Antonie van Leeuwenhoek (2014) 105:335–341 DOI 10.1007/s10482-013-0078-z
ORIGINAL PAPER
Vibrio cortegadensis sp. nov., isolated from clams Aide Lasa • Ana L. Die´guez • Jesu´s L. Romalde
Received: 23 June 2013 / Accepted: 15 November 2013 / Published online: 23 November 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract A group of four strains isolated from clams (Venerupis decussata and Venerupis philippinarum) in Galicia (NW Spain) were subjected to a polyphasic characterization, based on the phenotypic characteristics, the analysis of chemotaxonomic features, 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 isolates constitute a new species. The name Vibrio cortegadensis sp. nov. is proposed with C 16.17T (=CECT 7227T=LMG 27474T) as the type strain.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-013-0078-z) contains supplementary material, which is available to authorized users. A. Lasa A. L. Die´guez J. L. Romalde (&) Departamento de Microbiologı´a y Parasitologı´a, CIBUS, Universidad de Santiago de Compostela, Campus Sur s/n, 15782 Santiago de Compostela, Spain e-mail: [email protected]
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 including fish, molluscs and crustaceans (Beaz-Hidalgo et al. 2010) and, in addition, some of them have been described as pathogens to fish, molluscs and crustaceans (Farto et al. 2003; Gay et al. 2004; Go´mez-Leo´n 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
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(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 classification (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 decussata), 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 characterization 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 BeazHidalgo et al. (2008). Strain C 16.17T was isolated from Ria de Arousa (42°370 300 N/8°460 3800 W), strains CMJ 9.12 and CMJ 12.11 were isolated from Ria de Camarin˜as (43°80 1600 N/9°100 3700 W) and strain Rd 13.7 was isolated from Ria de Vigo (42°180 2700 N/ 8°370 1300 W). 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. anguillarum 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).
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Phenotypical characterization The four marine strains were subjected to the following phenotypic tests (MacFaddin 1993; Romalde and Toranzo 1991): cell morphology and motility, Gram stain, oxidase, catalase, 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, arginine dihydrolase test (Moeller’s medium), lysine and ornithine decarboxylation (Moeller’s medium), 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]) supplemented 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-diisopropylpteridine) (150 lg per disc) was determined on Mu¨eller– Hinton (Oxoid) agar. All media were supplemented with 1 % NaCl when required. Additional phenotypic characteristics were performed 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
Antonie van Leeuwenhoek (2014) 105:335–341
DNASTAR Lasergene SEQMAN program. Sequence similarities of 16S rRNA and housekeeping genes were determined using the EzTaxon-e server (www.eztaxone.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 stability 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 experiments were performed with the hydroxyapatite/microtitre 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
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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 phenotypic features for the four marine strains are shown in Table S1. Sequence similarity of the 16S rRNA gene indicated 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 representatives of Splendidus and Anguillarum clades (Figs. 1 and S1). Multilocus sequence analysis (MLSA) of housekeeping genes has been proposed as a useful tool to define the phylogenetic relationships among microorganisms (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
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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
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 demonstrated that the four clam strains represent a novel species within the genus Vibrio. The isolates from cultured clam can be differentiated 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-mannitol. 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 addition, 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
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Antonie van Leeuwenhoek (2014) 105:335–341
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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 substitutions per nucleotide position
cortegadensis sp. nov. is proposed for this new species.
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.
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 anaerobic metabolism. All strains are sensitive to the vibriostatic agent O/129, positive for arginine dihydrolase (Moeller’s medium), indole reaction,
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Table 1 Phenotypic characteristics for distinguishing V. cortegadensis sp. nov. from related Vibrio species Characteristics
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
ADH
?
-
-
-
?
?
?
?
-
?
?
?
?
?
-
-
?
Indole
?
?
-
?
?
?
?
-
?
?
?
?
?
?
?
?
?
TCBS Growth with
G
G
Y
Y
Y
G
Y
Y
G
Y
Y
G
G
Y
G
Y
Y
3 % NaCl
?
-
?
?
?
?
?
?
?
?
?
?
?
-
?
?
?
6 % NaCl
-
-
?
?
?
-
?
?
?
?
?
?
?
-
?
?
?
?
-
?
?
?
-
-
-
?
-
-
-
-
-
-
-
-
D-Galactose
V (?)
?
?
?
?
-
-
-
-
-
-
?
-
-
-
?
?
D-Mannose
V (-)
?
?
?
?
-
-
-
?
-
-
?
