest relatives. Based on the results of this polyphasic taxonomic study, strain M46T represents a novel species of the genus Photobacterium, for which the name Photobacterium aphoticum is proposed. The type strain is M46T ( = CECT 7614T = KCTC 23057T).

Vibrio2009, the third international conference on the biology of Vibrios, was held in Rio de Janeiro, Brazil, in November 2009. This conference united researchers studying various aspects of pathogenesis, symbiosis and environmental persistence of this diverse group of marine bacteria. Through many of the presentations, it became apparent how horizontal gene transfer and genetic flexibility has driven the incredible diversity of these microbes. Interestingly, unifying themes of behaviour could be seen in the interaction(s) of Vibrios with other organisms, such as with other bacteria, corals, invertebrates and humans. Presentations illuminated the idea that the path towards symbiosis is not that different from the path towards disease, and that alterations in environmental conditions, such as climate change, can tip the balance and change the Vibrio interactions from benign to pathogenic.

5 pages, 2 figures, 1 table, A novel heterotrophic, marine, strictly aerobic, motile bacterium was isolated from the Red Sea at a depth of 1 m. Analysis of its 16S rRNA gene sequence, retrieved from the whole-genome sequence, showed that this bacterium was most closely related to the genera Oleispira, Oceanobacter and Thalassolituus, each of which contains a single species, within the class Gammaproteobacteria. Phenotypic, genotypic and phylogenetic analyses supported the creation of a novel genus and species to accommodate this bacterium, for which the name Bermanella marisrubri gen. nov., sp. nov. is proposed. The type strain of Bermanella marisrubri is RED65T (=CECT 7074T =CCUG 52064T). © 2009 IUMS, This work was supported by the Swedish Science Council (project 621-2003-2692, to J. P.) and the Spanish Ministerio de Educación y Ciencia (project CGL-2005-02292, to M. J. P.). D. R. A. has a contract with the Universitat de València under the ‘Ramón y Cajal’ program (Ministerio de Educación y Ciencia, Spain)

The possible influence of the feeding regime (FR) on the immune system and pathological status of gilthead sea bream was studied. Two growth trials were performed starting at different seasons (trial 1 = March; trial 2 = June) under controlled experimental conditions. In both trials, FR-1 groups received a restricted amount of food, whereas FR-2 groups were fed to visual satiety. The pathology study included parasitological and bacteriological examination, and the immunological traits analysed were respiratory burst activity of head kidney leucocytes, serum lysozyme and alternative pathway complement activity (ACH50). The immunological status of gilthead sea bream not only was not impaired by the restricted feeding regime, but also seemed to be enhanced in some aspects, as the respiratory burst of FR-1 fish of trial 2 was significantly higher. No differences in the bacterial isolates were detected between the two feeding regimes, and Vibrio harveyi was the most prevalent species in both cases, especially in warm months. Also, fish under FR-1 regimes had significantly lower mortality, lower prevalence of infection of all the parasites except Cryptosporidium molnari, and less histopathological alterations in liver and intestine than those under FR-2 regimes. © 2003 Elsevier Science B.V. All rights reserved., This work was supported by research grants from the Spanish Ministerio de Educación, Cultura y Deporte under FEDER Program no. 1FD97-979-C02. M. Mingarro was recipient of a fellowship from the Diputación de Castellón. We are thankful to M.L. Alonso, M.A. González and G. Sanza for technical assistance in the samplings, and to J. Montfort for the histological processing.

Two strains of a novel bacterium were isolated independently of each other, from different depths in the Mediterranean Sea, within a time period of 7 months, using two different isolation approaches that were focused on different objectives. Both strains, designated ISO1 and ISO4T, were halophilic, Gram-negative, strictly aerobic, straight rods that were oxidase- and catalase-positive. Both strains produced mucoid colonies in some defined minimal media and were able to grow with organic acids and some alkanes; they were also able to accumulate intracellular poly-beta-hydroxybutyrate granules. The G + C content of the DNA of strain ISO4T was 66 mol%. Comparative analysis of 16S rRNA gene sequences showed that the closest described species to the novel strains were Alcanivorax borkumensis and Fundibacter jadensis, both of the gamma-Proteobacteria. Both of these recognized species were originally isolated from North Sea waters and are able to degrade aliphatic compounds, a property shared with strains ISO1 and ISO4T. However, strains ISO1 and ISO4T were different from A. borkumensis and F. jadensis, not only in their 16S rDNA sequences but also in the motility of their cells (by polar flagella) and by the presence of C19:Ocyclo in their cellular fatty acids, among other differential features. On the basis of biochemical and molecular data, it is suggested that strains ISO1 and ISO4T be recognized as a novel species of the genus Alcanivorax, for which the name Alcanivorax venustensis (ISO4T =DSM 13974T =CECT 5388T) is proposed. On the basis of its high phenotypic similarity and close phylogenetic relatedness to A. borkumensis, it is also proposed that F. jadensis (DSM 12178T) be reclassified as Alcanivorax jadensis in the genus Alcanivorax, and that the description of the genus Alcanivorax be emended.

