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 …

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 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, physi…

Immunological and pathological status of gilthead sea bream (Sparus aurata L.) under different long-term feeding regimes

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 …

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.

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

Summary We have compared the effectiveness of culture-based methods and a DNA-based method for the detection, of Vibrio vulnificus from seawater and three types of shellfish collected from the coastal waters of Valencia, Spain. For culture-based method, we used two selective media, thiosulphate-citrate-bile 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…

A Polyphasic Approach to Study the Intraspecif ic Diversity Amongst Vibrio vulnificus Isolates

Summary A polyphasic taxonomic approach using phenotypic and molecular genetic techniques, was carried out on the species Vibrio vulnificus in order to study its intraspecific diversity. Seven techniques, including phenotypic (API 20E, BIOLOG, total protein profiles, serotyping, ELISA), and genotypic methods (ribotyping and AFLP), were employed on 80 V. vulnificus strains of biotypes 1 and 2, including 9 reference cultures. The isolates came from different geographic origins (USA, Spain, Belgium, Sweden, Norway, Japan, Thailand) and types of samples (clinical, health/diseased fish, seafood, water). Diversity indexes calculated for strains of both biotypes revealed a higher phenotypic and ge…

Comparative study onSalmonellaisolation from sewage-contaminated natural waters

Alcaide, E., Martinez, J.P. & Garay, E. 1984. Comparative study on Salmonella isolation from sewage contaminated natural waters. Journal of Applied Bacteriology56, 365–371. A comparative study of five factors influencing the isolation of salmonellas from sewage-contaminated natural waters was carried out. The effect of pre-enrichment in buffered peptone water was compared with single-step enrichment in NR10 broth incubated at 43dC. A modification of NR10 has been compared with the original composition. Bismuth sulphite agar (BSA), Hektoen enteric agar (HE) and brilliant green agar (BGA) have been used as plating media. Other factors considered have been temperature of the water and sampling…

Marinomonas aquamarina sp. nov., isolated from oysters and seawater.

Abstract The characterization of three bacterial strains isolated from cultured oysters and seawater at the Spanish Mediterranean coast has been performed. Strains were phenotypically and genetically characterized and the results led us to identify them as members of the genus Marinomonas . A phylogenetic analysis based on the almost complete 16S rDNA sequences clustered all three strains together (with sequence similarities around 99.8%) in the vicinity of M. communis and M. vaga sequences and distantly related to the other four species of the genus. The most closely related species was M. communis that shared 97.4–97.6% with the Mediterranean strains. DNA–DNA hybridizations were performed…

First record of Vibrio vulnificus biotype 2 from diseased European eel, Anguilla anguilla L.

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.

Presence of a capsule in Vibrio vulnificus biotype 2 and its relationship to virulence for eels

Strains of Vibrio vulnificus biotype 2, isolated from internal organs of diseased European eels as pure cultures of opaque cells, together with some reference strains from Japanese eels, were used in this study. Spontaneous translucent-phase variants were obtained from the corresponding parent strains and compared for a variety of phenotypic traits related to virulence for eels. The rate of colony dissociation from opaque to translucent cells was higher (around 10(-2)) than that observed for translucent to opaque cells (10(-3) to 10(-4)). Electron microscopy with ruthenium red revealed the presence of a capsule of variable thickness on opaque cells, whereas translucent-type colonies had no …

Biochemical and toxigenic properties of Vibrio furnissii isolated from a European eel farm

Abstract The present study describes for the first time the isolation of Vibrio furnissii strains from a European eel culture system which are pathogenic for eels ( Anguilla anguilla ) (LD 50 dose, 10 6 cfu/fish). Biochemical characterization of the isolates was performed by API 20E system and by classical numerical taxonomy. Growth at 4 °C, and with 6–8% (w/v) NaCl in a broth medium were necessary to differentiate Vibrio furnissii from Aeromonas species when the API 20E system was used. Virulence for eels of V. furnissii isolates was demonstrated by intraperitoneal injection of living cells. The extracellular products (ECPs) produced by V. furnissii were lethal to elvers and induced some o…

In situ analysis of the bacterial communities associated to farmed eel by whole-cell hybridization.

