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1 CIAD/Mazatlán Unit for Aquaculture, AP. 711, Mazatlán Sinaloa, Mexico 82000
2 Laboratory for Microbiology, Ghent University, Ghent 9000, Belgium
3 BCCMTM/LMG Bacteria Collection, Laboratory for Microbiology, Ghent University, Ghent 9000, Belgium
Correspondence
B. Gomez-Gil
bruno{at}victoria.ciad.mx
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ABSTRACT |
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Published online ahead of print on 22 August 2003 as DOI 10.1099/ijs.0.02775-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of LMG 13240T clones 1 and 2 are AY254039 and AY254040, respectively.
A graph showing growth of Vibrio hispanicus sp. nov. at different NaCl concentrations is available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Artemia spp. cysts have been found to contain few bacteria, i.e. less than one bacterium per cyst (Austin & Allen, 1982; Dehasque et al., 1991); after hatching, bacterial density on TCBS agar (which presumably consists of vibrios) can increase dramatically to levels of 103 c.f.u. per nauplius (Sahul Hameed & Balasubramanian, 2000) or 106–108 c.f.u. (ml Artemia nauplii homogenate)-1 (López-Torres & Lizárraga-Partida, 2001), and up to 106 c.f.u. ml-1 in surrounding water (Austin & Allen, 1982; Dehasque et al., 1991). This suggests that Vibrio spp. originate in sea water and then colonize Artemia when it hatches, exposing itself for the first time to the surrounding environment.
The taxonomic composition of Artemia bacterial cultures has been reported to contain a very high proportion of vibrios. In three marine fish hatcheries, 58–87 % of isolates were identified as members of the genus Vibrio by fatty acid methyl ester analysis (Verdonck et al., 1994), although the proportion of vibrios in cysts is <2 % (Austin & Allen, 1982; López-Torres & Lizárraga-Partida, 2001) or non-existent (Igarashi et al., 1989). These facts also support the theory that many of the vibrios found in Artemia spp. cultures come from sea water and not from Artemia cysts.
Strains LMG 13240T (=CAIM 525T=VIB 213T) and LMG 13213 (=CAIM 524=VIB 186) were isolated from Artemia spp. during 1991. Strain LMG 13211 (=CAIM 523=VIB 184) was isolated from sea water in 1990. All strains were isolated in a fish hatchery in Barcelona, Spain, as described elsewhere (Verdonck et al., 1994). All strains have been deposited in the BCCM/LMG bacteria collection (Ghent, Belgium) and at the CAIM (Collection of Aquacultural Important Microorganisms; CIAD, A. C. Mazatlán, Mexico). These strains were previously analysed phenotypically (Austin et al., 1995; Vandenberghe et al., 2003) and with fluorescent AFLP (FAFLP) (Thompson et al., 2001). The patterns obtained by Austin et al. (1995) with the Biolog GN system clustered only with those of another four strains of the 260 Vibrio spp. strains that were analysed; one of these strains belongs to the recently described species Vibrio pacinii (Gomez-Gil et al., 2003), whereas Vandenberghe et al. (2003) clustered these strains only with the type strain of Vibrio gazogenes. All of these strains were shown to be pathogenic to the Atlantic salmon (Salmo salar) and had unique ribotyping patterns (Austin et al., 1995). Two of them (LMG 13213 and LMG 13240T) also carried a 4·4 kbp plasmid (Austin et al., 1995); analysis of these and many other Vibrio strains by FAFLP showed that these strains formed a unique cluster (A16; threshold delineation of 45 %) that was not associated with any type strain included in the analysis (Thompson et al., 2001).
Phenotypic characterization of the strains was performed as described previously (Gomez-Gil et al., 2003). Antibiotic sensitivity was estimated by the disc diffusion test (Bauer et al., 1966) on Iso-Sensitest agar (Oxoid) with 1·5 % (w/v) NaCl. GLC analysis of methylated fatty acids was performed as described by Osterhout et al. (1991), but cells were grown on tryptic soy agar (TSA; Difco) with 1·5 % (w/v) NaCl and incubated at 28 °C. Growth of the three strains at different NaCl concentrations was tested in a microtitre plate (five replicates) with peptone water (1·5 % Bactopeptone; Difco); inoculated plates were incubated for 48 h at 30 °C and turbidity (OD610) was measured.
Determination of the DNA G+C content of strain LMG 13240T was performed as described by Mesbah et al. (1989) and modified by Logan et al. (2000). DNA–DNA hybridization was done following the methodology described by Willems et al. (2001) at 39 °C.