-
-
-
?
?
D-Maltose
?
?
?
?
?
-
-
-
?
-
-
?
-
-
-
?
?
D-Glucose
?
?
?
-
?
?
-
?
?
-
?
?
?
-
?
?
?
D-Mannitol
-
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
Amygdalin
-
?
-
-
-
-
?
-
-
?
-
-
-
?
?
-
?
Fermentation of Glycerol
All data were obtained concurrently in this study. ? positive; - negative; V variable (in parenthesis is indicated the result for the type strain); G: green colonies on TCBS; Y: yellow colonies on TCBS. Taxa are indicated as: 1, V. cortegadensis (4 strains); 2, V. tapetis CECT 4600T; 3, V. atlanticus LMG 24300T; 4, V. artabrorum LMG 23865T; 5, V. celticus CECT 7224T; 6, V. chagassi LMG 21353T; 7, V. crassostreae LMG 20537T; 8, V. cyclitrophicus LMG 21359T; 9, V. gallaecicus LMG 24045T; 10, V. gigantis LMG 22741T; 11, V. kanaloae LMG 20539T; 12, V. lentus CECT 5110T; 13, V. pomeroyi LMG 20537T; 14, V. splendidus CECT 628T; 15, V. tasmaniensis LMG 20012T; 16, V. toranzoniae CECT 7225T; 17, V. anguillarum 19264T
All strains produce fermentation of D-glucose, N-acetylglucosamine, aesculin, D-maltose, D-trehalose, starch, glucogen and potassium 2-ketogluconate (weakly reaction), but not of erythritol, D-arabinose, L-xylose, D-adonitol, methyl-bDxylopyranoside, L-sorbose, L-rhamnose, dulcytol, inositol, D-sorbitol, methyl-aD-mannopyranoside, methyl-aD-glucopyranoside, amygdalin, arbutin, salicin, D-cellobiose, D-lactose, D-melibiose, D-sucrose, inuline, D-melezitose, D-raffinose, xylitol, gentiobiose, 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 arylamidase, 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 (comprising 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. D-fructose,
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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 Innovacio´n (Ministry of Science and Innovation) (Spain). A. L. acknowledges the Ministerio de Economı´a 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.
References Beaz-Hidalgo R, Cleenwerck I, Balboa S, De Wachter M, Thompson FL, Swings J, de Vos P, Romalde JL (2008) Diversity of Vibrios associated with reared clams in Galicia (NW Spain). Syst Appl Microbiol 3:215–222 Beaz-Hidalgo R, Balboa S, Romalde JL, Figueras MJ (2010) Diversity and pathogenicity of Vibrio species in cultured bivalve molluscs. Environ Microbiol Rep 2:34–43 Colwell RR (2006) A global and historical perspective of the genus Vibrio. In: Thompson FL, Austin B, Swings J (eds) The biology of Vibrios. American Society for Microbiology, Washington, DC, pp 3–11
Antonie van Leeuwenhoek (2014) 105:335–341 Farto R, Armada SP, Montes M, Guisande JA, Pe´rez MJ, Nieto TP (2003) Vibrio lentus associated with diseased wild octopus (Octopus vulgaris). J Invertebr Pathol 83:149–156 Gay M, Renault T, Pons AM, Le Roux F (2004) Two Vibrio splendidus related strains collaborate to kill Crassostrea gigas: taxonomy and host alterations. Dis Aquat Org 62:65–74 Go´mez-Leo´n J, Villamil L, Lemos ML, Novoa B, Figueras A (2005) Isolation of Vibrio alginolyticus and Vibrio splendidus from aquacultured carpet shell clam (Ruditapes decussatus) larvae associated with mass mortalities. Appl Environ Microbiol 71:98–104 Goris J, Suzuki K, de Vos P, Nakase T, Kersters K (1998) Evaluation of a microplate DNA–DNA hybridization method compared with the initial renaturation method. Can J Microbiol 44:1148–1153 Jensen S, Samuelsen OB, Andersen K, Torkildsen L, Lambert C, Choquet G, Paillard C, Bergh O (2003) Characterization of strains Vibrio splendidus and V. tapetis isolated from corkwing wrasse Symphodus melops suffering vibriosis. Dis Aquat Org 53:25–31 Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S, Chun J (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA Gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62:716–721 Kueh CSW, Chan KY (1985) Bacteria in bivalve shellfish with special reference to the oyster. J Appl Bacteriol 59:41–47 Lacoste A, Jalabert F, Malham S, Cueff A, Ge´le´bart F, Cordevant C, Lange M, Poulet SA (2001) A Vibrio splendidus strain is associated with summer mortality of juvenile oysters Crassotsrea gigas in the Bay of Morlaix (North Brittany, France). Dis Aquat Org 46:139–145 Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948 Le Roux F, Goubet A, Thompson FL, Faury N, Gay M, Swings J, Saulnier D (2005) Vibrio gigantis sp. nov., isolated from the haemolymph of cultured oysters (Crassostrea gigas). Int J Syst Evol Microbiol 55:2251–2255 Leano