Elsevier Editorial System(tm) for Systematic and Applied Microbiology Manuscript Draft Manuscript Number: SAM 3279R1 Title: Vibrio celticus sp. nov., a new Vibrio species belonging to the Splendidus clade with pathogenic potential for clams. Article Type: Full Length Papers Section/Category: Systematics Keywords: Vibrio celticus sp. nov. Splendidus clade MLSA phylogeny. Corresponding Author: Dr Jesus L. Romalde, Ph.D. Corresponding Author's Institution: Universidad de Santiago de Compostela First Author: Roxana Beaz-Hidalgo, Ph.D. Order of Authors: Roxana Beaz-Hidalgo, Ph.D.; Ana L Diéguez, M. Sc.; Ilse Cleewnwerck, Ph.D.; Sabela Balboa, M. Sc.; Alejandra Doce, M. Sc.; Paul de Vos, Ph.D.; Jesus L. Romalde, Ph.D. Abstract: A group of four motile facultative anaerobic marine isolates (Rd 8.15T [= CECT 7224T, = LMG 23850T], Rd 16.13, Rd 6.8 [= LMG 25696] and Rd2L5) were obtained from cultured clams (Ruditapes philippinarum and Venerupis pullastra) in Galicia, Northwestern Spain. They formed a tight phylogenetic group based on sequences of 16S rRNA gene and the four housekeeping genes rpoA (encoding the α−chain of RNA polymerase), rpoD (encoding the sigma factor of RNA polymerase), recA (encoding RecA protein), and atpA (encoding the α-subunit of bacterial ATP synthase). The phylogenies based on these sequences indicated that the four isolates represent a novel species in the genus Vibrio, and more precisely in the Splendidus clade. DNA-DNA hybridizations with the type strains of the species showing more than 98.6% 16S rRNA gene sequence similarity, revealed DNA-DNA relatedness below 70%. The isolates could be differentiated from the phylogenetically related Vibrio species on the basis of several phenotypic features. In addition, strain Rd 8.15T showed potential pathogenic activity for adult clam in the virulence assays. The name Vibrio celticus sp. nov. is proposed for this new taxon, with the type strain being Rd 8.15T (= CECT 7224T, = LMG 23850T).

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1

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Vibrio celticus sp. nov., a new Vibrio species belonging to the Splendidus clade with 4

pathogenic potential for clam. 5

6

7

Roxana Beaz-Hidalgo1†

, Ana L. Diéguez1, Ilse Cleenwerck

2, Sabela Balboa

1, 8

Alejandra Doce1, Paul de Vos

2 and Jesús L. Romalde

1*. 9

10

1 Departamento de Microbiología y Parasitología. Facultad de Biología. Universidad de 11

Santiago de Compostela. 15782, Santiago de Compostela. Spain. 12

2 BCCM/LMG Bacteria Collection, Laboratory of Microbiology, Ghent University, 13

Ghent, Belgium. 14

15

16

Submitted to: Systematic and Applied Microbiology. June 2010. 17

Revised Ms. SAM 3279 18

19

Running title: Vibrio celticus sp. nov. 20

21

* Corresponding author: 22

Phone: +34-981563100 # 16908 23

Fax: +34-981596904 24

E-mail: jesus.romalde@usc.es 25

26

27

† Present address: Departamento de Ciencias Médicas Básicas. Universidad Rovira i 28

Virgili, Reus, Spain. 29

30

R.B.H. and A.L.D. have contributed equally to this work. 31

32

Manuscript

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ABSTRACT 33

34

A group of four motile facultative anaerobic marine isolates (Rd 8.15T

[= CECT 7224T, 35

= LMG 23850T], Rd 16.13, Rd 6.8 [= LMG 25696] and Rd2L5) were obtained from 36

cultured clams (Ruditapes philippinarum and Venerupis pullastra) in Galicia, 37

Northwestern Spain. They formed a tight phylogenetic group based on sequences of 16S 38

rRNA gene and the four housekeeping genes rpoA (encoding the chain of RNA 39

polymerase), rpoD (encoding the sigma factor of RNA polymerase), recA (encoding 40

RecA protein), and atpA (encoding the -subunit of bacterial ATP synthase). The 41

phylogenies based on these sequences indicated that the four isolates represent a novel 42

species in the genus Vibrio, and more precisely in the Splendidus clade. DNA-DNA 43

hybridizations with the type strains of the species showing more than 98.6% 16S rRNA 44

gene sequence similarity, revealed DNA-DNA relatedness below 70%. The isolates 45

could be differentiated from the phylogenetically related Vibrio species on the basis of 46

several phenotypic features. In addition, strain Rd 8.15T showed potential pathogenic 47

activity for adult clam in the virulence assays. The name Vibrio celticus sp. nov. is 48

proposed for this new taxon, with the type strain being Rd 8.15T (= CECT 7224

T, = 49

LMG 23850T). 50

51

Keywords: Vibrio celticus sp. nov., Splendidus clade, MLSA, phylogeny. 52

53

54

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57

58

59

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INTRODUCTION 67

68

The genus Vibrio, ubiquitous in the aquatic environment, comprises nowadays more 69

than 70 bacterial species. These species have recently been classified into 14 clades 70

based on multilocus sequence analysis (MLSA) and other molecular techniques [27]. 71

Among these clades, the Splendidus clade contains the highest number of species (>10). 72

Moreover, the species of this clade are the dominant Vibrio species in coastal marine 73

sediments, seawater and bivalves in temperate climates [13, 28]. In addition, some of 74

these species have been associated with mortality of a wide range of marine animals 75

such as molluscs [7, 12, 16, 23, 30, 39], fish [11], shrimps [14] and octopus [5]. 76

