Vibrio celticus sp. nov., a new Vibrio species belonging to the Splendidus clade with pathogenic potential for clams

Systematic and Applied Microbiology 33 (2010) 311–315

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Vibrio celticus sp. nov., a new Vibrio species belonging to the Splendidus clade with pathogenic potential for clams Roxana Beaz-Hidalgo a,1,2 , Ana L. Diéguez a,2 , Ilse Cleenwerck b , Sabela Balboa a , Alejandra Doce a , Paul de Vos b , Jesús L. Romalde a,∗ a b

Departamento de Microbiología y Parasitología, Facultad de Biología, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain BCCM/LMG Bacteria Collection, Laboratory of Microbiology, Ghent University, Ghent, Belgium

a r t i c l e

i n f o

Article history: Received 23 February 2010 Keywords: Vibrio celticus sp. nov. Splendidus clade MLSA Phylogeny

a b s t r a c t 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, north-western Spain. They formed a tight phylogenetic group based on sequences of the 16S rRNA gene and the four housekeeping genes rpoA (encoding the ␣-chain of RNA polymerase), rpoD (encoding the sigma factor of RNA polymerase), recA (encoding RecA protein), and atpA (encoding the ␣-subunit of bacterial ATP synthase). The phylogenies based on these sequences indicated that the four isolates represented a novel species in the genus Vibrio, and more precisely in the Splendidus clade. DNA–DNA hybridizations with the type strains of species showing more than 98.6% 16S rRNA gene sequence similarity, revealed a 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 clams in 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 ). © 2010 Elsevier GmbH. All rights reserved.

Introduction The genus Vibrio, ubiquitous in the aquatic environment, nowadays comprises more than 70 bacterial species. These species have recently been classified into 14 clades based on multilocus sequence analysis (MLSA) and other molecular techniques [27]. Among these clades, the Splendidus clade contains the highest number of species (>10). Moreover, the species of this clade are the dominant Vibrio species in coastal marine sediments, seawater and bivalves in temperate climates [13,28]. In addition, some of these species have been associated with mortality of a wide range of marine animals, such as molluscs [7,12,16,23,30,39], fish [11], shrimps [14] and octopus [5]. The Splendidus clade is not considered to be a well-resolved group. It shows not only much phenotypical diversity, which makes species discrimination on the basis of biochemical tests diffi-

∗ Corresponding author. Tel.: +34 981563100x16908; fax: +34 981596904. E-mail address: [email protected] (J.L. Romalde). 1 Present address: Departamento de Ciencias Médicas Básicas, Universidad Rovira i Virgili, Reus, Spain. 2 These authors have contributed equally to this work. 0723-2020/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2010.06.007

cult, but also much genetic diversity. The latter was revealed by genotypic studies based on ribotyping, amplified fragment length polymorphism (AFLP), PCR-restriction fragment length polymorphism [18,33,38], and analysis of housekeeping gene sequences, such as recA, gyrB or gapA [27]. On the contrary, 16S rRNA gene sequences are extremely conserved within this group, and are therefore not useful for species differentiation. In the last 10 years, up to nine new species have been described within the Splendidus clade, including V. crassostreae, V. chagasii, V. cyclitrophicus, V. gallaecicus, V. gigantis, V. kanaloae, V. lentus, V. pomeroyi, and V. tasmaniensis [2,6,17,35,34]. A polyphasic approach, including MLSA analysis, DNA–DNA hybridization (DDH), chemotaxonomic techniques, and MALDI-TOF-MS, is currently considered an appropriate strategy for defining new species of the genus Vibrio [27,32]. During a previous study on the diversity of vibrios associated with reared clams in Galicia (Spain), a collection of 145 isolates were analyzed by AFLP, a technique that has proven useful for rapid and reliable species identification and classification of vibrios [1,33]. As a result of this analysis, a group of four strains (cluster 9) could not be ascribed to any known species [1]. Therefore, this study describes in detail further taxonomic characterization of these four strains obtained from clams, Ruditapes philippinarum and Venerupis