EM, Lavilla-Pitogo CR, Paner MG (1998) Bacterial flora in the hepatopancreas of pond reared Penaeus monodon juveniles with luminous vibriosis. Aquaculture 164:367–374 Lemos ML, Toranzo AE, Barja JL (1985) Modified medium for the oxidation–fermentation test in the identification of marine bacteria. Appl Environ Microbiol 49:1541–1543 MacFaddin JF (1993) Pruebas bioquı´micas para la Identificacio´n de Bacterias de Importancia Clı´nica (translation by Me´dica Panamericana SA). Williams & Wilkins, Baltimore (in Spanish) Nicolas JL, Corre S, Gauthier G, Robert R, Ansquer D (1996) Bacterial problems associated with scallop Pecten maximus larval culture. Dis Aquatic Org 27:67–76 Osorio CR, Collins MD, Toranzo AE, Romalde JL (1999) 16S rRNA gene sequence analysis of Photobacterium damselae
341 and nested-PCR method for rapid detection of fish pasteurellosis. Appl Environ Microbiol 65:2942–2946 Pascual J, Macia´n MC, Arahal DR, Garay E, Pujalte MJ (2010) Multilocus sequence analysis of the central clade of the genus Vibrio by using the 16S rRNA, recA, pyrH, rpoD, gyrB, rctB and toxR genes. Int J Syst Evol Microbiol 60:154–165 Prado S, Romalde JL, Montes J, Barja JL (2005) Pathogenic bacteria isolated from diseased outbreaks in shellfish hatcheries. First description of Vibrio neptunius as an oyster pathogen. Dis Aquat Org 67:209–215 Pujalte MJ (2011) La familia Vibrionaceae: una actualizacio´n taxono´mica. XXIII Congreso Nacional de la Sociedad Espan˜ola de Microbiologı´a, Salamanca Pujalte MJ, Ortigosa M, Urdaci MC, Garay E, Grimont PAD (1993) Vibrio mytili sp. nov., from mussels. Int Syst Bacteriol 43:358–362 Romalde JL, Toranzo AE (1991) Evaluation of the API 20E system for the routine diagnosis of the enteric redmouth disease. Bull Eur Assoc Fish Pathol 11:147–149 Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. Microbial ID Inc., Newark Sawabe T, Kita-Tsukamoto K, Thompson FL (2007) Inferring the evolutionary history of vibrios by means of multilocus sequence analysis. J Bacteriol 189:7932–7936 Stackebrandt E, Ebers J (2006) Taxonomic parameters revisited: tranished gold standards. Microbiol Today 33:152–155 Sugumar G, Nakai T, Hirata Y, Matsubara D, Helinski DR (1998) Vibrio splendidus biovar II as the causative agent of bacillary necrosis of Japanese oyster Crassostrea gigas larvae. Dis Aquat Org 33:111–118 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 Thompson FL, Swings J (2006) Taxonomy of the vibrios. In: Thompson FL, Austin B, Swings J (eds) The biology of Vibrios. American Society for Microbiology, Washington, DC, pp 29–43 Thompson CC, Thompson FL, Vandemeulebroecke K, Hoste B, Dawyndt P, Swings J (2004) Use of recA as an alternative phylogenetic marker in the family Vibrionaceae. Int J Syst Evol Microbiol 54:919–924 Thompson FL, Gevers D, Thompson CC, Dawyndt P, Naser S, Hoste B, Munn CB, Swings J (2005) Phylogeny and molecular identification of Vibrios on the basis of multilocus sequence analysis. Appl Environ Microbiol 71:5107–5115 Thompson CC, Thompson FL, Vicente AC, Swings J (2007) Phylogenetic analysis of vibrios and related species by means of atpA gene sequences. Int J Syst Evol Microbiol 57:2480–2484 Ziemke F, Ho¨fle MG, Lalucat J, Rossello´-Mora R (1998) Reclassification of Shewanella putrefaciens Owen’s genomic group II as Shewanella baltica sp. nov. Int J Syst Bacteriol 48:179–186
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ORIGINAL PAPER
Vibrio cortegadensis sp. nov., isolated from clams Aide Lasa • Ana L. Die´guez • Jesu´s L. Romalde
Received: 23 June 2013 / Accepted: 15 November 2013 / Published online: 23 November 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract A group of four strains isolated from clams (Venerupis decussata and Venerupis philippinarum) in Galicia (NW Spain) were subjected to a polyphasic characterization, based on the phenotypic characteristics, the analysis of chemotaxonomic features, 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 isolates constitute a new species. The name Vibrio cortegadensis sp. nov. is proposed with C 16.17T (=CECT 7227T=LMG 27474T) as the type strain.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-013-0078-z) contains supplementary material, which is available to authorized users. A. Lasa A. L. Die´guez J. L. Romalde (&) Departamento de Microbiologı´a y Parasitologı´a, CIBUS, Universidad de Santiago de Compostela, Campus Sur s/n, 15782 Santiago de Compostela, Spain e-mail: [email protected]