77

The Splendidus clade is considered to be a not well resolve group. It shows much 78

phenotypical diversity, which makes species discrimination on the basis of biochemical 79

tests difficult, but also much genetic diversity. The latter was revealed by genotypic 80

studies based on ribotyping, amplified fragment length polymorphism (AFLP), PCR-81

restriction fragment length polymorphism [18, 34, 38], and analysis of housekeeping 82

gene sequences such as recA, gyrB or gapA [27]. On the contrary, 16S rRNA gene 83

sequences are extremely conserved within this group, being not useful for species 84

differentiation. In the last 10 years, up to nine new species have been described within 85

the Splendidus clade including V. crassostreae, V. chagasii, V. cyclitrophicus, V. 86

gallaecicus, V. gigantis, V. kanaloae, V. lentus, V. pomeroyi, and V. tasmaniensis [2, 6, 87

17, 35, 36]. A polyphasic approach including MLSA analysis, DNA-DNA hybridization 88

(DDH), chemotaxonomic techniques, and MALDI-TOFF-MS, is currently considered 89

an appropriate strategy to define new species of the genus Vibrio [27, 37]. 90

91

During a previous study on the diversity of vibrios associated with reared clams in 92

Galicia (Spain), a collection of 145 isolates were analysed by AFLP, a technique that 93

has been proven useful for rapid and reliable species identification and classification of 94

vibrios [1, 34] As a result of that analysis, a group of four strains (cluster 9) could not 95

be ascribed to any known species [1]. This study, describes in detail further taxonomic 96

characterization of these four strains obtained from clams, Ruditapes philippinarum and 97

Venerupis pullastra, cultured in Galicia, and proves they belong to a new species of 98

Vibrio. 99

100

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MATERIALS AND METHODS 101

102

Bacterial strains and phenotypic tests. 103

During a sampling program performed in the years 2004 and 2005, a large collection of 104

marine bacterial isolaes were obtained from healthy clams, R. philippinarum (Adams & 105

Reeve, 1850) and V. pullastra (Montagu, 1803), cultured in different geographical sites 106

on the northwestern coast of Spain as previously described [1]. A representative number 107

of isolates from this collection (namely 145) were analyzed by AFLP and, in the present 108

study, a cluster with 4 strains that could not be identified, consisting of Rd 8.15T (= 109

CECT 7224T = LMG 23850

T), Rd 6.8 (= LMG 25696), and Rd 16.13 from R. 110

decussatus and Rd2L5 from V. pullastra [1], was further investigated. Reference strains 111

used in this study were obtained from Bacterial Culture Collections: V. chagasii LMG 112

21353T, V. crassostreae LMG 22240

T, V. cyclitrophicus, LMG 21359

T, V. gallaecicus 113

CECT 7244T, V. gigantis DSM 18531

T (= LMG 22741

T), V. kanaloae LMG 20539

T, V. 114

lentus CECT 5110T (= LMG 21359

T), V. pomeroyi LMG 20537

T, V. splendidus LMG 115

19031T, and V. tasmaniensis LMG 20012

T. All strains were cultured on plates of Marine 116

agar (MA; Pronadisa, Spain) at 24 ± 1ºC under aerobic conditions. Stock cultures were 117

maintained frozen at -80oC in Marine broth (MB; Oxoid Ltd., UK) supplemented with 118

15% glycerol (v/v). 119

120

The four marine strains from cluster 9 and the reference strains were subjected to the 121

following phenotypic tests as described previously [15, 20, 26, 40]: Gram stain, oxidase 122

activity, cell morphology and motility, oxidation/fermentation test, fermentation and 123

acid production from inositol, mannitol and sucrose, gas and acid production from 124

glucose, indole, methyl red, Voges-Proskauer reaction, utilization of citrate, 125

dihydrolation of arginine, decarboxylation of lysine and ornithine, nitrate reduction, 126

hydrolysis of gelatin, Tween 80, amylase, aesculin and alginate, DNase and urease 127

activities, use of 50 compounds as unique carbon source, salt tolerance tests (0, 0.5, 3, 6 128

and 8% of NaCl), growth at different temperatures (4ºC, 37ºC and 44ºC) and growth on 129

thiosulfate-citrate-bile salts sucrose (TCBS) agar (Oxoid). Sensitivity to the vibriostatic 130

agent O/129 (2,4-diamino-6,7-diisopropylpteridine) (150 µg per disc) and ampicillin (10 131

µg per disc) was determined in Müeller-Hinton (Oxoid) agar. Media were supplemented 132

with 1% NaCl when required. Additional phenotypic characteristics were determined 133

with API 20E and API ZYM (bioMerieux, France) using a saline solution (NaCl 0.85%) 134

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5

for the bacterial suspension. The study of the acid production from 50 carbon sources 135

was tested using the API 50CH (bioMerieux) with slight modifications [2]. Briefly, 136

bacterial suspensions were prepared in saline solution, adjusted to an optical density of 137

1.0 at 580 nm and mixed (1:90, v/v) with the ZOF medium [15] for inoculation 138

procedures. 139

140

Sequencing of 16S rRNA and housekeeping genes and phylogenetic analyses. 141

Genomic DNA extraction, PCR amplification and sequencing of the 16S rRNA gene 142

and amplification of the genes recA, rpoA, rpoD atpA and pyrH (encoding uridine 143

monophosphate kinase) were carried out as previously described [24, 32, 33, 37, 41]. 144

For the sequencing reactions, the kit GenomeLab DTCS-Quick Start Kit (Beckman 145

Coulter, Germany) was used. Sequence corrections and analysis were performed with 146

DNAstar Seqman program (Lasergene, USA). Sequences of phylogenetically related 147

species were obtained after BLAST searches against the latest EBI releases. 148

Phylogenetic trees were constructed using three different methods, neighbor joining, 149

maximum parsimony (Mega version 4.0) [31] and maximum likelihood (jModelTest, 150

http://darwin.uvigo.es/, FigTree v1.1.2, http://tree.bio.ed.ac.uk/) [25]. 151

152

DNA-DNA hybridization. 153

Genomic DNA for DNA-DNA hybridization experiments and G+C content 154

determinations was extracted as previously described [3]. DNA-DNA hybridizations 155

were performed at 39ºC in a hybridization solution containing 50% of formamide, 156

according to a modification [9] of the microplate method described by Ezaki et al. [4]. 157

