Photobacterium sanguinicancri sp. nov. isolated from marine animals.

Six strains were isolated from the hemolymph of the spider crab Maja brachydactyla, captured in Spain, and one from a diseased blue mussel, Mytilus edulis. The 16S rRNA gene sequences showed close similarity to the recently described Photobacterium swingsii (98.1 %) and to a lesser degree to Photobacterium aquimaris (97.8 %). MLSA analyses showed a monophyletic group including P. swingsii that form a new subclade. All genomic analyses (Average Nucleotide Identity, Average Amino Acid Identity, and in silico DNA-DNA) clearly separate the strains analysed from P. swingsii with values below the thresholds to delimit a new species. The phenotypic, genotypic and genomic data presented here clearly place these strains as a coherent group within the genus Photobacterium, for which we propose the name Photobacterium sanguinicancri sp. nov. Strain CAIM 1827(T) (=CECT 7579(T), =DSM 24670(T)) is proposed as the type strain of the species.

Evaluation of different culture media for the isolation and growth of the fastidious Vibrio tapetis, the causative agent of brown ring disease.

Thirteen culture media were evaluated at two temperatures for the growth and isolation of Vibrio tapetis. The bacterium showed similar growth dynamics at 15 °C or 25 °C, being faster at 15 °C regardless the general media employed. Best growth of V. tapetis was obtained on Agar Seawater (ASWT) (1.7 × 10(6)cfu/ml), Mannitol Marine Agar (MMA) (2.6 × 10(6)cfu/ml), and Mannitol Trypticase Soy Agar (MTSA-1) (1.9 × 10(6)cfu/ml), being slightly lower on Marine Agar (MA) (5.0 × 10(5)cfu/ml). Growth was poor on TCBS and nule in the other media containing bile salts, indicating their inhibitory effect on the V. tapetis growth. Recovery of V. tapetis from mixed Vibrio populations, differing in acid production from sucrose and mannitol, was only possible using the selective medium MMA at both temperatures. The use of ASWT or MA at 15 °C for the routinary growth of V. tapetis, and MMA for isolation of V. tapetis from bivalve samples is recommended.

Pseudomonas baetica sp. nov., a fish pathogen isolated from wedge sole, Dicologlossa cuneata (Moreau).

Five Gram-negative bacterial isolates, recovered from an outbreak that occurred in March 2006 in Huelva, Spain, affecting adult diseased cultured wedge sole [Dicologlossa cuneata (Moreau)], were characterized phenotypically and genotypically in order to clarify their taxonomic position. On the basis of 16S rRNA gene sequence analysis, the isolates were included in the genus Pseudomonas, within the Pseudomonas fluorescens-related species group, their closest relatives being the Pseudomonas jessenii and Pseudomonas koreensis subgroups. The highest sequence similarities were recorded with the type strains of Pseudomonas reinekei, P. moorei, P. umsongensis, P. jessenii and P. mohnii (99.4-99.3 % similarity). Sequence analysis of the housekeeping genes gyrB and rpoD clearly differentiated the isolates from currently described Pseudomonas species, the highest sequence similarities recorded to type strains being below 95 % for both genes. Phylogenetic analysis using concatenated sequences of the three genes showed Pseudomonas moraviensis DSM 16007T and P. koreensis DSM 16610T as the closest reference strains. DNA-DNA hybridization assays with related strains confirmed that these isolates belong to a novel species of the genus Pseudomonas, for which the name Pseudomonas baetica sp. nov. is proposed. The type strain is strain a390T (=CECT 7720T=LMG 25716T). The novel species could be easily distinguished from phylogenetically related species by several phenotypic characteristics, including gelatin hydrolysis, acid production from glucose and growth at 6 % NaCl. Virulence assays revealed that the novel species is pathogenic for wedge sole.

Evaluation of different species-specific PCR protocols for the detection of Vibrio tapetis.

In this study the specificity and sensitivity of three primer pairs, Jvt1-Jvt2, VtF-VtR and VtKF-VtKR, for the detection of Vibrio tapetis were evaluated in parallel using 23 V. tapetis strains isolated from different mollusc and fish species and with different geographical origin, as well as 29 representatives of related Vibrio species. The three primer pairs amplified all the V. tapetis strains, regardless of their host or geographical origin. However, with primer sets VtF-VtR and VtKF-VtKR amplification products of the expected size were obtained from chromosomal DNA of some of the non-V. tapetis bacteria tested. The sensitivity of the three PCR detection methods was also different. The detection limit obtained with primer pairs Jvt1-Jvt2 and VtF-VtR was between 1 and 10 pg DNA/PCR tube (2-20 bacterial cells per reaction). The primer set VtKF-VtKR showed a reduction of sensitivity in at least one order of magnitude. The results were highly reproducible with all primer sets when using the same thermal cycler, although some differences were observed in the results obtained in different PCR machines. Based on the findings reported here, we propose the Jvt1-Jvt2 PCR protocol as the most adequate for an accurate detection of V. tapetis in diagnostic pathology as well as in epidemiological studies of this clam pathogen.

Photobacterium swingsii sp. nov., isolated from marine organisms.