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 including fish, molluscs and crustaceans (Beaz-Hidalgo et al. 2010) and, in addition, some of them have been described as pathogens to fish, molluscs and crustaceans (Farto et al. 2003; Gay et al. 2004; Go´mez-Leo´n 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
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(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 classification (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 decussata), 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 characterization 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 BeazHidalgo et al. (2008). Strain C 16.17T was isolated from Ria de Arousa (42°370 300 N/8°460 3800 W), strains CMJ 9.12 and CMJ 12.11 were isolated from Ria de Camarin˜as (43°80 1600 N/9°100 3700 W) and strain Rd 13.7 was isolated from Ria de Vigo (42°180 2700 N/ 8°370 1300 W). 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. anguillarum 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).
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Phenotypical characterization The four marine strains were subjected to the following phenotypic tests (MacFaddin 1993; Romalde and Toranzo 1991): cell morphology and motility, Gram stain, oxidase, catalase, 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, arginine dihydrolase test (Moeller’s medium), lysine and ornithine decarboxylation (Moeller’s medium), 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]) supplemented 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-diisopropylpteridine) (150 lg per disc) was determined on Mu¨eller– Hinton (Oxoid) agar. All media were supplemented with 1 % NaCl when required. Additional phenotypic characteristics were performed 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
Antonie van Leeuwenhoek (2014) 105:335–341
DNASTAR Lasergene SEQMAN program. Sequence similarities of 16S rRNA and housekeeping genes were determined using the EzTaxon-e server (www.eztaxone.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 stability 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 experiments were performed with the hydroxyapatite/microtitre 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
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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 phenotypic features for the four marine strains are shown in Table S1. Sequence similarity of the 16S rRNA gene indicated 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 representatives of Splendidus and Anguillarum clades (Figs. 1 and S1). Multilocus sequence analysis (MLSA) of housekeeping genes has been proposed as a useful tool to define the phylogenetic relationships among microorganisms (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
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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
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 demonstrated that the four clam strains represent a novel species within the genus Vibrio. The isolates from cultured clam can be differentiated 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-mannitol. 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 addition, 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
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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 substitutions per nucleotide position
cortegadensis sp. nov. is proposed for this new species.
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.
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 anaerobic metabolism. All strains are sensitive to the vibriostatic agent O/129, positive for arginine dihydrolase (Moeller’s medium), indole reaction,
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Table 1 Phenotypic characteristics for distinguishing V. cortegadensis sp. nov. from related Vibrio species Characteristics
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
ADH
?
-
-
-
?
?
?
?
-
?
?
?
?
?
-
-
?
Indole
?
?
-
?
?
?
?
-
?
?
?
?
?
?
?
?
?
TCBS Growth with
G
G
Y
Y
Y
G
Y
Y
G
Y
Y
G
G
Y
G
Y
Y
3 % NaCl
?
-
?
?
?
?
?
?
?
?
?
?
?
-
?
?
?
6 % NaCl
-
-
?
?
?
-
?
?
?
?
?
?
?
-
?
?
?
?
-
?
?
?
-
-
-
?
-
-
-
-
-
-
-
-
D-Galactose
V (?)
?
?
?
?
-
-
-
-
-
-
?
-
-
-
?
?
D-Mannose
V (-)
?
?
?
?
-
-
-
?
-
-
?
-
-
-
?
?
D-Maltose
?
?
?
?
?
-
-
-
?
-
-
?