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pullastra, cultured in Galicia, and it has shown that they belong to a new species of Vibrio. Materials and methods Bacterial strains and phenotypic tests During a sampling program performed in 2004 and 2005, a large collection of marine bacterial isolates were obtained from healthy clams, R. philippinarum (Adams and Reeve, 1850) and V. pullastra (Montagu, 1803), cultured in different geographical sites on the north-western coast of Spain, as previously described [1]. A representative number of isolates from this collection (namely 145) was analyzed by AFLP and, in the present study, a cluster containing four strains that could not be identified, consisting of Rd 8.15T (=CECT 7224T , =LMG 23850T ), Rd 6.8 (=LMG 25696), and Rd 16.13 from R. decussatus and Rd2L5 from V. pullastra [1], was investigated further. Reference strains used in this study were obtained from bacterial culture collections: V. chagasii LMG 21353T , V. crassostreae LMG 22240T , V. cyclitrophicus, LMG 21359T , V. gallaecicus CECT 7244T , V. gigantis DSM 18531T (=LMG 22741T ), V. kanaloae LMG 20539T , V. lentus CECT 5110T (=LMG 21359T ), V. pomeroyi LMG 20537T , V. splendidus LMG 19031T , and V. tasmaniensis LMG 20012T . All strains were cultured on plates of Marine agar (MA; Pronadisa, Spain) at 24 ± 1 ◦ C under aerobic conditions. Stock cultures were maintained frozen at −80 ◦ C in Marine broth (MB; Oxoid Ltd., UK) supplemented with 15% glycerol (v/v). The four marine strains from cluster 9 and the reference strains were subjected to the following phenotypic tests, as described previously [15,20,26,40]: Gram stain, oxidase activity, cell morphology and motility, oxidation/fermentation test, fermentation and acid production from inositol, mannitol and sucrose, gas and acid production from glucose, indole, methyl red, Voges-Proskauer reaction, utilization of citrate, dihydrolation of arginine, decarboxylation of lysine and ornithine, nitrate reduction, hydrolysis of gelatine, Tween 80, amylase, aesculin and alginate, DNase and ureas activities, use of 50 compounds as unique carbon sources, salt tolerance tests (0%, 0.5%, 3%, 6% and 8% NaCl), growth at different temperatures (4 ◦ C, 37 ◦ C and 44 ◦ C), and growth on thiosulfatecitrate-bile salts sucrose (TCBS) agar (Oxoid). Sensitivity to the vibriostatic agent O/129 (2,4-diamino-6,7-diisopropylpteridine) (150 ␮g per disc) and ampicillin (10 ␮g per disc) was determined on Müeller-Hinton agar (Oxoid). Media were supplemented with 1% NaCl when required. Additional phenotypic characteristics were determined with API 20E and API ZYM (bioMérieux, France) using a saline solution (NaCl 0.85%) for the bacterial suspension. The study of the acid production from 50 carbon sources was tested using the API 50CH (bioMérieux) with slight modifications [2]. Briefly, bacterial suspensions were prepared in saline solution, adjusted to an optical density of 1.0 at 580 nm and mixed (1:90; v/v) with the ZOF medium [15] for inoculation procedures. Sequencing of 16S rRNA and housekeeping genes, and phylogenetic analyses

