vibrio hippocampi sp. nov., a new species isolated from...
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Vibrio hippocampi sp. nov., a new species isolated from wild seahorses 1
(Hippocampus guttulatus) 2
3 4 José Luis Balcázar1,2, José Pintado1, Miquel Planas1 5
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1Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas 7
(CSIC), c/. Eduardo Cabello 6, 36208 Vigo, Spain. 8
2Catalan Institute for Water Research (ICRA), Scientific and Technological Park of the 9
University of Girona, 17003 Girona, Spain. 10
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Correspondence: J. L. Balcázar. Tel: +34 986 214 457. Fax: +34 986 292 762. e-mail: 13
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Running title: Vibrio hippocampi sp. nov. 16
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and 25
the recA gene sequence of strain BFLP-4T are FN421434 and FN421435, respectively. 26
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Abstract 27
A Gram-negative, facultatively anaerobic, motile, and slightly curved rod-shaped 28
bacterium (BFLP-4T) was isolated from faeces of wild seahorses (Hippocampus 29
guttulatus) captured in northwest Spain (Toralla, Galicia). Strain BFLP-4T grew at 10–30
35 °C and pH 5–9 (optimally at 20 °C and pH 7.2) and at salt concentrations in the 31
range 0–7% (w/v) NaCl. The G+C content of the DNA was 49.3 mol%. Phylogenetic 32
analysis based on 16S rRNA gene sequence showed that strain BFLP-4T was a member 33
of the genus Vibrio, being most closely related to Vibrio ichthyoenteri (97.1%), V. 34
mediterranei (96.7 %), V. scophthalmi (96.7 %) and V. sinaloensis (96.6 %). A 35
phylogenetic analysis based on recA gene sequences also supported the affiliation of 36
strain BFLP-4T to the genus Vibrio. Strain BFLP-4T could be readily differentiated from 37
other closely related species by several phenotypic properties and fatty acid profiles. On 38
the basis of phenotypic, chemotaxonomic and phylogenetic data, strain BFLP-4T 39
represents a novel species within the genus Vibrio, for which the name Vibrio 40
hippocampi sp. nov. is proposed. The type strain is BFLP-4T (=DSM 22717T =LMG 41
25354T). 42
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Keywords: Vibrio hippocampi; polyphasic taxonomic analysis; seahorses 44
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Introduction 52
The genus Vibrio comprises a genetically diverse group of heterotrophic marine bacteria 53
that are found in a variety of aquatic environments (Thompson et al., 2004). Vibrio 54
species are commonly found as members of the normal microbiota in marine 55
invertebrates and fish; but they are also found to be etiological agents of several 56
diseases in humans and aquatic animals (Tantillo et al., 2004; Thompson et al., 2004; 57
Igbinosa & Okoh, 2008; Balcázar et al., 2010). 58
In the present study, we describe the physiological, chemotaxonomic and phylogenetic 59
characteristics of a Gram-negative, motile, facultatively anaerobic, and slightly curved 60
rod-shaped bacterium sharing the highest 16S rRNA gene sequence similarities to 61
Vibrio ichthyoenteri DSM 14397T, V. mediterranei CIP 103203T, V. scophthalmi A089T 62
and V. sinaloensis CAIM 797T. 63
Materials and methods 64
Isolation and culture conditions 65
During the characterization of organisms isolated from faeces of wild seahorses 66
(Hippocampus guttulatus), strain BFLP-4T was grown on tryptone soy agar (TSA) 67
supplemented with 1.5 % NaCl (w/v) at 20 ºC for 72 h. Subcultivation was done on the 68
same medium at 20 ºC for 48 h. 69
Phenotypic characterization 70
Gram reaction was determined using the non-staining (KOH) method as described by 71
Buck (1982). Cell morphology and motility were studied using phase-contrast 72
microscopy and electron microscopy as previously described by Herrera et al. (2007). 73
NaCl growth tolerance and requirements were investigated by using nutrient broth 74
[0.5% peptone from casein, 0.3% meat extract, 0.3% yeast extract, and adjusted to pH 75
7.2] supplemented with various concentrations of NaCl (0–15% at intervals of 1%). The 76