Bacterial communities in water samples and eel slime were investigated by fluorescence in situ hybridization of whole bacterial cells in an eel intensive culture system over 1 year. A newly developed probe, matching 27 Vibrio spp., and a specific probe for Vibrio vulnificus were used. Phylogenetic probes complementary to selected regions of the 16S and 23S ribosomal RNA revealed that Proteobacteria of the alpha and beta subclass were predominant in water and eel slime. Members of the gamma subclass (e.g. vibrios and aeromonads) were more abundant in eel slime, although no V. vulnificus was detected.

Tropicibacter multivorans sp. nov., an aerobic alphaproteobacterium isolated from surface seawater.

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 diva…

Pathogenicity of live bacteria and extracellular products of motileAeromonasisolated from eels

The pathogenic activities in vitro and in vivo of live bacteria and extracellular products (ECP) of 24 motile Aeromonas strains were investigated. Most Aer. hydrophila and Aer. jandaei isolates were pathogenic for eels (LD50 105·4‐107·6 cfu fish‐1) but no Aer. sobria, Aer. caviae and Aer. allosaccharophila caused mortality in eels at doses of > 108·4 cfu fish‐1. Of these Aeromonas strains, Aer. hydrophila and Aer. jandaei in particular produced elastases and haemolysins against fish erythrocytes. ECP from Aer. hydrophila and Aer. jandaei caused degenerative changes in fish cell lines and were strongly toxic for eels (LD50 1·0–3·2 μg (g fish)‐1) reproducing the symptoms associated with natur…

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%, respect…

Nested PCR method for rapid and sensitive detection of Vibrio vulnificus in fish, sediments, and water

A nested PCR for the detection of Vibrio vulnificus in fish farms was developed as an alternative to cultural methods by using universal primers flanking the V. vulnificus-specific sequences directed against 23S rRNA genes. This specific assay detected 10 fg of DNA or 12 to 120 cells in artificially inoculated samples without enrichment and within 24 h.

Euzebyella saccharophila gen. nov., sp. nov., a marine bacterium of the family Flavobacteriaceae.

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 containe…

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 bioty…

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 Vibr…

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

ABSTRACT 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 l…

Photobacterium aphoticum sp. nov., isolated from coastal water

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. ganghwens…

Physico-chemical and bacteriological parameters in a hypereutrophic lagoon (Albufera Lake, Valencia, Spain).

Several physico-chemical parameters related to water quality, as well as saprophytic and public health-related heterotrophic bacterial groups were studied in a hypereutrophic lake (Albufera, Valencia) at different seasons. Total microscopic and viable counts were compared, and, together with faecal indicators, were determined in water samples from different sites. Heterotrophic bacteria grown on nonselective medium were identified to genus level and a diversity index was calculated. Pseudomonas-Alcaligenes was the most frequently isolated group from all sampling sites along the study, following by Moraxella, Acinetobacter, Vibrio and Aeromonas. The relationships between all parameters were …

Description of Enterovibrio nigricans sp. nov., reclassification of Vibrio calviensis as Enterovibrio calviensis comb. nov. and emended description of the genus Enterovibrio Thompson et al. 2002

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-D…

Surface and virulence properties of environmental Vibrio cholerae non-O1 from Albufera Lake (Valencia, Spain).

