International Journal of Systematic and Evolutionary Microbiology (2003), 53, 245–252 DOI 10.1099/ijs.0.

02447-0

Vibrio neptunius sp. nov., Vibrio brasiliensis

sp. nov. and Vibrio xuii sp. nov., isolated from
the marine aquaculture environment (bivalves,
fish, rotifers and shrimps)
,
F.L. Thompson,1 2 Y. Li,3 B. Gomez-Gil,4 C. C. Thompson,1 B.
Hoste,2 K. Vandemeulebroecke,2 G. S. Rupp,5 A. Pereira,5 M. M.
,
De Bem,5 P. Sorgeloos6 and J. Swings1 2
Correspondence F. L.
1,2
Laboratory for Microbiology1 and BCCMTM/LMG Bacteria Collection2, Ghent
Thompson University, K. L. Ledeganckstraat 35, Ghent 9000, Belgium
Fabiano.Thompson@rug.ac.be 3
College of Marine Life Sciences, Ocean University of Qingdao, 5 Yushan Road, Qingdao,
266003, China
4
CIAD/Mazatla´n Unit for Aquaculture, AP. 711, Mazatla´n, Sinaloa, Mexico 82000
5
Laboratory for Culture of Marine Molluscs, Federal University of Santa Catarina,
Department of Aquaculture, Floriano´polis, Brazil
6
Laboratory of Aquaculture and Artemia Reference Center, Ghent University, Rozier 44, Ghent
9000, Belgium

The fluorescent amplified fragment length polymorphism (FAFLP) groups A5 (21 isolates),
A8 (6 isolates) and A23 (3 isolates) distinguished in an earlier paper (Thompson et al., Syst
Appl Microbiol 24, 520–538, 2001) were examined in more depth. These three groups were
phylogenetically related to Vibrio tubiashii, but DNA–DNA hybridization experiments proved
that the three AFLP groups are in fact novel species. Chemotaxonomic and phenotypic
analyses further revealed several differences among the 30 isolates and known Vibrio
species. It is proposed to accommodate these isolates in three novel species, namely Vibrio
neptunius (type strain LMG 20536T; EMBL accession no. AJ316171; G+C content of the type
strain 46?0 mol%), Vibrio brasiliensis (type strain LMG 20546T; EMBL accession no.
AJ316172; G+C content of the type strain 45?9 mol%) and Vibrio xuii (type strain LMG
21346T; EMBL accession no. AJ316181; G+C content of the type strain 46?6 mol%). These
species can be differentiated on the basis of phenotypic features, including fatty acid
composition (particularly 14 : 0 iso, 14 : 0 iso 3-OH, 16 : 0 iso, 16 : 0, 17 : 0 and 17 : 1v8c),
enzyme activities and utilization and fermentation of various carbon sources.

INTRODUCTION
Thompson et al., 2002a). Vibrios are highly abundant in
It is well recognized that bacteria play a pivotal role in the aquatic ecosystems, particularly in eutrophic environments,
4 5 21
cycling of dissolved and particulate organic matter in accounting for up to 14–45 % (i.e. 10 –10 cells ml ) of the
aquatic ecosystems (Sherr & Sherr, 2000). There has been culturable microbiota (Eilers et al., 2000; Suantika et al.,
increasing evidence that bacteria also fuel food webs in 2001). Moreover, vibrios are present in large numbers in a
marine aquaculture systems and influence the health of successful recirculating system for rotifers (Suantika et al.,
cultured marine organisms (Hansen & Olafsen, 1999; 2001) and are also part of the normal flora of penaeid

Published online ahead of print on 12 July 2002 as DOI 10.1099/ijs.0.02447-0.

Abbreviations: FAFLP, fluorescent amplified fragment length polymorphism; FAME, fatty acid methyl ester; TCBS, thiosulphate/citrate/bile
salts/ sucrose; TSA, tryptone soy agar.
T T T
The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA sequences of LMG strains 20536 , 20546 , 21346 , 20613, 20010 and
21347 are respectively AJ316171, AJ316172, AJ316181 and AJ490150–AJ490152.
Additional phenotypic features of the three novel species are listed as supplementary material in IJSEM Online (http://ijs.sgmjournals.org/).