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pH range for growth was determined in nutrient broth that was adjusted to various pH 77
values (pH 2.0–12.5 at intervals of 0.5 pH units). Anaerobic growth was assessed at 20 78
ºC in anaerobic chambers with an H2/CO2 atmosphere (bioMérieux). 79
Catalase activity was determined by assessing bubble production in 3 % (v/v) H2O2; 80
oxidase activity was determined using 1 % (w/v) tetramethyl-p-phenylenediamine as 81
described by Lim et al. (2008). Some physiological characteristics were performed 82
using API 20NE, API 50CH and API ZYM (bioMérieux). Cells for inoculation of the 83
strips were grown for 24 h at 20 ºC on TSA supplemented with 1.5 % NaCl and results 84
were visually interpreted according to the manufacturer’s instructions. 85
Genotypic characterization 86
Extraction and amplification of genomic DNA for 16S rRNA sequence analysis were 87
carried out as described previously (Balcázar et al., 2009), and the recA gene was 88
amplified and sequenced as described by Thompson et al. (2005). The sequences of 89
these genes were compared against the sequences available in the GenBank, EMBL and 90
DDBJ databases obtained from the National Center of Biotechnology Information 91
database using the BLASTN (Alschul et al., 1990). Phylogenetic analysis were 92
performed using the software MEGA version 4.0 (Tamura et al., 2007) after multiple 93
alignments of data by CLUSTAL X (Thompson et al., 1997). Distances (distance 94
options according to the Kimura two-parameter model) and clustering with the 95
neighbour-joining (Fig. 1) and maximum-parsimony (Supporting information Fig. S1) 96
methods were determined by using bootstrap values based on 1000 replications. 97
For base composition analysis, DNA was prepared according to Chun & Goodfellow 98
(1995). The G+C content of the DNA was determined by the thermal denaturation 99
method (Mandel & Marmur, 1968). DNA from Vibrio harveyi DSM 19623T was used 100
as reference for determination of the thermal-melting profile (Tm). 101
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Chemotaxonomic analysis 102
Whole-cell fatty acids from the isolate were extracted from biomass grown on nutrient 103
agar [0.5% peptone from casein, 0.3% meat extract, 0.3% yeast extract, 1.5% agar, and 104
adjusted to pH 7.2] supplemented with 1.5% NaCl and were analysed according to the 105
standard protocol of the Sherlock Microbial Identification System (MIDI version 4.5). 106
Results and discussion 107
Phenotypic characteristics 108
Phenotypically, strain BFLP-4T can be clearly assigned to the genus Vibrio (Noguerola 109
& Blanch, 2008). Cells of strain BFLP-4T were slightly curved rods (Fig. 2), Gram-110
negative, oxidase- and catalase-positive, motile, and facultatively anaerobic. The novel 111
strain also showed prolific growth on thiosulfate-citrate-bile salts-sucrose agar (TCBS), 112
forming green colonies. In addition, strain BFLP-4T could be differentiated from related 113
species on the basis of some biochemical properties such as negative utilization of D-114
fructose and D-mannose. Other physiological characteristics of strain BFLP-4T are 115
shown in Table 1 and also in the species description. 116
Phylogenetic analysis 117
The 16S rRNA sequence of strain BFLP-4T was a continuous stretch of 1417 bp. 118
Sequence similarity calculations after a neighbour-joining analysis indicated that the 119
closest relatives of strain BFLP-4T were Vibrio ichthyoenteri (97.1%), V. mediterranei 120
(96.7 %), V. scophthalmi (96.7 %) and V. sinaloensis (96.6 %). Similar results were 121
obtained for strain BFLP-4T when the maximum-parsimony algorithm was used 122
(Supporting information Fig. S1). The recA gene has also been proposed as an useful 123
marker in inferring bacterial phylogeny (Lloyd & Sharp, 1993; Eisen, 1995), and has 124
been used successfully to differentiate species of the genus Vibrio (Thompson et al. 125
2005). A pairwise analysis of the recA sequence of strain BFLP-4T also revealed low 126
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levels of similarity between this strain and several species from the genus Vibrio (Fig. 127