-
-
-
?
?
D-Glucose
?
?
?
-
?
?
-
?
?
-
?
?
?
-
?
?
?
D-Mannitol
-
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
Amygdalin
-
?
-
-
-
-
?
-
-
?
-
-
-
?
?
-
?
Fermentation of Glycerol
All data were obtained concurrently in this study. ? positive; - negative; V variable (in parenthesis is indicated the result for the type strain); G: green colonies on TCBS; Y: yellow colonies on TCBS. Taxa are indicated as: 1, V. cortegadensis (4 strains); 2, V. tapetis CECT 4600T; 3, V. atlanticus LMG 24300T; 4, V. artabrorum LMG 23865T; 5, V. celticus CECT 7224T; 6, V. chagassi LMG 21353T; 7, V. crassostreae LMG 20537T; 8, V. cyclitrophicus LMG 21359T; 9, V. gallaecicus LMG 24045T; 10, V. gigantis LMG 22741T; 11, V. kanaloae LMG 20539T; 12, V. lentus CECT 5110T; 13, V. pomeroyi LMG 20537T; 14, V. splendidus CECT 628T; 15, V. tasmaniensis LMG 20012T; 16, V. toranzoniae CECT 7225T; 17, V. anguillarum 19264T
All strains produce fermentation of D-glucose, N-acetylglucosamine, aesculin, D-maltose, D-trehalose, starch, glucogen and potassium 2-ketogluconate (weakly reaction), but not of erythritol, D-arabinose, L-xylose, D-adonitol, methyl-bDxylopyranoside, L-sorbose, L-rhamnose, dulcytol, inositol, D-sorbitol, methyl-aD-mannopyranoside, methyl-aD-glucopyranoside, amygdalin, arbutin, salicin, D-cellobiose, D-lactose, D-melibiose, D-sucrose, inuline, D-melezitose, D-raffinose, xylitol, gentiobiose, 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 arylamidase, 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 (comprising 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. D-fructose,
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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 Innovacio´n (Ministry of Science and Innovation) (Spain). A. L. acknowledges the Ministerio de Economı´a 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.
References Beaz-Hidalgo R, Cleenwerck I, Balboa S, De Wachter M, Thompson FL, Swings J, de Vos P, Romalde JL (2008) Diversity of Vibrios associated with reared clams in Galicia (NW Spain). Syst Appl Microbiol 3:215–222 Beaz-Hidalgo R, Balboa S, Romalde JL, Figueras MJ (2010) Diversity and pathogenicity of Vibrio species in cultured bivalve molluscs. Environ Microbiol Rep 2:34–43 Colwell RR (2006) A global and historical perspective of the genus Vibrio. In: Thompson FL, Austin B, Swings J (eds) The biology of Vibrios. American Society for Microbiology, Washington, DC, pp 3–11
Antonie van Leeuwenhoek (2014) 105:335–341 Farto R, Armada SP, Montes M, Guisande JA, Pe´rez MJ, Nieto TP (2003) Vibrio lentus associated with diseased wild octopus (Octopus vulgaris). J Invertebr Pathol 83:149–156 Gay M, Renault T, Pons AM, Le Roux F (2004) Two Vibrio splendidus related strains collaborate to kill Crassostrea gigas: taxonomy and host alterations. Dis Aquat Org 62:65–74 Go´mez-Leo´n J, Villamil L, Lemos ML, Novoa B, Figueras A (2005) Isolation of Vibrio alginolyticus and Vibrio splendidus from aquacultured carpet shell clam (Ruditapes decussatus) larvae associated with mass mortalities. Appl Environ Microbiol 71:98–104 Goris J, Suzuki K, de Vos P, Nakase T, Kersters K (1998) Evaluation of a microplate DNA–DNA hybridization method compared with the initial renaturation method. Can J Microbiol 44:1148–1153 Jensen S, Samuelsen OB, Andersen K, Torkildsen L, Lambert C, Choquet G, Paillard C, Bergh O (2003) Characterization of strains Vibrio splendidus and V. tapetis isolated from corkwing wrasse Symphodus melops suffering vibriosis. Dis Aquat Org 53:25–31 Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S, Chun J (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA Gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62:716–721 Kueh CSW, Chan KY (1985) Bacteria in bivalve shellfish with special reference to the oyster. J Appl Bacteriol 59:41–47 Lacoste A, Jalabert F, Malham S, Cueff A, Ge´le´bart F, Cordevant