Vibrio harveyi was isolated from internal organs or ulcers of diseased and apparently healthy gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) cultured in several fish farms located on the Spanish Mediterranean coast. The prevalence of the bacterium was significantly higher in European sea bass than in gilthead sea bream, and was closely related to the season in both fish species, occurring almost exclusively on warm months (June to November). After phenotypic characterization, a selection of forty five isolates from gilthead sea bream, sea bass, and several isolates previously obtained from common dentex (Dentex dentex) of the same area, were molecularly typed by automated ribotyping and random amplified polymorphic DNA (RAPD) analysis. Cluster analysis of data established 8 RAPD types and 13 ribotypes among wild isolates, and the combination of both techniques allowed to define fourteen different groups and a clear discrimination of all outbreaks and samplings. Several strains isolated from diseased gilthead sea bream and sea bass and also from asymptomatic sea bream, were tested for virulence in both fish species by intracoelomic injection. All the isolates (11) were pathogenic for sea bass, with nine out of the eleven LD50 values ranging from 1.5 × 105 to 1.6 × 106 cfu/fish. Gilthead sea bream was unaffected by the seven tested strains, even by those more virulent for sea bass, and only one strain caused a 10% mortality at 4.2 × 107 cfu/fish. This is the first report on virulence of V. harveyi for sea bass., This work was supported by research grants from the Spanish Ministerio de Educación, Cultura y Deporte MAR-98/1000 C02 and FEDER Program 1FD97-979-C02. We are thankful to M.L. Alonso for technical assistance in the samplings.

Strain R46 (CECT 7668; KCTC 23349), a nomenclatural type of Actibacterium mucosum , was isolated from surface seawater collected at Malvarrosa Beach (Valencia, Spain) in July 2008. The draft genome sequence of strain R46 (approximately 3.72 Mbp) contains 22 scaffolds and 3,619 protein-encoding genes, with a G+C content of 60.8 mol%.

Strain MD5T, an aerobic marine alphaproteobacterium, was isolated from Mediterranean seawater at Malvarrosa beach, Valencia, Spain. The strain was characterized in a polyphasic study and was placed phylogenetically within the Roseobacter clade in the family Rhodobacteraceae . Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MD5T is related to Tropicibacter naphthalenivorans C02T, Phaeobacter inhibens T5T, P. gallaeciensis BS107T and P. daeponensis TF-218T, with 96.9, 96.2, 96.1 and 96.1 % sequence similarity, respectively. Phylogenetic analyses also showed that strain MD5T forms a stable clade only with T. naphthalenivorans C02T. Strain MD5T requires Na+ plus a divalent cation (either Mg2+ or Ca2+) to grow, does not reduce nitrate to nitrite and uses a large number of carbohydrates as sole carbon sources. It is positive for β-galactosidase and urease activities and aesculin hydrolysis. Enzyme activities displayed in the API ZYM strip were alkaline phosphatase, leucine arylamidase, acid phosphatase and α-glucosidase. Major cellular fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c; 70.9 %) and C16 : 0 (8.2 %). The results of physiological and biochemical tests allowed clear phenotypic differentiation of this isolate from the only described species of the genus Tropicibacter . It is evident from the genotypic and phenotypic data obtained that the strain should be classified in a novel species in the genus Tropicibacter . The name Tropicibacter multivorans sp. nov. is proposed, with the type strain MD5T ( = CECT 7557T = KCTC 23350T).

