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Vibrio Tapetis Sp. Nov., the Causative Agent of the Brown Ring Disease Affecting Cultured Clams JUAN J

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Vibrio Tapetis Sp. Nov., the Causative Agent of the Brown Ring Disease Affecting Cultured Clams JUAN J INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1996, p. 480-484 Vol. 46, No. 2 0020-7713/96/$04.00+ 0 Copyright 0 1996, International Union of Microbiological Societies Vibrio tapetis sp. nov., the Causative Agent of the Brown Ring Disease Affecting Cultured Clams JUAN J. BORREGO,'" DOLORES CASTRO,* ANTONIO LUQUE,' CHRISTINE PAILLARD,2 PHILIPPE MARIA T. GARCIA,3 AND ANTONIO VENTOSA3 Department of Microbiology, Faculty of Sciences, University of Malaga, 29071 Malaga, and Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, 41 012 Seville, Spain, and U.R.A. Centre National de la Recherche ScientiJique 0-1513, Institut d'Etudes Marines, Gorgeu, 29287 Brest Cedex, France2 A taxonomic characterization was carried out on strains of the bacteria that cause the brown ring disease of clams. On the basis of their phenotypic and genotypic characteristics, these strains can be considered to constitute a new taxonomic unit, distinct from other Ebrio species. The guanine-plus-cytosinecontent of the strains ranged between 42.9 and 45.5 mol% (43.2 mol% for the proposed type strain). DNA-DNA hybridization studies showed 100% intragroup relatedness, but levels of genetic relatedness to the reference strains of different Ebrio species tested ranged between 15 and 58%. The strains have all the properties characteristic of the genus vibrio and can be clearly differentiated from other species of this genus by their growth at 4°C and their negative responses for growth at 30°C and in 6% NaCl, arginine dehydrolase, lysine decarboxylase, ornithine decarboxylase, and Voges-Proskauer reaction. The name yibrio tapetis is proposed for the new species; strain B1090 (CECT 4600) is the type strain. Since 1980, the genus Vibrio has been subject to an extensive yeast extract, 0.1%; NaCI, 2%) supplemented with 20% glycerol (volivol). For taxonomic revision, and 15 new species have been described comparative purposes, 15 culture collection strains of the genus Vibrio were used (Table 2). All of them are halophilic microorganisms frequently isolated from (12, 22). At present, the genus Vibrio includes more than 35 marine environment and/or from poikilotherms, including fish and shellfish (4), species, most of which are of aquatic origin. Some of them and they shared several biochemical and physiological characteristics with the have been demonstrated to be pathogenic for aquatic animals, strains of I/: tapetis. All strains were cultured on marine agar (Difco Laboratories, including fish (Vibrio anguillarum, V. alginolyticus, K damsela, Detroit, Mich.) at 20 -C 2°C for 24 to 36 h. Phenotypic characterization. All biochemical tests were performed at 20 2 K Jischeri, K ordalii, K salmonicida, V. splendidus, and I/: vul- 2°C using media supplemented with NaCl at a final concentration of 2%, except niJicus) and shellfish (V. haweyi, K pelagtus, K splendidus, and growth tests in NaCl. The isolates were identified by standard morphological, Il tubiashii) (1, 6, 9-11, 15, 18, 25, 30, 31). physiological, and biochemical tests (5, 14, 26, 33). Sensitivity to the vibriostatic Brown ring disease is the first epizootic infection described agent 0/129 (2,4 diamino-6,7-diisopropylpteridine)was determined after 24 h on marine agar plates by using 0/129 discs (150 pg) (Oxoid Ltd., Basingstoke, in Europe which affects cultured and wild populations of adult Hampshire, England). The utilization of substrates as sole carbon and energy clams, mainly manila clams (Tapes philippinarum) and fine clams (Tapes decussatus). This disease was firstly described in France by Paillard et al. (21), although further epidemiological studies have demonstrated its prevalence in other locations, TABLE 1. Designation and geographical sources mainly southwestern and northwestern Spain (2, 3, 23). of K tapetis strains tested The biochemical characterization of the causative agent of Strain designation Source Origin brown ring disease indicated that it belonged to the genus Kbrio, and it was tentatively named Vibrio group P1 (20). BlO9OT Cultured Tapes philippinarum Landeda, France However, on the basis of the response to routine biochemical IS-1 Cultured Tapes philippinarum Landeda, France tests, Vibrio group P1 does not match any of the presently IS-5 Cultured Tapes philippinarum Landeda, France described species of the genus Vibrio (4). IS-7 Cultured Tapes philippinarum Quiberon, France IS-8 Natural Venerupis aurea Quiberon, France We carried out a phenotypic and genetic characterization of IS-9 Natural Cerastodema edule Quiberon, France several strains belonging to Vibrio group P1 and compared 2.3 Cultured Tapes philippinarum Landeda, France them with other Vibrio strains frequently isolated from affected 8.1 Cultured Tapes philippinarum Landeda, France clams. The results obtained have alIowed us to propose a new 8.2 Cultured Tapes philippinarum Landeda, France species of the genus Vibrio, V. tapetis, on the basis of its phys- 8.3 Cultured Tapes philippinarum Landeda, France iological, biochemical, and genetic features. 