The 16S rRNA gene was amplified with primers V16S-9F (5'-AGAGTTTGATCATGGCTCAG-3') and V16S-1491R (5'-AGCGCTACCTTGTTACGACTT-3'). The PCR mix contained 18·46 µl water, 1·32 µl dNTP mix (2·5 mM each), 2·5 µl 10x PCR buffer (with 25 mM MgCl2), 0·26 µl each primer (0·25 µg µl-1), 0·2 µl Taq (5 U µl-1; Promega) and 2·0 µl DNA (final volume, 25 µl). Amplification parameters were 94 °C for 2 min followed by 35 cycles of 94 °C for 1 min, 60 °C for 1 min and 72 °C for 1 min, and a final extension of 72 °C for 5 min, rendering a 
1·5 kbp product. Purified PCR products were ligated into a pGEM-T cloning vector (Promega). Transformation was performed by heat shock at 37 °C for 20 s by using competent cells of Escherichia coli DH5
(Gibco/Invitrogen). Cells were then incubated at 37 °C in SOC medium for 1 h, centrifuged, plated on Luria–Bertani medium (Invitrogen) that contained ampicillin (60 µg ml-1) and incubated overnight at 37 °C. Plasmid extraction was performed by using a miniprep kit (Promega) according to the instructions of the manufacturer. Positive clones were identified by EcoRI digestion and gel electrophoresis. Bidirectional sequencing was carried out by using labelled T7/SP6 universal primers and a LiCor IR2 DNA sequencer, as described by the manufacturer.
The 16S rRNA gene sequence of LMG 13240T type 1 (GenBank accession no. AY254039) was also compared to the sequences deposited in GenBank (BLASTN; Altschul et al., 1990) and the Ribosomal Database Project II (RDP; Sequence Match version 2.7; Cole et al., 2003). Sequences were aligned with CLUSTALX (version 1.8; Thompson et al., 1997). Tree topology (neighbour-joining; Saitou & Nei, 1987) and stability of groupings (bootstrap analysis, 1000 replicates) of the closest phylogenetic species (with Vibrio cholerae El Tor strain as the outlier) and sequence similarities within the genus Vibrio (Jukes–Cantor model; 
=0·4; pairwise deletion; 1000 bootstrap replications; random number seed, 67 137) were calculated with the MEGA program (version 2.1; Kumar et al., 2001).
Phenotypic characters, including fatty acid proportions (Bertone et al., 1996), of the three strains analysed placed Vibrio hispanicus sp. nov. as a member of the genus Vibrio (see species description). Their phenotypic characteristics also permit clear differentiation from related species (Table 1), especially growth without NaCl, arginine dihydrolase and lysine and ornithine decarboxylase.
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), although V. fluvialis and V. furnissii may need small amounts of Na+ to grow (Farmer & Hickman-Brenner, 1992). Optimal Na+ concentration for many marine bacteria is 70–300 mM (Reichelt & Baumann, 1974), which is well below that of sea water (450–480 mM), although many vibrios have been isolated from hypersaline environments (4185 mM), from which Artemia cysts are also harvested (Straub & Dixon, 1993). The strains analysed here were able to grow at 1·0 mM NaCl, but best growth was obtained at 1024 mM and no growth was detected at 2048 mM (see Supplementary Figure in IJSEM Online).
16S rRNA gene sequences of the strains analysed placed them in the genus Vibrio; their closest phylogenetic neighbours were Vibrio proteolyticus (97·6 % BLASTN, 0·906 RDP), Vibrio diazotrophicus (97·9 % BLASTN, 0·900 RDP), Vibrio campbellii (96·8 % BLASTN, 0·857 RDP), Vibrio alginolyticus (96·8 % BLASTN, 0·876 RDP), Vibrio natriegens (96·7 % BLASTN, 0·867 RDP), Vibrio parahaemolyticus (96·6 % BLASTN, 0·871 RDP), Vibrio nigripulchritudo (96·6 % BLASTN, 0·882 RDP), Vibrio vulnificus (97·1 % BLASTN, 0·867 RDP) and Vibrio harveyi (96·4 % BLASTN, 0·875 RDP). Phylogenetic analysis with the neighbour-joining method (Fig. 1) clustered V. hispanicus sp. nov. strains close to the Vibrio core group (Dorsch et al., 1992). Strain LMG 13240T produced two different 16S rRNA gene sequences, which differed by 15 bases (0·99 %); five bases were within variable region (VR-) 1, five bases were within VR-7 and five bases were scattered in conserved regions.
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). DNA–DNA similarities of strains LMG 13240T and LMG 13211 with the species that was related most closely by 16S rRNA gene sequence similarity, V. proteolyticus LMG 3772T, gave 22·5 and 22·7 %, respectively. Hybridizations were performed with other closely related species: V. harveyi LMG 4044T (24·7 and 26·4 %, respectively), V. nigripulchritudo LMG 3896T (18·3 and 20·9 %, respectively) and V. natriegens LMG 10935T (23·9 and 24·2 %, respectively). Hybridization was 102·5 % between strains LMG 13240T and LMG 13211 and 30·6 % between V. proteolyticus LMG 3772T and V. harveyi LMG 4044T. The range of 16S rRNA gene sequence similarity within the Vibrio genus for V. hispanicus LMG 13240T was 89·1–96·8 %.
The data presented here, including phenotypic characteristics, 16S rRNA gene sequence analysis and DNA–DNA hybridization, and that of FAFLP fingerprinting that was published previously (Thompson et al., 2001), clearly support the identity of strains LMG 13240T, LMG 13211 and LMG 13213 as members of a novel species of the genus Vibrio, for which the name Vibrio hispanicus sp. nov. is proposed.