Strain 7SM30T , an aerobic marine, Gram-negative, heterotrophic and yellow- to orange-pigmented bacterium isolated from seawater from Castellón, Spain, was characterized using a polyphasic approach. Analysis of the 16S rRNA gene sequence showed that the isolate represented a novel lineage within the family Flavobacteriaceae. The most closely related genera were Pseudozobellia, Zobellia and Kriegella. Cells of strain 7SM30T were non-motile rods that required sea salts for growth, used a wide variety of carbohydrates as sole carbon and energy sources and, unlike species of the genera Pseudozobellia and Zobellia, did not possess flexirubin-type pigment or hydrolyse agar. Strain 7SM30T contained MK6 as the sole respiratory quinone. Phosphatidylethanolamine (PE) was the only identifiable polar lipid, although other lipids were also detected. The predominant cellular fatty acids were saturated C15 and monounsaturated C15. The DNA G+C content was around 40 mol%. On the basis of extensive phenotypic and phylogenetic comparative analysis, it is concluded that the new strain represents a novel genus and species, for which the name Euzebyella saccharophila gen. nov., sp. nov., is proposed. The type strain of the type species is 7SM30T (=CECT 7477T=KCTC 22655T).

Strain 7SM29T, an aerobic marine gammaproteobacterium isolated from seawater from Castellón, Spain, was characterized by classical phenotyping, chemotaxonomy and 16S rRNA gene sequence analysis. Strain 7SM29T was found to be closely related to strains in the genus Haliea and to Congregibacter litoralis KT71T, with which a genus-level cluster was formed within the NOR5/OM60 clade of the Gammaproteobacteria. Strain 7SM29T was a short, motile rod with a tuft of three polar flagella. The strain grew on marine agar and formed pale-yellow colonies. Strain 7SM29T required NaCl for growth, reduced nitrate to nitrite, degraded several polymers and showed a preference for organic acids and amino acids over carbohydrates as carbon and energy sources. Strain 7SM29T contained Q-8 as the sole respiratory quinone. The DNA G+C content was 62.1 mol%. Phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine were the major polar lipids. The major cellular fatty acids were unsaturated C16–C18 compounds. On the basis of extensive phenotypic and phylogenetic comparative analysis, it is concluded that the strain represents a novel species of the genus Haliea, for which the name Haliea mediterranea sp. nov. is proposed. The type strain is 7SM29T (=CECT 7447T =DSM 21924T).

The central clade of the genus Vibrio, also called the Vibrio core group, comprises six species that are tightly related (DNA–DNA reassociation values are very close to 70 % for most species pairs). Identification of novel strains to the species level within this group is troublesome and results are quite often dependent on the methodology employed. Therefore, this group represents an excellent framework to test the robustness of multilocus sequence analysis (MLSA) not only for inferring phylogeny but also as an identification tool without the need for DNA–DNA hybridization assays. The genes selected, 16S rRNA, recA, pyrH, rpoD, gyrB, rctB and toxR, were amplified by direct PCR from 44 Vibrio core-group strains. Subsequent analysis allowed us to recognize toxR and rpoD as the most resolving individual genes and showed that concatenated sequences of rpoD, rctB and toxR were more useful than concatenated sequences of all seven genes. To validate our conclusions, MLSA similarities have been correlated with DNA–DNA relatedness values obtained in this study and values taken from the literature. Although the seven concatenated genes gave the best correlation, the concatenated sequences of rpoD, rctB and toxR have the practical advantage of showing a considerable gap between the maximal interspecies similarity and the minimal intraspecies similarity recorded, meaning that they can be used quite conveniently for species identification of vibrios.

Nine bacterial strains were studied by means of rep-PCR, 16S rRNA gene sequence analysis, DNA-DNA hybridization and physiological characterization. Typing analysis by means of rep-PCR showed that all nine strains were highly homogeneous, with similarities above 94 %. The strains were isolated from the same geographical area (Mazatlan, Sinaloa state, Mexico) and the same type of host (cultured rose snapper, Lutjanus guttatus), although from different individuals and organs. Comparison of the almost-complete 16S rRNA gene sequences of five strains showed that they belonged to the genus Vibrio and are closely related to the type strains of Vibrio brasiliensis and Vibrio hepatarius, with similarity values ranging from 97.9 to 98.1 % and from 97.4 to 97.8 %, respectively. The DNA-DNA hybridization value of strain CAIM 797(T) with the type strain of V. brasiliensis (CAIM 495(T)) was 47.5 %, with a reciprocal value of 44.7 %. The main phenotypic features of the strains were in agreement with the phylogenetic and genomic data. The results presented here support the description of a novel species, for which the name Vibrio sinaloensis sp. nov. is proposed, with strain CAIM 797(T) (=CECT 7298(T)) as the type strain.