02447 G 2003 IUMS Printed in Great Britain 245

F. L. Thompson and others

shrimps (Gomez-Gil et al., 1998). Certain Vibrio strains the method described by Pitcher et al. (1989). All strains included in
stimulate reproduction and ameliorate growth rates of this study have been deposited in the BCCM/LMG Bacteria
Collection at Ghent University and in the CAIM collection of the
molluscs and rotifers and protect Artemia against bacterial Centre for Research on Nutrition and Development (CIAD) in
infections, whereas other Vibrio strains constitute serious Mazatlan,´ Mexico.
pathogens or potential pathogens for the same organisms
(Riquelme et al., 2001; Verschuere et al., 2000). Genotypic analyses. Selective amplification of restriction frag-ments
(FAFLP) and sequencing of almost complete 16S rDNA sequences were
Recently, we surveyed the genomic diversity of 506 strains accomplished essentially as described previously (Thompson et al.,
2001). Alignment of the 16S rDNA sequences, distance estimations
of the Vibrionaceae by means of the fluorescent ampli- (Jukes & Cantor, 1969), clustering by the neighbour-joining (Saitou &
fied fragment length polymorphism (FAFLP) technique Nei, 1987), maximum-likelihood and maximum-parsimony methods and
(Thompson et al., 2001). Many isolates from the aqua- analysis of the stability of clus-ters (bootstrap analysis with 1000
culture environment possess genomes that differ from replicates) were performed with the software BioNumerics 2.5 (Applied
Maths). DNA–DNA hybridi-zation experiments using photobiotin-
currently known Vibrio species and are thus potentially
novel species. In the present study, we describe additional
genomic and phenotypic characteristics of a subset of 30 ˚
labelled DNAs were run under stringent conditions (39 C) following the
isolates distributed in the FAFLP groups A5, A8 and A23. method of Willems et al. (2001). The G+C content of DNA was
determined by HPLC (Mesbah et al., 1989).
FAFLP cluster A5 represented mainly the dominant
culturable bacterial microflora of a recirculating system for
Phenotypic characterization. Biochemical characterization of the
rotifers (Suantika et al., 2001). Group A8 was abundant in isolates was performed using API 20E and API ZYM test strips
cultures of larvae of the bivalve Nodipecten nodosus at (bioMerieux)´ and metabolic fingerprinting was carried out by means
Floriano´polis, in southern Brazil, whereas group A23 was of Biolog GN2 microtitre plates. Preparations were done according to
found to be ubiquitous and in association with cultured the manufacturers’ instructions, with slight modifications (Thompson
et al., 2002b). Classical bacteriological tests were per-formed as
shrimps in China and Ecuador and in cultures of N. described previously (Baumann et al., 1984; Farmer & Hickman-
nodosus larvae in Brazil. Brenner, 1992; Thompson et al., 2002b; Vandamme et al., 1998).
Antibiograms were carried out using the disc-diffusion method (Acar
& Goldstein, 1996) with commercial discs (Oxoid). The inhibition
zone of each antibiotic was measured for strains grown on Iso-
METHODS sensitest agar (Oxoid) supplemented with 1?5 % (w/v) NaCl for 24 h
Bacterial strains, growth conditions and DNA isolation.
Strains used in this study are described in Table 1. Strains were grown
aerobically on tryptone soy agar (TSA; Oxoid) supplemented with 2 % (w/v)
˚
at 28 C. Fatty acid methyl ester (FAME) analysis was carried out as
described by Huys et al. (1994). Isolates were grown on trypticase
soy broth (Becton Dickinson) supplemented with
NaCl for 24 h at 28 ˚C. DNA was extracted following

Table 1. Strains included in this study

Abbreviations: LMG, BCCM/LMG Bacteria Collection, Ghent, Belgium; LCMM, Laboratory for Culture of Marine Molluscs, Floriano´polis,
Brazil; CENAIM, Center for Marine and Aquaculture Research, Guayaquil, Ecuador; ARC, Artemia Reference Center, Ghent, Belgium; CAIM,
Collection of Aquacultural Important Micro-organisms, Mazatla´n, Mexico.