neighbor joining, maximum parsimony (Mega version 4.0) [31] and maximum likelihood (jModelTest, http://darwin.uvigo.es/, FigTree v1.1.2, http://tree.bio.ed.ac.uk/) [25]. DNA–DNA hybridization Genomic DNA for DNA–DNA hybridization experiments and G+C content determinations was extracted as previously described [3]. DNA–DNA hybridizations were performed at 39 ◦ C in a hybridization solution containing 50% formamide, according to a modification [9] of the microplate method described by Ezaki et al. [4]. Reciprocal reactions (e.g. A × B and B × A) were performed and were generally within the limits of this method [9]. DNA relatedness values (%) reported were the means of a minimum of four hybridizations. Standard deviations, based on the reciprocal reactions, are also included. Additional reactions, including those between strain Rd 8.15T against the other strains in the cluster, were performed by the hydroxyapatite/microtitre plate method [42] with a hybridization temperature (Tm) of 60 ◦ C. The DNA G+C contents were determined using HPLC, as previously described [22]. MALDI-TOF-MS The protein analysis by MALDI-TOF-MS was performed in the mass unit of the University of Santiago de Compostela. Protein extraction was performed with ethanol, formic acid and acetonitrile (AN). Processed samples were placed in a 96-well plate, allowed to dry and covered with a matrix solution (␣-cyano4-hydroxycinnamic acid; HCCA). Mass spectra were obtained using a MALDI-TOF Autoflex mass spectrometer (Bruker Daltonik GmbH, Germany). The measured mass range of spectra was 2000–20,000 Da. Peak comparison was carried out using the database of Bruker Daltonik GmbH. The species-limit value considered was 2300. Identification to genus level was in the range 1700–1999. As a positive control, Escherichia coli CECT 433 was included in the analysis and protein profiles were compared with their own profiles. Reproducibility of the results was assessed by repetition of at least ten independent assays. Pathogenicity tests Experimental infections in healthy clams (R. philippinarum and R. decussatus) were performed with strain Rd 8.15T . The adult clams (mean size 30 mm) were kept for 2 days in tanks with aerated seawater (T = 19 ± 1 ◦ C; Sal. = 33‰) for acclimatization. Subsequently, three groups of 20 clams of each species were used for the infection experiments. Two groups were bath challenged for 3 h in noncirculating seawater conditions with two different bacterial doses (104 and 106 CFU ml−1 ), transferred to empty tanks for 1 h and, finally, to tanks containing aerated seawater at 19 ± 1 ◦ C. The third group was treated in the same way but adding sterile seawater instead of the Vibrio strain. Clams were monitored for mortality over a 14-day period. Mortalities were attributed to strain Rd 8.15T only if it could be re-isolated from the tissues of the dead clams. Results and discussion

Genomic DNA extraction, PCR amplification and sequencing of the 16S rRNA gene, and amplification of the genes recA, rpoA, rpoD, atpA and pyrH (encoding uridine monophosphate kinase) were carried out as previously described [24,32,36,37,41]. For the sequencing reactions, the GenomeLab DTCS-Quick Start kit (Beckman Coulter, Germany) was used. Sequence corrections and analysis were performed with the DNAstar Seqman program (Lasergene, USA). Sequences of phylogenetically related species were obtained after BLAST searches against the latest EBI releases. Phylogenetic trees were constructed using three different methods:

The four clam strains shared the properties of the genus Vibrio. They were motile, facultative anaerobic, Gram-negative and oxidase positive, susceptible to the vibriostatic agent O/129 (150 ␮g per disc), and capable of reducing nitrates to nitrites. Cells of strains were large regular rods of variable size, 1.3–1.5 ␮m long and 0.78–0.81 ␮m wide. The four strains showed high phenotypical homogeneity, although variable reactions were observed for the use of d-fructose, d-mannose, d-lactose, l-fucose, gluconic acid, citric acid, trans-aconitic acid, l-leucine, l-arginine, and l-histidine

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Fig. 1. Phylogenetic tree based on partial 16S rRNA gene sequences obtained by the neighbor joining method. Vibrio cholerae ATCC 14035T was used as an out-group. GenBank sequence accession numbers are given in parentheses. Numbers at the nodes show the percentage bootstrap values. The bar represents substitutions per nucleotide position.

as sole carbon sources, and acid production from l-arabinose, dmelibiose and potassium gluconate (Table S2). The four clam strains showed alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, trypsin, and acid phosphatase activities in the API ZYM tests. Variable results were obtained for valine arylamidase (negative for strain Rd2L5), and naphthol-AS-BI-phosphohydrolase (negative for strains Rd2L5 and Rd 8.15T ). Nearly complete 16S rRNA gene sequences were determined for the four clam strains. Phylogenetic analysis based on these sequences, using the neighbor joining, maximum parsimony (data not shown) and maximum likelihood methods, confirmed their position in the genus Vibrio, and allocated them to the Splendidus clade (Figs. 1 and S1). The four strains showed 99.9–100% 16S rRNA gene sequence similarity to each other, and more than 98.6% similarity [29] with eight species of the Splendidus clade (Table S1). These results supported those previously reported by other authors [2,6,10,19,30,33] on the extreme conservation of the 16S rRNA gene within the Splendidus clade, and its uselessness for differentiation among these species. MLSA has been proposed as a valuable technique for the identification of vibrios, and for studying their phylogeny [2,4,17,32].

