María J. Pujalte

The Family Rhodobacteraceae

The family Rhodobacteraceae can be considered a paradigm of modern taxonomy of prokaryotes. Taking into account the number of species and genera that conforms the family, together with the knowledge about their abundance and vast global distribution, it surprises that most of them have been described relatively recent to our days. Two notable exceptions are Rhodonostoc capsulatum (Molisch, Die purpurbakterien nach neuen untersuchungen, vols i–vii. G. Fischer, Jena, pp 1–95, 1907) and Micrococcus denitrificans Beijerinck and Minkman (Zentbl Bakteriol, Parasitenkd, Infektionskr Hyg. Abt II 25:30–63, 1910), early basonyms of Rhodobacter capsulatus and Paracoccus denitrificans, respectively. The fact that so many descriptions within this family are recent means that some studies have been concomitant and pose a challenge not only for pure taxonomic studies but also for interpreting other studies in which a rapidly evolving nomenclature had to be used anyway. The metabolic and ecological diversity of the group adds further complexity. In spite of all these difficulties, the picture is far from being a chaos and it can be considered an exciting and important bacterial group to study. Rhodobacteraceae are, fundamentally, aquatic bacteria that frequently thrive in marine environments. They comprise mainly aerobic photo- and chemoheterotrophs but also purple non-sulfur bacteria which perform photosynthesis in anaerobic environments. They are deeply involved in sulfur and carbon biogeochemical cycling and symbiosis with aquatic micro- and macroorganisms. One hundred genera are currently recognized as members of the family although the Stappia group, Ahrensia, Agaricicola, and Rhodothalassium do not belong, phylogenetically, to the family. The 90 other genera are distributed in 5 phylogenetic groups (the Rhodobacter, the Paracoccus, the Rhodovulum, the Amaricoccus, and the Roseobacter clades) that might be considered a family on its own.

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.

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

Abstract 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 (ACH 50 ). The immunological status of gilthead sea bream not only was not impaired by the restricted feeding regime, but also seemed to be enhanced in some aspects, as the respiratory burst of FR-1 fish of trial 2 was significantly higher. No differences in the bacterial isolates were detected between the two feeding regimes, and Vibrio harveyi was the most prevalent species in both cases, especially in warm months. Also, fish under FR-1 regimes had significantly lower mortality, lower prevalence of infection of all the parasites except Cryptosporidium molnari , and less histopathological alterations in liver and intestine than those under FR-2 regimes.

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 aestivus sp. nov. and Vibrio quintilis sp. nov., related to Marisflavi and Gazogenes clades, respectively.

Two new Vibrio species, Vibrio aestivus and Vibrio quintilis, are described after a polyphasic characterization of strains M22(T), M61 and M62(T), isolated from seawater collected off a beach on the East coast of Spain (Valencia). All three strains are Gram negative, mesophilic, slightly halophilic, fermentative rods. V. aestivus (M22(T)=CECT 7558(T)=CAIM 1861(T)=KCTC 23860(T) and M61=CECT 7559=CAIM 1862=KCTC 23861) is oxidase positive, reduces nitrates to nitrites, is negative for Voges Proskauer, arginine dihydrolase and indole and non hydrolytic on most substrates tested. The 16S rRNA gene sequences of M22(T) and M61 are most similar to Vibrio marisflavi (97.1-97.2%) but phylogenetic analysis using NJ, MP and ML methods display Vibrio stylophorae (96.2% similarity) as sibling species. The three species form a deep clade in the genus Vibrio. Average Nucleotide Identity (ANI) values, determined as a measure of overall genomic resemblance, confirmed that strains M22(T) and M61 are members of the same species, different to V. marisflavi CECT 7928(T). V. quintilis (M62(T)=CECT 7734(T)=CAIM 1863(T)=KCTC 23833(T)) is aerogenic, arginine dihydrolase and Voges Proskauer positive, oxidase negative and unable to reduce nitrate, traits shared by most species in the Gazogenes clade. It is unpigmented and does not grow on TCBS Agar. 16S rRNA gene similarities to its nearest species, Vibrio aerogenes and Vibrio mangrovi, are 97.6% and 96.0% respectively. Strain M62(T) and V. aerogenes CECT 7868(T) display ANI values well below the 95% boundary for genomic species.