Strain(s) Location and date Source

of isolation

Vibrio neptunius sp. nov. (FAFLP group A5)

T T T
LMG 20536 (=CAIM 532 =INCO 17 ) LCMM, 1998 Bivalve larvae (Nodipecten nodosus)
LMG 20610 ARC, 1999 Culture water of rotifers
LMG 20611, R-15119, R-15120, R-15121 ARC, 1999 Rotifer in recirculation system (Brachionus plicatilis)
LMG 20612 ARC, 1996 Gut of turbot larvae (Scophthalmus maximus)
LMG 20613, R-15113, R-15116, R-15117 ARC, 1999 Rotifer in recirculation system (B. plicatilis)
LMG 20614, R- 15118, R-15108, R-15111, R-15112 ARC, 1999 Rotifer in recirculation system (B. plicatilis)
LMG 20615 LCCM, 1998 Diseased bivalve larvae (N. nodosus)
R-1575, R-1579, R-1592 ARC, 1997 Gut of turbot larvae (S. maximus)
R-15123 ARC, 1999 Healthy rotifer (B. plicatilis)
Vibrio brasiliensis sp. nov. (FAFLP group A8)
T T T
LMG 20546 (=CAIM 495 =INCO 317 ), LMG 20010 LCMM, 1999 Bivalve larvae (N. nodosus)
(=INCO 320), R-15002, R-15003, R-15004, R-15005
Vibrio xuii sp. nov. (FAFLP group A23)
T T T
LMG 21346 (=CAIM 467 =STD3-1071 ) Dahua (China), 1995 Shrimp culture water
LMG 21347 (=CAIM 568 =STD3-1204) CENAIM, 1995 White shrimp (Litopenaeus vannamei)
LMG 20011 (=INCO 167) LCMM, 1998 Bivalve larvae (N. nodosus)

246 International Journal of Systematic and Evolutionary Microbiology 53

 Three novel Vibrio species

1?5 % (w/v) Bacto agar (Becton Dickinson) and 1?5 % (w/v) NaCl at
The value of AFLP in determining genome divergence and
˚
28 C for 24 h. Approximately 50 mg cells was harvested and the species delineation for other bacterial genera, e.g.
fatty acids were isolated following the recommendations of the Agrobacterium and Xanthomonas, has also been
manufacturer using the Microbial Identification System manual and
software, version 3.9 (Microbial ID). appreciated (Mougel et al., 2002; Rademaker et al., 2000).
Mougel et al. (2002) calculated that strains belonging to
the same species of Agrobacterium would have about 86
% FAFLP band pattern similarity, while Rademaker et al.
RESULTS AND DISCUSSION (2000) found about 65 % AFLP pattern similarity between
The 30 Vibrio isolates formed three groups by FAFLP strains of the same species.
fingerprinting analysis. FAFLP groups A5, A8 and A23 had
complex band patterns, respectively consisting of 126±14, The 16S rDNA sequences of two representative isolates of
115±7 and 83±14 bands (50–536 bp) (Fig. 1). The three each FAFLP group were determined and were allocated to the
T
FAFLP groups were clearly different from known Vibrio genus Vibrio by the FASTA program. Isolates LMG 20536
species (Thompson et al., 2001), suggesting that they (EMBL accession no. AJ316171; 1468 bp) and LMG 20613
represent novel species. Isolates of group A5 shared at least (AJ490150, 681 bp) had 99?9 % 16S rDNA simila-rity,
T
75 % pairwise pattern similarity and showed less than 71 % whereas LMG 20546 (AJ316172; 1504 bp) and LMG 20010
pairwise pattern similarity towards other Vibrio species. (AJ490151, 467 bp) had 99?3 % similarity. Strains LMG
Surprisingly, these strains, which were isolated over a 4-year T
21346 (AJ316181; 1435 bp) and LMG 21347 (AJ490152,
period and from different places, showed remarkable genome 1123 bp) had 99?2 % similarity. Clustering obtained by the
T
resemblance. For instance, strains LMG 20536 , isolated in neighbour-joining, maximum-likelihood and maximum-
1998 at Floriano´polis island (Brazil), and LMG 20612, parsimony methods was in agreement and the closest
isolated in 1996 at the ARC (Belgium), had 87?5 % pattern phylogenetic neighbours of the three novel Vibrio species
similarity. Some strains, e.g. pairs LMG 20614 and R-15108 were Vibrio tubiashii (98–98?8 %), Vibrio nereis (97?6–98?
and R-15111 and R-15112, clustered at the reproducibility 8 %), Vibrio coralliilyticus (96?8–98?5 %), Vibrio mytili (96?
level of FAFLP (i.e. ¢88 % pattern similarity) and were thus 8–98?2 %) and Vibrio diabolicus (97?1–98?1 %) (Fig. 2). V.
T
indistinguishable by FAFLP. Isolates of FAFLP groups A8 and coralliilyticus and LMG 20536 were closely related, having
A23 respectively showed mutual similarities of at least 82 and T
98?2 % 16S rDNA similarity, and so were LMG 20546 and
62 % and simila-rity levels below 73 and 54 % towards other T T
LMG 21346 (98?4 %). Strain LMG 20536 had 97?2 % 16S
Vibrio species.
rDNA similarity towards strains