Partial sequences of rpoA, rpoD, recA, atpA, and pyrH were determined for the four strains, and sequence similarities above 99.9%, 99.6%, 98.7%, 99.9% and 96.4% were obtained for each of these genes, respectively (Table S1). As these values are higher than the limits for species delineation previously described for these genes [32], these results indicated that the four strains probably represented a single novel species in the genus Vibrio, thus supporting the previous AFLP data. Phylogenetic trees based on the housekeeping genes recA, rpoA, rpoD, and atpA separately (Fig. S2) confirmed the close relationship of the four clam strains. In these trees, the strains formed a tight group, whereas in the pyrH gene gene sequence-based tree the four strains were split over two subgroups (Fig. S2e). The latter tree topology can be explained by recombination events, which have been detected using the RDP3 beta41 software [8,21]. Phylogenetic trees based on concatenated sequences (4060 nt) of the 16S rRNA gene and the four housekeeping genes recA, rpoA, rpoD, and atpA also confirmed the close relationship between the four strains, and supported their distinction from the closest phylogenetic neighbors based on 16S rRNA gene sequences (Fig. S3). When pyrH gene sequences were included (concatenated sequences of 4477 nt), the tight relationship among the four clam

Fig. 2. Phylogenetic tree based on concatenated sequences of the housekeeping genes recA, rpoA, rpoD, atpA, and pyrH, and the 16S rRNA gene obtained by the neighbor joining method. Numbers at the nodes show the percentage bootstrap values. The bar represents substitutions per nucleotide position. GenBank sequence accession numbers for individual gene sequences are given in Table S3 and Fig. S2.

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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* Susceptibility to O/129 (150 ␮g) Growth at 35 ◦ C 6% NaCl 8% NaCl Hydrolysis of Gelatine Tween 80* Use as sole carbon source of N-acetyl-d-glucosamine

+ − +

+ − −

− + −

+ − −

+ + −

− − +

− + +

+ − −

+ − −

− + −

− − −

+ + V−(1)

− − +

+ − +

− − +

− + +

− − +

− − −

− − +

− ND ND

− + +

− − +

+

+

+

+

+

+

−

V

+

+

+

− + −

− − −

V − V

− + −

− + +

+ V −

− + −

− + V

− + V

+ + +

− − −

+ +

+ +

+ +

+ +

+ −

− −

+ +

+ +

+ +

+ +

+ +

−

+

ND

+

+

+

ND

+

ND

+

ND

+, Positive; −, negative; V, variable; V−, variable but the type strain is negative; ND, not determined. The number in parenthesis indicates the number of strains giving a positive result. Taxa are indicated as: 1, V. celticus (four strains); 2, V. gigantis DSM 18531T ; 3, V. splendidus, CECT 528T ; 4, V. crassostreae, LMG 22240T ; 5, V. pomeroyi, LMG 20537T ; 6, V. tasmaniensis, LMG 20012T ; 7, V. lentus CECT 5110T ; 8, V. kanaloae LMG 20539T ; 9, V. chagasii LMG 21353T ; 10, V. cyclitrophicus, LMG 21359T ; 11, V. gallaecicus LMG 7244T . Data for the reference strains are taken from the literature [2,4,10,17,19,35,36], except when indicated by * to show data derived from tests performed within the framework 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.

