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1 CIAD/Mazatlán Unit for Aquaculture, AP 711, Mazatlán Sinaloa, México 82000
2 Laboratory for Microbiology, Ghent University, Ghent 9000, Belgium
3 BCCM/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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30 % DNA–DNA similarity to its closest phylogenetic neighbours. Vibrio pacinii sp. nov. is proposed, with LMG 19999T (=CAIM 530T=STD3-1057T; DNA G+C content, 44·9 mol%) as the type strain.
The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequence of strain LMG 19999T is AJ316194.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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), crustaceans (Lightner, 1993) and molluscs (Austin, 1988). Some Vibrio species may also have a beneficial effect in host organisms, either as symbionts [e.g. of squids (McFall-Ngai, 2002)] or probionts (Gomez-Gil et al., 2000). Several studies have investigated phenotypic diversity of vibrios in aquaculture environments (Vandenberghe et al., 1998, 2003). Recently, genotypic techniques have also been employed to better characterize the diversity of vibrios: Thompson et al. (2001) characterized 506 strains by using fluorescent amplified fragment length polymorphism (FAFLP) analysis and found many strains that did not cluster with any known type strains and might thus be considered as potential novel species of the genus Vibrio. This analysis disclosed a group (A47) of three tightly related strains (LMG 13245, LMG 19999T and LMG 21514). These strains were grouped apart from known type strains of Vibrio species, with >70 % FAFLP pattern similarity between them but only 52 % similarity to that of their closest FAFLP neighbour, strain LMG 10953 (FAFLP group A1). Phenotypic analysis of 1476 Vibrio isolates with the Biolog GN system clustered strains LMG 19999T and LMG 13245 in a separate group (STD3-1057) that was related to Vibrio logei, and strain LMG 21514 clustered in the Vibrio ordalii group (Vandenberghe et al., 2003). Austin et al. (1995) reported that strain LMG 13245 had unique ribotype and lipopolysaccharide profiles and no plasmid content, belonged to an unknown serogroup and was pathogenic for Atlantic salmon (Salmo salar). Phenotypic and genotypic methodologies clearly show that bacterioflora, particularly vibrios, found in aquaculture environments represent a highly diverse group with many potential novel species that are yet to be detected and described.
Strain LMG 19999T (=CAIM 530T=STD3-1057T) was isolated from healthy shrimp larvae (Penaeus chinensis) in the Dahua hatchery in Laizhou (Shandong Province, China) during the spring of 1996 (Vandenberghe et al., 1998). Strain LMG 13245 (=CAIM 526=VIB 218) was isolated from sea bass (Dicentrarchus labrax) in Spain 1991, while strain LMG 21514 (=CAIM 466=VIB 847) was isolated from Atlantic salmon (Salmo salar) in Tasmania during the 1990s. All strains have been deposited at the BCCM/LMG bacteria collection and at the CAIM (Collection of Aquacultural Important Microorganisms, CIAD, A. C. Mazatlán, México).
Strains were grown aerobically on tryptone soy agar (TSA; Oxoid) supplemented with 2 % (w/v) NaCl for 24 h at 28 °C unless otherwise stated. Phenotypic characterization was done with the API 20E system (bioMérieux) to determine the biochemical and nutritional properties of the strains tested, the API ZYM system (bioMérieux) for evaluation of enzymes produced by the strains and the Biolog GN2 system to test the strains' ability to utilize different carbon sources. All systems were used according to the manufacturers' instructions, but sterile saline solution [1·5 % (w/v) NaCl] was used to prepare the inocula in every case. Additional phenotypic tests and confirmation of doubtful test results obtained by these systems were performed by following the methodologies of Lanyi (1987) and Austin & Lee (1992). 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 the cells were grown on TSA (Difco) with 1·5 % (w/v) NaCl and incubated at 28 °C. Determination of G+C content of the DNA of strain LMG 19999T was determined according to Mesbah et al. (1989) and modified as described by Logan et al. (2000). Strain LMG 19999T (GenBank/EMBL accession no. AJ316194) was analysed further by sequencing the 16S rDNA as described by Thompson et al. (2001). Sequence similarities were obtained with the Jukes–Cantor model [gamma 0·4, pairwise deletion; standard error (SE) estimated by bootstrap method, 1000 replications and random number seed=67137] with the MEGA program (version 2.1; Kumar et al., 2001). The LMG 19999T sequence was also compared to the sequences in GenBank (BLASTN; Altschul et al., 1990) and the Ribosomal Database Project (RDP; Maidak et al., 1999). Sequences of the closest species and of isolate LMG 19999T were aligned with CLUSTAL X (version 1.8; Thompson et al., 1997). Tree topology (neighbour-joining; Saitou & Nei, 1987) and stability of groupings (bootstrap analysis, 1000 replicates) were performed with PHYLO_WIN software (Galtier et al., 1996) with Vibrio cholerae as the outlier. DNA–DNA hybridization was done following the methodology described by Willems et al. (2001) at 39 °C.
