ORIGINAL PAPER

Vibrio cortegadensis sp. nov., isolated from clams

Aide Lasa • Ana L. Dieguez • Jesus L. Romalde

Received: 23 June 2013 / Accepted: 15 November 2013 / Published online: 23 November 2013

� Springer Science+Business Media Dordrecht 2013

Abstract A group of four strains isolated from

clams (Venerupis decussata and Venerupis philippin-

arum) in Galicia (NW Spain) were subjected to a

polyphasic characterization, based on the phenotypic

characteristics, the analysis of chemotaxonomic fea-

tures, the sequencing of the 16S rRNA and five

housekeeping (atpA, pyrH, recA, rpoA and rpoD)

genes, as well as DNA–DNA hybridization (DDH).

The analysis of the phenotypic and chemotaxonomic

characteristics and the results of a phylogenetic study,

based on the 16S rRNA gene sequence analysis and

multilocus sequence analysis, clearly indicated that

these strains belong to the genus Vibrio and were

allocated between the Splendidus and Anguillarum

clades showing a close relationship with the type

strains of Vibrio tapetis (98.8 %), Vibrio pomeroyi

(98.0 %) and Vibrio crassostreae (97.9 %). DNA–

DNA hybridization results confirmed that these iso-

lates constitute a new species. The name Vibrio

cortegadensis sp. nov. is proposed with C 16.17T

(=CECT 7227T=LMG 27474T) as the type strain.

Keywords Vibrio � V. cortegadensis sp. nov. �MLSA � DDH

Introduction

The genus Vibrio comprises of a large number of

species that are common inhabitants of aquatic

environments such as estuarine, coastal waters and

sediments (Colwell 2006; Thompson and Swings

2006). Several species of this genus have been

associated with marine eukaryotic organisms includ-

ing fish, molluscs and crustaceans (Beaz-Hidalgo et al.

2010) and, in addition, some of them have been

described as pathogens to fish, molluscs and crusta-

ceans (Farto et al. 2003; Gay et al. 2004; Gomez-Leon

et al. 2005; Jensen et al. 2003; Kueh and Chan 1985;

Lacoste et al. 2001; Le Roux et al. 2005; Leano et al.

1998; Nicolas et al. 1996; Pujalte et al. 1993; Sugumar

et al. 1998).

The increasing number of environmental studies

and the introduction of molecular techniques in

bacterial taxonomy, such as DNA–DNA hybridization

(DDH), multilocus sequence analysis (MLSA) and

amplified fragment length polymorphism (AFLP)

(Beaz-Hidalgo et al. 2008; Colwell 2006; Thompson

and Swings 2006), have enhanced the understanding of

the family Vibrionaceae taxonomic structure and

phylogeny.

Nowadays, there are 98 validly described species

of the genus Vibrio, including two subspecies

Electronic supplementary material The online version ofthis article (doi:10.1007/s10482-013-0078-z) contains supple-mentary material, which is available to authorized users.

A. Lasa � A. L. Dieguez � J. L. Romalde (&)

Departamento de Microbiologıa y Parasitologıa, CIBUS,

Universidad de Santiago de Compostela, Campus Sur s/n,

15782 Santiago de Compostela, Spain

e-mail: jesus.romalde@usc.es

123

Antonie van Leeuwenhoek (2014) 105:335–341

DOI 10.1007/s10482-013-0078-z

(http://www.vibriobiology.net), that have been

grouped into 14 clades (Sawabe et al. 2007). The

large number of the species described in the last

6 years, together with the proposal of the new

clades (i.g. Marisflavi and Comitans) (Pujalte 2011),

have led to an update of the intra-genus classifica-

tion (Gomez-Gil, personal communication).

In a previous study on the diversity of vibrios

conducted in 2004 and 2005, a collection of isolates were

obtained from reared clams, Manila clam (Venerupis

philippinarum) and carpet–shell clam (Venerupis decuss-

ata), aquacultured in different geographical sites of the

coast of Galicia (NW Spain). A representative number of

isolates of this collection were analysed by AFLP and a

group of four strains (cluster 68) could not be assigned to

any of the currently known species of the genus Vibrio

(Beaz-Hidalgo et al. 2008). In the present study, a

polyphasic approach was employed for the characteriza-

tion of the cluster of four strains isolated from the clams.