Reciprocal reactions (e.g. A xB and B x A) were performed and were generally within 158

the limits of this method [9]. DNA relatedness values (%) reported are the means of 159

minimum four hybridizations. Standard deviations, based on the reciprocal reactions, 160

are included. Additional reactions, including those between the strain Rd 8.15 T

against 161

the other strains in the cluster, were performed by the hydroxyapatite/microtitre plate 162

method [42] with a hybridization temperature (Tm) of 60ºC. The DNA G+C contents 163

were determined using HPLC as previously described [22]. 164

165

MALDI-TOF-MS 166

The protein analysis by MALDI-TOF-MS was performed in the mass unit of the 167

University of Santiago de Compostela. Protein extraction was performed with ethanol, 168

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6

formic acid and acetonitrile (AN). Processed samples were placed in a 96 well plate, 169

allowed to dry and covered with a matrix solution (-cyano-4-hydroxycinnamic acid; 170

HCCA). Mass spectra were obtained using a MALDI-TOF Autoflex mass spectrometer 171

(Bruker Daltonik GmbH, Germany). The measured mass range of spectra was 2000-172

20,000 Da. Peak comparison was done with the data base of Bruker Daltonik GmbH. 173

The species-limit value considered was 2,300. Identification to genus level was in the 174

range 1,700-1,999. As a positive control, Escherichia coli CECT 433, was included in 175

the analysis and protein profiles were compared with their own profiles. Reproducibility 176

of the results was assessed by repetition of at least ten independent assays. 177

178

Pathogenicity tests. 179

Experimental infections in healthy clams (R. philippinarum and R. decussatus) were 180

performed with strain Rd 8.15T. The adult clams (mean size 30 mm) were kept for 2 181

days in tanks with aerated seawater (T = 19±1ºC; Sal. = 33%o) for acclimatization. 182

Subsequently, three groups of 20 clams of each species were used for the infection 183

experiments. Two groups were bath challenged for 3 h in non-circulating seawater 184

conditions with two different bacterial doses (104 and 10

6 CFU/ml), transferred to 185

empty tanks for 1 h and, finally, to the tanks containing aerated seawater at 19±1ºC. The 186

third group was treated in the same way but adding sterile seawater instead of the Vibrio 187

strain. Clams were monitored for mortality over a 14-day period. Mortalities were 188

attributed to the strains Rd 8.15T only if it could be re-isolated from the tissues of the 189

dead clams. 190

191

RESULTS AND DISCUSSION 192

193

The four clam strains shared the properties of the genus Vibrio. They were motile, 194

facultative anaerobic, Gram-negative and oxidase positive, susceptible to the 195

vibriostatic agent O/129 (150 µg per disc), and capable to reduce nitrates to nitrites. 196

Cells of strains were large regular rods of variable size, 1.3-1.5 µm long and 0.78-0.81 197

µm wide. The four strains showed high phenotypical homogeneity, although variable 198

reactions were observed for the use of D-fructose, D-mannose, D-lactose, L-fucose, 199

gluconic acid, citric acid, trans-aconitic acid, L-leucine, L-arginine, and L-histidine as 200

sole carbon source, and the acid production from L-arabinose, D-melibiose and 201

potassium gluconate (Table S2). The four clam strains showed alkaline phosphatase, 202

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esterase, esterase lipase, leucine arylamidase, trypsin, and acid phosphatase activities in 203

the API ZYM tests. Variable results were obtained for valine arylamidase (negative for 204

strain Rd2L5), and Naphthol-AS-BI- phosphohydrolase (negative for strains Rd2L5 and 205

Rd 8.15T). 206

207

Nearly complete 16S rRNA gene sequences were determined for the four clam strains. 208

Phylogenetic analysis based on these sequences, using the neighbor joining, maximum 209

parsimony (data not shown) and maximum likelihood methods, confirmed their position 210

in the genus Vibrio, and allocated them to the Splendidus clade (Figs. 1 and S1). The 211

four strains showed 99.9-100% 16S rRNA gene sequence similarity to each other, and 212

more than 98.6% similarity [29] with eight species of the Splendidus clade (Table S1). 213

These results support those previously reported by other authors [2, 6, 10, 19, 30, 34] on 214

the extreme conservation of the 16S rRNA gene within the Splendidus clade, and its 215

uselessness for differentiation among these species. 216

217

MLSA has been proposed as a valuable technique for the identification of vibrios, and 218

for studying their phylogeny [2, 4, 17, 37]. Partial sequences of rpoA, rpoD, recA, atpA, 219

and pyrH were determined for the four strains, and sequence similarities above 99.9%, 220

99.6%, 98.7%, 99.9% and 96.4% were obtained for each of these genes, respectively 221

(Table S1). As these values are higher than the limits for species delineation previously 222

described for these genes [37], these results indicated that the four strains probably 223

represent a single novel species in the genus Vibrio, supporting hereby the previous 224

AFLP data. Phylogenetic trees based on the housekeeping genes recA, rpoA, rpoD, and 225

atpA separately (Fig. S2), confirm the close relationship of the four clam strains. In 226

these trees, the strains form a tight group, whereas in the pyrH gene gene sequence 227

based tree the four strains are split over two subgroups (Fig. S2e). The latter tree 228

topology can be explained by recombination events, which have been detected using the 229

RDP3 beta41 software [8, 21]. 230

Phylogenetic trees based on concatenated sequences (4060 nt) of the 16S rRNA gene 231

and the 4 housekeeping genes recA, rpoA, rpoD, and atpA also confirmed the close 232

relationship between the four strains, and support their distinction from the closest 233

phylogenetic neighbours based on 16S rRNA gene sequences (Fig. S3). When pyrH 234

gene sequences were included (concatenated sequences of 4477 nt), the tight 235

relationship among the four clam strains was confirmed but the tree topology was 236