During two long-term bacteriological surveys in European sea bass (Dicentrarchus labrax L.) and gilthead sea bream (Sparus aurata L.) culture facilities on the Spanish Mediterranean coast, four different species of Pseudoalteromonas were found in the internal organs of animals. Pseudoalteromonas undina, Pseudoalteromonas haloplanktis, P. espejiana and Pseudoalteromonas sp. were isolated from 9% out of 177 European sea bass and 11.7% out of 547 gilthead sea bream and they were recovered as pure cultures in 50% of isolations. Among fish with positive bacterial growth, Pseudoalteromonas was more prevalent in sea bass showing clinical signs (27.7%) and in gilthead sea bream without clinical signs (22.5%). The possible pathogenicity of seven strains of three Pseudoalteromonas spp., selected by their higher frequency in fish with clinical signs, was tested in both sea bass and sea bream. None of them was virulent for gilthead sea bream at any of the assayed doses. Only one strain, - initially isolated from a diseased sea bass and named U58 - was weakly virulent for this fish species, as it killed 100% of experimentally inoculated fish at a dose of 107 cfu/fish (LD50 = 1.3 × 107 cfu/fish). This U58 strain was further characterized as Gram-negative, motile, non-fermenting and non-pigmented rod that requires Na+ ion for growth. A further phenotypical (including Biolog GN) and phylogenetical (16S rDNA) characterization identified it as P. undina. This is the first report on P. undina as a pathogen for marine fish., This work was supported by project AGL2002-04075-C02-01 and -02 of the Spanish Ministerio de Ciencia y Tecnología. The authors are thankful to M. Alonso and P. Cabrera for skillful assistance in virulence tests.

6 pages, 2 figures, 2 tables, A novel heterotrophic, moderately halophilic, strictly aerobic, motile bacterium was isolated from a seawater sample collected at the Blanes Bay Microbial Observatory in the north-western Mediterranean Sea. Analysis of its 16S rRNA gene sequence, retrieved from the whole-genome sequence, showed that this bacterium was most closely related to the single-species genera Reinekea and Saccharospirillum (95 and 94 % sequence similarity, respectively) within the class Gammaproteobacteria. The data from phenotypic, genotypic, chemotaxonomic and phylogenetic analyses supported the creation of a novel species of the genus Reinekea to accommodate this bacterium, for which the name Reinekea blandensis sp. nov. is proposed. The type strain is MED297T (=CECT 7120T =CCUG 52066T), This work was supported by the Swedish Science Council (project 621-2003-2692, to J. P.), the Spanish Ministerio de Educación y Ciencia (project CGL-2005-02292, to M. J. P.) and by the Consellería d'Empresa, Universitat i Ciencia (project ACOMP2006/177, to M. J. P.). D. R. A. has a contract with the Universitat de València under the Ramón y Cajal program (Ministerio de Educación y Ciencia)

7 pages, 1 figure, 1 table, A Gram-negative, slightly halophilic, strictly aerobic, motile chemoorganotrophic bacterium, strain MED92(T), was isolated from a surface water sample from the eastern Mediterranean Sea. Phylogenetic analysis based on its 16S rRNA gene sequence, retrieved from the whole-genome sequence, demonstrated that this isolate is unique, showing, This work was supported by the Spanish Ministerio de Ciencia y Tecnología (project CGL-2005-02292 to M. J. P.), Conselleria d’Empresa, Universitat i Ciencia (project ACOMP2006/177 to M. J. P.) and by the Swedish Science Council (project 621-2003-2692, to J. P.). D. R. A. has a contract with the Universitat de València under the Ramón y Cajal program (Ministerio de Ciencia y Tecnología)

