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Vibrio Inhibens Sp. Nov., a Novel Bacterium with Inhibitory Activity Against Vibrio Species

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Vibrio Inhibens Sp. Nov., a Novel Bacterium with Inhibitory Activity Against Vibrio Species The Journal of Antibiotics (2012) 65, 301–305 & 2012 Japan Antibiotics Research Association All rights reserved 0021-8820/12 www.nature.com/ja ORIGINAL ARTICLE Vibrio inhibens sp. nov., a novel bacterium with inhibitory activity against Vibrio species Jose´ Luis Balca´zar1,2, Miquel Planas1 and Jose´ Pintado1 Strain BFLP-10T, isolated from faeces of wild long-snouted seahorses (Hippocampus guttulatus), is a Gram-negative, motile and facultatively anaerobic rod. This bacterium produces inhibitory activity against Vibrio species. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain BFLP-10T was a member of the genus Vibrio and was most closely related to Vibrio owensii (99%), Vibrio communis (98.9%), Vibrio sagamiensis (98.9%) and Vibrio rotiferianus (98.4%). However, multilocus sequence analysis using gyrB, pyrH, recA and topA genes revealed low levels of sequence similarity (o91.2%) with these closely related species. In addition, strain BFLP-10T could be readily differentiated from other closely related species by several phenotypic properties and fatty acid profiles. The G þ C content of the DNA was 45.6 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain BFLP-10T represents a novel species within the genus Vibrio, for which the name Vibrio inhibens sp. nov. is proposed. The type strain is BFLP-10T (¼ CECT 7692T ¼ DSM 23440T). The Journal of Antibiotics (2012) 65, 301–305; doi:10.1038/ja.2012.22; published online 4 April 2012 Keywords: polyphasic taxonomic analysis; seahorses; Vibrio inhibens INTRODUCTION Physiological and biochemical characterization The family Vibrionaceae currently comprises six validly published Gram reaction was determined using the non-staining (KOH) method.13 Cell genera: Vibrio,1 Photobacterium,2 Salinivibrio,3 Enterovibrio,4 Grimontia5 morphology and motility were studied using phase-contrast microscopy and 14 and Aliivibrio.6 Vibrio species are common inhabitants of aquatic electron microscopy as previously described by Herrera et al. NaCl growth environments, and they can be isolated under a wide range of salinity tolerance and requirements were investigated by using nutrient broth (0.5% peptone from casein, 0.3% meat extract, 0.3% yeast extract, and adjusted to and temperature conditions from oysters, clams, mussels, and fish, as pH 7.2) supplemented with various concentrations of NaCl (0–15% at well as from sediment and plankton.7,8 intervals of 1%). The pH range for growth was determined in marine broth Some species have been reported to cause infections in humans and that was adjusted to various pH values with acetic acid-sodium acetate (pH 9,10 aquatic animals, whereas a small number of other species have 4.0–4.5, 100 mM), MES (pH 5.0–6.0, 50 mM), MOPS (pH 6.5, 50 mM), Tris (pH 11,12 been used as probiotics in aquaculture. In the present study, a 7.0–9.0, 50 mM) or CHES (pH 9.5–10.0, 50 mM). Growth temperature between Gram-negative bacterium (BFLP-10T), with inhibitory activity against 5and401C was tested in nutrient broth supplemented with 2% w/v NaCl for Vibrio species, was isolated from faeces of wild long-snouted seahorses 48 h with shaking. Anaerobic growth was assessed at 22 1Cinanaerobic (H. guttulatus) captured in northwest Spain. The phylogenetic chambers with an H2/CO2 atmosphere (BioMe´rieux, Marcy l’Etoile, France). analysis based on the 16S rRNA gene of BFLP-10T indicated that it Catalase activity was determined by assessing bubble production in 3% (v/v) H2O2; oxidase activity was determined using 1% (w/v) tetramethyl-p- is closely related to members of the Harveyi clade. Thus, we 15 characterize strain BFLP-10T and describe the identification of this phenylenediamine as described by Lim et al. Some physiological characteristics were performed using API 20E, API 20NE and API ZYM novel species. (BioMe´rieux). Cells for inoculation of the strips were grown for 24 h at 22 1C on marine agar and the results were visually interpreted according to the manufacturer’s instructions. MATERIALS AND METHODS Antimicrobial activity was determined as described by Balca´zar et al.16 Culture conditions Briefly, strain BFLP-10T was grown in 100 ml of marine broth without Strain BFLP-10T was isolated from faeces of wild long-snouted seahorses agitation at 22 1C for 48 h. After incubation, bacteria were removed by by using the standard dilution plating method on marine agar at 20 1C centrifugation (2000 g), and cell-free culture supernatant was recovered by for 72 h. The strain was subcultured on the same medium at 22 1C for 24 h. passage through 0.22 mm pore size filters. The cell-free culture supernatant was Stock cultures were stored at À80 1C in marine broth with 30% (v/v) adjusted to pH 6.5 with 5M NaOH to eliminate the inhibitory effects produced glycerol. by organic acids. Moreover, sensitivity of cell-free culture supernatant to 1Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Cientı´ficas (IIM-CSIC), c/. Eduardo Cabello 6, Vigo, Spain and 2Catalan Institute for Water Research (ICRA), Scientific and Technological Park of the University of Girona, Girona, Spain Correspondence: Dr JL Balca´ zar, Catalan Institute for Water Research (ICRA), Scientific and Technological Park of the University of Girona, Emili Grahit 101, Girona 17003, Spain. E-mail: [email protected] Received 24 November 2011; revised 14 February 2012; accepted 5 March 2012; published online 4 April 2012 Vibrio inhibens sp. nov. JL Balca´zar et al 302 trypsin and proteinase K (Sigma Chemical Co., St Louis, MO, USA) at a final Table 1 Characteristics of strain BFLP-10T and some related species concentration of 1.0 mg ml À1 was also tested in buffers recommended by the supplier. Samples with and without enzymes were incubated at 37 1Cfor2h Characteristic 1 2 3 4 5 6 7 and residual activity was determined. To exclude potential inhibition by hydrogen peroxide, we added catalase (Sigma Chemical Co.) at a final Lysine decarboxylase þþþÀþÀþ concentration of 0.5 mg ml À1 and incubated at 37 1C for 30 min. All assays Ornithine decarboxylase þþþÀþÀþ were independently repeated at least two times for reproducibility. Urease þÀÀÀþÀÀ Five strains, Vibrio alginolyticus N26-1, Vibrio harveyi HT351, Vibrio Acetoin production ÀÀþ ÀÀÀ ichthyoenteri HT21, Vibrio parahaemolyticus HT352 and Vibrio splendidus Assimilation of: HT29, which have been isolated and identified in previous studies, were used L-Arabinose ÀÀÀÀþw À 10,16 as indicator bacteria. Briefly, bacterial strains were grown in 5 ml of marine D-Mannitol þþþÀÀþþ broth at 22 1C for 24 h. The cells were harvested by centrifugation (2000 g), D-Mannose ÀþþÀÀþþ washed twice with sterile saline solution and resuspended in 5 ml of the same Citrate ÀÀÀÀÀÀw solution. The bacterial suspensions were spread on marine agar plates. Four Enzyme activities of: wells were made in each agar plate with a sterile Pasteur pipette, and cell-free b-galactosidase þÀþÀÀÀþ T culture supernatants (10 ml) from strain BFLP-10 were placed into each well. Lipase (C14) þÀþþÀþþ 1 The plates were incubated aerobically at 22 C for 24 h and then examined for N-acetyl-b-glucosaminidase þÀþÀÀþÀ zones of inhibition. Fermentation of: Amygdalin þþþÀþþÀ Genotypic characterization Melibiose ÀÀÀÀþÀþ Genomic DNA extraction, PCR amplification and sequencing of the 16S Sucrose ÀþþÀþÀþ rRNA, DNA gyrase B subunit (gyrB), urydilate kinase (pyrH), recombination Abbreviations: þ , positive; À, negative; w, weak reaction. Strains: (1) V. inhibens sp. nov. repair protein (recA) and topoisomerase I (topA), were carried out as described BFLP-10T;(2)V. owensii LMG 25443T (Cano-Go´mez et al.32); (3), V. communis R-40496T 33 T 34 17 18 (Chimetto et al. ); (4) V. sagamiensis LC2-047 (Yoshizawa et al. ); (5) V. rotiferianus LMG previously by Sawabe et al. and Balca´zar et al. The sequences obtained were 21460T;(6)Vibrio campbelllii LMG 11216T;(7)V. harveyi LMG 4044T. compared against the sequences available in the GenBank, EMBL and DDBJ databases obtained from the National Center for Biotechnology Information using the BLASTN.19 Phylogenetic analysis was performed using the software MEGA version 4.0 (Center for Evolutionary Medicine and Informatics, Tempe, Phylogenetic analysis AZ, USA) after multiple alignments of data by CLUSTAL X.20,21 Distances The 16S rRNA gene sequence of strain BFLP-10T was a continuous (distance options according to the Kimura two-parameter model) and stretch of 1484 bp. In the neighbour-joining phylogenetic tree based clustering with the neighbour-joining and maximum-parsimony methods on 16S rRNA gene sequences (Figure 1), strain BFLP-10T was closely were determined using bootstrap values based on 1000 replications. related to V. owensii DY05T (99.0%), V. communis R-40496T (98.9%), For base composition analysis, DNA was prepared according to Chun & V. sagamiensisT (98.9%) and V. rotiferianus LMG 21460T (98.4%). 22 Goodfellow. The G þ C content of the DNA was determined using the Similar results were obtained with the maximum-parsimony algo- thermal denaturation method.23 DNA from Escherichia coli ATCC 11775T and rithm (Supplementary Information, Figure S1). Multilocus sequen- Vibrio azureus LMG 25266T were used as a reference for determination of the cing analysis of housekeeping genes has been used previously for thermal-melting profile (Tm). inferring evolutionary relationships among members of the genus Vibrio.17,26 In the present study, we used four housekeeping genes, Chemotaxonomic analysis gyrB, pyrH, recA and topA, in order to establish the taxonomic Whole-cell fatty acids from the isolate were extracted from biomass grown on position of strain BFLP-10T. The phylogenetic trees based on gyrB, tryptone soy agar (TSA) supplemented with 1.5% NaCl (w/v) and analysed pyrH, recA and topA gene sequences revealed low levels of similarity according to the standard protocol of the Sherlock Microbial Identification (o91.2%) between strain BFLP-10T and the most closely related System, version 4.5 (MIDI Inc., Newark, DE, USA).
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