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somewaht distorted (Figs. 2 and S4) because of the detected recombination events in the 237

pyrH gene. 238

239

Levels of DNA relatedness were determined between the four clam strains, and between 240

strain Rd 8.15T and the type strains of the Splendidus clade to which it showed more 241

than 98.5% 16S rRNA sequence similarity [29]. The DNA relatedness values among the 242

four clam strains ranged from 88% to 94.2%, while DNA relatedness values between 243

strain Rd 8.15T and the tested type strains were below 70%, namely, 52±19% with V. 244

crassostreae LMG 22240T, 55±4% with V. cyclitrophicus LMG 21359

T, 50±13% with 245

V. gigantis LMG 22741T, 63±6% with V. kanaloae LMG 20539

T, 50±18% with V. 246

lentus LMG 21359T, 46±8% with V. pomeroyi LMG 20537

T, 53±2% with V. 247

psplendidus LMG 19031T, and 46±8% with V. tasmaniensis LMG 20012

T. These values 248

are in the range of those obtained between other species of the Splendidus clade [2, 6, 249

17, 19, 35, 36], and prove that the four strains represent a single novel species in the 250

genus Vibrio. The DNA G+C content of strain Rd 8.15T is 46.6 mol%. 251

252

Several differentiating phenotypic characteristics were found between the presumptive 253

novel species and closely related Vibrio species (Table 1). Useful phenotypic traits to 254

differentiate the four clam isolates from other species within the Splendidus clade 255

include the positive reaction for the Voges Proskauer test, their ability to produce acid 256

from L-rhamnose and D-lactose and their inability to produce acid from potassium 257

gluconate or to use N-acetyl-D-glucosamine as a sole carbon source. 258

259

MALDI-TOFF-MS for strain Rd 8.15T resulted in a protein profile that was different 260

from the profiles of the type strains of closely related species present in the data base 261

(Fig. S5). The closest species defined by MALDI-TOF-MS were V. gigantis (score = 262

2,140) and V. pomeroyi (score = 2,000). For the other species of the Splendidus clade 263

values ranged from 1,403 and 1,885. A correct identification of E. coli CECT 433 was 264

obtained, with profiles always having a score above 2,300. 265

266

In the virulence assays, strain Rd 8.15T produced cummulative mortalities of 100% of 267

the inoculated adult clams, regardless the dose assayed or the bivalve species (R. 268

philippinarum or R. decussatus). In the control tanks mortalities were always lower than 269

15%. Strain Rd 8.15T were re-isolated from all the dead inoculated animals, but not 270

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from dead control clams. These results suggest the pathogenic potential of strain Rd 271

8.15T for clams. 272

273

In conclusion, this polyphasic study clearly demonstrates that the four isolates represent 274

an undescribed species of the genus Vibrio potentially pathogenic for clam. The name 275

Vibrio celticus sp. nov. is proposed with Rd 8.15T (=CECT 7224

T =LMG 23850

T) as the 276

type strain. 277

278

279

280

Description of Vibrio celticus sp nov. 281

Vibrio celticus [cel’ti.cus. L. masc. adj. celticus, pertaining to the celtics, here pertaining 282

to the pre-romanic inhabitants of Galicia]. 283

284

Cells are Gram-negative rods, motile and facultative anaerobic. Colonies are round with 285

smooth margins, beige in color and non-swarming on MA plates. Strains grow on TCBS 286

agar forming yellow colonies after incubation for 1 day at 24ºC. Glucose metabolism is 287

fermentative without gas production. Strains reduce nitrates to nitrites. Oxidase and 288

catalase tests are positive. Strains are sensitive to the vibriostatic agent O/129 (150µg 289

per disc). All strains grow at 4ºC and require NaCl for growth. The optimal NaCl 290

concentration for growth is 1 to 3% (wt/vol) and strains are able to grow at 6% NaCl but 291

not at higher salinity concentrations. Not luminescent. All strains are positive for 292

arginine dihidrolase, and negative for lysine and ornitine decarboxylases, ONPG, use of 293

citrate and hydrolysis of urea. Indole, Voges-Proskauer, and methyl red reactions are 294

positive. All strains hydrolyse starch, gelatin, aesculin and Tween 80. Acid is produced 295

from D-lactose and L-rhamnose, but not from potassium gluconate (except strain 296

Rd2L5). None of the strains utilized N-acetyl-D-glucosamine as sole carbon source. The 297

strains showed alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, 298

trypsin and acid phosphatase activities. Other properties are given in Tables 1 and S2. 299

300

The type strain Rd 8.15T (=CECT 7224

T, =LMG 23850

T) was isolated from clam 301

Ruditapes philippinarum in northwestern coast of Spain. Its DNA G+C content is 44.6 302

mol%. 303

304

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10

305

The GenBank accession numbers for the 16S rRNA, recA, rpoA, rpoD, atpA and pyrH 306

gene sequences of Vibrio celticus sp. nov. strains, for rpoD gene sequence of V. 307

gallaecius CECT 7244T, and for pyrH gene sequence of V. kanaloae 20539

T are listed 308

in Supplementary Table S3. 309

310

311

ACKNOWLEDGEMENTS 312

This work was supported in part by a grants AGL2003-09307-C02-01 and AGL2006-313

13208-C02-01, from the Ministerio de Ciencia y Tecnología and grant 314

PGIDIT04PXIC20001PN from the Xunta de Galicia (Spain). R.B.H. and S.B. 315

acknowledge the Ministerio de Ciencia y Tecnología (Spain), and A.D. the Xunta de 316

Galicia (Spain) for research fellowships. The authors thank E. Guitián from the RIAIDT 317

(USC) for the mass spectrometry analysis and Bruker Daltonik GmbH for the data-base 318

availability. The resarch was also supported bt the Prime Minister’s Services, Federal 319

Office for Scientific, Technical and Culture Affairs, Belgium. The authors wish to thank 320

Katrien Engelbeen for performing the DNA-DNA hybridizations. 321

322

323

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REFERENCES 324

325

[1] R. Beaz-Hidalgo, I. Cleenwerck, S. Balboa, M. De Wachter, F. Thompson, J. 326