A pathological study was conducted on laboratory-reared juvenile common dentex (Dentex dentex) suffering trickling and continuous mortalities. During a 3-month period (October-December), water temperature, clinical signs and mortalities were recorded. Moribund or dead fish were examined for bacteria and parasites. Bacteria were isolated from head kidney and external ulcers, and samples from the gills, intestine, stomach, trunk kidney, gall bladder and liver were taken for histology. Cumulative mortality reached 73%, and 80% of fish examined were positive for bacteria (102 isolates). Vibrio splendidus was the most prevalent in head kidney (59.7%) and ulcers (88.9%), and it was frequently isolated as pure culture (74.7% and 100% respectively). This is the first report of this bacterium in association with mortality in common dentex. Vibrio scophthalmi was the second most prevalent bacterium (29.2%) and accounted for 63.6% of the mixed infections with V. splendidus, V. harveyi, Pseudoalteromonas spp. and other species were rarely isolated. No parasites were found in histological sections. Bacteria were frequently observed in the lumen of the intestine and stomach, destroying the epithelium. A cellular reaction was suggested by the high numbers of rodlet cells (RC) in the intestinal epithelium, and the abundance of eosinophilic granular cells (EGC) in the intestinal lamina propria. In the stomach, vacuolized cells containing unidentified debris, sometimes of crystalloid appearance, were very common. The high prevalence of V. splendidus in pure cultures and the absence of other aetiological agents suggest that V. splendidus is substantially involved in the registered mortalities, although the implication of V. scophthalmi and even of some nutritional factors cannot be discounted., Funding for this study was provided by JACUMAR-Generalitat Valenciana (project no. GCI 0870/2002).The authors are grateful to P. Cabrera for excellent as-sistance in pathogen isolation and to J. Monfort forhistological processing at IATS. The authors are in-debted to Dr A. Estevez (Centre d’Aquicultura, IRTA,Tarragona) for providing animals.

The possible interaction of Cryptosporidium molnari and bacteria in gilthead sea bream (Sparus aurata) was studied. Epidemiological data from a pathological survey under farm conditions were analyzed. In addition, parasite and bacteria burdens were studied in experimental models in which naturally and experimentally parasitized fish were challenged with a particular strain of Vibrio harveyi (H57). All the bacteria species present were studied. Under farm conditions, the parasite was more prevalent when mortality or morbidity cases (study C) occurred than in randomly sampled fish (study B). In study C, parasite abundance was significantly higher in bacteria-negative fish, and total bacteria abundance was significantly higher within non-parasitized fish. V. harveyi and V. splendidus were the most prevalent among bacteria carriers in studies B and C, respectively. In study C, among bacteria carriers, most isolates were slightly more prevalent in parasitized than in non-parasitized fish. Two groups (G1, G2) of naturally parasitized fish were inoculated with H57 by intracoelomic injection (ICI) and by oral intubation (OI). H57 was recovered only in G1 inoculated fish, which had a significantly higher basal abundance of total bacteria, and where the only ones with mortalities. In G1, the mortality rate and the prevalence of other V. harveyi strains different from the H57 molecular type were higher in ICI than in OI fish, and the total bacteria abundance was also significantly higher in ICI fish. C. molnari abundance was significantly higher in G1 than in G2, and also in OI than in ICI fish within G1. When H57 was IC inoculated to fish (G3, from the same farm as G2) experimentally infected with C. molnari, H57 was not recovered from any fish. A low mortality was recorded, and only in those fish inoculated with both pathogens. Also in these fish, the prevalence of infection of C. molnari was higher and histopathological damage to the stomach was greater than in fish inoculated only with the parasite. Therefore, the impact of the parasite would be reduced notably when the bacterial burden or the intensity of parasite infection are low (G2, G3)., Funding for this study was provided by the Spanish Ministerio de Ciencia y Tecnología (project no. AGL-2002-0475-C02). We thank two anonymous reviewers for their valuable comments on the manuscript. The authors are also grateful to M. Alonso and P. Cabrera for excellent assistance in experimental infections, to J. Monfort for histological processing, and to Tinamenor S.A. for providing parasite-free fish.