C, Lange M, Poulet SA (2001) A Vibrio splendidus strain is associated with summer mortality of juvenile oysters Crassotsrea gigas in the Bay of Morlaix (North Brittany, France). Dis Aquat Org 46:139–145 Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948 Le Roux F, Goubet A, Thompson FL, Faury N, Gay M, Swings J, Saulnier D (2005) Vibrio gigantis sp. nov., isolated from the haemolymph of cultured oysters (Crassostrea gigas). Int J Syst Evol Microbiol 55:2251–2255 Leano EM, Lavilla-Pitogo CR, Paner MG (1998) Bacterial flora in the hepatopancreas of pond reared Penaeus monodon juveniles with luminous vibriosis. Aquaculture 164:367–374 Lemos ML, Toranzo AE, Barja JL (1985) Modified medium for the oxidation–fermentation test in the identification of marine bacteria. Appl Environ Microbiol 49:1541–1543 MacFaddin JF (1993) Pruebas bioquı´micas para la Identificacio´n de Bacterias de Importancia Clı´nica (translation by Me´dica Panamericana SA). Williams & Wilkins, Baltimore (in Spanish) Nicolas JL, Corre S, Gauthier G, Robert R, Ansquer D (1996) Bacterial problems associated with scallop Pecten maximus larval culture. Dis Aquatic Org 27:67–76 Osorio CR, Collins MD, Toranzo AE, Romalde JL (1999) 16S rRNA gene sequence analysis of Photobacterium damselae
341 and nested-PCR method for rapid detection of fish pasteurellosis. Appl Environ Microbiol 65:2942–2946 Pascual J, Macia´n MC, Arahal DR, Garay E, Pujalte MJ (2010) Multilocus sequence analysis of the central clade of the genus Vibrio by using the 16S rRNA, recA, pyrH, rpoD, gyrB, rctB and toxR genes. Int J Syst Evol Microbiol 60:154–165 Prado S, Romalde JL, Montes J, Barja JL (2005) Pathogenic bacteria isolated from diseased outbreaks in shellfish hatcheries. First description of Vibrio neptunius as an oyster pathogen. Dis Aquat Org 67:209–215 Pujalte MJ (2011) La familia Vibrionaceae: una actualizacio´n taxono´mica. XXIII Congreso Nacional de la Sociedad Espan˜ola de Microbiologı´a, Salamanca Pujalte MJ, Ortigosa M, Urdaci MC, Garay E, Grimont PAD (1993) Vibrio mytili sp. nov., from mussels. Int Syst Bacteriol 43:358–362 Romalde JL, Toranzo AE (1991) Evaluation of the API 20E system for the routine diagnosis of the enteric redmouth disease. Bull Eur Assoc Fish Pathol 11:147–149 Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. Microbial ID Inc., Newark Sawabe T, Kita-Tsukamoto K, Thompson FL (2007) Inferring the evolutionary history of vibrios by means of multilocus sequence analysis. J Bacteriol 189:7932–7936 Stackebrandt E, Ebers J (2006) Taxonomic parameters revisited: tranished gold standards. Microbiol Today 33:152–155 Sugumar G, Nakai T, Hirata Y, Matsubara D, Helinski DR (1998) Vibrio splendidus biovar II as the causative agent of bacillary necrosis of Japanese oyster Crassostrea gigas larvae. Dis Aquat Org 33:111–118 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 Thompson FL, Swings J (2006) Taxonomy of the vibrios. In: Thompson FL, Austin B, Swings J (eds) The biology of Vibrios. American Society for Microbiology, Washington, DC, pp 29–43 Thompson CC, Thompson FL, Vandemeulebroecke K, Hoste B, Dawyndt P, Swings J (2004) Use of recA as an alternative phylogenetic marker in the family Vibrionaceae. Int J Syst Evol Microbiol 54:919–924 Thompson FL, Gevers D, Thompson CC, Dawyndt P, Naser S, Hoste B, Munn CB, Swings J (2005) Phylogeny and molecular identification of Vibrios on the basis of multilocus sequence analysis. Appl Environ Microbiol 71:5107–5115 Thompson CC, Thompson FL, Vicente AC, Swings J (2007) Phylogenetic analysis of vibrios and related species by means of atpA gene sequences. Int J Syst Evol Microbiol 57:2480–2484 Ziemke F, Ho¨fle MG, Lalucat J, Rossello´-Mora R (1998) Reclassification of Shewanella putrefaciens Owen’s genomic group II as Shewanella baltica sp. nov. Int J Syst Bacteriol 48:179–186
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