System. Appl. Microbiol. 26, 65–69 (2003) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/sam

Vibrio tasmaniensis sp. nov., isolated from Atlantic Salmon (Salmo salar L.)

F. L. Thompson1,2, C. C. Thompson1,2, and J. Swings1,2

1Laboratory for Microbiology, 2 BCCMTM/LMG Collection, Ghent University, K.L. Ghent, Belgium

Received: November 14, 2002

Summary

We describe the polyphasic characterization of four isolates which formed a tight AFLP group in a former study. The group was closely related to V. cyclitrophicus, V. lentus and V. splendidus (98.2–98.9% similarity) on the basis of the 16S rDNA sequence analysis, but by DNA-DNA hybridisation experiments it had at maximum 61% DNA similarity towards V. splendidus. Thus, we propose that the isolates represent a new Vibrio i.e. V. tasmaniensis (LMG 20012T; EMBL under the accession numbers AJ316192; mol% G+C of DNA of the type strain is 44.7). Useful phenotypical features for discrimination of V. tasmaniensis from other Vibrio species include gelatinase and β- galactosidase activity, fatty acid composition (particularly 14:0), utilisation and fermentation of different compounds (e.g. sucrose, melibiose and D-galactose) as sole carbon source.

Keywords: γ- – – V. tasmaniensis – Atlantic Salmon (Salmo salar)

Introduction

The description of bacterial diversity has attracted been examined in several studies, but mainly with the aim much attention in the last years, and an increasing number of identifying known bacterial species which have of new species has been proposed [20]. Recent estimations presumptive probiotic properties for fish [18, 19]. of the bacterial diversity in marine ecosystems by means of In this study we report on the taxonomic characteri- 16S rDNA similarity have revealed the existence of about sation of the AFLP cluster A45 consisting of four isolates 1200 bacterial species [7]. It has also been concluded that originated from Atlantic salmon reared in Tasmania [24]. much of the species richness in these systems is already We propose that these isolates represent a new Vibrio sampled [7]. On the other hand, more detailed studies species i.e. V. tasmaniensis. based on whole genome analyses and phenotypic characterisation clearly demonstrated that many of the culturable bacteria (including ) isolated from the Material and Methods marine environment are yet to be characterised [13, 24]. Indeed novel bacterial species found in the environment Bacterial strains, growth conditions and DNA isolation and in association with marine animals have recently been Strains characterised in this study were: Vibrio tasmaniensis proposed [3, 25]. In this respect it is clear now that LMG 20012T (VIB 836T) LMG 21574 (VIB 840), LMG 21575 coevolved animal-bacterial partnerships have been (VIB 848) and LMG 21576 (VIB 842); V. lentus LMG 21355 happening throughout the evolution of life of marine (R-3884) and LMG 21356 (R-3912); V. cyclitrophicus LMG organisms. Benign e.g. V. fischeri-squid or V. halioticoli- 21579 (R-14870), LMG 21580 (R-14874) and LMG 21581 (R- abalone and pathogenic e.g. V. corallyliticus-coral or V. 1556). Strains were grown aerobically on tryptone soy agar (TSA; Oxoid) supplemented with 2% (w/v) NaCl for 24 h at harveyi-white shrimp interactions among animals and 28 °C. DNA was extracted following the methodology vibrios have been documented, but understanding of described by Pitcher et al. (1989). All strains included in this animal-bacterial interactions is yet to be established and study are deposited in the BCCMTM/LMG Bacteria Collection at may depend also on a better overview of the bacterial Ghent University and in the CAIM collection of the Centre for diversity associated with host animals [3, 14, 22]. The Research on Nutrition and Development (CIAD) in Mazatlán, bacterial microflora associated with Atlantic salmon has México.

0723-2020/03/26/01-065 $ 15.00/0 66 F. L. Thompson et al.

