Genome sequencing identify chromosome 9 inversions disrupting ENG in 2 unrelated HHT families.

Hereditary hemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber disease, is a dominant inherited vascular disorder. The clinical diagnosis is based on the Curaçao criteria and pathogenic variants in the ENG and ACVRL1 genes are responsible for most cases of HHT. Four families with a negative targeted gene panel and selected by a multidisciplinary team were selected and whole-genome sequencing was performed according to the recommendations of the French National Plan for Genomic Medicine. Structural variations were confirmed by standard molecular cytogenetic analysis (FISH). In two families with a definite diagnosis of HHT, we identified two different paracentric inversions of chromosome 9, both disrupting the ENG gene. These inversions are considered as pathogenic and causative for the HHT phenotype of the patients. This is the first time structural variations are reported to cause HHT. As such balanced events are often missed by exon-based sequencing (panel, exome), structural variations may be an under-recognized cause of HHT. Genome sequencing for the detection of these events could be suggested for patients with a definite diagnosis of HHT and in whom no causative pathogenic variant was identified.

[Analysis of the status of cardiac rehabilitation in Spain: an exploratory review].

Little is known about the characteristics of the cardiac rehabilitation programs currently implemented in Spain, the aim was therefore to know more about the effectiveness of cardiac rehabilitation in patients with ischemic heart disease in Spain, through an exploratory systematic review of the scientific literature on cardiac rehabilitation carried out in different databases from 2010 to 2018. We reviewed 35 articles whose objective was to measure the effectiveness of cardiac rehabilitation programs from different perspectives. More than twice as many men as women participated and only 8.6% were clinical trials. The studies showed improvements in relation to mortality and physiological and quality of life variables, but with discrepant results in morbidity, moods, maintenance long-term healthy habits and profitability of the programs.

Interactions between bacteria and Cryptosporidium molnari in gilthead sea bream (Sparus aurata) under farm and laboratory conditions.

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).

A comparison of strategies for the detection and recovery of Vibrio vulnificus from marine samples of the western Mediterranean coast.

We have compared the effectiveness of culture-based methods and a DNA-based method for the detection, of Vibrio vulnificus from a seawater and three types of shellfish collected from the costal waters of Valencia, Spain. For culture-based method, we used two selective media, thiosulphate-citrate-salts-sucrose (TCBS), and cellobiose-polymyxin B-colistin (CPC) agars with and without previous enrichment in alkaline-saline-peptone-water (APWS). Presumptive colonies were confirmed as V. vulnificus by the polymerase chain reaction (PCR) using previously described 23S rRNA V. vulficus-specific sequences as primers (Dvu 9V and Dvu 45R). Direct detection was accomplished by a nested-PCR procedure developed for environmental samples, with the above mentioned primers for the second amplification. Of 32 seawater samples, only one yielded positive results by direct detection by PCR, whereas five were positive by culture methods. Of the 32 bivalve samples, two were positive by PCR and five by culture methods. From a total of 675 presumptive colonies selected on the two media, only 48 (20 from seawater and 28 from bivalves) were confirmed as V. vulnificus by PCR. Forty-six V. vulnificus isolates were obtained after enrichment and only two after direct inoculation of CPC. Except for one sampling, positive results by direct detection did not correlate with confirmed strains obtained from culture media. API 20E profiles were recorded for all isolates previously identified as V. vulnificus, revealing that around 20% of the strains were sucrose-positive. For our samples, the best strategy consisted in the combination of culture based methods (3 h enrichment in APWs at 40 degrees C followed by CPC at the same temperature) and DNA-based procedures (specific PCR amplification of the presumptive colonies with primers Dvu 9V and Dvu 45 R), which allowed the detection and accurate identification of V. vulnificus in less than 48h. This is the first report on the detection of cells of V. vulnificus naturally present in seawater and edible shellfish in the Spanish Mediterranean coast.

Vibrio ponticus sp. nov., a neighbour of V fluvialis-V. furnissii clade, isolated from gilthead sea bream, mussels and seawater.

