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Aeromonas piscicola sp. nov. , isolated from diseased fish.7
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R. Beaz-Hidalgo 1, A. Alperi 2, M. J. Figueras 2 & J. L. Romalde 1
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1 Departamento de Microbiología y Parasitología. Facultad de Biología. Universidad de11
Santiago de Compostela. Santiago de Compostela, Spain.12
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2 Departamento de Ciencias Médicas Básicas. Universidad de Rovira I Virgili, Reus,14
Spain.15
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Submitted to: Systematic and applied Microbiology. March 2009.21
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Running title: Aeromonas piscicola sp. nov.23
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* Corresponding author:27
Phone: +34-981563100 # 128
Fax: +34-98159690429
E-mail: jesus.romalde@usc.es30
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ABSTRACT37
Four Aeromonas strains (S1.2, EO-0505, TC1 and TI 1.1) isolated from moribund fish38
in Spain showed a restriction fragment length polymorphism (RFLP) pattern related to39
strains of A. salmonicida and A. bestiarum but their specific taxonomic position was40
unclear. Multilocus sequence analysis (MLSA) of housekeeping genes rpoD, gyrB, recA41
and dnaJ confirmed the allocation of these isolates in an unknown genetic lineage42
within the genus Aeromonas with A. salmonicida, A. bestiarum and A. popoffii as the43
phylogenetically nearest neighbours. Furthermore, a strain biochemically labelled as A.44
hydrophila (AH-3), showing a pattern of A. bestiarum in basis to 16S rDNA-RFLP, also45
clustered with the unknown genetic linage. The genes rpoD and gyrB proved to be the46
best phylogenetic markers to differentiate these isolates from their neighbour species.47
Useful phenotypic features for differentiating the novel species from other known48
Aeromonas species include their ability to produce acid from glycerol and salicin, their49
inability to use L-lactate as a carbon source and their capacity to hydrolyze elastin. A50
polyphasic approach using phenotypic characterization, phylogenetic analysis of the51
16S rRNA gene and of 4 housekeeping genes including, DNA-DNA hybridization52
studies and an analysis of the protein profiles by MALDI-TOF-MS showed that these53
strains represent a novel species for which the name Aeromonas piscicola sp. nov. is54
proposed with isolate S1.2T (=CECT 7443T= LMG 24783T) as the type strain.55
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Keywords: Aeromonas piscicola sp. nov., DNA-DNA hybridization, 16S RDNA,66
housekeeping genes, MALDI-TOF-MS.67
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INTRODUCTION79
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Several approaches have been attempted to clarify the phylogenetic relationships among81
the species of the genus Aeromonas but its taxonomy remains complex and appears to82
be continuously changing due to the addition of new described species. At present the83
genus comprises 19 species: A. hydrophila, A. bestiarum, A. salmonicida, A. caviae84
(synonym is A. punctata) A. media, A. eucrenophila, A. sobria, A. veronii (synonyms85
are A. ichthiosmia and A. culicicola), A. jandaei, A. schubertii, A. trota (synonym is A.86
enteropelogenes), A. allosaccharophila, A. encheleia, A. popoffii, A. simiae, A.87
molluscorum, A. bivalvium, A. aquariorum (synomym of A. hydrophila subsp.88
dhakensis), A. tecta, and two DNA homology groups, Aeromonas sp. HG11 (proposed89
to be the synonym of A. encheleia [10]) and HG13 (Enteric group 501), which remain90
without a species name [5, 6, 8, 11, 12, 18, 21, 23, 25, 26, 30]. One of the most evident91
problems is the delineation of the species within A. hydrophila complex which includes92
A. hydrophila, A. bestiarum, A. salmonicida and A. popoffii [22]. Particularly difficult is93
the separation between A. bestiarum and A. salmonicida. The gene 16S rRNA has been94
found to be highly conserved and unable to differentiate members of the latter species95
[20, 22]. Moreover, A. salmonicida is divided into four psycrophilic subspecies isolated96
from fish A. salmonicida subsp. salmonicida, A. salmonicida subsp. achromogenes, A.97
salmonicida subsp. masoucida and A. salmonicida subsp. smithia, and one mesophilic98
subspecies A. salmonicida subsps. pectinolytica isolated from water [18, 29]. Other99
motile A. salmonicida mesophilic strains have been described and can be misidentified100