3). For example, BFLP-4T exhibited 90.5% similarity to V. harveyi LMG 4044T, 90.2% 128
to V. rotiferianus LMG 21460T and 89.5% to V. campbellii LMG 11216T. 129
Chemotaxonomic characteristics and DNA base composition 130
The major fatty acids in strain BFLP-4T were summed feature 3 (comprising C16:1ω7c 131
and/or C15:0 iso 2-OH), C16:0, C18:1ω7c and C14:0, which comprise approximately 80.7 % 132
of the cellular fatty acids extracted. Fatty acids C16:1ω7c and/or C15:0 iso 2-OH, C16:0, 133
C18:1ω7c, C14:0, C12:0 and C16:0 iso are typically the major fatty acids found in Vibrio 134
species (Thompson and Swings, 2006). However, strain BFLP-4T and most closely 135
related type strains, V. ichthyoenteri V. mediterranei, V. shilonni and V. sinaloensis, 136
could be clearly distinguished from each other based on the relative fatty acid 137
concentration. The DNA G+C content was calculated as 49.3 mol%. This value is 138
within the range for the genus Vibrio (Farmer, 1992). 139
Therefore, the phenotypic and genotypic properties of strain BFLP-4T support its 140
description as a novel species within the genus Vibrio, for which the name Vibrio 141
hippocampi sp. nov. is proposed. 142
Description of Vibrio hippocampi sp. nov. 143
Vibrio hippocampi (hip.po.cam’pi. L. gen. n. hippocampi, of the seahorse, isolated from 144
Hippocampus guttulatus). Cells are Gram-negative, motile, facultatively anaerobic, and 145
slightly curved rod-shaped (1.0 × 2.0–2.5 µm). Colonies on TSA supplemented with 146
1.5% (w/v) NaCl are cream coloured, circular and 1.5–2.0 mm in diameter. Optimum 147
growth temperature is 20 °C. No growth occurs below 10 °C or above 35 °C. Growth 148
occurs at pH 5.5–9.0, but not below pH 5.0 or above pH 9.0. Growth occurs at NaCl 149
concentrations between 0 and 7 % (w/v), but not in the presence of 8 % (w/v) NaCl. 150
Positive for catalase, oxidase; nitrate reduction to nitrite; N-acetyl-D-glucosamine; 151
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assimilation of D-glucose and D-maltose. Negative for indole production; arginine 152
dihydrolase; urease; aesculin; gelatine hydrolysis; assimilation of L-arabinose, D-153
mannose, D-mannitol, potassium gluconate, caprate, adipate, malate, citrate and phenyl-154
acetate. Acid is produced from D-glucose, D-mannitol, D-cellobiose, D-maltose and D-155
trehalose, but not from glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, D-156
xylose, L-xylose, D-adonitol, methyl β-D-xylopyranoside, D-galactose, D-fructose, D-157
mannose, L-sorbose, L-rhamnose, dulcitol, myo-inositol, D-sorbitol, methyl α-D-158
mannopyranoside, methyl α-D-glucopyranoside, amygdalin, arbutin, salicin, D-lactose, 159
D-melibiose, D-saccharose, inulin, D-melezitose, D-raffinose, amidon, glycogen, 160
xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, R-arabitol 161
and L-arabitol. API ZYM tests show activities for esterase (C4), leucine arylamidase 162
and acid phosphatase. Alkaline phosphatase, esterase lipase (C8), lipase (C14), valine 163
arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, naphthol-AS-BI-164
phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-165
glucosidase, α-mannosidase and α-fucosidase activities are not observed. The fatty acid 166
profile consists of C12:0 (3.8 %), C11:0 3-OH (0.2 %), C13:0 (0.2 %), C12:0 2-OH (0.1 %), 167
C12:0 3-OH (2.5 %), C14:0 (7.8 %), C15:1ω8c (0.2 %), C15:1ω6c (0.2 %), C15:0 (2.38 %), 168
C16:1ω7c (0.2 %), summed feature 2 (2.7 %; comprising C14:0 3-OH and/or C16:1 Iso I), 169
summed feature 3 (41.6 %; comprising C16:1ω7c and/or C15:0 iso 2-OH), C16:1ω5c (0.3 170
%), C16:0 (19.7 %), C17:1ω8c (0.6 %), C17:1ω6c (0.5 %), C17:0 (0.9 %), C18:1ω9c (0.1 %), 171
C18:1ω7c (11.6 %), C18:1ω6c (2.2%), and C18:0 (0.4 %). The DNA G+C content is 49.3 172
mol%. 173
The type strain is BFLP-4T (=DSM 22717T =LMG 25354T), isolated from faeces of 174
wild seahorses captured in northwest Spain (Toralla, Galicia). 175
Acknowledgements 176
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This study was financed by the Spanish Ministry of Science and Technology 177