Fig. 1. Dendrogram of FAFLP patterns of 30

marine aquaculture Vibrio isolates. V.
tubiashii and V. nereis were included as out-
groups. A band-based (Dice) cluster analysis
(Ward) was used.

http://ijs.sgmjournals.org 247
F. L. Thompson and others

The 30 Vibrio isolates examined in this study had the main

phenotypic and chemotaxonomic features of the genus
Vibrio (Bertone et al., 1996; Farmer & Hickman-Brenner,
1992; Lambert et al., 1983). They were slightly curved
rods, Gram-negative, oxidase- and catalase-positive and
motile by means of at least one polar flagellum. The major
fatty acids were summed feature 3 (comprising 16 : 1v7c
and/or 15 : 0 iso 2-OH), 16 : 0, 18 : 1 v7c and 14 : 0,
accounting for ¢68 % of the total fatty acids (Table 3).
These facultatively anaerobic isolates grew on
thiosulphate/citrate/bile salts/ sucrose (TCBS) agar,
forming yellow colonies, but they did not grow without
Fig. 2. Phylogenetic tree with the estimated positions of Vibrio NaCl or in presence of the vibriostatic agent O/129 at 10 or
neptunius sp. nov., Vibrio brasiliensis sp. nov. and Vibrio xuii T
150 mg per disc (except LMG 21346 ). Prolific growth
sp. nov., using the neighbour-joining method based on the
almost complete 16S rDNA sequences. Bootstrap analyses
were made with 1000 cycles; values greater than 50 % are occurred in media containing 2?5 % (w/v) NaCl at 28 C. ˚
indicated on the branching nodes. Bar, 1 % estimated None of the isolates fermented inositol, sorbitol, rhamnose
sequence divergence. or melibiose. All isolates utilized dextrin, N-acetyl-D-
glucosamine, D-fructose, a-D-glucose, maltose, D-mannose,
T T psicose, D-trehalose, DL-lactic acid, succinic acid, L-alanine, L-
LMG 20546 and LMG 21346 . Similarity levels of the alanyl glycine, L-asparagine, L-aspartic acid, L-glutamic acid,
three proposed novel species towards other genera of the glycyl L-aspartic acid, L-serine, ino-sine, uridine and
family Vibrionaceae were below 95 %. thymidine as sole carbon sources. None of the isolates utilized
adonitol, D-arabitol, i-erythritol, L-fucose, m-inositol, a-
Two representative isolates of each FAFLP group were chosen lactose, a-D-lactose lactulose, D-melibiose, D-raffinose, L-
for DNA–DNA hybridization experiments. The levels of DNA T
rhamnose, xylitol, cis-aconitic acid (except LMG 21346 ),
relatedness within each FAFLP group were ¢93 %, but less citric acid, formic acid, D-galactonic acid lactone, D-
than 67 % towards other phylogenetic related Vibrio species galacturonic acid, D-glucosaminic acid, D-glucuronic acid, a-
(Table 2). DNA–DNA hybridiza-tions confirmed the FAFLP hydroxybutyric acid, itaconic acid, a-ketovaleric acid, malonic
grouping and also revealed other interesting relationships. For acid, quinic acid, D-saccharic acid, sebacic acid, succinamic
instance, FAFLP group A5 was found to be highly related acid, glucurona-mide, L-histidine, L-leucine, L-pyroglutamic
(64–66 %) to the recently described coral-pathogenic species acid, DL-carnitine, urocanic acid or phenyl ethylamine. None
V. coralliilyticus (Ben-Haim et al., 2003); V. coralliilyticus of the isolates was luminescent, but they reduced nitrate and
belongs to FAFLP clusters A1–A3 (Thompson et al., 2001). were Voges–Proskauer and methyl red positive. The 30
isolates produced indole, alkaline phosphatase, esterase

Table 2. DNA–DNA similarity and DNA G+C content of marine aquaculture Vibrio isolates and related Vibrio species

Strain G+C content (mol%) DNA–DNA hybridization with DNA from:

