The taxonomic positions of three representative strains of Vibrio hollisae (LMG 17719T, LMG 21416 and LMG 21538) were investigated by means of 16S rDNA sequences and phenotypic data. V. hollisae strains (GenBank/EMBL accession nos AJ514909–AJ514911) shared 99·5 % 16S rDNA sequence similarity, but had only 94·6 % similarity to their closest phylogenetic neighbour, Enterovibrio norvegicus. 16S rDNA sequence similarity of V. hollisae and Vibrio cholerae was only 91 %. These results suggest that V. hollisae should be placed into a novel genus, for which the name Grimontia gen. nov. is proposed

Hoste, B.

Swings, J.

Thompson, F.L.

Vandemeulebroecke, K.

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Reclassification of Vibrio hollisae as Grimontia
hollisae gen. nov., comb. nov.
Fabiano L. Thompson,1,2 Bart Hoste,2 Katrien Vandemeulebroecke2
and Jean Swings1,2
Correspondence
Fabiano L. Thompson
Fabiano.Thompson@ugent.be
1Laboratory for Microbiology, Ghent University, K. L. Ledeganckstraat 35, Ghent 9000, Belgium
2BCCMTM/LMG Bacteria Collection, Laboratory for Microbiology, Ghent University, K. L.
Ledeganckstraat 35, Ghent 9000, Belgium
The taxonomic positions of three representative strains of Vibrio hollisae (LMG 17719T, LMG
21416 and LMG 21538) were investigated by means of 16S rDNA sequences and phenotypic
data. V. hollisae strains (GenBank/EMBL accession nos AJ514909–AJ514911) shared 99?5%
16S rDNA sequence similarity, but had only 94?6% similarity to their closest phylogenetic
neighbour, Enterovibrio norvegicus. 16S rDNA sequence similarity of V. hollisae and Vibrio
choleraewas only 91%. These results suggest that V. hollisae should be placed into a novel genus,
for which the name Grimontia gen. nov. is proposed.
Vibrio hollisae was first described by Hickman et al. (1982).
This organism produces a number of toxins and also invades
host epithelial cells (Miliotis et al., 1995), causing both
gastroenteritis and septicaemia (Abbott & Janda, 1994).
Phenotypically, V. hollisae strains are atypical of the genus
Vibrio, as they are arginine dihydrolase-, lysine and
ornithine decarboxylase-negative and cannot grow on
thiosulphate/citrate/bile salts/sucrose (TCBS) agar. FAFLP
(fluorescent amplified fragment length polymorphism)
fingerprinting revealed that V. hollisae is quite different
from other vibrios, as it remained unclustered when 506
Vibrio strains were grouped by FAFLP pattern similarity
(Thompson et al., 2001). Dorsch et al. (1992) analysed the
phylogenetic positions of several Vibrio species by means of
16S rRNA gene sequences and concluded that V. hollisae
should be considered as a novel genus, although these
authors highlighted the need for more representative 16S
rRNA gene sequences of diverse Vibrio branches to
determine whether V. hollisae is actually a novel genus.
In a comprehensive phylogenetic study of the families
Vibrionaceae, Aeromonadaceae and Plesiomonadaceae based
on 16S rRNA gene sequences, Ruimy et al. (1994) showed
that V. hollisae in fact lies at the outskirts of the genus Vibrio,
being as closely related to this genus as to the genera
Photobacterium and Salinivibrio. Analyses of toxR and gyrB
gene sequences have pointed out the very low similarity
between V. hollisae and other Vibrio species (Osorio &Klose,
2000; Vuddhakul et al., 2000). More recently, on the basis of
16S rDNA sequence analysis, we found that V. hollisae
branched between Photobacterium species and a newly
described genus, Enterovibrio, rather than with other vibrios
(Thompson et al., 2002).
In this study, we further analysed the phylogenetic positions
of three representative strains of V. hollisae (LMG 17719T,
LMG 21416 and LMG 21538) that were associated with cases
of gastroenteritis in the USA. Strains LMG 17719T (=ATCC