Swings, P. De Vos, J.L. Romalde, Diversity of vibrios associated with reared clams 327

in Galicia (NW of Spain). Syst. Appl. Microbiol. 31 (2008) 215-222. 328

[2] R. Beaz-Hidalgo, A. Doce, J. Pascual, A.E.Toranzo, J.L.Romalde, Vibrio 329

gallaecicus sp. nov. isolated from cultured clams in north-western Spain. Syst. Appl. 330

Microbiol. 32 (2009) 111–117. 331

[3] I. Cleenwerck, K. Vandemeulebroecke, D. Janssens, J. Swings, Re-examination of 332

the genus Acetobacter, with descriptions of Acetobacter cerevisiae sp. nov. and 333

Acetobacter malorum sp. nov. Int. J. Syst. Bacteriol. 52 (2002) 1551-1558. 334

[4] T. Ezaki, Y. Hashimoto, E. Yabuuchi, Fluorometric deoxyribonucleic acid-335

deoxyribonucleic acid hybridization in microdilution wells as an alternative to 336

membrane filter hybridization in which radioisotopes are used to determine genetic 337

relatedness among bacterial strains. Int. J. Syst. Bacteriol. 39 (1989) 224-229. 338

[5] R. Farto, S.P. Armada, M. Montes, J. Guisande, M.J. Pérez, T.P. Nieto, Vibrio lentus 339

associated with diseased wild octopus (Octopus vulgaris). J Invert. Pathol. 83 340

(2003)149-156. 341

[6] N. Faury, D.Saulnier, F.L. Thompson, M. Gay, J. Swings, F. Le Roux, Vibrio 342

crassostreae sp. nov isolated from the haemolymph of oysters (Crassostrea gigas). 343

Int. J. Syst. Evol. Microbiol. 54 (2004) 2137-2140. 344

[7] M. Gay, T. Renault, A. Pons, F. Le Roux, Two Vibrio splendidus related strains 345

collaborate to kill Crassostrea gigas: taxonomy and host alterations. Dis. Aquat. 346

Org. 62 (2004) 65-74. 347

[8] M.J. Gibbs, J.S. Armstrong, A.J. Gibbs, Sister-scanning: a Monte Carlo procedure 348

for assessing signals in recombinant sequences. Bioinformatics 16 (2000) 573-582. 349

[9] J. Goris, K. Suzuki, P. De Vos, T. Nakase, K. Kersters, Evaluation of a microplate 350

method DNA-DNA hybridization method compared with the initial renaturation 351

method. Can. J. Microbiol. 44 (1998) 1148-1153. 352

[10] B.P. Hedlund, J.T. Staley, Vibrio cyclotrophicus sp. nov., a polycyclic aromatic 353

hydrocarbon (PAH) degrading marine bacterium. Int. J. Syst. Evol. Microbiol. 51 354

(2001) 61-66. 355

356

357

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

12

[11] S. Jensen, O.B. Samuelsen, K. Andersen, L. Torkildsen, C. Lambert, G. Choquet, 358

C. Paillard, O. Berg, Characterization of strains of Vibrio splendidus and V. tapetis 359

isolated from corwing wrasse Symphodus melops suffering vibriosis. Dis. Aquat. 360

Org. 53 (2003) 25-31. 361

[12] A. Lacoste, S. Jalabert, S. Malham, A. Cueff, F. Gelebart, C. Cordevant, M. Lange, 362

S. A. Poulet, A Vibrio splendidus strain is associated with summer mortality of 363

juvenile oysters Crassostrea gigas in the Bay of Morlaix (North Brittany, France). 364

Dis. Aquat. Org. 46 (2001) 139-145. 365

[13] C. Lambert, J.L. Nicolas, V. Cilia, S. Corre, Vibrio pectenicida sp. nov., a 366

pathogen of scallop (Pecten maximus) larvae. Int. J. Syst. Bacteriol. 48 (1998) 481-367

487. 368

[14] E.M. Leano, C.R. Lavilla-Pitogo, M.G. Paner, Bacterial flora in the hepatopancreas 369

of pond reared Penaeus monodon juveniles with luminous vibriosis. Aquaculture 370

164 (1998) 367-374. 371

[15] M.L. Lemos, A.E. Toranzo, J.L. Barja, Modified medium for the oxidation-372

fermentation test in the identification of marine bacteria. Appl. Environ. Microbiol. 373

49 (1985) 1541-1543. 374

[16] F. Le Roux, M. Gay, C. Lambert, M. Waechter, S. Poubalanne, B. Chollet, J.L. 375

Nicolas, F. C. J. Berthe, Comparative analysis of Vibrio splendidus- related strains 376

isolated during Crassostreae gigas mortality events. Aquat. Living Resour. 15 (2002) 377

251-258. 378

[17] F. Le Roux, A. Goubet, F.L. Thompson, N. Faury, M. Gay, J. Swings, D. Saulnier, 379

Vibrio gigantis sp. nov. isolated from the haemolymph of cultured oysters 380

(Crassostrea gigas). Int. J. Syst. Evol. Microbiol. 55 (2005) 2251-2255. 381

[18] M.C. Macián, E. Garay, F. Gonzalez- Candelas, M.J. Pujalte, R. Aznar, Ribotyping 382

of vibrio populations associated with cultured oysters (Ostrea edulis). Sys. Appl. 383

Microbiol. 23 (2000) 409-417. 384

[19] M.C. Macián, W. Ludwig, R. Aznar, P.A.D Grimont, K.H. Schleifer, E. Garay, M.J. 385

Pujalte, Vibrio lentus sp. nov., isolated from Mediterranean oysters. Int. J. Syst. 386

Evol. Microbiol. 51 (2001) 1449-1456. 387

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

13

[20] J.F. MacFaddin, Pruebas bioquímicas para la identificación de bacterias de 388

importancia clínica. The William & Wilkins Company. Baltimore. (1993). 389

Translation by Médica Panamericana S.A. 390

[21] D.P. Martin, C. Williamson, D. Posada, RDP2: recombination detection and 391

analysis from sequence alignments. Bioinformatics 21(2005) 260-262. 392

[22] M. Mesbah, U. Premachandran, W.B. Whitman, Precise measurement of the G+C 393

content of deoxyribonucleic acid by high-performance liquid chromatography. Int. J. 394