98.2% 16S rDNA similarity towards V. cyclitrophicus, Genotypic analyses V. lentus and V. splendidus type strains, respectively. Sequencing of the almost complete 16S rDNA sequences was Strains LMG 21581 (AJ 316203) and LMG 2/355 accomplished essentially as described previously [24]. Alignment (AJ 316200) had 99.1 and 98.7% 16S rDNA similarity of the 16S rDNA sequences, distance estimations [10], clustering towards V. cyclitrophicus and V. lentus, respectively. The by neighbour joining [21], maximum likehood and maximum parsimony methods and stability of the clusters (Bootstrap 16S rDNA similarity of the representative strains towards analysis with 1000 replicates) were performed with the software known Vibrio species was above the level proposed BioNumerics 2·5 (Applied Maths). DNA-DNA hybridisation recently as the intraspecies variability (i.e. ≥98%) [23] experiments using photobiotin-labelled DNAs were run at and thus the 16S rDNA did not provide discrimination stringent conditions (39 °C) following the methodology between the strains at species level. Nevertheless, it is well described by Willems et al. [27]. Hybridisations were performed known that the 16S rDNA similarity between certain in four replicates. DNA binding values are the mean of Vibrio species (e.g. V. harveyi and V. campbellii, V. reciprocal and non-reciprocal reactions. The mol% G+C of anguillarum and V. ordalii) is nearly 100%, indicating a DNA was determined by HPLC [15]. need for applying other molecular techniques in order to Phenotypic analyses better discriminate such species. Phenotypic characterisation of the isolates was performed using API20E and Biolog GN metabolic fingerprinting kits following the instructions of the manufacturers, with slight modifications [26]. Classical phenotypic tests were performed as described previously [2, 6, 16, 26]. Antibiograms were carried out using the disc diffusion methodology [1] using commercial antibiotic-impregnated discs (Oxoid). The inhibition zone of each antibiotic was determined on Iso-sensitest agar (Oxoid) supplemented with 1.5% (w/v) NaCl for 24 hours at 28 °C. Fatty acid methyl esters (FAME) analysis was carried out as described by Huys et al. [9]. Isolates were grown on Trypticase Soy Broth (TSB; Becton Dickinson) supplemented with 1.5% (w/v) Bacto agar (Becton Dickinson) and 1.5% (w/v) NaCl at 28 °C for 24 hours. Approximately 50 mg of cells were harvested and the fatty acid were isolated following the recommendations of the manufacturer using the Microbial Identification System manual and software package, version 3⋅9 (Microbial ID).

Results and Discussion

Phylogenetic analysis of representative strains of each Fig. 1. Phylogenetic tree with the estimated position V. tasma- AFLP cluster (i.e. A45, A55, and A61) using almost niensis, using the neighbour joining method based on the almost complete 16S rDNA sequences clearly showed that these complete 16S rDNA sequences. Bootstrap values (>50%) after strains branch within the V. splendidus phylogenetic 1000 simulations are shown. Bar, 1% estimated sequence branch (Fig 1). Strain LMG 20012T had 98.9, 98.8 and divergence.

Table 1. DNA-DNA binding values and mol% G+C of DNA of Vibrio strains examined

T T T T Mol% of G+C LMG 20012 LMG 21356 LMG 21355 LMG 21580 LMG 20581 LMG 21579 LMG 19031 LMG 16751 LMG 21034 LMG 21359

V. tasmaniensis (A45) LMG 20012T 100 44.7 (A55) LMG 21356 100 44.5 LMG 21355 77 100 44.3 (A61) LMG 21580 100 44.3 LMG 20581 92 100 44.3 LMG 21579 94 99 100 44.1 V. splendidus LMG 19031T 61 56 62 61 64 65 100 45.0 LMG 16751 61 63 70 100 44.5 V. lentus LMG 21034T 55 78 81 52 55 56 57 62 100 45.2 V. ciclytrophicus LMG 21359T 57 57 59 92 91 93 61 59 58 100 44.2 Vibrio tasmaniensis sp. nov. 67

Table 2. Useful differentiating features among V. tasmaniensis and closely related Vibrio species.

