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1 Laboratory for Microbiology, Ghent University, K. L. Ledeganckstraat 35, Ghent 9000, Belgium
2 Laboratory for BCCMTM/LMG Bacteria Collection, Ghent University, K. L. Ledeganckstraat 35, Ghent 9000, Belgium
3 College of Marine Life Sciences, Ocean University of Qingdao, 5 Yushan Road, Qingdao 266003, China
4 CIAD/Mazatlán Unit for Aquaculture, AP. 711, Mazatlán, Sinaloa, Mexico 82000
Correspondence
Fabiano L. Thompson
Fabiano.Thompson{at}rug.ac.be
| ABSTRACT |
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-galactosidase activity and fatty acid content (particularly of 12 : 0, 14 : 0, 14 : 0 iso and 16 : 0 iso).
Published online ahead of print on 4 October 2002 as DOI 10.1099/ijs.0.02490-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA sequences of strains LMG 20539T, LMG 21522, LMG 21523, LMG 21353T, LMG 13237, LMG 21354, LMG 20537T, LMG 21351 and LMG 21352 are respectively AJ316193, AJ490153, AJ490154, AJ316199, AJ490157, AJ490158, AJ491290, AJ316197 and AJ490152.
Results of repetitive extragenic palindrome-PCR fingerprinting of V. kanaloae, V. pomeroyi, V. chagasii and the most closely related Vibrio species and variable features/differentiating characteristics for V. kanaloae, V. pomeroyi and V. chagasii are available as supplementary data in IJSEM Online.
| INTRODUCTION |
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| METHODS |
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Phenotypic analyses.
Phenotypic characterization of the isolates was performed using API 20E and API ZYM (bioMérieux) and BIOLOG GN metabolic fingerprinting according to the instructions of the manufacturers, with slight modifications (Thompson et al., 2002
). Classical phenotypic tests were performed as described previously (Baumann et al., 1984
; Farmer & Hickman-Brenner, 1992
; Murray et al., 1994
; Thompson et al., 2002
; Vandamme et al., 1998
). Antibiograms were carried out using disc-diffusion methodology (Acar & Goldstein, 1996
) with commercial discs (Oxoid). The inhibition zone of each antibiotic was measured on strains grown on Iso-sensitest agar (Oxoid) supplemented with 1·5 % (w/v) NaCl for 24 h at 28 °C. Fatty acid methyl ester analysis was carried out as described by Huys et al. (1994)
. Isolates were grown on trypticase soy broth (Becton Dickinson) supplemented with 1·5 % (w/v) Bacto agar (Becton Dickinson) and 1·5 % (w/v) NaCl at 28 °C for 24 h. Approximately 50 mg cells was harvested and the fatty acids were isolated, according to the recommendations of the manufacturer, using the Microbial Identification System manual and software, version 3.9 (Microbial ID).
| RESULTS AND DISCUSSION |
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99 %. Isolates LMG 20539T, LMG 21522 and LMG 21523 shared 99·5 % 16S rDNA similarity, while LMG 20537T, LMG 21351 and LMG 21352 shared 99·4 %. Isolates LMG 21353T and LMG 13237 showed 99·7 % similarity. The similarity between representative isolates of each FAFLP group was at least 97·4 %. Phylogenetic trees based on almost-complete sequences and using neighbour-joining, maximum-likelihood and maximum-parsimony methods were all in agreement and revealed that the three novel Vibrio species are closely related to V. splendidus (respectively 98·0, 99·1 and 98·5 % similarity), Vibrio lentus (97·8, 98·4 and 98·2 %), Vibrio cyclitrophicus (97·0, 98·3 and 97·7 %), Vibrio mediterranei (95·7, 97·2 and 97·8 %) and Vibrio orientalis (96·0, 97·1 and 97·6 %) (Fig. 2
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65 % with other Vibrio species. Thus, these results confirm their status as novel species (Table 2
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7c and/or 15 : 0 iso 2-OH), 16 : 0, 18 : 1
7c, 14 : 0 and 12 : 0.