Syst. Bacteriol. 39 (1989) 159-167. 395

[23] J.L. Nicolas, S. Corre, G. Gauthier, R. Robert, D. Ansquer, Bacterial problems 396

associated with scallop Pecten maximus larval culture. Dis. Aquat. Org. 27 (1996) 397

67-76. 398

[24] C.R. Osorio, M.D. Collins, A.E. Toranzo, J.L. Romalde, 16S rRNA gene sequence 399

analysis of Photobacterium damselae and nested-PCR method for rapid detection of 400

fish pasteurellosis. Appl. Environ. Microbiol. 65 (1999) 2942-2946. 401

[25] D. Posada, jModelTest: Phylogenetic Model Averaging. Mol. Biol. Evol. 25 (2008) 402

1253-1256. 403

[26] J.L. Romalde, A.E. Toranzo, Evaluation of the API 20E system for the routine 404

diagnosis of the enteric redmouth disease. Bull. Eur. Ass. Fish. Pathol. 11 (1991) 405

147-149. 406

[27] T. Sawabe, K. Kita-Tsukamoto, F.L. Thompson, Inferring the evolutionary history 407

of vibrios by means of multilocus sequence analysis. J. Bacteriol. 189 (2007) 7932-408

7936. 409

[28] P.A. Sobecky, T.J. Mincer, M.C. Chang, A. Toukdarian, D.R. Helinski, Isolation of 410

broad- host- range replicons from marine sediment bacteria. Appl. Environ. 411

Microbiol. 64 (1998) 2822-2830. 412

[29] E. Stackebrandt, J. Ebers, Taxonomic parameters revisited: tarnished gold 413

standards. Microbiol. Today. 33 (2006) 152-155. 414

[30] G. Sugumar, T. Nakai, Y. Hirata, D. Matsubara, K. Muroga, Vibrio splendidus 415

biovar II as the causative agent of bacillary necrosis of Japanese oyster Crassostrea 416

gigas larvae. Dis. Aquat. Org. 33 (1998) 111-118. 417

[31] K. Tamura, J. Dudley, M. Nei, S. Kumar, MEGA4: Molecular Evolutionary 418

Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24 (2007) 1596-419

1599. 420

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

14

[32] C.C. Thompson, F.L. Thompson, K. Vandemeulebroecke, B. Hoste, P. Dawyndt, J. 421

Swings Use of recA as an alternative phylogenetic marker in the family 422

Vibrionaceae. Int. J. Syst. Evol. Microbiol. 54 (2004) 919-929. 423

[33] C.C. Thompson, F.L. Thompson, A.C. Vicente. J. Swings, Phylogenetic analysis of 424

vibrios and related species by means of atpA gene sequences. Int. J. Syst. Evol. 425

Microbiol. 57 (2007) 2480-2484. 426

[34] F.L. Thompson, B. Hoste, K. Vandemeulebroecke, J. Swings, Genomic diversity 427

amongst Vibrio isolates from different source determined by fluorescent amplified 428

fragment length polymorphism. System. Appl. Microbiol. 24 (2001) 520-538. 429

[35] F.L. Thompson, C.C. Thompson, J. Swings, Vibrio tasmaniensis sp. nov. isolated 430

from Atlantic salmon (Salmo salar L.). Syst. Appl. Microbiol. 26 (2003a) 65-69. 431

[36] F.L. Thompson, C.C. Thompson, Y. Li, B. Gomez-Gil, J. Vanderberghe, B. Hoste, 432

J. Swings, Vibrio kanaloae sp. nov., Vibrio pomeroyi sp. nov. and Vibrio chagasii 433

sp. nov., from sea water and marine animals. Int. J. Syst. Evol. Microbiol. 53 (2003b) 434

753-759. 435

[37] F.L. Thompson, D. Gevers, C.C. Thompson, P. Dawyndt, S. Naser, B. Hoste, C.B. 436

Munn, J. Swings, Phylogeny and molecular identification of Vibrios on the basis of 437

Multilocus Sequence Analysis. Appl. Environ. Microb. 71 (2005) 5107-5115. 438

[38] H. Urakawa, K. Kita-Tsukamoto, K. Ohwada, 16S rDNA restriction fragment 439

length polymorphism analysis of psychrotophic vibrios from Japanese coastal water. 440

Can. J. Microbiol. 45 (1999) 1001-1007. 441

[39] M. Waechter, F. Le Roux, J.L. Nicolas, E. Marissal, F. Berthe, Characterization of 442

pathogenic bacteria of the cupped oyster Crassostrea gigas. C. R. Biol. 325 (2002) 443

231-238. 444

[40] P.A. West, P. R. Brayton, T.N. Bryant, R.R. Colwell, Numerical taxonomy of 445

vibrios isolated from aquatic environments. Int. J. Syst. Bacteriol. 36 (1986) 531-446

543. 447

[41] S. Yamamoto, H. Kasai, D.L. Arnold, R.W. Jackson, A. Vivian, S. Harayama. 448

Phylogeny of the genus Pseudomonas: intrageneric structure reconstructed from the 449

nucleotide sequences of gyrB and rpoD genes. Microbiology 146 (2000) 2385-2394. 450

[42] F. Ziemke, M.G. Hofle, J. Lalucat, R. Roselló-Mora, Reclassification of 451

Shewanella putrefaciens Owen´s genomic group II as Shewanella baltica sp. nov. 452

Int. J. Syst. Bacteriol. 48 (1998) 179-186. 453

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

15

Table 1. Phenotypic characteristics for distinguishing V. celticus from phenotypically and phylogenetically related Vibrio species.