33564T) and LMG 21416 (=ATCC 33565=JCM 1284)
were analysed in detail in the original description of
V. hollisae (Hickman et al., 1982), while strain LMG 21538
(=CIP 104354) was isolated and studied by Carnahan et al.
(1994). 16S rDNA sequence analysis was performed as
described previously (Thompson et al., 2001). 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 uC for 24 h. Cells (approx. 50 mg) were harvested
and fatty acids were isolated, following the recommenda-
tions of the manufacturer, by using the Microbial
Identification system and software package, version 3.9
(MIDI).
The results of our phylogenetic analysis are depicted in
Fig. 1. The three V. hollisae strains shared 99?5% 16S rDNA
sequence similarity, but had only 94?6% similarity to their
closest phylogenetic neighbour, Enterovibrio norvegicus. A
maximum-parsimony tree gave a very similar branching
pattern to the neighbour-joining tree shown in Fig. 1. 16S
rDNA sequence similarity of V. hollisae to the genera
Photobacterium and Salinivibrio was respectively 93 and
91?2%whereas similarity betweenV. hollisae andV. cholerae
Abbreviation: FAFLP, fluorescent amplified fragment length polymorphism.
Published online ahead of print on 11 April 2003 as DOI 10.1099/
ijs.0.02660-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA
sequences of strains LMG 17719T, LMG 21416 and LMG 21538 are
AJ514909–AJ514911.
02660 G 2003 IUMS Printed in Great Britain 1615
International Journal of Systematic and Evolutionary Microbiology (2003), 53, 1615–1617 DOI 10.1099/ijs.0.02660-0
Downloaded from www.microbiologyresearch.org by
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was only 90?8%, clearly indicating that V. hollisae belongs to
a different genus within the family Vibrionaceae.
In our phylogenetic analyses we included representative
16S rDNA sequences of all six genera currently assigned
to the family Vibrionaceae, i.e. Allomonas, Enhydrobacter,
Listonella, Photobacterium, Salinivibrio and Vibrio [see
outline of Bergey’s Manual of Systematic Bacteriology
(2002) at http://dx.doi.org/10?1007/bergeysoutline200210].
We also included representative sequences of the newly
described genus Enterovibrio (Thompson et al., 2002). 16S
rDNA sequences of Allomonas enterica LMG 21754T
(GenBank/EMBL accession no. AJ550855, 1468 bp) and
Enhydrobacter aerosaccus LMG 21877T (AJ550856, 1484 bp)
were determined in the course of this study. The genera
Allomonas (Kalina et al., 1984) and Enhydrobacter (Staley
et al., 1987) were allocated tentatively to the family
Vibrionaceae based on phenotypic features, but so far
there had been no confirmation of their phylogenetic
positions based on 16S rDNA sequence data (Farmer, 1986;
Farmer & Hickman-Brenner, 1992). According to our 16S
rDNA data, A. enterica and E. aerosaccus are highly related
(with almost 100% 16S rDNA sequence similarity) to
Vibrio fluvialis (X74703) and Moraxella osloensis (X74897,
AF005191 and AY043376), respectively (Pettersson et al.,
1998; Coenye et al., 2002). Further experiments with a more
comprehensive collection of reference strains of Allomonas
and Enhydrobacter are needed to clarify the taxonomic
affiliations of these genera.
The three 16S rDNA sequences of V. hollisae determined
in the present study (GenBank accession nos AJ514909–
AJ514911) were highly related (99?3%) to sequence
X74707, determined by Ruimy et al. (1994) for the type
strain of V. hollisae. The four above-mentioned sequences
were ~4% different from sequence X56583, proposed by
Dorsch et al. (1992). Mellado et al. (1996) had already
highlighted the need for an explanation of such a difference
in sequence data. For their comparisons, Mellado et al.
(1996) used the sequence of Dorsch et al. (1992), although
