Pujalte Mj

Multilocus sequence analysis of the central clade of the genus Vibrio by using the 16S rRNA, recA, pyrH, rpoD, gyrB, rctB and toxR genes.

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.

Numerical Taxonomy of Vibrionaceae Isolated from Oysters and Seawater Along an Annual Cycle

Summary A numerical taxonomic study on Gram negative heterotrophic facultative anaerobic bacteria isolated from marine samples (oysters and seawater of Western Mediterranean Sea) was performed. Three hundred sixty eight strains, including reference strains of most species of the Vibrionaceae , were characterized (96 tests per strain). Cluster analysis of similarity matrices obtained with S SM and S J coefficients was performed and S J -based tree and 0.75 S level selected for definition of phena. Larger phena corresponded to non-luminescent Vibrio splendidus biotype 1 and V. harveyi . The species V. tubiashii (an oyster larvae pathogen), V. pelagius, V. mediterranei, V. orientalis and Photobacterium angustum were also represented. Minor phena corresponded to V. damsella, V. fisheri, V. alginolyticus and several unidentifiable phena that might represent new species of Vibrionaceae . The absence of human pathogens such as V. parahaemolyticus and V. vulnificus was noticeable. Some identification problems concerning xanthine and arginine dihydrolase (ADH) tests were highlighted by the analysis.

Vibrio lentus sp. nov., isolated from Mediterranean oysters

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.

Low incidence of Vibrio vulnificus among Vibrio isolates from sea water and shellfish of the western Mediterranean coast

A specific search for Vibrio vulnificus in natural marine samples from the Spanish Mediterranean Sea was carried out by nested PCR and cultural approaches using thiosulphate‐citrate‐bile salts‐sucrose agar (TCBS) and cellobiose‐polymixin B‐colistin agar (CPC), incubated at 40 °C, as selective media. Presumptive colonies were identified by PCR using specific primers against 23S rRNA sequences. This species was isolated from sea water and edible bivalves, mainly after pre‐enrichment in alkaline peptone water (APW) at 40 °C followed by CPC agar. None of the V. vulnificus isolates identified corresponded to serovar E. Dominant Vibrio species on directly inoculated TCBS plates incubated at 25 °C were V. splendidus below 20 °C and V. harveyi and V. mediterranei above that temperature. Low percentages of several pathogenic vibrios were recorded but V. vulnificus was never recovered at this incubation temperature. The incidence of this species in the samples studied was lower than that described for other geographical areas, probably due to the high salinity values of the Mediterranean Sea.

Vibrio Species in Seawater and Mussels: Abundance and Numerical Taxonomy

Summary Qualitative and quantitative studies were performed on Vibrio species from seawater and mussel samples at a hatchery in Valencia harbour, and from market mussels. Vibrio alginolyticus and V. harveyi were the most abundant species in all samples. Other species identified were V. pelagius, V. mediterranei, V. tubiashii, V. damsella, V. splendidus, V. fluvialis, V. parahaemolyticus, V. anguillarum, V. cholerae and Aeromonas spp . The abundance of V. harveyi and V. mediterranei was positively correlated with temperature whereas V. pelagius counts showed negative correlation with this parameter. The pathogenic species were recovered in very low numbers. A numerical taxonomic study was performed on several representative isolates. Separation between V. parahaemolyticus and V. alginolyticus was not possible at species level. Two phena were phenotypically different from any described Vibrio species.

Thalassomonas viridans gen. nov., sp. nov., a novel marine gamma-proteobacterium.

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).

Numerical taxonomy of aerobic, Gram-negative bacteria associated with oysters and surrounding seawater of the Mediterranean coast

Abstract A numerical taxonomic study was performed on 245 strains of heterotrophic, aerobic, marine bacteria, plus 26 reference strains. The isolates were obtained from oysters and seawater sampled monthly over one year, by direct plating on Marine Agar. The strains were characterised by 93 morphological, biochemical, physiological and nutritional tests. Clustering yielded 46 phena at 0.60 S level (S J coefficient). Some could be identified as species of Alteromonas, Shewanella, Deleya, Flavobacterium, Oceanospirillum, Pseudomonas and marine Agrobacterium -like organisms, others were unidentified groups. Several phena seem to correspond to as yet undescribed taxa.

Virulence and molecular typing of Vibrio harveyi strains isolated from cultured dentex, gilthead sea bream and European sea bass.

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 x 10(5) to 1.6 x 10(6) 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 x 10(7) cfu/fish. This is the first report on virulence of V. harveyi for sea bass.

Vibrios isolated from the cultured manila clam (Ruditapes philippinarum): numerical taxonomy and antibacterial activities

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.

Vibrio agarivorans sp. nov., a novel agarolytic marine bacterium.

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).