The three novel species fermented D-glucose and mannitol but not inositol or rhamnose. Strains of the novel species utilized
-D-glucose, dextrin, glycogen, N-acetyl-D-glucosamine, D-fructose, maltose, D-trehalose, DL-lactic acid, succinic acid, L-alanyl glycine, L-asparagine, L-aspartic acid, L-glutamic acid, monomethyl succinate, glycyl L-aspartic acid, L-threonine, inosine and glycerol as sole carbon sources. None of the novel species utilized N-acetyl-D-galactosamine, adonitol, D-arabitol, i-erythritol, L-fucose, m-inositol,
-lactose, D-melibiose, D-raffinose, L-rhamnose, turanose, xylitol, citric acid, D-galactonic acid lactone, D-galacturonic acid, D-glucosaminic acid,
-hydroxybutyric acid, p-hydroxyphenylacetic acid, itaconic acid,
-ketobutyric acid,
-ketovaleric acid, malonic acid, L-leucine, L-pyroglutamic acid, DL-carnitine,
-aminobutyric acid, urocanic acid, phenylethylamine, 2-aminoethanol or 2,3-butanediol. Strains of the three species produced indole, alkaline phosphatase, esterase (C4), esterase lipase (C8) and naphthol-AS-BI-phosphohydrolase but not lysine or ornithine decarboxylase, H2S, urease, cystine arylamidase,
-galactosidase,
-glucuronidase,
- or
-glucosidase,
-mannosidase or
-fucosidase. The three novel species were non-luminescent, reduced nitrate and were methyl-red-positive (A46 isolates were methyl-red-negative). FAFLP groups A51 and A52/A53 were susceptible to polymixin B (300 U), tetracycline (30 µg per disc) and chloramphenicol (30 µg per disc), but were resistant to ampicillin (25 µg per disc). None of the 23 isolates grew on 0 or
10·0 % NaCl or at
35 °C. Additional phenotypical features found to be variable among the three novel Vibrio species are available as supplementary data in IJSEM Online.
Genomic and phenotypic evidence presented in this study clearly indicates that the 23 isolates should be accommodated in three novel Vibrio species. Although the novel species have the main phenotypic traits of the genus Vibrio, several useful differentiating features were found that distinguish them from known Vibrio species (available as supplementary material in IJSEM Online).
Description of Vibrio kanaloae sp. nov.
Vibrio kanaloae (ka.na.lo'a.e. N.L. gen. n. kanaloae of Kanaloa, the Hawaiian god of the sea and seamen).
Cells are slightly curved, 1 µm wide by 23 µm long and motile by at least one polar flagellum. They form translucent, convex, non-swarming, smooth, rounded colonies with entire margins; colonies are beige in colour and about 5 mm in diameter on TSA after 48 h incubation at 28 °C. Strains form yellow, translucent, 510 mm colonies on TCBS agar. All strains ferment sucrose and arabinose but not sorbitol, melibiose or amygdalin. Cells grow at 4 °C but not in the absence of NaCl. All strains utilize Tween 40, D-mannitol, sucrose, monomethyl succinate,
-ketoglutaric acid, D-alanine, L-alanine, L-ornithine, L-proline, L-serine and L-threonine. None of the strains utilizes cellobiose, D-sorbitol, D-saccharic acid, sebacic acid, succinamic acid, hydroxy-L-proline, L-phenylalanine or DL-
-glycerol phosphate. Strains produce leucine arylamidase, trypsin, tryptophan deaminase, acetoin and gelatinase, but not
-chymotrypsin,
-galactosidase,
-galactosidase or lysine. Arginine dihydrolase activity is variable, but is positive for the type strain. The major fatty acids are summed feature 3 (39·2±0·2 %, comprising 16 : 1
7c and/or 15 : 0 iso 2-OH), 16 : 0 (25·6±1·0 %), 14 : 0 (5·0±0·3 %), 12 : 0 (4·2±0·1 %), 18 : 1
7c (10·2±1·0 %), summed feature 2 (2·1±0·6 %, comprising 14 : 0 3-OH and/or 16 : 1 iso I and/or unidentified fatty acid with equivalent chain-length value of 10·928 and/or 12 : 0 ALDE), 12 : 0 3-OH (3·4±0·1 %), 18 : 0 (1·0±0·1 %) and 16 : 0 3-OH (0·3±0·1 %). Additional phenotypical features are listed as supplementary material in IJSEM Online.
The type strain, strain LMG 20539T (=CAIM 485T=INCO 191T), was isolated from diseased oyster (Ostrea edulis) larvae in France. The G+C content of the DNA of the type strain is 44·5 mol%.
