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

Strain R46(T), 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 R46(T) 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 C(18:1)ω7c/C(18:1)ω6c 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 R46(T) ( = CECT 7668(T) = KCTC 23349(T)).

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 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 ß-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).

Pseudomonas litoralis sp. nov., isolated from Mediterranean seawater.

Strains 2SM5(T) and 2SM6, two strictly aerobic chemo-organotrophic gammaproteobacteria, were isolated from Mediterranean seawater off the coast of Vinaroz, Castellón, Spain, in February, 1990. They were extensively characterized by a polyphasic study that placed them in the genus Pseudomonas. Phylogenetic analysis of 16S rRNA gene sequences showed that both strains shared 100 % sequence similarity and were closely related to members of the Pseudomonas pertucinogena clade, with less than 97.3 % similarity to strains of established species; Pseudomonas xiamenensis was the closest relative. Analysis of sequences of three housekeeping genes, rpoB, rpoD and gyrB, further confirmed the phylogenetic assignment of the Mediterranean isolates. Chemotaxonomic traits such as quinone and polar lipid composition also corroborated the placement of strains 2SM5(T) and 2SM6 in the gammaproteobacteria. Other phenotypic traits, including fatty acid composition, enabled clear differentiation of both isolates from other species of Pseudomonas. We therefore conclude that strains 2SM5(T) and 2SM6 represent a novel species of Pseudomonas, for which the name Pseudomonas litoralis is proposed; the type strain is 2SM5(T) ( = CECT 7670(T) = KCTC 23093(T)).

Photobacterium aphoticum sp. nov., isolated from coastal water.

A facultatively anaerobic marine gammaproteobacterium, designated strain M46(T), 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 M46(T) was closely related to P. rosenbergii CECT 7644(T), P. halotolerans CECT 5860(T) and P. ganghwense CECT 7641(T), 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 7641(T) with 87.4 and 85.0 % sequence similarity, respectively. Regardless of the gene used in phylogenetic analysis, strain M46(T) always formed a separate and stable clade containing these three species of the genus Photobacterium. Strain M46(T) 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 M46(T) was positive for arginine dihydrolase (ADH), ß-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 C(18) and C(16), as in other members of the genus Photobacterium, but their relative amounts and the presence or absence of other fatty acids differentiated strain M46(T) from its closest relatives. Based on the results of this polyphasic taxonomic study, strain M46(T) represents a novel species of the genus Photobacterium, for which the name Photobacterium aphoticum is proposed. The type strain is M46(T) ( = CECT 7614(T)  = KCTC 23057(T)).

Evaluation of a real-time PCR assay for the detection and quantification of Bacillus cereus 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 infant formula was about 4 spores per reaction or 60 spores per ml. The newly developed methodology based on the DNeasy tissue kit and an SYBR Green quantitative real-time PCR assay is very suitable for the rapid and accurate detection and quantification of B. cereus group strains and their spores in food samples.

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 exploitation of microbial resources by offering the guarantee of accessibility and of transparency of supply, taking into account all relevant regulations and stakeholders' rights, as required by the Convention on Biological Diversity (CBD). Scientists are encouraged to deposit researched microbial material at public BRCs to contribute to the Science (semi-) Commons and maximize the impact of prior knowledge. BRCs are essential infrastructures supporting the future of life sciences and biotechnology.

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

Strain 7SM30(T)(,) 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 7SM30(T) 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 7SM30(T) 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 C(15) and monounsaturated C(15). 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 7SM30(T) (=CECT 7477(T)=KCTC 22655(T)).

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 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).

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.

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-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.

Value of recN sequences for species identification and as a phylogenetic marker within the family "Leuconostocaceae".

The genera Leuconostoc, Oenococcus, and Weissella (family "Leuconostocaceae") constitute a group of lactic acid bacteria of great interest in food microbiology. From the taxonomic point of view, they are considered phylogenetically coherent according to their 16S rRNA gene sequences and other macromolecules. These three genera were the focus of the present study; specifically, the resolution and discriminatory power of recN (encoding a DNA repair and genetic recombination protein) as a molecular marker at the species level were investigated. For this purpose, partial sequences (about 1200 nt) were obtained from 23 type strains and from several additional strains following direct amplification of recN and subsequent sequencing. Phylogeny was evaluated according to different treeing methods (neighbor joining, maximum likelihood, and maximum parsimony) and the inclusion of variability filters. The results showed that recN, used either alone or in combination with 16S rRNA data, can serve as a phylogenetic marker as well as a tool for species identification.

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 (Mazatlán, 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.

Value of recN sequences for species identification and as a phylogenetic marker within the family «Leuconostocaceae»

The genera Leuconostoc, Oenococcus, and Weissella (family «Leuconostocaceae») constitute a group of lactic acid bacteria of great interest in food microbiology. From the taxonomic point of view, they are considered phylogenetically coherent according to their 16S rRNA gene sequences and other macromolecules. These three genera were the focus of the present study; specifically, the resolution and discriminatory power of recN (encoding a DNA repair and genetic recombination protein) as a molecular marker at the species level were investigated. For this purpose, partial sequences (about 1200 nt) were obtained from 23 type strains and from several additional strains following direct amplification of recN and subsequent sequencing. Phylogeny was evaluated according to different treeing methods (neighbor joining, maximum likelihood, and maximum parsimony) and the inclusion of variability filters. The results showed that recN, used either alone or in combination with 16S rRNA data, can serve as a phylogenetic marker as well as a tool for species identification (AU)

No disponible

Stappia alba sp. nov., isolated from Mediterranean oysters.

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 5OM6T and S. aggregata CECT 4269T as reference DNAs confirmed the independent status at species level of the oyster isolates. Phenotypically, they can be distinguished from the closest relatives by the ionic requirements, growth temperatures and use of carbon compounds. We propose these oyster strains constitute a new species of Stappia, for which the name Stappia alba sp. nov. has been chosen, and strain 5OM6T (= CECT 5095T = CIP 108402T) as its type strain.

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

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 to clarify the taxonomic position of our isolates and the results confirmed their specific isolation, with interspecific binding ratios below 59%. We propose the bacteria to constitute a new Marinomonas species, i.e. M. aquamarina and strain 11SM4T (CECT 5080T, CIP 108405T, CCUG 49439T) as the type strain.

Description of Alcanivorax venustensis sp. nov. and reclassification of Fundibacter jadensis DSM 1 21 78T (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 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.

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.