A Gram-negative, slightly halophilic, non-pigmented, strictly aerobic, chemo-organotrophic bacterium was isolated from Mediterranean sea water off the Spanish coast near Valencia. This strain was poorly reactive, being unable to grow in most carbon sources analysed in minimal medium. However, good growth was observed when more complex media and longer incubation times were used. Phylogenetic analysis based on an almost complete 16S rRNA gene sequence placed strain 2SM4T within the Roseobacter group, in the vicinity of uncultured bacteria described as gall symbionts of several species of the red alga Prionitis. Sequence similarity values between strain 2SM4T and the closest neighbouring species were below 95·0 %. The cellular fatty acid composition of the Mediterranean strain confirmed its position within the ‘Alphaproteobacteria’, sharing 18 : 1ω7c as the major cellular fatty acid. The phylogenetic distance from any taxon with a validly published name and also a number of distinguishing features support the designation of strain 2SM4T as representing a novel genus and species, for which the name Nereida ignava gen. nov., sp. nov. is proposed. The type strain is 2SM4T (=CECT 5292T=DSM 16309T=CIP 108404T=CCUG 49433T).

A Gram-negative, slightly halophilic, strictly aerobic, chemo-organotrophic bacterium was isolated from Mediterranean sea water near Valencia (Spain). Comparison of the almost complete 16S rRNA gene sequence showed that strain 4SM3T belonged to the Roseobacter group, with Jannaschia helgolandensis as its closest relative, with a similarity of 98·7 %. DNA–DNA hybridization analysis showed that the Mediterranean isolate had a level of relatedness of less than 42 % with J. helgolandensis and therefore that it represented a novel species of the genus Jannaschia. Phenotypic characteristics gave further evidence that the two organisms are not related at the species level. Isolate 4SM3T grows on solid media as irregular pink–red colonies that penetrate into the agar. Cells are rods, motile by a tuft of polar flagella. The DNA base composition is 64·6 mol% G+C. Morphological, physiological and genotypic differences from related species support the description of a novel species, Jannaschia rubra sp. nov., with strain 4SM3T (=CECT 5088T=DSM 16279T) as the type strain.

A Gram-negative, slightly halophilic, strictly aerobic, chemo-organotrophic bacterium was isolated from Mediterranean sea water near Valencia (Spain). 16S rRNA gene sequence comparisons showed that the isolate represented a separate branch within the α-3 subclass of the Proteobacteria, now included within the order ‘Rhodobacterales’. Jannaschia helgolandensis was the closest relative, but their low sequence similarity and other features indicated that they were not related at the genus level. Isolate 5SM22T produced bacteriochlorophyll a and grew on solid media as regular salmon-pink colonies. Cells are motile rods, with polar flagella. The DNA G+C content is 59·1 mol%. Morphological, physiological and genotypic differences from related, thus far known genera support the description of Thalassobacter stenotrophicus gen. nov., sp. nov. with strain 5SM22T (=CECT 5294T=DSM 16310T) as the type strain.

A bacteriological survey of gilthead sea bream Sparus aurata from different fish farms and culture systems on the Spanish Mediterranean coast was conducted. Three different studies were performed. Study A included hatchery-reared larvae; Study B, periodic examination of randomly sampled growing fish; and Study C, growing fish sampled only during mortality/morbidity events. In Studies B and C, sea cages, earth ponds and indoor tanks were surveyed, and in both cases diseased (showing clinical signs) and non-diseased fish were included. In Study A, a shift from Vibrio spp. (30 d after hatching) to oxidative species (60 d after hatching) was detected, and no mortality events were registered. The percentage of fish yielding bacterial growth were similar in Studies B and C, reaching 57.4 and 61.3%, respectively. A statistically significant relationship between the bacterial carriage and the type of facility was only found in Study B, showing that fish from sea cages had a higher bacterial occurrence than fish from other facilities. A statistically significant relationship between bacterial carriage and signs of disease was found, although the pattern differed in each study. Thus, in Study B only 36.2% of fish yielding abundant bacterial growth were diseased, versus 68.0% in Study C. In total, 25.0% of the fish examined were diseased. Bacterial species composition was similar in asymptomatic and diseased fish, except for a group of V. ichthyoenteri-like isolates that occurred almost exclusively in asymptomatic fish. Dominant bacterial species were V. harveyi and V. splendidus, followed by V. ichthyoenteri-like isolates, Photobacterium damselae ssp. damselae and V. fisheri. Non-fermenters were less frequent but, among them, unidentified halophilic Cytophaga-Flavobacterium isolates and Pseudoalteromonas haloplanktis were the most abundant. An association of individual species with disease was not clear, which suggests the involvement of mixed infections., This work was supported by research grants from the Spanish Ministerio de Educación, Cultura y Deporte MAR98-1000-C02-02 and FEDER Program 1FD97- 979-C02. We are thankful to M. L. Alonso for technical assistance in the samplings.