Bermanella marisrubri gen. nov., sp. nov., a genome-sequenced gammaproteobacterium from the Red Sea

A novel heterotrophic, marine, strictly aerobic, motile bacterium was isolated from the Red Sea at a depth of 1 m. Analysis of its 16S rRNA gene sequence, retrieved from the whole-genome sequence, showed that this bacterium was most closely related to the genera Oleispira, Oceanobacter and Thalassolituus, each of which contains a single species, within the class Gammaproteobacteria. Phenotypic, genotypic and phylogenetic analyses supported the creation of a novel genus and species to accommodate this bacterium, for which the name Bermanella marisrubri gen. nov., sp. nov. is proposed. The type strain of Bermanella marisrubri is RED65(T) (=CECT 7074(T) =CCUG 52064(T)).

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.

Multilocus sequence analysis of putative Vibrio mediterranei strains and description of Vibrio thalassae sp. nov.

A multilocus sequence analysis based on partial gyrB, mreB, rpoD and pyrH genes was undertaken with 61 putative Vibrio mediterranei/V. shilonii strains from different hosts (mussels, oysters, clams, coral, fish and plankton) or habitat (seawater and sediment) and geographical origins (Mediterranean, Atlantic and Pacific). A consistent grouping was obtained with individual and concatenated gene sequences, and the clade, comprising 54 strains, was split into three subclades by all methods: subclade A (40 strains, including AK1, the former type strain of Vibrio shilonii), subclade B (8 strains) corresponding to the species V. mediterranei, and subclade C (six strains) representing a new species, V. thalassae sp. nov., with strain MD16(T) (=CECT 8203(T)=KCTC 32373(T)) as the proposed type strain. Average nucleotide identity (ANI) values, determined as a measure of genomic similarity, confirmed these assignments, and supported that strains in subclade C were a different species from V. mediterranei, with ANIb and ANIm figures lower than 90.0%. The synonymy of V. shilonii and V. mediterranei was also stressed by both MLSA and ANI determinations (97.0% between both type strains). No connection was found between geographic origin or sample type and MLSA grouping.

An MLSA approach for the taxonomic update of the Splendidus clade, a lineage containing several fish and shellfish pathogenic Vibrio spp.

A multilocus sequence analysis was undertaken in order to redefine the Splendidus clade of the genus Vibrio, a large group of species containing several pathogenic members that affect fish and shellfish, and are difficult to identify through both phenotypic and genotypic approaches. The study included analysis of partial sequences of recA, gyrB, mreB, rpoD and pyrH genes, as well as the 16S rRNA gene. Seventeen type strain species were included that were complemented with other reference strains and a collection of isolates tentatively identified as members of this clade, as well as a set of other Vibrio species. The clade was well defined and stable in all analyses, and was confirmed to contain V. celticus, V. atlanticus, V. artabrorum, V. toranzoniae and V. hemicentroti, in addition to the twelve previously recognized species. While some species were well-defined members (e.g. Vibrio cyclitrophicus, V. chagasii) others formed tight groups that were related by sequence similarities and lineage topology, which suggested a synonymy among their members, particularly the V. splendidus-V. hemicentroti pair. Most of the isolates were related to two major groups: the V. celticus-V. crassostreae-V. gigantis subclade that contained all isolates from oysters sampled in the cold season, and V. chagasii that included oyster isolates from warm months. This suggested a sharp seasonal occurrence for these species. None of the single genes were able to mimic the resolving power of the five-gene MLSA and none worked well for the identification of the whole group of species in the clade.

Description of Tropicibacter mediterraneus sp. nov. and Tropicibacter litoreus sp. nov.

Four strains (M15∅_3, M17(T), M49 and R37(T)) were isolated from Mediterranean seawater at Malvarrosa beach, Valencia, Spain. Together with an older preserved isolate (strain 2OM6) from cultured oysters at Vinaroz, Castellón, Spain, the strains were thoroughly characterized in a polyphasic study and were placed phylogenetically within the Roseobacter clade in the family Rhodobacteraceae. Highest 16S rRNA sequence similarities of the five strains to the types of any established species corresponded to Tropicibacter multivorans (95.8-96.4%), Phaeobacter inhibens (95.9-96.3%) and Phaeobacter gallaeciensis (95.9-96.2%). On the other hand, whole genome (ANI) and protein fingerprinting (MALDI-TOF) data proved: (i) non clonality among the strains, and (ii) the existence of two genospecies, one consisting of strains M15∅_3, M17(T), M49 and 2OM6 and another one consisting of strain R37(T). Phenotypic traits determined allow differentiating both genospecies from each other and from closely related taxa. In view of all data collected we propose to accommodate these isolates in two species as members of the genus Tropicibacter, Tropicibacter mediterraneus sp. nov. (type strain M17(T)=CECT 7615(T)=KCTC 23058(T)) and Tropicibacter litoreus sp. nov. (type strain R37(T)=CECT 7639(T)=KCTC 23353(T)).