(Hippocampus CGL2005-05927-C03-01). J.L.B. was supported by a postdoctoral I3P 178
contract from the Spanish Council for Scientific Research (CSIC). We thank P. Quintas, 179
A. Chamorro, M. Cueto and S. Otero for skilful technical assistance. 180
References 181
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determining the guanine plus cytosine content of DNA. Methods Enzymol 12B: 195–207
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dichotomous keys. J Appl Microbiol 105: 175–185. 210
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(Thompson FL, Austin B & Swings J, eds), pp. 29–43. ASM Press, Washington, DC. 216
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Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F & Higgins DG (1997) The 222
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Table 1. Characteristics of strain BFLP-4T and some related Vibrio species 243
Characteristic 1 2 3 4 5 6
Growth on 6% NaCl + + + – – +
Growth on TCBS G Y Y Y Y Y
Gelatine hydrolisis – – – – + +
Utilization of:
Amygdalin – – – – + –
D-fructose – + + + + +
D-lactose – – – – + –
D-mannose – + + + + +
L-rhamnose – – – – + –
myo-inositol – – – – + –
244 Strains: 1, Vibrio hippocampi sp. nov. BFLP-4T; 2, Vibrio ichthyoenteri DSM 14397T; 245
3, V. mediterranei CECT 621T; 4, V. scophthalmi CECT 4638T; 5, V. shilonii AK1T; 6, 246
V. sinaloensis CAIM 797T. All data are from this study. +, Positive; –, negative; TCBS, 247
thiosulfate-citrate-bile salts-sucrose agar; G, green colonies; Y, yellow colonies. 248
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V. rotiferianus LMG 21460T (AJ316187)
V. campbellii ATCC 25920T (X74692)
V. harveyi ATCC 14126T (X74706)
V. parahaemolyticus ATCC 17802T (X74720)
V. alginolyticus ATCC 17749T (X74690)
V. natriegens ATCC 14048T (X74714)
V. diabolicus HE800T (X99762)
V. tubiashii ATCC 19109T (X74725)
V. sinaloensis CAIM 797T (DQ451211)
V. brasiliensis LMG 20546T (AJ316172)
V. mediterranei CIP 10320T (X74710)
V. shilonii AK1T (AF007115)
V. hippocampi BFLP-4T (FN421434)
V. scophthalmi A089T (U46579)
V. ichthyoenteri DSM 14397T (AJ421445)
V. aestuarianus ATCC 35048T (X74689)
V. pectenicida A365T (Y13830)
V. ordalii ATCC 33509T (X70641)
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P. phosphoreum ATCC 11040T (D25310)
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Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, 278
showing the position of strain BFLP-4T (in bold) among other Vibrio species. Numbers 279
at nodes indicate bootstrap values (expressed as percentages of 1000 replications); only 280
values >50% are given. Photobacterium phosphoreum ATCC 11040T was used as an 281
outgroup. Bar, 0.01 substitutions per nucleotide position. 282
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Fig. 2. Scanning electron micrograph of strain BFLP-4T showing a slightly curved, rod-291
shaped morphology (1.0 × 2.0–2.5 µm). Bar, 1.0 µm. 292
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V. campbellii LMG 11216T (AJ842377)
V. rotiferianus LMG 21460T (AJ842501)
V. harveyi LMG 4044T (AJ842440)
V. alginolyticus LMG 4409T (AJ842373)
V. diabolicus LMG 19805T (AJ842407)
V. parahaemolyticus LMG 2850T (AJ842490)
V. natriegens LMG 10935T (AJ842473)
V. hippocampi BFLP-4T (FN421435)
V. shilonii LMG 19703T (AJ842507)
V. mediterranei LMG 11258T (AJ842459)
V. sinaloensis R-732 (EU717074)
V. tubiashii LMG 10936T (AJ842518)
V. brasiliensis LMG 20546T (AJ842376)
V. scophthalmi LMG 19158T (AJ842505)
V. ichthyoenteri LMG 19664T (AJ842446)
V. aestuarianus LMG 7909T (AJ842369)
V. ordalii LMG 13544T (AJ842482)
V. pectenicida LMG 19642T (AJ842491)
P. phosphoreum ATCC 11040T (EF415550)
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Fig. 3. Neighbour-joining phylogenetic tree based on recA gene sequences, showing the 300
position of strain BFLP-4T (in bold) among other Vibrio species. Numbers at nodes 301
indicate bootstrap values (expressed as percentages of 1000 replications); only values 302
>50% are given. Photobacterium phosphoreum ATCC 11040T was used as an outgroup. 303
Bar, 0.02 substitutions per nucleotide position. 304
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