Description of Vibrio pomeroyi sp. nov.
Vibrio pomeroyi (po.me.roy'i. N.L. gen. n. pomeroyi of Pomeroy, in honour of the North American microbial ecologist L. R. Pomeroy).
Cells are slightly curved, 1 µm wide by 23 µm long and motile by at least one polar flagellum. They form translucent, convex, non-swarming, smooth, rounded colonies with entire margins; colonies are beige in colour and about 3 mm in diameter on TSA after 48 h incubation at 28 °C. Strains (except LMG 20537T) form yellow, translucent colonies on TCBS agar. Cells grow at 4 °C but not in the absence of NaCl. All strains utilize D-galactose, cellobiose, monomethyl succinate, sucrose, glycyl L-glutamic acid, L-serine, L-threonine, inosine, uridine and thymidine. None of the strains utilizes
-cyclodextrin, gentiobiose,
-D-lactose lactulose, putrescine, formic acid, D-glucuronic acid,
-hydroxybutyric acid,
-ketoglutaric acid, quinic acid, D-saccharic acid, sebacic acid, succinamic acid, glucuronamide, L-histidine, hydroxy-L-proline, L-leucine, L-phenylalanine, L-pyroglutamic acid, D-serine, DL-carnitine or DL-
-glycerol phosphate. Strains produce
-galactosidase and acid phosphatase, but not lipase (C14), tryptophan deaminase or valine arylamidase. Arginine dihydrolase activity is variable, but is positive for the type strain. The major fatty acids are summed feature 3 (32·9±1·6 %), 16 : 0 (29·2±1·7 %), 14 : 0 (10·5±0·4 %), 12 : 0 (8·9±1·2 %), 18 : 1
7c (7·6±1·8 %), summed feature 2 (4·1±0·6 %), 12 : 0 3-OH (3·9±0·6 %), 18 : 0 (1·6±0·2 %) and 16 : 0 3-OH (0·7±0·1 %). Additional phenotypic features are available as supplementary material in IJSEM Online.
The type strain, LMG 20537T (=CAIM 578T=INCO 62T), was isolated from bivalve (Nodopecten nodosus) larvae in southern Brazil. The G+C content of the DNA of the type strain is 44·1 mol%.
Description of Vibrio chagasii sp. nov.
Vibrio chagasii (cha.ga'si.i. N.L. gen. n. chagasii of Chagas, in honour of the Brazilian physician and microbiologist C. Chagas).
Cells are slightly curved, 1 µm wide by 23 µm long and motile by means of at least one polar flagellum. They form opaque, convex, non-swarming, smooth, rounded colonies with entire margins; colonies are dark beige in colour and 34 mm in diameter on TSA after 48 h incubation at 28 °C. Does not grow in the absence of NaCl. All strains (except LMG 13251) form green, transparent colonies on TCBS agar. All strains utilize Tweens 40 and 80, cellobiose, L-alanine, D-mannitol, psicose and
-ketoglutaric acid as sole carbon sources. None of the strains utilizes L-arabinose, methyl
-D-glucoside,
-cyclodextrin, gentiobiose,
-D-lactose, lactulose, putrescine,
-hydroxybutyric acid, S-saccharic acid, sebacic acid, L-ornithine or L-phenylalanine. Strains do not produce tryptophan deaminase The most abundant fatty acids are summed feature 3 (38·4±3·5 %), 16 : 0 (22·4±3·9 %), 18 : 1
7c (9·7±1·6 %), 14 : 0 (7·2±3·5 %), 16 : 0 iso (5·2±2·6 %), 12 : 0 (3·8±2·0 %), summed feature 2 (3·3±1·3 %), 12 : 0 3-OH (2·7±1·4 %), 18 : 0 (1·1±0·5 %), 14 : 0 iso (1·1±0·7 %), 15 : 0 (0·6±0·3 %), 17 : 0 (0·5±0·3 %) and 14 : 0 iso 3-OH (0·5±0·3 %). Additional phenotypic features are available as supplementary material in IJSEM Online.
The type strain, LMG 21353T (=CAIM 431T=R-3712T), was isolated from the gut of turbot larvae (Scophthalmus maximus) in Norway. The G+C content of the DNA of the type strain is 44·5 mol%.
| ACKNOWLEDGEMENTS |
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