Aims: A numerical taxonomic study of halophilic Vibrio isolated from healthy and brown ring disease (BRD) affected manila clams (Ruditapes philippinarum), harvested from the Atlantic coast of south-western Spain, was performed. Methods and Results: Characterization of 123 presumptive Vibrio spp. was carried out using 94 phenotypic tests. Simple matching and Jaccard similarity coefficients were used for numerical analysis. Cluster analysis by the unweighted pair group method with arithmetic averages yielded 15 phena defined at 0.81 similarity. Large phena corresponded to Vibrio tubiashii, V. splendidus biotype I and V. harveyi (phena 1, 5 and 9, respectively). The species V. splendidus biotype II, V. natriegens, V. mediterranei and V. alginolyticus were also represented. The inhibitory effect of diffusible extracellular products of the isolates against 27 strains of V. tapetis, the aetiological agent of BRD, was also investigated. Only five V. tubiashii isolates inhibited the growth of V. tapetis strains. The antimicrobial effect was inhibited by heating and depended on the culture medium. Conclusions: The main Vibrio species associated with manila clams were V. tubiashii, V. spendidus and V. harveyi. The antagonistic relationship established between V. tapetis and the Vibrio spp. clam microbiota may explain the failure of isolation in plating medium of V. tapetis from BRD-affected clams on the south Atlantic coast of Spain. Significance and Impact of the Study: Some of the strains isolated from manila clams correspond to agarolytic strains that constitute phenon 7 and they do not fit into any of the currently described Vibrio species.

Two Gram-negative, aerobic, heterotrophic, marine bacteria, isolated from Mediterranean sea water off the coast near Valencia (Spain), were the object of this study. These non-motile, yellow-pigmented, rod-shaped strains have been studied by means of DNA-DNA hybridization, 16S rRNA sequencing and cultural and physiological features. Phylogenetic analysis showed that both strains belong to the phylum Cytophaga-Flavobacterium-Bacteroides, and their closest neighbour is the psychrophilic bacterium Gelidibacter algens. The two strains differ from G. algens in their mesophilic behaviour, hydrolytic pattern and use of different carbon sources. There is 31% DNA-DNA hybridization between the proposed type strain and G. algens, and both isolates show 97.5% 16S rDNA similarity to G. algens. They represent a novel species of the genus Gelidibacter, for which the name Gelidibacter mesophilus sp. nov. is proposed, with strain 2SM29T (= CECT 5103T = DSM 14095T) as the type strain.

It is proposed that the new Vibrio species Vibrio agarivorans accommodates two agarolytic, halophilic, fermentative bacterial strains isolated from Mediterranean sea water. The cells were gram-negative, oxidase-positive, polarly flagellated bacilli that fermented glucose without gas production and that produced no decarboxylases. They used a wide range of compounds as sole carbon and energy sources. The DNA G+C content was 44.8 mol%. Phylogenetic analysis based on complete 16S and 23S rDNA sequences revealed that the strains belong to the gamma-Proteobacteria, and are specifically related to Vibrio species. Their nearest relatives were species of the Vibrio fischeri group, sharing 16S rDNA sequence similarities below 97% with the agarolytic strains. The type strain is 289T (= CECT 5085T = DSM 13756T).

A new genus and species are proposed for two halophilic, strictly aerobic, chemo-organotrophic, marine bacterial strains. These bacteria are gram-negative, motile rods isolated from oysters cultivated off the Mediterranean coast at Valencia (Spain). They produce green/blue-green diffusible pigment. The G+C content of the DNA of the proposed type strain (XOM25T) is 48.4 mol %. A 16S rRNA gene sequence analysis of the two strains has shown that the new isolates represent a branch within the gamma-Proteobacteria, close to the genus Colwellia. The type species of the new genus is Thalassomonas viridans gen. nov., sp. nov., with the type strain XOM25T (= CECT 5083T = DSM 13754T).

