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1 CIAD/Mazatlán Unit for Aquaculture, AP. 711, Mazatlán Sinaloa, Mexico 82000
2 Laboratory for Microbiology, K. L. Ledeganckstraat 35, Ghent University, Ghent 9000, Belgium
3 BCCMTM/LMG Bacteria Collection, K. L. Ledeganckstraat 35, Ghent University, Ghent 9000, Belgium
Correspondence
B. Gomez-Gil
bruno{at}victoria.ciad.mx
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ABSTRACT |
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99 % similarity) to Vibrio campbellii, Vibrio harveyi, Vibrio alginolyticus and Vibrio parahaemolyticus. However, DNA hybridization experiments revealed similarity values below 70 % with its closest species V. campbellii and V. harveyi. Additionally, the analysed strains differ from related Vibrio species by the utilization of melibiose and production of acid from L-arabinose and amygdalin. Among the strains analysed, differences were observed in some phenotypic characters, particularly susceptibility to ampicillin, polymyxin B and amikacin, and urease activity. The major fatty acids identified were 16 : 0, 18 : 1
7c, 14 : 0, 12 : 0 3-OH and 18 : 0. Vibrio rotiferianus sp. nov. is proposed, with type strain LMG 21460T (=CAIM 577T); it has a DNA G+C content of 44·5±0·01 mol%.
Published online ahead of print on 12 July 2002 as DOI 10.1099/ijs.0.02430-0.
The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequence of Vibrio rotiferianus LMG 21460T is AJ316187.
Present address: Laboratory for Microbiology, K. L. Ledeganckstraat 35, Ghent University, Ghent 9000, Belgium. 
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), and thus may cause poor survival and growth of the fish larvae (Gatesoupe, 1989). Other bacteria may enhance the growth of rotifers (Douillet, 2000) and of fish larvae (Skjermo & Vadstein, 1999). The principal genera identified in rotifer cultures have been Pseudomonas, Vibrio, Moraxella and Flavobacterium (Verdonck et al., 1994, 1997). Vibrio was the dominant genus in rotifer cultures, constituting up to 56 % of the bacterial community, with Vibrio anguillarum, Vibrio alginolyticus, Vibrio diazotrophicus, Vibrio mediterranei and Vibrio tubiashii-like as representative species (Verdonck et al., 1997). Understanding the bacterial composition of rotifers and rotifer cultures is important for the aquaculture industry.
Several bacteria were isolated from rotifers and from the water of a flow-through rotifer system during August 1999 at the Artemia Reference Centre, University of Ghent, Belgium. The rotifer rearing system and bacterial isolation procedures have been described by Suantika et al. (2001). Samples of rotifer culture water and from rotifers + water were homogenized and serially diluted in sterile saline solution (SSS; 1·5 % NaCl, w/v), plated onto marine agar (Difco) and thiosulphate-citrate-bile salts-sucrose agar (TCBS; Difco), and incubated for 24–48 h at 25 °C. Five isolates (LMG 21456, LMG 21457, LMG 21458, LMG 21459 and LMG 21460T) were analysed by Thompson et al. (2001) by fluorescent amplified fragment length polymorphism (FAFLP) and 16S rDNA sequencing. They showed (1) that these strains formed a tight cluster, and (2) that no known Vibrio type species grouped into this cluster. Therefore, all five isolates were considered as potentially novel species of Vibrio.