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…

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 degrees 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 preenrichment in alkaline peptone water (APW) at 40 degrees C followed by CPC agar. None of the V. vulnificus isolates identified corresponded to serovar E. Dominant Vibrio species on directly inoculated TCBS plates incub…

Actibacterium mucosum gen. nov., sp. nov., a marine alphaproteobacterium from Mediterranean seawater

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. …

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 type…

Role of iron, capsule, and toxins in the pathogenicity of Vibrio vulnificus biotype 2 for mice

The virulence mechanisms of Vibrio vulnificus biotype 2 have been studied and compared with those of biotype 1 in mice as the experimental animals. Biotype 2 isolates from European eels were as virulent for mice as biotype 1 strains (50% lethal dose, about 10(5) CFU per mouse); a septicemic infection developed in less than 24 h. These strains had several properties in common with biotype 1 organisms including capsule expression, uptake of various iron sources, and production of exoproteins, whose role in mouse virulence has been demonstrated. We also discuss the implication of biotype 2 strains in human infections.

Vibrio sinaloensis sp. nov., isolated from the spotted rose snapper, Lutjanus guttatus Steindachner, 1869.

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 simila…

The genus thiobacillus: Physiology and industrial applications

This review deals with different aspects concerning the genus Thiobacillus as an obligat chemoautotroph able to oxidize reduced anorganic sulphur compounds. There are given the following topics: physiology of the genus (nutritional requirements, enzymatic complexes for sulphur oxidation, energy generation, reductive CO2 assimilation and growing in presence of organic compounds). The ecological significance of Thiobacilli in the sulphur cycle in Nature is presented. The industrial applications of Thiobacilli in respect to the leaching processes that allow the recovery of metals through oxidation and solubilization from low-grade ores are discussed, with special reference to copper and uraniu…

Comparison of outer membrane protein profiles of Vibrio vulnificus biotypes 1 and 2.

The outer membrane proteins of 17 Vibrio vulnificus biotype 2 strains from Japanese and European eels, and 12 biotype 1 strains from clinical and environmental sources have been compared. The overall profile in both biotypes was similar, and a major protein band of molecular mass 36 kDa was detected in the majority of the strains. Differences in the minor bands allowed differentiation of strains from different origins, suggesting that outer membrane protein profiles could be useful as epidemiological markers in the species V. vulnificus. Immunoblotting with antisera to whole cells of selected strains of biotypes 1 and 2 showed a strong antigenic response to outer membrane proteins 66, 60, 4…

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 h…

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 Photo…

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 conclude…

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 …

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. Th…

Occurrence and virulence of Pseudoalteromonas spp. in cultured gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.). Molecular and phenotypic characterisation of P. undina strain U58

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 sig…

Bacteria associated with winter mortalities in laboratory-reared common dentex (Dentex dentex L.)

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 iso…

Thalassobacter stenotrophicus Macián 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 22015T) and Thalassobacter stenotrophicus (CECT 5294T) 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 22015T showed DNA–DNA relatedness levels of 72 % when hybridized with the genomic DNA of T. stenotrophicus CECT 5294T. 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 wer…

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 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 spec…

Haliea mediterranea sp. nov., a marine gammaproteobacterium.

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 acid…

Description of Alcanivorax venustensis sp. nov. and reclassification of Fundibacter jadensis DSM 12178T (Bruns and Berthe-Corti 1999) as Alcanivorax jadensis comb. nov., members of the emended genus Alcanivorax

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 seq…

The role of public biological resource centers in providing a basic infrastructure for microbial research

Public collections of microorganisms have been established since the late 19th century, and currently 573 service collections are registered at the World Data Center for Microorganisms (www.wdcm.org). All together, they hold more than 1.5 million microorganisms. By implementing guidelines compiled by the Organisation for Economic Co-operation and Development (OECD), many public service collections evolve into professional ex situ repositories of biodiversity and distribution nodes for known, validated and precisely identified microbial resources and associated information to legitimate end-users. These Biological Resource Centers (BRCs) may be the preferred mechanism for the appropriate exp…

Amplified fragment length polymorphism (AFLP) and biochemical typing of Photobacterium damselae subsp. damselae.