as A. bestiarum or A. hydrophila [2, 14, 22]. A number of articles have described101
atypical A. salmonicida strains that cannot be included in any of the described102
subspecies due to the presence of atypical phenotypic or genetic characteristics [1, 9, 16,103
40].104
A previously described molecular method based on 16S rDNA RFLP analysis has105
provided species specific profiles enabling the identification of most species of106
Aeromonas [7]. The five fish strains analyzed in this study displayed mixed patterns107
between A. salmonicida and A. bestiarum when RFLP analysis was performed. In108
previous studies, strains with such behaviour could be assigned to either one of these109
two species using gyrB and rpoD genes [22]. Recently, other studies have contributed to110
the clarification of controversial relationships in the genus Aeromonas and have111
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demonstrated that the genes dnaJ and recA can also be powerful tools for interspecies112
differentiation [27, 33]. In the present study, the five Aeromonas isolates were subjected113
to a polyphasic approach including phylogenetic analysis derived from 16S rRNA and 4114
housekeeping genes (gyrB, rpoD, dnaJ and recA), DNA-DNA hybridization, MALDI-115
TOF-MS analysis and extensive biochemical tests in order to determine their taxonomic116
positions. Based on the reported findings, we describe a novel species of the genus117
Aeromonas.118
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MATERIALS AND METHODS120
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Bacterial strains and phenotypic tests122
The strains used in this study had been previously classified as belonging to the genus123
Aeromonas based on phenotypic features. Four strains S1.2, EO-0505, TC1 and TI 1.1124
were isolated from diseased fish; strains S1.2 and EO-0505 from salmon (Salmo salar)125
in 2005 and strains TC1 and TI 1.1 from trout (Oncorhynchus mykiss) in 2007, and all126
belonged to the Collection of the University of Santiago de Compostela. Strain AH-3,127
originally identified as A. hydrophila and isolated from gold fish (Carassius auratus)128
[24] was kindly supplied by Dr. Tomás (Universidad de Barcelona, Spain). The isolates129
were grown in Trypticase Soy Agar (TSA) (Pronadisa) and incubated at 25ºC for 24h.130
Stock cultures were maintained frozen at -80ºC in tryptone soy broth (TSB) with 15%131
(v/v) glycerol.132
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Physiological and biochemical characterization of the five isolates was performed by134
standard techniques, following the methodologies described in previous studies [15, 17,135
39]. Incubation was performed at 25ºC. The following tests were performed: Gram-136
staining, motility, cytochrome oxidase, catalase activity, nitrate reduction, susceptibility137
to the vibriostatic agent (150!g), glucose oxidation-fermentation test, production of a138
brown diffusible pigment, gas production from D-glucose, methyl red (MR) and Voges-139
Proskauer (VP) reactions, arginine dihydrolase (ADH), lysine and ornithine140
decarboxylases (LDC, ODC) activity (Moeller´s method), indole production, hydrolysis141
of gelatin, starch, esculin, Tween 80, elastin and DNA and !-hemolysis on Columbia142
sheep blood agar (CBA, Oxoid). Temperature ranges for growth were assayed in TSA at143
4, 37 and 44ºC. Salt tolerance was determined in nutrient broth agar containing 0, 0.5, 3,144
and 6% (w/v) NaCl. Utilization of the following substrates as carbon sources were145
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assayed: L-arabinose, sucrose, cellobiose, L-rhamnose, D-mannitol, D-sorbitol, m-146
inositol, L-lactate, raffinose and salicine. The test !-galactosidase (ONPG) was147
performed using API 20E (BioMérieux) following the instructions of the manufacturer,148
with the exception of using saline solution (0.85%) for the preparation of the inocule.149
Hydrogen production from cysteine was performed in the A. hydrophila medium [13].150
Fermentation of forty-nine carbohydrate substrates were tested by using API 50CH151
(BioMérieux) at 25ºC for 24h, following the manufacturer’s instructions.152
Antibiotic susceptibility tests were undertaken by the disc method on Müeller-Hinton153
agar plates (Oxoid, UK). After 24h of incubation at 25ºC, organisms were classified as154
susceptible (S), intermediate (I) or resistant (R) according to the guidelines of the155
Clinical and Laboratory Standards Institute [4]. The antibiotic containing discs (!g ml-1)156
were the following: ampicillin (AMP10), amoxicillin (AML25), amoxicillin + clavulanic157