Genetic relationships among 132 strains of Vibrio vulnificus (clinical, environmental, and diseased-eel isolates from different geographic origins, as well as seawater and shellfish isolates from the western Mediterranean coast, including reference strains) were analyzed by random amplified polymorphic DNA (RAPD) PCR. Results were validated by ribotyping. For ribotyping, DNAs were digested with Kpn I and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Random amplification of DNA was performed with M13 and T3 universal primers. The comparison between ribotyping and RAPD PCR revealed an overall agreement regarding the high level of homogeneity of diseased-eel isolates in contrast to the genetic heterogeneity of Mediterranean isolates. The latter suggests the existence of autochthonous clones present in Mediterranean coastal waters. Both techniques have revealed a genetic proximity among Spanish fish farm isolates and a close relationship between four Spanish eel farm isolates and some Mediterranean isolates. Whereas the differentiation within diseased-eel isolates was only possible by ribotyping, RAPD PCR was able to differentiate phenotypically atypical isolates of V. vulnificus . On the basis of our results, RAPD PCR is proposed as a better technique than ribotyping for rapid typing in the routine analysis of new V. vulnificus isolates.

Eleven strains of halophilic, facultative anaerobes isolated from healthy and diseased Dentex dentex and Sparus aurata (bony fishes) cultured in Spanish Mediterranean fisheries have been studied by a polyphasic approach that included a wide phenotypic characterization, DNA-DNA hybridization and phylogenetic analysis using 16S rRNA, recA and rpoD gene sequences. All strains were phylogenetically related to Enterovibrio species and Vibrio calviensis. On the basis of sequence analysis and DNA-DNA hybridization data, eight of the strains were identified as Enterovibrio coralii. The remaining three strains formed a tight, independent clade in all sequence analyses and showed less than 70 % DNA-DNA hybridization with strains of the closest Enterovibrio species, from which they could be differentiated by several phenotypic traits. We conclude that these three strains represent a novel species in the genus Enterovibrio and we thus propose the name Enterovibrio nigricans sp. nov., with strain DAl 1-1-5(T) (=CECT 7320(T) =CAIM 661(T)) as the type strain. In addition, we propose the reclassification of Vibrio calviensis Denner et al. 2002 as Enterovibrio calviensis comb. nov. (type strain RE35F/12(T) [corrected] =CIP 107077(T) =DSM 14347(T) =CECT 7414(T)) and we provide an emended description of the genus Enterovibrio.

A total of 140 environmental Vibrio cholerae non-O1 isolates, together with several culture collection strains from both environmental and clinical sources, were studied in relation to hemagglutination, surface hydrophobicity, and the enzymatic, hemolytic, cytotoxic, and enterotoxic activities of their extracellular products. A total of 78 and 62% of the strains produced hemagglutinins and exohemagglutinins, respectively. Four different hemagglutinating and two exohemagglutinating activities were found by using eight sugars in the inhibition assays. Cell-bound mannose-sensitive hemagglutination was detected mainly in chicken blood, whereas fucose-sensitive hemagglutination was recorded only in human blood. Cell-bound hemagglutinin resistant to all sugars tested was the only one related to surface hydrophobicity. The surface properties varied along the growth curves. The non-O1 strains displayed strong enzymatic and hemolytic activities, except for esculin hydrolysis. Of 26 non-O1 isolates selected for cytotoxin and enterotoxin production, 23 showed a wide spectrum of cytotoxic effects on cell lines of poikilothermic and homoiothermic species, but they were weakly enterotoxigenic in the infant mouse test. All extracellular products of cytotoxic strains were proteolytic, lipolytic, and hemolytic, and a high percentage produced hemagglutination of chicken blood. The cytotoxic factors in the non-O1 strains analyzed were not R plasmid mediated.

Twelve phenotypically similar marine bacteria have been studied by means of ribotyping, DNA-DNA hybridization and cultural and physiological characterization. Phylogenetic analysis has been performed of the 16S and 23S rRNA genes of two representative strains. Phylogenetically, they belong to the Vibrio/Photobacterium branch of the gamma-Proteobacteria and they share all of the properties that define the genus Vibrio. The strains represent a new Vibrio species that is phenotypically similar to Vibrio splendidus. However, resistance to the vibriostatic agent 0129 and production of acid from several carbohydrates allow differentiation between V. splendidus and the proposed new species. The DNA G+C content of the proposed type strain is 44.0 mol %. The name Vibrio lentus sp. nov. is proposed for the new species and strain 40M4T (= CECT 5110T = DSM 13757T) is the type strain.