Aims: The aim of the present study was to characterize subspecifically Photobacterium damselae subsp. damselae strains isolated from cultured Sparus aurata and Dicentrarchus labrax by means of phenotypic and molecular typing techniques (amplified fragment length polymorphism, AFLP). Methods and Results: Seventy-one strains of P. damselae subsp. damselae were isolated from 38 cultured fishes at different fish farms located on the Mediterranean coast near Valencia, Spain. Most fish studied were asymptomatic and some were recovered during infectious outbreaks. Phenotypic characterization revealed a considerable degree of variability within the subspecies, including some characters, such as pro…

Numerical Taxonomy of Pseudomonads Isolated from Water, Sediment and Eels

Summary A numerical taxonomic study was performed on 129 pseudomonad isolates from aquatic environments. 67 out of 98 field strains were isolated from water and sediment samples from Albufera lake, a hypereutrophic lake near Valencia, and 31 strains came from an eel hatchery. 10 clusters were formed at 84% similarity level when the Sokal-Michener similarity coefficient was used. Five clusters were designated as Pseudomonas putida (16 strains), P. fluorescens biotype I (9 strains), P. pseudoalcaligenes (11 strains), P. stutzen (4 strains) and Shewanella putrefaciens (3 strains). A subcluster of 7 strains (out of 15) was ascribed to the species Xanthomonas maltophilia , while the other strain…

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 …

Evaluation of the API 20E system for identification and discrimination of Vibrio vulnificus biotypes 1 and 2

Improved Salmonella recovery from moderate to highly polluted waters.

A new enrichment procedure for the recovery of salmonellas from aquatic environments is proposed. It has been tested in a eutrophic lake showing moderate to high faecal contamination levels (the Albufera lake near Valencia, Spain), and in effluents coming into a wastewater treatment plant. The new method consists of the addition of sodium novobiocin to a modification of Rappaport's medium (R10/43°C). The new medium (NR10/43°C) allows a better recovery of salmonellas from water than selenite broth.

Evaluation of a Real-Time PCR Assay for the Detection and Quantification of Group Spores in Food

A procedure based on quantitative real-time PCR was evaluated for the detection and quantification of Bacillus cereus spores. Several methods for DNA isolation, such as various heat treatments and germination solutions, were evaluated on spore suspensions of representative strains of the B. cereus group. Overall, the commercially available DNeasy tissue kit yielded the maximum amount of DNA. The procedure also was used to construct calibration curves for different food matrices, with a wide spore quantification range of 5 log units using serial dilutions of spore suspensions of B. cereus CECT 148T. The detection limit for B. cereus in artificially contaminated liquid egg and reconstituted i…

Stappia alba sp. nov., isolated from Mediterranean oysters

Abstract Three bacterial strains isolated from oysters recovered at the Spanish Mediterranean coast have been phenotypically and genetically characterized. The results of the phylogenetic analysis based on almost complete 16S rDNA sequences clustered all three strains together with 99.9% average sequence similarity and situated them in the neighbourhood of the genera Stappia , Roseibium and Pannonibacter , Stappia aggregata being their closest neighbour with sequence similarities between 98.8% and 98.9%. DNA–DNA hybridization experiments using DNA of strains 5OM6 T and S. aggregata CECT 4269 T as reference DNAs confirmed the independent status at species level of the oyster isolates. Phenot…

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

Lipopolysaccharides (LPS) of 11 strains of Vibrio 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,…

R plasmids in environmental Vibrio cholerae non-O1 strains.

The occurrence of drug resistance and its plasmid-mediated transferability was investigated in 140 environmental strains of Vibrio cholerae non-O1 and 6 strains of Vibrio cholerae, both O1 and non-O1, of clinical origin. Of the 146 strains tested, 93% were resistant to at least one drug and 74% were resistant to two or more antibiotics. The O1 strains were susceptible to all antibiotics used. A total of 26 of 28 selected resistant wild strains carried R plasmids that were transferable by intraspecific and intergeneric matings. The most common transmissible R factor determined resistance to ampicillin, amoxicillin, and sulfanilamide (30%), followed by resistance to ampicillin and amoxicillin…

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 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 s…