1 2 3 4 5 6 7 8 9 10 11 12
T
1. V. mediterranei LMG 11258 43?8 100
T
2. V. mytili LMG 19157 44?6 21 100
T
3. V. diabolicus LMG 19805 45?6 22 37 100
T
4. V. coralliilyticus LMG 20984 46?2 19 22 23 100
T
5. V. nereis LMG 3895 45?9 25 30 34 32 100
T
6. V. tubiashii LMG 10936 44?8 22 35 25 30 34 100
V. neptunius sp. nov.
T
7. LMG 20536 46?0 20 25 26 64 34 34 100
8. LMG 20613 45?3 24 27 27 66 37 38 93 100
V. brasiliensis sp. nov.
T
9. LMG 20546 45?9 17 20 22 25 32 34 32 29 100
10. LMG 20010 45?9 16 21 22 26 32 34 32 28 100 100
V. xuii sp. nov.
T
11. LMG 21346 46?6 ND ND ND ND 50 31 ND ND ND ND 100
12. LMG 21347 47?1 16 28 26 24 41 27 29 26 25 26 94 100

ND, Not done.

248 International Journal of Systematic and Evolutionary Microbiology 53

 Three novel Vibrio species

Table 3. Fatty acid compositions of the novel Vibrio species Cells are 1 mm wide and 2?3–3 mm long. Forms translucent,
convex, non-swarming, smooth-rounded colonies with entire
Values are percentages (means±SD) of total fatty acids. Only values margins, beige in colour and about 3 mm in diameter on TSA
above 0?1 % are included.