Materials and methods

Bacterial isolates

Bacterial strains C 16.17T (=CECT 7227T=LMG

27474T), CMJ 9.12 (=CECT 8125=LMG 27475), CMJ

12.11 (=LMG 27477) and Rd 13.7 (=LMG 27476) were

corresponded to the clam isolates of cluster 68 of Beaz-

Hidalgo et al. (2008). Strain C 16.17T was isolated from

Ria de Arousa (42�370300N/8�4603800W), strains CMJ

9.12 and CMJ 12.11 were isolated from Ria de

Camarinas (43�801600N/9�1003700W) and strain Rd 13.7

was isolated from Ria de Vigo (42�1802700N/

8�3701300W). The four isolates were characterized in

comparison with the following reference strains obtained

from bacterial culture collections: V. atlanticus CECT

7223T, V. artabrorum CECT 7226T, V. celticus CECT

7224T, V. chagasii LMG 21353T, V. crassostreae CAIM

1405T, V. cyclitrophicus LMG 21359T, V. gallaecicus

CECT 7244T, Vibrio gigantis LMG 22741T, V. kanaloae

LMG 20539T, V. lentus CECT 5110T, V. pomeroyi LMG

20537T, V. splendidus CECT 628T, V. tasmaniensis

LMG 20012T, V. tapetis CECT 4600T and V. anguilla-

rum ATCC 19264T. All strains were cultured on plates of

Marine agar (MA, Difco) at 24 ± 1 �C for 24 h. Stock

cultures were maintained frozen at -80 �C in Marine

broth (MB, Pronadisa, Spain) supplemented with 15 %

of glycerol (v/v).

Phenotypical characterization

The four marine strains were subjected to the following

phenotypic tests (MacFaddin 1993; Romalde and Tora-

nzo 1991): cell morphology and motility, Gram stain,

oxidase, catalase, oxidation/fermentation test, fermenta-

tion and acid production from inositol, mannitol and

sucrose, gas and acid production from glucose, indole,

methyl red, Voges–Proskauer reaction, utilization of

citrate, arginine dihydrolase test (Moeller’s medium),

lysine and ornithine decarboxylation (Moeller’s med-

ium), nitrate reduction, hydrolysis of gelatin, Tween 80,

amylase and aesculin. Salt tolerance test was performed

on Basal medium agar (BMA, neopeptone [4 g/l], yeast

extract [1 g/l], bacteriological agar [15 g/l]) supple-

mented with 0, 0.5, 1, 3, 6, 8 and 10 % NaCl. Growth at

different temperatures (4, 20, 25, 30, 37 and 44 �C), pH

(4–10), and on thiosulfate–citrate–bile sucrose (TCBS)

agar (Oxoid) were also determined. Sensitivity to the

vibriostatic agent O/129 (2,4-diamino-6,7-diisopropylp-

teridine) (150 lg per disc) was determined on Mueller–

Hinton (Oxoid) agar. All media were supplemented with

1 % NaCl when required.

Additional phenotypic characteristics were per-

formed using API 20 NE, API 50CH and API ZYM

miniaturized systems (BioMerieux, France) using

Saline solution (SS, 0.85 % NaCl) to prepare the

bacterial suspensions. API 50CH was used with the

slight modifications described by Prado et al. (2005).

Briefly, bacterial suspensions were prepared in SS,

adjusted to an OD580 of 1.0 and mixed (1:10, v/v) with

ZOF medium (without agar) (Lemos et al. 1985) for

the inoculation of the strips. With the exception of the

growth at different temperatures, all phenotypic tests

were performed at 24 ± 1 �C.

16S rRNA and housekeeping genes sequencing

Genomic DNA for sequencing was obtained as described

previously (Osorio et al. 1999). Amplification and

sequencing of the 16S rRNA gene and the housekeeping

genes atpA (ATP synthase alpha subunit gene), recA

(recombinase A gene), pyrH (uridine monophosphate

kinase gene), rpoA (RNA polymerase alpha subunit

gene) and rpoD (RNA polymerase sigma factor gene)

were performed according to Thompson et al. (2004,

2005, 2007) and Pascual et al. (2010). For reference

strains, sequences were acquired from GenBank/EMBL/

DDBJ. Sequence analyses were performed using the

336 Antonie van Leeuwenhoek (2014) 105:335–341

123

DNASTAR Lasergene SEQMAN program. Sequence

similarities of 16S rRNA and housekeeping genes were

determined using the EzTaxon-e server (www.eztaxon-

e.ezbiocloud.net; Kim et al. 2012) and the BLASTN

program respectively. Sequences were aligned using

CLUSTAL W tool (Larkin et al. 2007), and phylogenetic

trees were reconstructed using the neighbour-joining and

maximum-likelihood algorithms (MEGA version 5.05)

(Tamura et al. 2011). Distance matrices were calculated

by using Kimura’s two-parameter correction and stabil-

ity of the groupings was estimated by bootstrap analysis

(1,000 replicates) using the MEGA version 5.0 (Tamura

et al. 2011).

DNA–DNA hybridization (DDH)

Genomic DNA for DDH experiments was extracted

using the commercial DNeasy Blood & Tissue kit

(QIAGEN), following the manufacturer’s protocol.