strains was confirmed but the tree topology was somewhat distorted (Figs. 2 and S4) because of the detected recombination events in the pyrH gene. Levels of DNA relatedness were determined between the four clam strains, and between strain Rd 8.15T and the type strains of the Splendidus clade to which it showed more than 98.5% 16S rRNA sequence similarity [29]. The DNA relatedness values among the four clam strains ranged from 88% to 94.2%, while DNA relatedness values between strain Rd 8.15T and the tested type strains were below 70%, namely, 52 ± 19% with V. crassostreae LMG 22240T , 55 ± 4% with V. cyclitrophicus LMG 21359T , 50 ± 13% with V. gigantis LMG 22741T , 63 ± 6% with V. kanaloae LMG 20539T , 50 ± 18% with V. lentus LMG 21359T , 46 ± 8% with V. pomeroyi LMG 20537T , 53 ± 2% with V. splendidus LMG 19031T , and 46 ± 8% with V. tasmaniensis LMG 20012T . These values are in the range of those obtained between other species of the Splendidus clade [2,6,17,19,35,34], and proved that the four strains represented a single novel species in the genus Vibrio. The DNA G+C content of strain Rd 8.15T was 46.6 mol%. Several differentiating phenotypic characteristics were found between the presumptive novel species and closely related Vibrio species (Table 1). Useful phenotypic traits to differentiate the four clam isolates from other species within the Splendidus clade included the positive reaction for the Voges-Proskauer test, their ability to produce acid from l-rhamnose and d-lactose, and their inability to produce acid from potassium gluconate or to use Nacetyl-d-glucosamine as a sole carbon source. MALDI-TOF-MS for strain Rd 8.15T resulted in a protein profile that was different from the profiles of the type strains of closely related species present in the database (Fig. S5). The closest species defined by MALDI-TOF-MS were V. gigantis (score = 2140) and V. pomeroyi (score = 2000). For the other species of the Splendidus clade, values ranged from 1403 to 1885. A correct identification of E. coli CECT 433 was obtained, with profiles always having a score above 2300. In the virulence assays, strain Rd 8.15T produced cumulative mortalities of 100% in the inoculated adult clams, regardless of the dose assayed or the bivalve species (R. philippinarum or R. decussatus). In the control tanks, mortalities were always lower than 15%. Strain Rd 8.15T was re-isolated from all the dead inoculated animals, but not from dead control clams. These

results suggested the pathogenic potential of strain Rd 8.15T for clams. In conclusion, this polyphasic study clearly demonstrated that the four isolates represented an undescribed species of the genus Vibrio potentially pathogenic for clam. The name Vibrio celticus sp. nov. is proposed with Rd 8.15T (= CECT 7224T , = LMG 23850T ) as the type strain.

Description of Vibrio celticus sp. nov. Vibrio celticus [cel’ti.cus. L. masc. adj. celticus, pertaining to the celtics, here pertaining to the pre-romanic inhabitants of Galicia]. Cells are Gram-negative rods, motile and facultative anaerobic. Colonies are round with smooth margins, beige in color and nonswarming on MA plates. Strains grow on TCBS agar and form yellow colonies after incubation for 1 day at 24 ◦ C. Glucose metabolism is fermentative without gas production. Strains reduce nitrates to nitrites. Oxidase and catalase tests are positive. Strains are sensitive to the vibriostatic agent O/129 (150 ␮g per disc). All strains grow at 4 ◦ C and require NaCl for growth. The optimal NaCl concentration for growth is 1–3% (w/v) and strains are able to grow at 6% NaCl but not at higher concentrations. Non-luminescent. All strains are positive for arginine dihydrolase, and negative for lysine and ornithine decarboxylases, ONPG, use of citrate and hydrolysis of urea. Indole, Voges-Proskauer, and methyl red reactions are positive. All strains hydrolyze starch, gelatine, aesculin and Tween 80. Acid is produced from d-lactose and l-rhamnose, but not from potassium gluconate (except strain Rd2L5). None of the strains utilized N-acetyl-d-glucosamine as a sole carbon source. The strains showed alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, trypsin and acid phosphatase activities. Other properties are given in Tables 1 and S2. The type strain Rd 8.15T (= CECT 7224T , = LMG 23850T ) was isolated from the clam R. philippinarum on the north-western coast of Spain. The DNA G+C content is 44.6 mol%. The GenBank accession numbers for the 16S rRNA, recA, rpoA, rpoD, atpA and pyrH gene sequences of Vibrio celticus sp. nov. strains, for the rpoD gene sequence of V. gallaecius CECT 7244T , and for the pyrH gene sequence of V. kanaloae 20539T are listed in Supplementary Table S3.

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Acknowledgements This work was supported in part by grants AGL2003-09307C02-01 and AGL2006-13208-C02-01 from the Ministerio de Ciencia y Tecnología and grant PGIDIT04PXIC20001PN from the Xunta de Galicia (Spain). R.B.H. and S.B. acknowledge the Ministerio de Ciencia y Tecnología (Spain), and A.D. the Xunta de Galicia (Spain) for research fellowships. The authors thank E. Guitián from the RIAIDT (USC) for the mass spectrometry analysis and Bruker Daltonik GmbH for the database availability. The research was also supported by the Prime Minister’s Services, Federal Office for Scientific, Technical and Culture Affairs, Belgium. The authors wish to thank Katrien Engelbeen for performing the DNA–DNA hybridizations. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.syapm.2010.06.007.

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