The three isolates grew as round, yellow, bright colonies on TCBS (thiosulfate/citrate/bile/sucrose) agar; no swarming or luminescence was observed. The isolates were arginine dihydrolase-positive and lysine and ornithine decarboxylase-negative (A+, L-, O-). A+, L-, O- vibrios represent a diverse group that includes many species (Alsina & Blanch, 1994a, b). According to the identification scheme provided by Alsina & Blanch (1994b), strains LMG 19999T, LMG 21514 and LMG 13245 would be identified as Vibrio splendidus II or Vibrio furnissii. All three isolates presented many characters that differentiated them from other A+, L-, O- vibrios, e.g. a positive Voges–Proskauer reaction (except for LMG 13245; positive only in Vibrio anguillarum, Vibrio metschnikovii and Vibrio tapetis); negative for indole production (also negative for Vibrio mytili and V. tapetis, variable for V. splendidus II); and utilization of amygdalin (also by Vibrio diabolicus) but not of D-mannitol (also negative in Vibrio nereis and V. tapetis) (Table 1). Fatty acid content of the strains varied considerably: 28 were detected in strain LMG 13245, 23 in LMG 19999T and only 10 in LMG 21514 (see species description and Table 2). The fatty acid methyl ester (FAME) profiles are in good agreement with those of representatives of the genus Vibrio (Bertone et al., 1996), although two fatty acids were present in higher concentrations. Fatty acids useful for differentiation are shown in Table 1.
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), including V. metschnikovii CIP 69.14T (d=0·028, SE=0·005; 97·4 % BLASTN; 87·0 % RDP, 1360 uniquely occurring oligomers), Vibrio kanaloae LMG 20539T (d=0·029, SE=0·005; 96·6 % BLASTN), Vibrio pomeroyi LMG 20537T (d=0·029, SE=0·005; 96·4 % BLASTN), Vibrio aestuarianus ATCC 35048T (d=0·031, SE=0·005; 97·7 % BLASTN; 87·7 % RDP, 1345 uniquely occurring oligomers), V. tapetis CECT 4600T (d=0·033, SE=0·005; 97·3 % BLASTN; 87·2 % RDP, 1387 uniquely occurring oligomers) and V. furnissii ATCC 35016T (d=0·035, SE=0·005; 96·6 % BLASTN; 87·2 % RDP, 1453 uniquely occurring oligomers). 16S rDNA sequence distance within the Vibrio genus for the isolates was 0·028–0·091.
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Collectively, phenotypic characterization, 16S rDNA sequences, DNA–DNA similarity and FAFLP fingerprinting (Thompson et al., 2001) provide solid evidence to support the proposal of strains LMG 19999T, LMG 21514 and LMG 13245 as members of a novel species of the genus Vibrio, for which the name Vibrio pacinii is proposed.
Description of Vibrio pacinii sp. nov.
Vibrio pacinii (pa.ci'ni.i. N.L. gen. n. pacinii of Pacini, named after the Italian anatomist Filipo Pacini, who first discovered the causal agent of cholera).