Characteristic 1 2 3 4 5 6 7 8 9 10 11

ADH + + – + + – – + + – –

ONPG* – – + – + – + – – + –

Voges Proskauer + – – – – + + – – – –

Acid from:

L-rhamnose*

D-lactose*

Potassium gluconate*

+

+

V– (1)

–

–

+

+

–

+

–

–

+

–

+

+

–

–

+

–

–

–

–

–

+

–

ND

ND

–

+

+

–

–

+

Susceptibility to:

O/129 (150µg)

+

+

+

+

+

+

–

V

+

+

+

Growth at:

35ºC

6% NaCl

8% NaCl

–

+

–

–

–

–

V

–

V

–

+

–

–

+

+

+

V

–

–

+

–

–

+

V

–

+

V

+

+

+

–

–

–

Hydrolysis of:

Gelatin

Tween 80*

+

+

+

+

+

+

+

+

+

–

–

–

+

+

+

+

+

+

+

+

+

+

Use as sole carbon source of:

N-acetyl-D-glucosamine

–

+

ND

+

+

+

ND

+

ND

+

ND

+, positive; –, negative; V, variable; V–, variable but the type strain is negative. The number in parenthesis indicates the number of strains giving a positive

result. Taxa are indicated as: 1, V. celticus (4 strains); 2. V. gigantis DSM 18531T; 3, V. splendidus, CECT 528

T; 4, V. crassostreae, LMG 22240

T; 5, V,

pomeroyi, LMG 20537T; 6, V. tasmaniensis, LMG 20012

T; 7, V. lentus CECT 5110

T; 8, V. kanaloae LMG 20539

T; 9, V. chagasii LMG 21353

T; 10, V.

cyclitrophicus, LMG 21359T; 11, V. gallaecicus LMG 7244

T. Data for the reference strains are taken from the literature [2, 4, 10, 17, 19, 35, 36], except when

indicated by *. Those data derive from tests performed in the frame of this study on the type strains. All taxa are negative for lysine and ornithine

decarboxylase, acid from inositol and D–sorbitol. All taxa are positive for indole and hydrolysis of starch.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

16

Figure legends

Fig. 1. Phylogenetic tree based on partial 16S rRNA gene sequences, and obtained by

the Neighbor joining method. Vibrio cholerae ATCC 14035T

was used as out–group.

GeneBank sequence accession numbers are given in parentheses. Numbers at the nodes

show the percentages bootstrap values.

Fig. 2. Phylogenetic tree based on concatenated sequences of the housekeeping genes

recA, rpoA, rpoD, atpA, and pyrH, and the 16S rRNA gene, and obtained by the

Neighbor joining method. Numbers at the nodes show the percentages bootstrap values.

GeneBank sequence accession numbers for individual gene sequences are given in

Table S3 and Fig. S2.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

17

Fig. 1.- Beaz-Hidalgo et al.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

18

Fig. 2.- Beaz-Hidalgo et al.

Vibrio celticus sp. nov., a new Vibrio species belonging to the

Splendidus clade with pathogenic potential for clams.

Roxana Beaz-Hidalgo1†

, Ana L. Diéguez1, Ilse Cleenwerck

2, Sabela Balboa

1,

Alejandra Doce1, Paul de Vos

2 and Jesús L. Romalde

1*.

Supplementary Material

Supplementary Material

Table S1. Sequence similarity values between 16S rRNA, recA, rpoA, rpoD, atpA and

pyrH gene sequences of V. celticus type strain Rd 8.15T (=CECT 7224

T =LMG 23850

T)

and related Vibrio species.

Type strains 16S recA rpoA rpoD atpA pyrH

Vibrio celticus

Rd 6.8

Rd 16.13

Rd2L5

V. splendidus LMG 19031T

V. gigantis DSM 18531T

V. pomeroyi LMG 20537T

V. crassostreae LMG 22240T

V. lentus CECT 5110T

V. tasmaniensis LMG 20012T

V. cyclitrophicus LMG 21359T

V. kanaloae LMG 20539T

99.9

100

99.9

99.2

99.9

99.6

99.7

98.6

99.3

98.7

99.0

99.2

98.7

100

93.8

95.7

96.0

97.1

93.7

96.0

93.2

98.7

99.9

100

100

99.3

100

99.6

99.6

98.3

98.6

99.3

98.2

100

99.6

100

95.2

97.9

95.0

98.0

92.2

95.0

92.8

95.2

99.9

100

100

97.4

99.1

97.6

99.3

97.3

98.9

99.1

99.5

96.4

99.8

96.4

95.9

98.1

95.7

96.4

96.6

94.0

94.5

94.7

V. chagasii LMG 21353T 98.4 90.7 99.0 94.8 94.4 94.3

V. gallaecicus CECT 7244T 97.3 87.1 97.5 89.4 98.9 91.8

Table S2.- Variable phenotypic characteristics of Vibrio celticus sp. nov.a

a Acid is produced from: D-glucose, D-fructose, D-galactose, D-mannose, D-ribose, D-mannitol, glycerol,

N-acetyl-glucosamine, D-cellobiose, D-maltose, D-trehalose, glycogen, starch, D-sucrose, D-lactose, L-

rhamnose, and D-xylose. None of the strains produce acid from erythritol, D-arabinose, L-xylose, D-

adonitol, methyl-D-xylopranoside, L-sorbose, dulcitol, inositol, D-sorbitol, methyl-D-

mannopyranoside, methyl-D-glucopyranoside, amygdalin, arbutin, aesculin, salicin, inulin, D-

melezitose, D-raffinose, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-

arabitol, L-arabitol, potassium 2-ketogluconate and potassium 5-ketogluconate. All strains utilized as sole

carbon source D-glucose, D-trehalose, D-maltose, D-cellobiose, D-sucrose, D-mannitol, glycerol, sodium

acetate, propionic acid, succinic acid, glycine, L-serine, L-threonine, L-glutamic acid, D-alanine, L-

aspartic acid, -ketoglutaric acid, fumaric acid, and N-acetyl-D-glucosamine, but not D-ribose, L-

arabinose, D-xylose, D-galactose, L-rhamnose, D-melibiose, D-salicin, D-amygdaline, D-glucuronic acid,

D-galacturonic acid, D-sorbitol, myo-inositol, butiric acid, L-tirosine, L-lysine, L-ornitine, L-citruline,

amino-N-butiric acid, and N-acetyl-D-glucosamine.