Description of Vibrio hispanicus sp. nov.
Vibrio hispanicus (his.pa'ni.cus. L. masc. adj. hispanicus from Spain).
Gram-negative, small (2·2±0·659 µm), motile, curved rods with polar flagellation. Bright yellow, small (1–3 mm), circular colonies are formed on TCBS agar. Non-luminescent, translucent and non-swarming colonies are formed on marine agar. Growth occurs in media that contain 0, 2·5, 6·0, 8·0 and 10 % NaCl (w/v), but not in 12·0 % NaCl. Growth occurs at 4, 30, 35 and 40 °C. Positive for oxidase, indole, citrate, methyl red, nitrite reduction and -galactosidase. Fermentative, arginine dihydrolase-positive, lysine and ornithine decarboxylase- and L-tyrosine-negative. Tryptophan deaminase, H2S, gas from glucose production, Voges–Proskauer reaction, gelatinase and urease are negative. Susceptible to vibriostatic agent O/129 at 10 and 150 µg and to polymixin B at 300 U, but resistant to streptomycin (25 µg) and gentamicin (10 µg). Positive for -D-glucose, 
-hydroxybutyric acid, methyl -D-glucoside, D-glucuronic acid (LMG 13213 is weakly positive), cellobiose, DL-lactic acid, dextrin, D-fructose, D-galactose, D-gluconic acid, D-mannitol, D-mannose, D-raffinose (LMG 13240T and LMG 13213 are weakly positive), D-saccharic acid (LMG 13213 is weakly positive), D-trehalose, gentiobiose, inosine, L-asparagine, L-aspartic acid (LMG 13240T is weakly positive), L-glutamic acid, L-rhamnose, L-serine, maltose, methyl pyruvate, N-acetyl-D-glucosamine, psicose, sucrose, thymidine and uridine utilization as sole carbon sources. Negative for 2,3-butanediol, 2-aminoethanol, acetic acid, adonitol, alaninamide, -cyclodextrin, -D-lactose lactulose, -hydroxybutyric acid, -ketobutyric acid, -ketoglutaric acid, -ketovaleric acid, bromosuccinic acid, cis-aconitic acid, citric acid, DL--glycerol phosphate, DL-carnitine, D-alanine, D-arabitol, D-galactonic acid lactone, D-galacturonic acid, D-glucosaminic acid, D-melibiose, D-serine, D-sorbitol, formic acid, 
-aminobutyric acid, -hydroxybutyric acid, glucose 1-phosphate, glucose 6-phosphate, glucuronamide, glycogen, glycyl L-aspartic acid, glycyl L-glutamic acid, hydroxy L-proline, i-erythritol, itaconic acid, L-alanine, L-alanylglycine, L-fucose, L-histidine, L-leucine, L-ornithine, L-phenylalanine, L-proline, L-pyroglutamic acid, L-threonine, malonic acid, myo-inositol, monomethyl succinate, N-acetyl-D-galactosamine, phenylethylamine, p-hydroxyphenylacetic acid, propionic acid, putrescine, quinic acid, sebacic acid, succinamic acid, turanose, Tween 40, Tween 80, urocanic acid and xylitol as sole carbon sources. Acid and alkaline phosphatases, -glucosidase, esterase, esterase lipase, leucine arylamidase and naphthol-AS-BI-phosphohydrolase are positive; -chymotrypsin, -fucosidase, -glucosidase, -mannosidase, -galactosidase, -glucuronidase, cystine arylamidase, lipase, N-acetyl--glucosaminidase, trypsin and valine arylamidase are negative. Most abundant fatty acids (% of total fatty acids) are, in descending order (mean of three strains, minimum and maximum): summed feature 3 (C16 : 1
7c and/or iso-C15 : 0 2-OH; 37·2, 35·8–39·1), C16 : 0 (25·7, 22·8–27·9), C18 : 17c (16·7, 15·5–17·8), C14 : 0 (5·9, 5·3–6·5), C12 : 0 (4·4, 4·2–4·8), summed feature 2 (C14 : 0 3-OH and/or iso-C16 : 1 I; 3·2, 3·1–3·4), C12 : 0 3-OH (2·6, 2·5–2·8) and C18 : 0 (1·1, 0·9–1·2). Differences between strains analysed are: LMG 13240T does not utilize -lactose, LMG 13211 does not utilize glycerol or amygdalin and LMG 13213 does not utilize succinic acid. LMG 13213 is sensitive to ampicillin (30 µg; the others are intermediately resistant) and is intermediately resistant to kanamycin (30 µg; the others are resistant) and oxytetracycline (30 µg; the others are resistant); LMG 13211 is resistant to amikacin (30 µg; the others are intermediately resistant). DNA G+C content is 42·8 mol%.
The type strain is LMG 13240T (=CAIM 525T=VIB 213T) and reference strains are LMG 13211 (=CAIM 523) and LMG 13213 (=CAIM 524). All were isolated from Artemia sp. and its culture water in Barcelona, Spain.
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ACKNOWLEDGEMENTS |
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