Fatty acid V. neptunius V. brasiliensis V. xuii ˚

after 48 h incubation at 28 C; colonies are yellow, umbonate,
round, entire, smooth, shiny and transparent and 2–3 mm in
12:0 1?9±0?3 1?4±0?2 3?2±0?0
14:0 5?5±0?6 4?6±0?1 3?5±0?3
15:0 1?7±0?3 1?0±0?1 0?9±0?1 ˚
size on TCBS after 24 h at 28 C. No growth in the absence
16:0 18?0±0?8 11?3±0?3 12?5±0?6 of NaCl or in the presence of ¢ 8?0 % (w/v) NaCl. No growth
17:0 2?3±0?2 0?6±0?1 0?5±0?1
13 : 0 iso 1?0±0?0 1?0±0?0 – ˚
at 4 or ¢40 C. Strains are facultatively anaerobic and ferment
14 : 0 iso 0?2±0?1 3?3±0?4 1?2±0?1 D-glucose and sucrose. None of the strains ferments mannitol
15 : 0 iso 1?2±0?3 1?8±0?1 1?1±0?1 or amygdalin. All strains utilize citrate, glycogen, D-mannose,
16 : 0 iso 0?5±0?1 10?5±0?6 5?5±0?4 methyl b-D-glucoside, sucrose, D-serine, L-threonine, glucose
17 : 0 iso 1?5±0?1 1?4±0?1 – 1-phosphate and glucose 6-phosphate as sole carbon sources.
18 : 0 iso – 1?1±0?0 – None of the strains utilizes Tween 80, N-acetyl-D-
galactosamine, L-arabinose, cellobiose, D-galactose,
12 : 0 3-OH 2?1±0?6 1?5±0?3 1?4±0?0
gentiobiose, D-mannitol, D-sorbitol, turanose, monomethyl
14 : 0 iso 3-OH 0?1±0?1 1?3±0?2 0?9±0?1 succinate, D-gluconic acid, b-hydroxybutyric acid, c-
16 : 1v7c alcohol 0?9±0?3 0?3±0?0 – hydroxybutyric acid, p-hydroxyphenylacetic acid, hydroxy-L-
17 : 1v6c 1?2±0?1 0?3±0?1 0?2±0?0 proline, L-phenyla-lanine, DL-carnitine, c-aminobutyric acid,
17 : 1v8c 2?1±0?1 0?7±0?1 – putrescine or 2, 3-butanediol as a sole carbon source. Strains
18 : 1v7c 17?8±1?6 17?3±0?3 21?0±2?4 produce gelatinase, tryptophan deaminase, trypsin and N-
acetyl-b-glucosaminidase, but they do not produce cystine
11-methyl 18 : 1v7c 0?6±0?3 – –
arylami-dase, acid phosphatase, naphthol-AS-BI-
Summed feature 2* 2?4±0?3 1?8±0?3 2?6±0?0 phosphohydrolase, b-galactosidase or a-glucosidase. Arginine
Summed feature 3* 35?7±0?9 34?7±1?0 38?7±1?5 dihydrolase is variable, but positive for the type strain. The
major fatty acids are summed feature 3 (35?7±0?9 %), 16 : 0
*Summed feature 2: one or more of 14 : 0 3-OH, 16 : 1 iso I, an (18?0± 0?8 %) and 18 : 1v7c (17?8±1?6 %) (Table 3). Strains
unidentified fatty acid with equivalent chain-length of 10?928 and/or
are resistant to ampicillin (25 mg per disc). Additional
phenotypic features are listed as supplementary material in
12 : 0 ALDE. Summed feature 3: 16 : 1v7c and/or 15 : 0 iso 2-OH.
IJSEM Online (http://ijs.sgmjournals.org/). The type strain of
T T
this species is LMG 20536 (=CAIM 532 ), isolated from
larvae of the bivalve Nodipecten nodosus in the south of
(C4), esterase lipase (C8), lipase (C14), leucine arylami-dase Brazil. The G+C content of the type strain is 46?0 mol%.
and valine arylamidase (except LMG 20613), but they did not
produce urease, H2S, lysine or ornithine decarbo-xylases, a- Description of Vibrio brasiliensis sp. nov.
chymotrypsin, a-galactosidase, b-glucuronidase, b-
glucosidase or a-fucosidase. The 30 isolates were sensitive to Vibrio brasiliensis (bra.si.li.en9sis. N.L. masc. adj.
chloramphenicol (30 mg per disc) (except LMG 21346 ),
T brasiliensis from Brazil).
tetracycline (30 mg per disc) and polymyxin B (300 U) and Cells are 1 mm wide and 2?5–3 mm long. Forms translucent,
resistant to kanamycin (30 mg per disc). convex, smooth-rounded colonies with entire margins, beige
in colour and 2?5–3 mm in size on TSA after 48 h incubation
We propose to accommodate the 30 Vibrio isolates exa-mined
in the present study in three novel species, Vibrio neptunius ˚
at 28 C. Colonies are yellow, umbonate, wavy, shiny,
sp. nov., Vibrio brasiliensis sp. nov. and Vibrio xuii sp. nov. translucent, round with scalloped margins and about 3 mm in
The three novel Vibrio species can be differentiated from each size on TCBS after 24 h incubation at
other and from other Vibrio species by a number of
phenotypic features (Table 4). Quantitative and qualita-tive ˚
28 C. No growth in the absence of NaCl or in the presence
differences were detected in the fatty acid compositions of
these novel species. Of special interest were the fatty acids ˚
of ¢8?0 % NaCl. No growth at 4 or ¢45 C. Facultatively
14 : 0 iso, 14 : 0 iso 3-OH and 16 : 0 iso, which appeared at a anaerobic and ferments D-glucose, sucrose, mannitol and
higher concentration in group A8, and the fatty acids 16 : 0, amygdalin. None of the strains ferments arabinose. All strains
17 : 0 and 17 : 1 v8c, which were present at a higher utilize a-cyclodextrin, glycogen, cellobiose, gentiobiose, D-
galactose, gentiobiose, a-D-glucose, D-mannitol, methyl b-D-
concentration in group A5. glucoside, sucrose, methyl pyruvate, b-hydroxybutyric acid,
bromosuccinic acid, L-glutamic acid, glycyl L-aspartic acid,
glycyl L-glutamic acid, L-ornithine, L-proline, D-serine, L-
threonine and glycerol as sole carbon sources. None of the
Description of Vibrio neptunius sp. nov. strains utilizes N-acetyl-D-galactosamine, adonitol, c-
hydroxybutyric acid, p-hydroxyphenylacetic acid, a-
Vibrio neptunius (nep.tu9ni.us. L. masc. adj. neptunius ketoglutaric acid, a-ketovaleric acid, alanina-mide, L-
of Neptune, the Roman god of the sea). phenylalanine, 2-aminoethanol, 2,3-butanediol,

http://ijs.sgmjournals.org 249
F. L. Thompson and others

Table 4. Features useful in differentiating V. neptunius, V. brasiliensis and V. xuii spp. nov. from closely related Vibrio species