TEST Strain

Rd 8.15T Rd 6.8 Rd 16.13 Rd2L5 Production of acid from:

L- arabinose – – – + D-melibiose + – – – Potassium gluconate – – – +

Use as sole carbon source: + + + + D-fructose + + – + D-mannose + + – + D-lactose – – – + L-fucose – – – + Gluconic acid – – – + Citric acid + + – + Trans-aconitic acid + – – – L-leucine – – – + L-arginine + – + + L-histidine – – – +

Table S3. GenBank accession numbers of the 16S rRNA, recA, rpoA, rpoD, atpA, and

pyrH gene sequences of the four strains of Vibrio celticus sp. nov. investigated in this

study.

Strain 16rRNA recA rpoA rpoD atpA pyrH

RD 8.15T EF599162 EU541590 EU541570 FN582243 FN582232 FN582244

Rd6.8 FN582228 FN582236 FN582237 FN582242 FN582231 FN582245

Rd16.13 FN582227 FN582234 FN582239 FN582240 FN582230 FN582247

Rd2L5 FN582229 FN582235 FN582238 FN582241 FN582233 FN582246

V. gallaecicus

CECT 7244T

FN908850

V. kanaloae

LMG 20539T

FN908851

Fig. S1. Phylogenetic tree based on partial 16S rRNA gene sequences, and obtained by

the maximum likelihood method (GTR+I+G model). The Vibrio choleare ATCC

14035T

was used as out-group. GeneBank sequence accession numbers are given in

parenthesis. Numbers above the branches show the phylogenetic distance.

Fig. S2. Phylogenetic trees based on partial housekeeping gene sequences, and obtained

by the Neighbor joining method. a) recA, b) rpoA, c) rpoD, d) atpA, and e) pyrH genes.

The Vibrio cholerae LMG 21698T was used as out-groups. GeneBank sequence

accession numbers are given in parentheses. Numbers at the nodes show the percentages

bootstrap values.

A

B

C

D

E

Fig. S3. Phylogenetic tree based on concatenated sequences of the housekeeping genes,

recA, rpoA, rpoD, and atpA and the 16S rRNA gene, and obtained by Neighbor Joining

(A) the maximum likelihood (GTR+I+G model)(B) methods. GeneBank accession

numbers of individual gene sequences are given in Table S3 and Fig. S2. Numbers in

nodes in A indicate bootstrap values and above branches in B show the phylogenetic

distance.

A

B

Fig. S4. Phylogenetic tree based on concatenated sequences of the housekeeping genes,

recA, rpoA, rpoD, atpA, and pyrH and the 16S rRNA gene, and obtained by the

maximum likelihood method (GTR+I+G model). GeneBank accession numbers of

individual gene sequences are given in Table S3 and Fig. S2. Numbers above branches

show the phylogenetic distance.

Fig S5. Protein profile of type strain of V. celticus sp. nov., Rd 8.15T obtained by the

technique MALDI-TOF-MS.

Dr. Ramón Roselló-Mora

Executive editor

Systematic and Applied Microbiology

Dear Dr. Roselló-Mora:

Querido Ramón:

Thank you very much for the useful comments on the manuscript SAM 3279 entitled

“Vibrio celticus sp. nov., a new Vibrio species belonging to mthe Splendidus clade qith

pathogenic potential for clams", by Beaz-Hidalgo et al. We are submitting a revised

version, which was modified taking into account the reviewers’ suggestions and your

comments.

Answers to editor:

1. The meaning of the adjective “celticus –a –um” was changed according to your

suggestions to .”cel’ti.cus. L. masc. adj. celticus, pertaining to the celtics, here

pertaining to the pre-romanic inhabitants of Galicia”

Answers to reviewer # 1:

1. Vibrio kanaloae, V. chagasii and V. gallaecicus were included in MLSA

analysis and the correspondent independent and concatenated trees. New

sequences were obtained for some genes and deposited in the GenBank, and the

accession numbers included in Table S3. To avoid a hugh table, the inclusion of

all the accession numbers was not perfoemed. Instead size source in tress was

increased to make easy their reading.

2. The number of Vibrio species was modified (p. 3, line 71) and V. cyclitrophicus

and V. lentus included in the paragraph (p. 3, lines 86-88).

3. The list of genes was corrected (p. 7, line 218).

4. Line 258 (p. 8), the sentence was modified to clarify its meaning.

*Detailed Response to Reviewers

5. At the end of the description, reference was made to the tables to complete the

characteristics of the new species.

6. Table 1 footnote was corrected.

7. The reviewer was right indicating the mistakes in the similarities included in

Table S1. All similarity values were checked out and corrected if needed. The

corrected values match well with the tree topologies.

Answers to reviewer # 2:

1. Regarding t the pathogenicity tests, this approach has been used widely in the

literature. The doses utilized are the usual for this type of experiment. Although

not included in this work, the pathogenicity tests were performed for a high

number of vibrios in paralell using the same doses, and for ost of them no

mortality was detected, excluding therefore the septic shock mentioned by the

reviewer. The transfer of the clams to a empty container for 1 h is to avoid the

dilution factor that would happen if the clams were put straight into the water.

Since the clams are intertidal organisms this period has no effects on them.

The reviewer is right in the fact that disease signs are not clear in molluscs, but

in most works the re-isolation of the inoculated bacterium is enough to prove the

pathogenic potential, if the bacterium is not detected in the controls. It is also

true that V. celticus was isolated from periodical samplings and theoretically

healthy clams, but as many other vibrios it can be an opportunistic pathogen

from the normal microbiota of the clams, which what we want to pointed out in

the manuscript.

2. Line 75, we agree with the reviewer and the sentence was changed

3. Lines 79, 93, 96 and 97, all the suggestions were accepted and the text modified

acordingly.

4. The taxonomic authorities for clam species were included in the text.

5. The other typographical and style corrections suggested by the reviewer were

included in the text.

We hope that you find this revision suitable for publication.

Sincerely,

Dr. Jesus L. Romalde