The five strains were phenotypically analysed by API 20E and API ZYM (bioMérieux) and Biolog GN2 according to the manufacturers' instructions, except that SSS was used to prepare the inocula. Other phenotypic tests were performed following the methodologies of Lanyi (1987). Presence of flagella was determined with Gray's stain (Murray et al., 1994). Antibiotic sensitivity was estimated by the disk diffusion test (Bauer et al., 1966) in Iso-sensitest agar (Oxoid)+1·5 % NaCl (w/v). Fatty acid analysis was performed as described by Osterhout et al. (1991), except that the cells were grown on Tryptone Soya Agar (TSA; Oxoid)+1·5 % NaCl (w/v) and incubated at 28 °C for 24 h. The 16S rDNA sequence of strain LMG 21460T (GenBank/EMBL accession no. AJ316187) was compared with sequences deposited in EMBL (FASTA; Pearson & Lipman, 1988) and in the Ribosomal Database Project (RDP; Maidak et al., 1999) to specify the closest related species. Sequences of relevant taxa and of strain LMG 21460T were aligned by means of CLUSTAL X version 1.8 (Thompson et al., 1997). Distance estimations (Jukes & Cantor, 1969), tree topology [neighbour-joining, Saitou & Nei (1987), with 0·4 gamma correction and pairwise deletion] and stability of groupings (Bootstrap analysis, 1000 replicates) were performed with MEGA version 2.1 software (Kumar et al., 2001) with Vibrio cholerae as outgroup. The DNA G+C content was determined as described by Mesbah et al. (1989) using the modifications proposed by Logan et al. (2000). DNA–DNA hybridization analysis was carried out at stringent conditions (39 °C) following the methodology described by Willems et al. (2001).
All five isolates grew well on TCBS agar as bright non-luminescent yellow colonies and unpigmented translucent colonies in marine agar. Phenotypically, the five strains can be clearly assigned to the genus Vibrio (Alsina & Blanch, 1994), and present many characters that clearly distinguish them from similar species (Table 1 and description). Of particular interest is the capacity to utilize melibiose, a feature only observed in Vibrio nigripulchritudo, Vibrio agarivorans and some strains of Vibrio natriegens, but in none of the arginine-dihydrolase-negative, lysine- and ornithine-decarboxylase-positive species. Differences were observed in the phenotypic characters among the five strains analysed (see Table 2).
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7c was 10·79 % (max. 12·34 %, min. 9·13 %) against 21·05 and 22·55 %, respectively. For other fatty acids, see species description, but no clear differences with the other type strains were observed. In general, the identified fatty acids of strain LMG 21460T were in agreement with the fatty acid signature of the genus Vibrio; only 14 : 0 was slightly above the maximum reported for the genus (8·63 %) (Bertone et al., 1996).
The 16S rDNA sequence clearly classified strain LMG 21460T in the genus Vibrio. The closest phylogenetic neighbours were V. campbellii (99·86 % FASTA and 99·2 % RDP) and V. harveyi (99·11 and 96·7 %) (Fig. 1). Phylogenetic analysis with maximum-likelihood and maximum-parsimony treeing methods produced congruent results with the neighbour-joining method regarding the positioning of the type strain LMG 21460T. Strain LMG 21460T clustered within the group of Vibrio species called the V. harveyi group (Reichelt et al., 1976), and later called the core group of the Vibrio genus (Dorsch et al., 1992). This group has had little taxonomic change over time; the last species described was Vibrio vulnificus (Farmer, 1980).
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). Strain LMG 21460T was hybridized with its two closest neighbours (by 16S rDNA) V. campbellii (LMG 11216T) and V. harveyi (LMG 4044T) showing 65 and 66 % reassociation, respectively. The DNA reassociation between V. campbellii and V. harveyi was 69 %, a similar result to the 65 % obtained by Reichelt et al. (1976).
These results clearly showed that strain LMG 21460T is closely related to V. campbellii and V. harveyi, but it can be differentiated from these taxa by means of FAFLP (Thompson et al., 2001), DNA–DNA similarity, as well as by several phenotypic traits, i.e. utilization of melibiose and acid formation of L-arabinose and amygdalin (Table 1
).
Description of Vibrio rotiferianus sp. nov
Vibrio rotiferianus (ro.ti.fer.i.a'nus. English n. rotifer; L. masc. suff. -ianus pertaining to; N.L. masc. adj. rotiferianus pertaining to rotifers).