A new Vibrio species, Vibrio ponticus, is proposed to accommodate four marine bacteria isolated from sea water, mussels and diseased sea bream (Sparus aurata), at the Mediterranean coast of Spain. Strains are Gram negative, slightly halophilic bacteria that require Na+ ion for growth, oxidase and catalase positive, negative for arginine dihydrolase and ornithine decarboxylase but positive for lysine decarboxylase and indole, and utilize beta-hydroxybutyrate as a sole carbon source. Phylogenetic analysis locate these marine bacteria in the vicinity of the V. fluvialis-V. furnissii clade, sharing with these two species 16S rDNA sequence similarities slightly above 97% (97.1 and 97.3%, respectively). DNA-DNA hybridisation values confirm that the four strains form a genospecies and represent a new species in the genus Vibrio. We propose strain 369T (CECT 5869T, DSM 16217T) as the type strain.

Vibrio pelagius: differences of the type strain deposited at various culture collections.

A critical evaluation of published and own taxonomic and phylogenetic studies on Vibrio pelagius showed substantial diversity of strains received as type strains from various Culture Collections. The comparison of data based upon 16S rRNA sequence analyses, earlier genomic DNA-DNA similarity studies as well as physiological investigations and the original description indicate that Vibrio pelagius strains CECT 4202T and ATCC 25916T really represent the originally described type species whereas strains NCIMB 1900T and CIP 102762T highly likely are representatives of Vibrio natriegens.

Ribotyping of vibrio populations associated with cultured oysters (Ostrea edulis).

The intraspecific variability of Vibrio splendidus, V. harveyi and V. tubiashii recovered from oysters (Ostrea edulis) collected at the Mediterranean coast near Valencia, Spain, was analyzed by ribotyping. The two former species represented the most abundant ones, and the third one was the only species described as pathogenic for oysters. A total of 115 environmental strains were studied, 84 of V. splendidus, 23 of V. harveyi and 8 of V. tubiashii. Chromosomal DNA was digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Ribotyping among natural populations of the three species rendered 5 to 9 bands, and showed a high genetic diversity, with a ratio no. of strains/no. of ribotypes between 1.1 and 1.5. Cluster analysis of V. splendidus ribotypes suggests a seasonal pattern of incidence, with those ribotypes corresponding to winter and spring samples being maintained in the oysters over the year.

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.

Carriage of potentially fish-pathogenic bacteria in Sparus aurata cultured in Mediterranean fish farms.

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.

Thalassobius mediterraneus gen. nov., sp. nov., and reclassification of Ruegeria gelatinovorans as Thalassobius gelatinovorus comb. nov.

A Gram-negative, slightly halophilic, non-pigmented, strictly aerobic, chemo-organotrophic bacterium was isolated from sea water off the western Mediterranean coast near Valencia (Spain). This strain was able to grow on several organic acids and amino acids added to a minimal medium as carbon sources, but used few carbohydrates or yielded slight growth when sugars were used. Phylogenetic analysis based on an almost complete 16S rRNA gene sequence revealed that strain XSM19T was a member of the Roseobacter group within the 'Alphaproteobacteria', with its closest phylogenetic neighbour being Ruegeria gelatinovorans (97.6 % sequence similarity). Following a polyphasic approach, it was concluded that strain XSM19T represents a new genus and novel species, for which the name Thalassobius mediterraneus sp. nov. is proposed. The type strain is XSM19T (=CECT 5383T=CIP 108400T=CCUG 49438T). It is also proposed that R. gelatinovorans (Rüger & Höfle 1992) Uchino et al. 1999 is reclassified as Thalassobius gelatinovorus comb. nov.

Thalassobacter stenotrophicus Macian et al. 2005 is a later synonym of Jannaschia cystaugens Adachi et al. 2004, with emended description of the genus Thalassobacter.

The type strains of Jannaschia cystaugens (LMG 22015(T)) and Thalassobacter stenotrophicus (CECT 5294(T)) were analysed by means of genomic DNA-DNA hybridization, comparison of 16S rRNA gene sequences and phenotypic properties determined under the same methodological conditions. J. cystaugens LMG 22015(T) showed DNA-DNA relatedness levels of 72% when hybridized with the genomic DNA of T. stenotrophicus CECT 5294(T). Sequence comparisons revealed that the 16S rRNA genes of the two strains had a similarity of 99.8%. The cellular fatty acid and polar lipid compositions of the two strains and their DNA mol% G+C contents were almost identical. Bacteriochlorophyll a (Bchl a) and polyhydroxybutyrate were produced by both strains under the same culture conditions. Their closest phylogenetic neighbours were Jannaschia helgolandensis and Jannaschia rubra; however, the low sequence similarity values (95.7-95.9%) and several important differences in phenotypic traits (ionic requirements, Bchl a production and polar lipids) support the distinction between the genera Thalassobacter and Jannaschia. Thus, we propose the unification of J. cystaugens (LMG 22015(T)) and T. stenotrophicus (CECT 5294(T)) as Thalassobacter stenotrophicus (type strain, CECT 5294(T)=DSM 16310(T)). An emended description of the genus Thalassobacter is also presented.