DDH experiments were undertaken between the strain

C16.17T and the type strains of the species with highest

similarities in the 16S rRNA gene (V. tapetis, V.

pomeroyi and V. crassostreae) and a representative of

the Anguillarum clade (V. anguillarum). DDH experi-

ments were performed with the hydroxyapatite/micro-

titre plate method (Ziemke et al. 1998) using a

hybridization temperature (Tm) of 60 �C. Reciprocal

reactions (i.e. A 9 B and B 9 A) were performed and

were generally within the limits of this method (Goris

et al. 1998).

Fatty acids analysis

Chemotaxonomic features were studied by the analyses

of fatty acid methyl esters (FAME). FAME were

extracted and prepared from 24 h cultures on MA

incubated at 24 ± 1 �C as described by Sasser et al.

(1990) according to the MIDI Microbial Identifications

System (MIDI, Newark, DE, USA). The two closest

species in the MLSA were analysed in parallel for

comparison.

Results and discussion

The four marine strains were facultative anaerobic,

motile, Gram-negative rods. Positive for oxidase

production and reduction of nitrates to nitrites, only

one of the strains (CMJ 12.11) was positive in the

catalase test. They required salt for growth (optimal

range 1–3 %), were able to grow at 4 �C but not at 37

or 44 �C (optimal range 20–25 �C). Optimal pH was

found to be in the range of 6–9. The strains C 16.17T

and CMJ 9.12 grew on TCBS (Oxoid) but not the

strains CMJ 12.11 and Rd 13.7. They were sensitive to

the vibriostatic agent O/129. Differentiating pheno-

typic features for the four marine strains are shown in

Table S1.

Sequence similarity of the 16S rRNA gene indi-

cated that the isolates belonged to the genus Vibrio.

Isolate C16.17T showed highest sequence similarities

with the species Vibrio tapetis (98.7 %), Vibrio

pomeroyi (98.0 %), and Vibrio crassostreae

(97.9 %). Phylogenetic analysis based on 16S rRNA

gene sequences of the isolates, employing both NJ or

ML approaches, showed that the four marine strains

present an intermediate position between the repre-

sentatives of Splendidus and Anguillarum clades

(Figs. 1 and S1).

Multilocus sequence analysis (MLSA) of house-

keeping genes has been proposed as a useful tool to

define the phylogenetic relationships among microor-

ganisms (Stackebrandt and Ebers 2006). In the genus

Vibrio, several genes have been studied for delineating

new species, such as gyrB, atpA, recA, pyrH or dnaJ

(Pascual et al. 2010; Sawabe et al. 2007; Thompson

et al. 2004, 2005, 2007). In this study, sequences of the

genes atpA (1,300 bp), pyrH (575 bp), recA (765 bp),

rpoA (875 bp) and rpoD (842 bp) were obtained for

the clam isolates and compared with the closest

relatives. Each housekeeping gene pointed different

species of the genus Vibrio as the closest relative, with

similarity values lower than 93 % in all cases (Fig.

S2). Phylogenetic trees based on each housekeeping

gene and on concatenated sequences of the five

housekeeping genes, not only showed that the four

clam isolates form a tight group, but also suggested a

closer relationship with the Splendidus clade (Figs. 2,

S2 and S3). Further studies will confirm the inclusion

of this group of isolates in a specific clade of the genus.

The GenBank accession numbers for the 16S rRNA,

atpA, pyrH, recA, rpoA and rpoD gene sequences

obtained for the four clam strains are listed in

Supplementary Table S2.

The type strain C16.17T showed levels of DNA

relatedness of 53 % (reciprocal 45 %) with V. tapetis

CECT 4600T, 41 % (38 %) with V. pomeroyi LMG

20537T, 41 % (44 %) with V. crassostreae CAIM

Antonie van Leeuwenhoek (2014) 105:335–341 337

123

1405T, and 35 % (39 %) with V. anguillarum ATCC

19264T. All these DDH values are below 70 %, the

threshold accepted to delimit species. On the other

hand, the four clam isolates showed DDH values of at

least 80 % (data not shown). These results demon-

strated that the four clam strains represent a novel

species within the genus Vibrio.

The isolates from cultured clam can be differenti-

ated from the phylogenetically related species of the

genus Vibrio, by several phenotypic features

(Table 1). Strains can be distinguished from V. tapetis

by their ability in the arginine dyhydrolsis, the

fermentation of glycerol and D-maltose, and the lack

of the fermentation of D-mannitol and amygdalin. At

the same time, the clam isolates can be differentiated

from V. pomeroyi by their ability in fermentation of

glycerol and inability in the fermentation of D-manni-

tol. These strains can be differentiated from V.

crassostreae by their inability to grow at 6 % of NaCl,

the fermentation of D-maltose and inability in the

fermentation of D-mannitol and amygdalin. In addi-

tion, the analyses of FAMEs of the type strain C16.17T

showed its distinct FA profile (Table S3).