acid (AMC30), tricarcillin (TIC75), cephalotin (KF30), gentamicin (CN10), norfloxacin158
(NOR10), erythromycin (E5), trimethoprin-sulfamethoxazole (SXT25), chloramphenicol159
(C30), streptomycin (S10), penicillin (P10), tetracycline (TE30) and nalidixic acid (NA30).160
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Electron microscopy162
Flagellar arrangement and cellular size were determined by transmission electron163
microscopy (TEM). Cells were stained with phosphotungstic acid (2%) and processed164
samples were visualized by an electron microscope Philips CM-12.165
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16S rRNA and housekeeping analyses167
Genomic DNA extraction was prepared using the InstaGene Matrix (Bio-Rad)168
according to the instructions of the manufacturer. 16S rDNA sequencing was performed169
using primers and conditions as previously described [20, 28]. Amplification and170
sequencing reactions of the housekeeping genes gyrB, rpoD, dnaJ and recA were171
carried out by the methods previously described [27, 34, 37, 41]. PCR products were172
purified using the QIAquick PCR Purification Kit (Quiagen). Sequencing reactions173
were performed using the kit GenomeLab DTCS-Quick Start Kit (Beckman Coulter).174
Sequences were analyzed in an Automatic DNA Sequencer (model 373A, Applied175
Biosystems). Sequencing was also performed for 3 strains of A. bestiarum (116p, 117p176
and J4N 98) for the genes 16S rRNA, recA and dnaJ and for 3 strains of A. popoffii177
(LMG 17543, LMG 17545 and LMG 17546) for the genes 16S rRNA (except strain178
LMG 17543), recA and dnaJ in order to have more related sequences to compare with179
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the fish strain sequences. Additional sequencing was performed for type strains absent180
in the NCIMB/BLAST release server: A. salmonicida subsp. achromogenes CECT181
895T, A. salmonicida subsp masoucida CECT 896T and A. salmonicida subsp182
petinolytica 34melT for the gene recA, A. bivalvium CECT 7113T, A. aquariourum183
CECT 7289T and A. tecta CECT 7082T for the genes dnaJ and recA; and the gene rpoD184
for the latter species.185
Sequence corrections and analysis were performed with DNAstar Seqman program186
(Lasergene) and AutoAsembler 1.40 (Applied Biosystems). Alignments using the above187
mentioned sequences and those already available were used to construct the188
phylogenetic trees by neighbour-joining (NJ) with the program Mega version 4.0. [36]189
and maximum likelihood (ML) (jModelTest, http://darwin.uvigo.es/, [31], FigTree190
v1.1.2, http://tree.bio.ed.ac.uk)191
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DNA-DNA hybridization193
Genomic DNA for DNA-DNA hybridizations was prepared using the Easy DNA194
(Invitrogen) kit. DNA-DNA hybridization experiments were done with the195
hydroxiapatite/microtitre plate method [42] with a hybridization temperature of 60ºC.196
Reciprocal reactions (eg. A x B and B x A) were performed and the DNA homology197
percentage reported are the means of a minimum of 3 hybridizations.198
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MALDI-TOF-MS200
The protein analysis by MALDI-TOF-MS was performed in the mass unit of the201
University of Santiago de Compostela. Protein extraction was performed with ethanol,202
formic acid and acetonitrile (AN). Processed samples were placed in a 96 well plate,203
allowed to dry and covered with a matrix solution ("-cyano-4-hydroxycinnamic acid;204
HCCA). Mass spectra were obtained using a MALDI-TOF Autoflex mass spectrometer205
(Bruker Daltronics). The measured mass range of spectra was 2.000-20.000 Da. Peak206
comparison was done with the data base of Bruker Daltronics. As a positive control207
Escherichia coli strain CECT 433 was included in the analysis, also protein profiles208
were compared with their own profiles as controls. In order to determine significant209
differences in the profiles, reproducibility of the results were assessed by repetition of at210
least 10 independent assays and reciprocal data was considered.211
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RESULTS AND DISCUSSION214
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Optimum growth temperature for the novel species is 25-30ºC. The novel mesophilic216
species showed several differentiating features in relation to other Aeromonas species217
(Table 1). Our isolates are readily distinguished from typical psychrophilic A .218
salmonicida by its motility, inability to produce brown pigment and growth at 37ºC219
[29]. The major characteristics that differentiate the new proposed species from the220