Thalassobacter stenotrophicus gen. nov., sp. nov., a novel marine alpha-proteobacterium isolated from Mediterranean sea water.

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 alpha-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.

Nereida ignava gen. nov., sp. nov., a novel aerobic marine alpha-proteobacterium that is closely related to uncultured Prionitis (alga) gall symbionts.

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 2SM4(T) 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 2SM4(T) 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 : 1omega7c 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 2SM4(T) as representing a novel genus and species, for which the name Nereida ignava gen. nov., sp. nov. is proposed. The type strain is 2SM4(T) (=CECT 5292(T)=DSM 16309(T)=CIP 108404(T)=CCUG 49433(T)).

Jannaschia rubra sp. nov., a red-pigmented bacterium isolated from sea water.

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 4SM3(T) 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 4SM3(T) 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 4SM3(T) (=CECT 5088(T)=DSM 16279(T)) as the type strain.

The annual cycle of zooplankton-associated Vibrio cholerae and related vibrios in Albufera lake and its coastal surrounding waters (Valencia, Spain).

Most probable numbers of zooplankton-associated Vibrio spp. and V. cholerae were determined in Albufera lake, Spain, and in its coastal receiving waters throughout a year. Highest counts of 10(5) bacterial cells/g of plankton were associated to high temperatures and were also related to the kind of water. All isolates were non-01 serovars, and most belonged to Heiberg groups I and II.

[DNA-DNA homology and phenotypic characterization of environmental strains of Vibrio parahaemolyticus and Vibrio pelagius]. / Homología ADN-ADN y caracterización fenotípica de cepas ambientales de Vibrio parahaemolyticus y Vibrio pelagius.

A taxonomic study by means of DNA homology and the most recent phenetic criteria has been carried out in 11 environmental strains of Vibrio parahaemolyticus and 4 of Vibrio pelagius, previously ascribed to the respective phenons in a numerical taxonomy study. The strains of V. parahaemolyticus showed genetic homogeneity, a guanine plus cytosine content (G + C) of 46% and 61% homology with V. parahaemolyticus type strain ATTC 17802. They exhibited phenetic atypicalities but could be ascribed to the species V. parahaemolyticus. The strains of V. pelagius behaved similarly in regard to the phenotype, but were genetically heterogeneous; they showed a % G + C of 44-46 and were assigned to V. splendidus-V. pelagius homology group.

Enumeration of salmonellas by most probable number method from sewage and sewage-polluted natural waters, comparing the use of preenrichment versus direct enrichment in NR10 at different temperatures.

Crude sewage and sewage-contaminated natural waters were analysed for the presence of Salmonella by Most Probable Number Procedures, Direct enrichment in NR10 at 37 degrees C and 43 degrees C was compared with preenrichment in buffered peptone water at these two temperatures, followed by selective enrichment in NR10 at 37 degrees C and 43 degrees C. The growth of competing bacteria on Hektoen agar plates was also recorded and quantified. Direct enrichment in NR10 gave higher numbers in both types of waters. The temperature of 37 degrees C was more favourable for the less heavily contaminated waters, whereas 43 degrees C was more efficient for crude sewage. The growth of competing bacteria was restricted at 43 degrees C and enhanced at 37 degrees C. With direct enrichment in NR10/43 degrees C, pure Salmonella cultures can be obtained after 48 h incubation.

Gram-positive bacteria of marine origin: a numerical taxonomic study on Mediterranean isolates.

A numerical taxonomic study was performed on 65 Gram-positive wild strains of heterotrophic, aerobic, marine bacteria, and 9 reference strains. The isolates were obtained from oysters and seawater sampled monthly over one year, by direct plating on Marine Agar. The strains were characterized by 96 morphological, biochemical, physiological and nutritional tests. Clustering yielded 13 phena at 0.62 similarity level (Sl coefficient). Only one of the seven phena containing wild isolates could be identified (Bacillus marinus). A pronounced salt requirement was found in most isolates.

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).

Genetic relatedness among environmental, clinical, and diseased-eel Vibrio vulnificus isolates from different geographic regions by ribotyping and randomly amplified polymorphic DNA PCR.

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 KpnI 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.