Species are identified as: 1, V. neptunius sp. nov. (n=21); 2, V. brasiliensis sp. nov. (n=6); 3. V. xuii sp. nov. (n=3); 4, Vibrio aes-tuarianus; 5,
Vibrio anguillarum; 6, Vibrio cyclitrophicus; 7, V. coralliilyticus; 8, V. diabolicus; 9, Vibrio diazotrophicus; 10, Vibrio fluvialis; 11, Vibrio
lentus; 12, V. mediterranei; 13, V. mytili; 14, V. nereis; 15, Vibrio splendidus; 16, V. tubiashii. Phenotypic data for reference species were
obtained from Ben-Haim et al. (2003); Baumann et al. (1984); Farmer & Hickman-Brenner (1992); Hedlund & Staley (2001); Macia´n et al.
(2001); Pujalte et al. (1993) and Rague´ne`s et al. (1997). Fatty acid profiles of known Vibrio species (type strains) are from our own database.
ND, No data; V, variable.

Feature 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Utilization of:
Cellobiose 2 + + + + + 2 2 + V + + + 2 V +
D-Galactose 2 + 2 + V + + + + + + + + 2 V +
Gentiobiose 2 + V + + ND 2 2 + + ND V + 2 2 2
b-Hydroxybutyric acid 2 + + 2 2 2 2 ND 2 + ND 2 2 + V V
Growth on 8 % (w/v) NaCl 2 2 + V 2 + 2 ND + V ND V + + V V
Fermentation of:
Mannitol 2 + + + + + 2 + + + + + + 2 + +
Amygdalin 2 + + + + ND 2 2 + + ND + + 2 + 2
Melibiose 2 2 2 2 2 ND 2 2 2 2 + V 2 2 + V
Enzyme activity:
Gelatinase + + 2 + + + + + 2 + + V 2 V + 2
b-Galactosidase 2 + 2 + + 2 + 2 + V ND ND + 2 V +
N-Acetyl-b-glucosaminidase + 2 2 + + ND 2 2 2 + ND + + ND 2 +
FAME composition:*
14 : 0 iso 0?2±0?1 3?3±0?4 1?2±0?1 0?3 0?0 0?0 0?5 0?2 0?3 1?4 0?0 1?8 0?0 0?2 0?0 0?0
14 : 0 iso 3-OH 0?1±0?1 1?3±0?2 0?9±0?1 0?2 0?0 0?0 0?3 0?3 0?3 0?8 0?0 0?5 0?3 0?3 0?0 0?0
16:0 18?0±0?8 11?3±0?3 12?5±0?6 23?2 28?6 30?5 15 14?4 24?5 16?1 24?7 10?9 18?8 12?9 20?8 17?3
16 : 0 iso 0?5±0?1 10?5±0?6 5?5±0?4 2?2 0?4 0?0 0?8 1?7 2?2 4?9 0?0 4?3 1?5 1?1 0?0 0?0
17:0 2?3±0?2 0?6±0?1 0?5±0?1 0?3 0?0 0?1 2?5 1?6 0?3 0?7 0?0 0?0 0?0 1?9 0?0 0?1
17 : 1v8c 2?1±0?1 0?7±0?1 0?5 0?5 0?0 0?0 1?8 2?5 0?3 0?9 0?0 0?2 0?0 4?6 0?0 0?1
17 : 1v6c 1?2±0?1 0?3±0?1 0?2 0?0 0?0 0?0 0?6 0?7 0?0 0?2 0?0 0?0 0?0 1?3 0?0 0?0
18 : 1v7c 17?8±1?6 17?3±0?3 21?0±2?4 15?9 12?5 7?5 18?2 17?4 16?5 16?8 8?7 17?2 19?9 22?6 12?0 25?4

*Means±SD as percentages of total fatty acids.