Gram-negative rods, motile, with polar flagella. Non-luminescent, non-pigmented, translucent colonies on marine agar with no swarming. Round, firm, yellow bright colonies on TCBS agar, 1·5–2·8 mm in diameter. Grows in the presence of 1·5, 2·5, 6·0 and 8·0 % NaCl but not at 0, 10·0 or 12·0 %; can grow at 4, 30 and 35 °C but not at 40 °C in TSB. Oxidase- and catalase-positive, ferments glucose and lactose; arginine and L-tyrosine dihydrolase-positive, lysine and ornithine decarboxylase-negative. Positive for Voges–Proskauer test (except for strain LMG 13245), methyl red, tryptophan deaminase and nitrate reduction; negative for indole production, H2S and urease. Test for citrate utilization is weakly positive, except for strain LMG 13245, which is strongly positive. Positive activity of acid phosphatase (LMG 13245 is weak), 
-glucosidase (LMG 21514 is weak), alkaline phosphatase, esterase (C4), esterase lipase (C8) (LMG 13245 is weak), leucine arylamidase, naphthol-AS-BI-phosphohydrolase and valine arylamidase (LMG 13245 and LMG 21514 are weak). Negative activity of -chymotrypsin (LMG 19999T is weakly positive), -fucosidase, -galactosidase, -mannosidase, 
-glucosidase, -glucuronidase, lipase (C14), N-acetyl--glucosaminidase and trypsin. All strains utilize -D-glucose, cellobiose, dextrin, D-fructose, D-mannitol, D-trehalose, gentiobiose, inosine, L-asparagine, L-glutamic acid, L-serine, maltose, N-acetyl-D-glucosamine, sucrose, thymidine and uridine as sole sources of carbon. None of the strains utilized 2,3-butanediol, 2-aminoethanol, acetic acid, adonitol, alaninamide, -D-lactose lactulose, -hydroxybutyric acid, -ketobutyric acid, -ketoglutaric acid, -ketovaleric acid, -lactose, -hydroxybutyric 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-gluconic acid, D-glucosaminic acid, D-glucuronic acid, D-mannose, D-melibiose, D-raffinose, D-saccharic acid, D-serine, formic acid, 
-aminobutyric acid, -hydroxybutyric acid, glucose 1-phosphate, glucose 6-phosphate, glucuronamide, glycerol, glycyl L-glutamic acid, hydroxy L-proline, i-erythritol, itaconic acid, L-arabinose, L-fucose, L-histidine, L-leucine, L-ornithine, L-phenylalanine, L-proline, L-pyroglutamic acid, L-rhamnose, L-threonine, malonic acid, m-inositol, N-acetyl-D-galactosamine, phenylethylamine, p-hydroxyphenylacetic acid, propionic acid, putrescine, quinic acid, sebacic acid, succinamic acid, turanose, Tween 40, Tween 80, urocanic acid or xylitol. The following cellular fatty acids are present in decreasing order (mean percentage of the three strains analysed, minimum and maximum of total fatty acid content): C16 : 0 (18·7 %, 14·1–25·5), C18 : 1
7c (11·1 %, 9·9–13·4), iso-C16 : 0 (9·0 %, 8·4–9·7, not detected in LMG 21514), C14 : 0 (6·9 %, 4·7–11·2), C12 : 0 (3·7 %, 1·9–5·2), C12 : 0 3-OH (3·1 %, 1·5–4·9), iso-C14 : 0 3-OH (1·9 %, 1·1–2·7, not detected in LMG 21514), iso-C17 : 0 (1·4 %, 0·9–2·0, not detected in LMG 21514), C16 : 17c and/or iso-C15 : 0 2-OH (summed feature 3) (38·7 %, 37·8–39·2) and C14 : 0 3-OH and/or iso-C16 : 1 I (summed feature 2) (2·9 %, 1·5–3·6). Eighteen other fatty acids are present in percentages below 1·0, these are: iso-C18 : 0, iso-C14 : 0, C16 : 17c alcohol, iso-C13 : 0, iso-C15 : 0, anteiso-C17 : 0, C18 : 0, C15 : 0, C17 : 0, 11-methyl C18 : 17c, C17 : 18c, iso-C13 : 0 3-OH, iso-C15 : 0 3-OH, iso-C12 : 0, C12 : 0 2-OH, anteiso-C15 : 0, C15 : 18c and C16 : 15c. Antibiotic susceptibility was observed to chloramphenicol (30 µg), oxolinic acid (2 µg), oxytetracycline (30 µg), polymyxin B (300 U), tetracycline (30 µg); resistant to gentamicin (10 µg) (except for LMG 21514, which is intermediate), kanamycin (30 µg), streptomycin (25 µg) and vibriostatic agent O/129 at 10 and 150 µg. Additional phenotypic features are listed in Table 2
. G+C content of the DNA is 44·9 mol%.
The type strain, LMG 19999T (=CAIM 530T), was isolated from shrimp larvae (Penaeus chinensis) in the Dahua hatchery in Laizhou (Shandong Province, China). Reference strains are LMG 13245 (=CAIM 526) and LMG 21514 (=CAIM 466).
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ACKNOWLEDGEMENTS |
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