Six Gram-negative coccobacilli were isolated from Pacific oysters (Crassostrea gigas) from Mexico and haemolymph of spider crabs (Maja brachydactyla) from Spain. All of the isolates grew as small green colonies on thiosulphate-citrate-bile salts-sucrose (TCBS) agar and were facultatively anaerobic, oxidase-positive and sensitive to the vibriostatic agent O/129. Repetitive palindromic PCR analysis revealed a high degree of genomic homogeneity among the isolates. Several phenotypic traits differentiated the isolates from the type strains of species of the genus Photobacterium. DNA-DNA relatedness between two representative isolates and their closest phylogenetic neighbours by 16S rRNA gene sequence similarity, Photobacterium aplysiae CAIM 14(T) and Photobacterium frigidiphilum CAIM 20(T), was 44.01-53.85 %. We propose a novel species of the genus Photobacterium to accommodate the six isolates, with the name Photobacterium swingsii sp. nov. The type strain is CAIM 1393(T) (=CECT 7576(T)).

Pseudo-membranes on internal organs associated with Rhodococcus qingshengii infection in Atlantic salmon (Salmo salar).

This paper describes a pathological condition in intensive reared Atlantic salmon (Salmo salar), restricted to the appearance of pseudo-membranes covering internal organs (i.e. spleen, liver, heart and others) associated with the presence of large numbers of a Gram-positive bacteria. Isolate 79043-3, obtained as pure culture from affected fish, was subjected to a polyphasic taxonomic study in order to determine its exact taxonomic position, as well as to experimental challenges leading to determine its pathogenic potential for cultured fish. Based on this characterization, we report the first isolation of Rhodococcus qingshengii, from a farmed population of Atlantic salmon in Chile. Virulence studies demonstrated that the isolate fulfilled the Koch's postulates, suggesting that this bacterial species could be considered as an opportunistic pathogen for Atlantic salmon.

Vibrio celticus sp. nov., a new Vibrio species belonging to the Splendidus clade with pathogenic potential for clams.

A group of four motile facultative anaerobic marine isolates (Rd 8.15(T) [=CECT 7224(T), =LMG 23850(T)], Rd 16.13, Rd 6.8 [=LMG 25696] and Rd2L5) were obtained from cultured clams (Ruditapes philippinarum and Venerupis pullastra) in Galicia, north-western Spain. They formed a tight phylogenetic group based on sequences of the 16S rRNA gene and the four housekeeping genes rpoA (encoding the α-chain of RNA polymerase), rpoD (encoding the sigma factor of RNA polymerase), recA (encoding RecA protein), and atpA (encoding the α-subunit of bacterial ATP synthase). The phylogenies based on these sequences indicated that the four isolates represented a novel species in the genus Vibrio, and more precisely in the Splendidus clade. DNA-DNA hybridizations with the type strains of species showing more than 98.6% 16S rRNA gene sequence similarity, revealed a DNA-DNA relatedness below 70%. The isolates could be differentiated from the phylogenetically related Vibrio species on the basis of several phenotypic features. In addition, strain Rd 8.15(T) showed potential pathogenic activity for adult clams in virulence assays. The name Vibrio celticus sp. nov. is proposed for this new taxon, with the type strain being Rd 8.15(T) (=CECT 7224(T), =LMG 23850(T)).

Aliivibrio finisterrensis sp. nov., isolated from Manila clam, Ruditapes philippinarum and emended description of the genus Aliivibrio.

Four strains isolated from cultured Manila clam, Ruditapes philippinarum, in the north-western coast of Spain were characterized phenotypically and genotypically. Phylogenetic analyses based on the 16S rRNA gene sequences indicated that these bacteria were closely related to Aliivibrio wodanis, Aliivibrio salmonicida, Aliivibrio fischeri and Aliivibrio logei with sequence similarities between 98.1 and 96.0 %. Phylogenetic analysis based on the RNA polymerase alpha chain (rpoA), RecA protein (recA), the alpha-subunit of bacterial ATP synthase (atpA) and the uridine monophosphate (UMP) kinase (pyrH) genes and fluorescent amplified fragment length polymorphism experiments clearly showed that these novel isolates form a tight genomic group different from any currently known Aliivibrio species. On the basis of phylogenetic analysis and phenotypic data, the four strains represent a novel taxon, for which the name Aliivibrio finisterrensis sp. nov. is proposed. Several phenotypic features were revealed that discriminate A. finisterrensis from other Aliivibrio species. The type strain is CMJ 11.1(T) (=CECT 7228(T)=LMG 23869(T)).

Vibrio gallaecicus sp. nov. isolated from cultured clams in north-western Spain.

A group of three motile facultative anaerobic marine bacteria were isolated from cultured Manila clams (Ruditapes philippinarum) in Galicia, north-western Spain. The strains were characterized phenotypically and genotypically. Phylogenetic analysis of the 16S rRNA gene and four housekeeping genes, RNA polymerase alpha-chain (rpoA), RecA protein (recA), the alpha-subunit of bacterial ATP synthase (atpA) and the uridine monophosphate (UMP) kinase (pyrH), indicated that these strains were closely related to the Vibrio splendidus clade. The amplified fragment length polymorphism (AFLP) fingerprints, DNA-DNA hybridizations and phylogenies of the housekeeping and 16S rRNA gene sequences showed that the three strains represented a different species from all currently described vibrios. The new species could be differentiated from its nearest neighbours on the basis of several phenotypic features. The three strains are therefore a novel species within the genus Vibrio, for which the name Vibrio gallaecicus is proposed, with the type strain being VB 8.9T(=CECT 7244T=LMG 24045T).