V. tasmaniensis V. cyclitrophicus V. lentus V. splendidus

Growth at /in: 35 °C + + – V 10% NaCl – + – –

Susceptibility to O/129 + + – +

β-galactosidase – – + + Gelatinase – + + +

Fermentation of: Sucrose – + – – Melibiose – V + –

Utilisation of: Sucrose – + – – Methyl pyruvate – V V + L-proline – V – + D-galactose – + + +

Fatty acid composition: 14:0 9.5–12.4 6.2–8.2 4.8–8.8 6.3–8.7 Summed feature 3 34.4–36.1 37.1–38.3 33.3–41.7 39.4–40.4

Phenotypic data were obtained from Baumann et al. (1984); Farmer III and Hickman-Brenner (1992); Hedlund and Staley (2001) and Macián et al. (2001). Fatty acids are minimum and maximum. Fatty acid profiles of known Vibrio species are from our own database. V, variable feature.

The results of DNA-DNA hybridisation experiments phenotypical and chemotaxonomic features of the genus are summarised in the Table 1. Strain LMG 20012T had Vibrio [4, 6, 11]. The four isolates were facultative at maximum 61% DNA similarity towards V. splendidus. anaerobic, catalase and oxidase positive and showed Additionally, DNA similarities towards recently described prolific growth on thiosulfate-citrate-bile salts-sucrose species V. kanaloae, V. pomeroyi and V. chagassi were agar (TCBS), forming green. Isolates were slightly curved below 70%. Thus V. tasmaniensis can be considered a rods, motile by at least a polar flagellum, susceptible to the new species in the genus Vibrio. On the other hand A55 vibriostatic agent O/129, and did not grow without NaCl. representative strains LMG 21356 and LMG 21355 Genomic and phenotypic data described in this study clearly belong to V. lentus while A61 representative clearly indicate that the four isolates should be strains LMG 21579, LMG 21580 and LMG 20581 accommodate in a new Vibrio species, namely V. belong to V. cyclitrophicus. As already pointed out by tasmaniensis. Although the new species had the main Macián et al. [12], DNA-DNA similarity levels within phenotypical traits of the genus Vibrio, several useful this phylogenetic branch are high. These authors found differentiating features were disclosed which discriminate 59% DNA-DNA similarity between V. lentus and V. it from other related Vibrio species (Table 2). splendidus. The discrimination of different Vibrio species which are highly related on the basis of both 16S rDNA Description of V. tasmaniensis and DNA-DNA hybridisation seems to be more appropriate and reliable by applying genomic Vibrio tasmaniensis (tas.ma.nien'sis M. L. adj. tasma- fingerprinting techniques e.g. AFLP and rep-PCR which niensis of Tasmania, where the organism was isolated). can unambiguously identify the different Vibrio species Cells are slightly curved, 1 µm in width and 2–3 µm in [24]. The A45 isolates were clearly distinguishable from length. They form translucent, convex, non-swarming, other closely phylogenetic neighbours by means of AFLP smooth-rounded colonies with entire margin, beige in analysis. Another elegant alternative would be the colour and of about 4 mm in diameter on TSA after 48 h application of Multilocus sequence typing (MLST) to incubation at 28 °C. Strains formed green, translucent, unravel the structure of the genus Vibrio. Attempts have smooth-rounded colonies of 4–5 mm on TCBS. All been made in this respect, particularly on the study of the strains have a facultative anaerobic metabolism and population structure of V. cholerae [5]. By analysing six ferment glucose, mannitol and amygdalin, but not house keeping genes spaced around the genome of inositol, sorbitol, rhamnose, sucrose, melibiose and V. cholerae strains these authors concluded that this arabinose. Growth occurs at 4 to 35 °C. No growth in bacterium forms a metapopulation which consists of the absence of NaCl or in media with ≥ 8% (w/v) NaCl. several ecological populations of environmental and Prolific growth occurs at 28 °C in media containing 2.5% pathogenic strains. (w/v) NaCl. The following tests are positive for all The novel Vibrio species represented by the four isolates strains: Oxidase, catalase, tryptophane deaminase, of A45 examined in this study shared the main indole, NO3 reduction and Voges-Proskauer reaction. 68 F. L. Thompson et al.

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