8.4 Cultured Tapes philippinarum Landeda, France 8.5 Cultured Tapes philippinarum Landeda, France 8.6 Cultured Tapes philippinarum Landeda, France MATERIALS AND METHODS 8.7 Cultured Tapes philippinarum Landeda, France 8.17 Cultured Tapes philippinarum Landeda, France Bacterial strains. Twenty-four strains of Vibrio group P1 constituting the 8.19 phenon 6 described by Castro (2), isolated from cultivated and natural popula- Cultured Tapes philippinarum Landeda, France tions of diseased clams between 1988 and 1991, were studied (Table 1). They 9.3 Cultured Tapes philippinarum Landeda, France were maintained as working stocks at -20°C in basal medium (peptone, 0.4%; 9.4 Cultured Tapes philippinarum Landeda, France 9.5 Cultured Tapes philippinarum Landeda, France 9.7 Cultured Tapes philippinarum Landeda, France 11.1 Cultured Tapes philippinarum Landeda, France * Corresponding author. Mailing address: Departamento de Micro- 11.2 Cultured Tapes philippinarum Landeda, France biologia, Facultad de Ciencias, Universidad de Mhlaga, Campus Uni- 11.3 Cultured Tapes philippinarum Landeda, France versitario Teatinos, 29071 Mdaga, Spain. Phone: 34-5-2131893. Fax: 34- 11.4 Cultured Tapes philippinarum Landeda, France 5-2132000. Electronic mail address: [email protected]. 480 VOL.46, 1996 TAXONOMIC DESCRIPTION OF VTIBRlO TAPETIS SP. NOV. 481 TABLE 2. Phenotypic characteristics of Vibrio species studied, including V. tapetis Result for species”: Test 123 4 5 6 7 8 910111213141516 Swarming Indole production Acetoin production Growth at (“C): 4 +-+- - - - - - - - - - - - - 18 ++++++++++++++++ 22 ++++++++++++++++ 30 -+++++++++++++++ 35 -+++++- - +++++-++ Growth in NaCl (%): 0 - - - - - - - - - - - - - - - - 1.5 ++++++++++++++++ 6 -++++++++++++- ++ 8 -+- -+- - - - +++- - - - 10 Production of: Alginase - - - - - - - - - - - -+- - - Amylase ++++- - ++- - -+-+ ++ Arginine dehydrolase - -+- ++- - - -+- -+ +- Catalase ++++++++-+++++ ++ P-Galactosidase +-+- -+-+-+- - - + ++ Gelatinase ++++- - -+-+-+-+ ++ Lipase ++++- - ++++- +++++ Lysine decarboxylase -+-+- -+++- - +- - -+ Nitrate reduction ++++-+++++++++ ++ Ornithine decarboxylase Hydrolysis of - - Esculin - - - -+-+- ++- -+- Casein +++- -+-+- - ++- +++ Urea Utilization of Acetate Aconitate D-Alanine L-Alanine y-Arninobutyrate Amygdalin L-Arabinose L- Aspart a te Cellobiose Citrate D-Fructose D-Galactose D-Galacturonate D-Gluconate a-D-Glucose D-Glucuronate L-Glutamate Glycerol Glycine L-Histidine P-Hydroxybutyrate myo-Inositol a-Ketoglu tarate DL-Lacta te Lactose L-Leucine - - + ++- - - - DL-Malate +++++- ++- Maltose ++++++ +++ D-Mannitol +++-++ ++- D-Mannose ++- -++ +++ Melibiose - -+ -+- N-Acetylglucosamine ++++++ +++ L-Ornithine - -+ +- + -+- L-Proline +++ +++ +++ Propionate +-+ ++++-+ Putrescine - +++++ -+- Continued on following page 482 BORREGO ET AL. INT. J. SYST. BACTERIOL. TABLE 2 - Continued Result for species": Test 123 4 5 6 7 8 910111213141516 Pyruvate ++ -+ - + ++++ ++ Quinate -+ - + - - - - +- L-Rhamnose - - - + - - - - - - L-Serine -+++ +- -+ ++++ ++ +- D-Sorbitol - -+- - - +- - - - - - - Succinate ++++++ -+ ++++ ++++ Sucrose - ++- +- -+ +++- +- +- D-Trehalose ++++ +- -+ ++ ++ ++++ L-Threonine - +++- - -+ ++++ ++ ++ Tween 40 ++++++ ++ ++ ++ ++++ Tween 80 - +++++ -+ -+ -+ ++ ++ 1, K tapetis B1090T; 2, K alginolyricus CECT 521*; 3, K anguillamm CECT 522T; 4, K campbellii CECT 523T; 5, K costicola NCIMB 1281T; 6, K danzsela ATCC 33539; 7, I/. fischen' CECT 524T; 8, K haweyi CECT 525T; 9, K meditevanei CECT 621T; 10,V natriegens CECT 526T; 11, K nereis CECT 595T; 12, K parahuemolyticus CECT 588; 13, V. pelagius ATCC 25916T; 14, K splendidus CECT 52gT; 15, K tubiashii CECT 631; 16, K vulniJicus CECT 529T. sources was performed by using GN Microplates (Biolog, Inc., Hayward, Calif.). to hydrolyze gelatin, casein, lecithin, and starch; and inability All the biochemical tests were repeated twice. to ferment sucrose, D-mannitol, or amygdalin. Clustering analysis. Tests were coded as 1 (positive result), 0 (negative result), and 9 (no growth or equivocal result). Test error was determined by the method On the basis Of profiles, group was of Sneath and Johnson (28). Clustering analysis was performed using 73 traits included previously in the T/. splendidus-like group (2, 4). HOW- determined for 15 reference strains of known Kbrio species and 10 strains of ever, V&io group P1 is clearly distinguished from this group by Kbrio group P1. The simple matching similarity coefficient (27) and unweighted pair
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