The analysis of the polyphasic study clearly

indicated that strains of the cluster 68 represent a

new taxon within the genus Vibrio. The name Vibrio

Fig. 1 Phylogenetic position of the four isolates according to

16S rRNA gene sequence analysis. The tree is a NJ tree; Vibrio

cholera was used as an outgroup. GeneBank sequence accession

numbers are given in parentheses. Numbers at the nodes show

the percentage bootstrap values (only values higher than 50 %

are shown). Bar 0.002 substitutions per nucleotide position

338 Antonie van Leeuwenhoek (2014) 105:335–341

123

cortegadensis sp. nov. is proposed for this new

species.

Description of Vibrio cortegadensis sp. nov

Vibrio cortegadensis [cor.te.ga.den0.sis. N.L. gen. n.

cortegadensis intended to mean that the type strain

was isolated from Cortegada Island in Carril (Galicia,

Spain)].

Gram-negative motile rods with facultative anaer-

obic metabolism. All strains are sensitive to

the vibriostatic agent O/129, positive for arginine

dihydrolase (Moeller’s medium), indole reaction,

production of oxidase, lipase and amylase, and for

the reduction of nitrates to nitrites. They are negative

for the decarboxylation of lysine and ornithine,

Voges–Proskauer reaction, utilization of citrate, and

the hydrolysis of aesculin and urea. Variable reaction

was observed for the catalase test, showing only CMJ

9.12 strain a positive reaction. Some strains positive

for the ONPG test. The ability to grow on TCBS is

variable, with strains including the type strain able to

grow on this medium as green colonies (sucrose

negative). Strains show growth from 1 to 3 % NaCl,

but not in the absence of NaCl or at salinities higher

than 6 % NaCl. Able to grow from 4 to 30 �C, but not

at 37 and 44 �C.

Fig. 2 Phylogenetic position of the four isolates according to

MLSA of the five housekeeping genes atpA, pyrH, recA, rpoA

and rpoD, and the 16S rRNA gene. The tree is a NJ tree.

Numbers at the nodes show the percentage bootstrap values

(only values higher than 50 % are shown). Bar 0.02 substitu-

tions per nucleotide position

Antonie van Leeuwenhoek (2014) 105:335–341 339

123

All strains produce fermentation of D-glucose,

D-fructose, N-acetylglucosamine, aesculin, D-malt-

ose, D-trehalose, starch, glucogen and potassium

2-ketogluconate (weakly reaction), but not of eryth-

ritol, D-arabinose, L-xylose, D-adonitol, methyl-bD-

xylopyranoside, L-sorbose, L-rhamnose, dulcytol,

inositol, D-sorbitol, methyl-aD-mannopyranoside,

methyl-aD-glucopyranoside, amygdalin, arbutin, sal-

icin, D-cellobiose, D-lactose, D-melibiose, D-sucrose,

inuline, D-melezitose, D-raffinose, xylitol, gentiobi-

ose, D-turanose, D-lyxose, D-tagatose, D-fucose,

L-fucose, D-arabitol, L-arabitol, potassium gluconate

and potassium 5-ketogluconate. In the API ZYM

system, all strains show positive reactions for alkaline

phosphatase, esterase, esterase lipase, leucine aryl-

amidase, valine arylamidase, acid phosphatase,

naftol-AS-BI-phosphohydrolase. The major fatty

acids of the type strain C16.17T are C12:0 3OH

(4.3 %), C16:0 (22.6 %), summed feature in 3 (com-

prising C16:1 x7c and/or C16:1 x6c) (47.9 %), and

summed feature 8 (comprising C18:1 x7c and/or

C18:1 x6c) (9.9 %).

The type strain C16.17T (=CECT 7227T=LMG

27474T) was isolated in the north-western coast of Spain

(Galicia), from healthy cultured clams, V. decussata.

Isolates CMJ 9.12 (=CECT 8125=LMG 27475),

CMJ 12.11 (=LMG 27477) and Rd 13.7 (=LMG

27476) were also deposited at culture collections as

reference strains of the species.

Acknowledgments This work was supported in part by grant

AGL-2010-18438 from the Ministerio de Ciencia e Innovacion

(Ministry of Science and Innovation) (Spain). A. L.

acknowledges the Ministerio de Economıa y Competitividad

(Ministry of Economy and Competitiveness) (Spain) for a

research fellowship.

Conflict of interest The authors declare that they have no

conflict of interest.

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ADH ? - - - ? ? ? ? - ? ? ? ? ? - - ?

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