our results show clearly that the most accurate sequence
is in fact that of Ruimy et al. (1994) or any of the three
sequences determined in this study.
V. hollisae can be also differentiated from other members
of the family Vibrionaceae by several phenotypic features
(Table 1). V. hollisae has higher levels of the fatty acids
C18 : 1v9c and C16 : 1v9c and does not grow on TCBS, as most
vibrios can. V. hollisae is arginine dihydrolase-negative and
nitrate reduction-positive, by which it differs from the
genera Enterovibrio, Photobacterium and Salinivibrio.
On the basis of the phylogenetic and phenotypic data
presented here, we propose to accommodate V. hollisae in a
novel genus, Grimontia gen. nov. The type species of the
novel genus is Grimontia hollisae gen. nov., comb. nov.
Description of Grimontia gen. nov.
Grimontia (Gri.mon9ti.a. N.L. gen. n. Grimontia of
Grimont, after the Frenchmicrobiologist P. A. D. Grimont).
Fig. 1. Phylogenetic tree with estimated positions of V. hollisae,
Allomonas enterica and Enhydrobacter aerosaccus by using
the neighbour-joining method, based on almost-complete 16S
rDNA sequences (positions 10–1415). Bootstrap percentages
after 1000 simulations are shown. Bar, 2% estimated sequence
divergence.
Table 1. Features that differentiate Grimontia gen. nov.
from other genera of the family Vibrionaceae
Taxa: 1, Grimontia; 2, Enterovibrio (Thompson et al., 2002);
3, Photobacterium (Alsina & Blanch, 1994); 4, Salinivibrio
(Mellado et al., 1996); 5, Vibrio (Alsina & Blanch, 1994). Fatty
acid data (%) are from our own database. TCBS medium was
obtained from Oxoid.
Feature 1 2 3 4 5
Growth on/in:
TCBS 2 + + + +*
12% NaCl 2 2 2 + 2
Production of:
Acetoin 2 2 +/2 + 2*
Indole + + 2 2 +/2
Nitrate reduction + 2 +/2 2 +*
Arginine dihydrolase 2 + + + +/2
Fatty acids:
C18 : 1v9c 4–6 3 0 <1 0–5
C16 : 1v9c 4 4 0 1–2 0–2
*Over 85% of species show this feature.
1616 International Journal of Systematic and Evolutionary Microbiology 53
F. L. Thompson and others
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Cells are Gram-negative, motile by a polar flagellum and
oxidase-positive. Strains have a DNA G+C content of
48?5–51?0 mol%. Most abundant fatty acids are summed
feature 3 (31±1%; comprising C16 : 1v7c and/or iso-C15 : 0
2-OH), C18 : 1v7c (23±2%), C16 : 0 (14±1%), C12 : 0
(5±1%), C18 : 1v9c (5±1%), summed feature 2 (5±2%;
comprising C14 : 0 3-OH and/or iso-C16 : 1 I and/or uniden-
tified fatty acid with equivalent chain-length value of
10?928 and/or C12 : 0 ALDE), C16 : 1v9c (4±0%), C12 : 0
3-OH (4±2%), C14 : 0 (3±0%) and C18 : 0 (2±1%).
Chemoheterotrophic, mesophilic and moderately halo-
philic. Strains are negative for Voges–Proskauer reaction,
arginine dihydrolase, lysine and ornithine decarboxylase,
but indole production and nitrate reduction are positive.
16S rDNA sequences of Grimontia strains have typical
signatures at positions 970–971 (TC instead of AG) and
1107–1108 (CG instead of AA), which differ from those of
other members of the family Vibrionaceae.
The type species is Grimontia hollisae (LMG 17719T;
GenBank/EMBL accession no. AJ514909, DNA G+C
content is 48?5 mol%). Description of Grimontia hollisae
gen. nov., comb. nov. is based on the original description of
Hickman et al. (1982).
Acknowledgements
F. L. T. has a PhD scholarship (no. 2008361/98-6) from Conselho
Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq),
Brazil. J. S. acknowledges grants from the Fund for Scientific Research
(FWO), Belgium. The authors acknowledge the suggestions of the two
reviewers.
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Grimontia hollisae gen. nov., comb. nov.


Reclassification of <i>Vibrio hollisae</i> as <i>Grimontia hollisae</i> gen. nov., comb. nov.

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