Gram-negative curved rods (0·8–1·2x2·0–3·5 µm), facultative anaerobic, motile by means of more than one polar flagella. Non-pigmented, translucent, non-luminescent colonies on marine agar with no swarming. Bright, round, 2–3 mm yellow colonies, with umbilicated growth in TCBS agar. No growth occurs without NaCl ions in the culture medium; growth occurs in the presence of 1·5, 3·0 and 6·0 % NaCl (w/v), but not at 8 or 10 %; grows at 28–40 °C, but not at 4 °C. Susceptible to chloramphenicol (30 µg), tetracycline (30 µg), oxolinic acid (2 µg), oxytetracycline (30 µg), and to the vibriostatic agent O/129 at 10 and 150 µg; resistant to kanamycin (30 µg), streptomycin (25 µg) and gentamicin (10 µg). Arginine-dihydrolase-negative, lysine- and ornithine-decarboxylase-positive, ferments glucose without producing gas; positive for indole, oxidase, urease, tryptophan deaminase and gelatinase. Voges–Proskauer-, H2S- and citrate-negative. Phenotypic differences are observed between the strains (Table 2). Utilizes the following substrates as sole carbon source: alaninamide, 
-cyclodextrin, -D-glucose, methyl 
-D-glucoside, cellobiose, dextrin, D-fructose, D-galactose, D-gluconic acid, D-glucuronic acid, D-mannose, D-melibiose, D-raffinose, D-serine, D-trehalose, gentiobiose, glucose 6-phosphate, glycogen, glycyl-L-aspartic acid, inosine, L-alanine (LMG 21460T and LMG 21458 weakly positive), L-alanine-glycine (LMG 21460T and LMG 21458 weakly positive), L-arabinose, L-asparagine, L-aspartic acid, L-serine, maltose, N-acetyl-D-glucosamine, psicose, sucrose, thymidine and uridine. None of the strains utilizes the following carbon sources: 2,3-butanediol, 2-aminoethanol, acetic acid, adonitol, -D-lactose lactulose, -hydroxybutyric acid, -ketobutyric acid, -ketoglutaric acid, -ketovaleric acid, -hydroxybutyric acid, cis-aconitic acid, citric acid, DL--glycerol phosphate, DL-carnitine, D-alanine, D-arabitol, D-galactonic acidolactone, D-galacturonic acid, D-glucosaminic acid, D-mannitol, D-saccharic acid, D-sorbitol, formic acid, 
-aminobutyric acid, -hydroxybutyric acid, glucose 1-phosphate, glucuronamide, glycerol, glycyl-L-glutamic acid, L-hydroxyproline, i-erythritol, itaconic acid, L-fucose, L-histidine, L-leucine, L-ornithine, L-phenyl alanine, L-proline, L-pyro glutamic acid, L-rhamnose, malonic acid, methyl pyruvate, meso-inositol, monomethyl succinate, N-acetyl-D-galactosamine, phenyl ethylamine, p-hydroxyphenylacetic acid, propionic acid, putrescine, quinic acid, sebacic acid, succinamic acid, succinic acid, turanose, Tween 40, Tween 80, urocanic acid or xylitol. All are weakly positive for DL-lactic acid (except LMG 21457, positive). All strains have activities of alkaline phosphatase, esterease (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, trypsin, -chymotrypsin, acid phosphatase and naphthol-AS-BI-phosphohydrolase. None showed activity of lipase (C14), cystine arylamidase, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase. The following cellular fatty acids are present in descending order (mean percentage of the five strains analysed; maximum, minimum of the total fatty acid content): 16 : 0 (25·40; 28·47, 21·18), 18 : 17c (10·79; 12·34, 9·13), 14 : 0 (9·52; 10·31, 8·89), 12 : 0 3-OH (2·91; 3·84, 2·33), 18 : 0 (1·10; 1·35, 0·75). Undefined fatty acids are also observed, summed feature 3 (16 : 17c and/or 15 iso 2-OH – 37·14; 39·77, 34·79), summed feature 2 (14 : 0 3-OH and/or 16 : 1 iso I – 7·05; 8·65, 5·98) and one unknown (0·74; 0·98, 5·98). The G+C content of the DNA is 44·5 mol%. The type strain is LMG 21460T (=CAIM 577T), reference strains are LMG 21456, LMG 21457, LMG 21458 and LMG 21459; isolated from a rotifer (Brachionus plicatilis) flow-through culture system.
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ACKNOWLEDGEMENTS |
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