Esperanza Garay

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.

Development of a real-time PCR assay for detection and quantification of enterotoxigenic members of Bacillus cereus group in food samples

A highly sensitive real-time PCR (qPCR) procedure, targeting the phosphatidylcholine-specific phospholipase C gene (pc-plc), was developed for specific detection and quantification of strains belonging to Bacillus cereus group. The target region was selected based on the enterotoxigenic profiles of 75 Bacillus strains. The inclusivity and exclusivity of the RTi-PCR assay were assessed with 59 isolates of the B. cereus group, 16 other Bacillus spp., and 4 non-Bacillus strains. The assay was also used to construct calibration curves for different food matrices, and it had a wide quantification range of 6 log units using both serial dilutions of purified DNA and calibrated cell suspensions of B. cereus CECT 148(T). The detection limit for B. cereus in artificially contaminated liquid egg and reconstituted infant formula was about 3CFU per reaction or 60CFU/ml of food, with a relative accuracy of 86.27% to 116.12% in artificially contaminated liquid egg. Naturally contaminated food samples were tested for the presence of B. cereus with the standard method, a conventional PCR and the new developed RTi-PCR assay. Results showed that the new developed RTi-PCR assay is very suitable for detection and quantification of strains of B. cereus group in food samples without an enrichment step.

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.

Thalassospira lucentensis gen. nov., sp. nov., a new marine member of the alpha-Proteobacteria.

A novel bacterium from the Mediterranean Sea was isolated under oligotrophic conditions at in situ temperature after prolonged continuous culture. The isolates were initially characterized by partial 16S rRNA gene sequencing. Similarity searches of one of the isolates, QMT2T, indicated high sequence identity to the well-characterized Rhodospirillum rubrum, [Aquaspirillum] itersonii and [Oceanospirillum] pusillum micro-organisms, which are representatives of the alpha-subclass of the Proteobacteria. The highest level of similarity of the complete 165 rRNA gene with respect to these microorganisms was 89%. Features such as the low similarities of 165 rRNA of QMT2T with its phylogenetically close neighbours, the distinct G+C content, and the differences in phenotypic features, including pigmentation, fatty acid composition, salt tolerance, the lack of bacteriochlorophyll a, and the capacity to use carbohydrates as carbon sources, are indicative of the novel nature of the isolate QMT2T among the alpha-Proteobacteria. This report describes the classification of strain QMT2T (= DSM 14000T = CECT 5390T) as a new genus and species, Thalassospira lucentensis gen. nov, sp. nov., in the family Rhodospirillaceae.

Electrophoretic analysis of heterogeneous lipopolysaccharides from various strains ofVibrio vulnificus biotypes 1 and 2 by silver staining and immunoblotting

Lipopolysaccharides (LPS) of 11 strains ofVibrio vulnificus biotypes 1 and 2, isolated from an eel farm, and of 10 reference strains, were examined by SDS-polyacrylamide gel electrophoresis coupled with silver staining and immunoblotting. LPS samples were obtained from whole-cell lysates, outer membrane fragments, and extracellular products. By silver staining, only a diffuse band of low-molecular weight could be visualized in all cases except for a biotype 1 strain isolated from water. However, immunoblotting with antisera obtained against strains of biotypes 1 and 2 from eels allowed visualization of multiple O-polysaccharide chains. All biotype 2 strains, independently of their origins, belonged to the same serotype and presented the same LPS profile, whereas eel isolates of biotype 1 were serologically identical and different from the rest of tested strains of biotype 1. This is the first report of LPSs with a ladder-like structure inVibrio vulnificus.

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.