DL-a-glycerol phosphate, glucose 1-phosphate or glucose 6- ˚

incubation at 28 C. Colonies are yellow, convex, round,
phosphate as a sole carbon source. All strains produce entire, shiny, translucent and about 2 mm in size on TCBS
arginine dihydrolase, b-galactosidase and gelatinase. None of
the strains produces trypsin, acid phosphatase, a-glucosidase
or N-acetyl-b-glucosaminidase. The most abundant fatty acids
˚
after 24 h incubation at 28 C. No growth in the absence of
are summed feature 3 (34?7±1?0 %), 18 : 1v7c (17?3±0?3 %), NaCl or in the presence of ¢10?0 % NaCl. No growth at 4
16 : 0 (11?3±0?3 %) and 16 : 0 iso (10?5±0?6 %) (Table 3).
Additional phenotypic features are listed online in the ˚
or ¢45 C. Facultatively anaerobic organism that ferments
supplementary material. Isolated from larvae of the bivalve N. glucose, mannitol, sucrose, amygdalin and arabinose.
nodosus in the south of Brazil. The type strain is strain LMG Utilizes a-cyclodextrin, Tweens 40 and 80, N-acetyl-D-
T T
20546 (=CAIM 495 ). The G+C content of the type strain is galactosamine, L-arabinose, cellobiose, D-mannitol, D-
mannose, D-sorbitol, sucrose, methyl pyruvate, monomethyl
45?9 mol%. succinate, acetic acid, D-gluconic acid, b-hydroxybutyric acid,
c-hydroxybutyric acid, p-hydroxy-phenylacetic acid, a-
ketoglutaric acid, D-alanine, glycyl L-glutamic acid, L-proline,
L-threonine, 2,3-butanediol, glycerol and DL-a-glycerol
Description of Vibrio xuii sp. nov. phosphate as sole carbon sources. Does not utilize D-
Vibrio xuii (xu9i.i. N.L. gen. n. xuii of Xu, in honour of the galactose, gentiobiose, methyl b-D-glucoside, D-raffinose, a-
ketobutyric acid, propionic acid, D-serine, quinic acid, sebacic
microbiologist H. Xu). acid, hydroxy-L-proline, 2-aminoethanol or glucose 1-
phosphate as a sole carbon source. Produces arginine
Cells are 1 mm wide and 2–3 mm long. Forms translucent, dihydrolase, acid phosphatase, naphthol-AS-BI-
convex, smooth-rounded colonies with entire margins, phosphohydrolase and tryptophan deami-nase. Does not
beige in colour and 3–4 mm in size on TSA after 48 h produce cystine arylamidase, trypsin,

250 International Journal of Systematic and Evolutionary Microbiology 53

 Three novel Vibrio species

b-galactosidase, a-glucosidase, N-acetyl-b-glucosaminidase or and hybridization groups in the genus Aeromonas. Int J Syst
gelatinase. The major fatty acids are summed feature Bacteriol 44, 651–658.
3 (38?7±1?5 %), 18 : 1v7c (21?0±2?4 %) and 16 : 0 Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In
(12?5±0?6 %) (Table 3). Sensitive to ampicillin (25 mg per Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro.
disc). Additional phenotypic features are listed online in New York: Academic Press.
T
the supplementary material. The type strain, LMG 21346 Lambert, M. A., Hickman-Brenner, F. W., Farmer, J. J., III & Moss,
T C. W. (1983). Differentiation of Vibrionaceae species by their cellular
(=CAIM 467 ), was isolated from shrimp culture water in
fatty acid composition. Int J Syst Bacteriol 33, 777–792.
China. The G+C content of the type strain is
46?6 mol%. Macia´n, M. C., Ludwig, W., Aznar, R., Grimont, P. A. D., Schleifer,
K. H., Garay, E. & Pujalte, M. J. (2001). Vibrio lentus sp. nov.,
isolated from Mediterranean oysters. Int J Syst Evol Microbiol 51,
1449–1456.
ACKNOWLEDGEMENTS Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise
measurement of the G+C content of deoxyribonucleic acid by high-
F. L. T. has a PhD scholarship (no. 2008361/98-6) from Conselho performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq),
Brazil. J. S. acknowledges grants from the Fund for Scientific
Research (FWO), Belgium. G. S. R., A. P. and M. M. B. acknowledge Mougel, C., Thioulouse, J., Perrie`re, G. & Nesme, X. (2002). A
support of the EU project INCO Scallop (no. ERBIC 18 CT 970188). mathematical method for determining genome divergence and species
The authors are indebted to J. Goris, A. Balcaen and L. Lebbe for delineation using AFLP. Int J Syst Evol Microbiol 52, 573–586.
their skilful assistance during the DNA–DNA hybridizations and
G+C measurements. The excellent remarks of two anonymous Murray, R. G. E., Doetsch, R. N. & Robinow, C. F. (1994).
reviewers are gratefully acknowledged. Determinative and cytological light microscopy. In Methods for
Gene-ral and Molecular Bacteriology, pp. 21–41. Edited by P.
Gerhardt. Washington, DC: American Society for Microbiology.
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