mesophilic species belonging to the A. hydrophila complex is their ability to produce221
acid from glycerol unlike A. bestiarum, A. popoffii and A. salmonicida strains, and their222
inability to use of L-lactate as a carbon source unlike A. hydrophila strains. Three or223
more tests allowed differentiation from all known Aeromonas species. Useful224
phenotypic tests to differentiate this group of strains from all Aeromonas taxa described225
up to date were hydrolysis of elastin and acid production of salicin (Table 1). The226
isolates studied were phenotypically homogeneous except for 4 variable traits, i.e. strain227
S1.2 uses cellobiose as a carbon source and does not produce acid from L-arabinose,228
strains S1.2 and TC1 do not produce acid from L-rhamnose and strain AH-3 produces229
acid from D-sorbitol. The latter strain has been proven to bare an important determinant230
of virulence in several studies [3, 24, 38]. Is important to consider that antibiotic231
susceptibility tests showed that the strains analysed were susceptible dose dependent to232
streptomycin (10!g) and sensitive to gentamicin (10!g), norfloxacin (10!g),233
trimethoprim + sulfamethoxazole (25!g) and chloramphenicol (10!g).234
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Previous phylogenetic analyses of A . be stiarum and A. salmonicida strains236
demonstrated that the gene 16S rRNA is not sufficiently discriminative to clearly237
differentiate some strains of both species [19]. The type strain of A. bestiarum is238
identical to that of A. salmonicida subsp achromogenes and A. salmonicida subsp.239
masoucida and only shows two nucleotide differences (positions 1011 and 1018) from240
that of A. salmonicida subsp. salmonicida [22]. Similar results were obtained for the241
strains analyzed in this study. Strains S1.2, TC1, TI 1.1 and AH-3 cluster with the type242
strain of A. bestiarum (CECT 5227T), A. salmonicida subsp. masoucida (CECT 896T)243
and A. salmonicida subsp. achromogenes (CECT 895T), but strain EO-0505 clusters244
with the type strain of A. salmonicida subsp. salmonicida (CECT 894T) (Figs. 1 and245
S1). Phylogenetic analysis of the isolates examined in this study based on the genes246
rpoD and gyrB showed that, although genetically related to A. bestiarum and A.247
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salmonicida, they formed a clear separated cluster with considerable phylogenetic248
divergence (Fig. S2). Phylogenetic trees based on rec A and dnaJ sequences also249
differentiate this new Aeromonas cluster although less phylogenetic divergence was250
appreciated with the closest neighbour A. bestiarum (Fig. S3). As has already been251
proved gyrB and rpoD genes are excellent molecular markers for assessing the252
phylogeny of the A. bestiarum / A. salmonicida [22], the present study confirms that the253
fish strains grouped in a cluster supported by a high bootstrap value (100%). Multilocus254
sequence analysis (MLSA) on the four concatenated genes reinforces the above results255
(Figs. 2 and S4).256
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The inter-species percentage similarities of the genes obtained in this study show that258
the closest phylogenetic relative to all fish strains is A. bestiarum (Table 2). Rates of259
nucleotide substitutions at the inter-species level have been described to be 3% or260
higher for the gene rpoD and 1.8-4.3% for gyrB [34]; the five strains examined were261
within these proposed limits. For the dnaJ and recA genes the inter-species limits found262
between the fish strains and A. bestiarum were lower than the limits described in263
previous studies (5.2% and 7-8% respectively) [27, 33]. Is important to consider that264
only sequences from type strains were used in the study proposing the limits for the265
dnaJ gene, where as for the recA gene the sequence fragment examined was 272 bp in266
comparison with 559 bp used in the present work.267
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Levels of DNA-DNA relatedness determined between the strain S1.2 of the269
presumptive novel species and type strains of A. salmonicida, A. popoffii, and A .270
bestiarum were 49.9%, 60.6% and 65.4% respectively, below the suggested level (i.e.271
70%) for species delineation [35].272
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MALDI-TOF-MS mass spectra of cell extracts confirmed the differentiation of strain274
S1.2 from the type strains of the phylogenetically closest species A. bestiarum, A.275
salmonicida and A. popoffii (see Supplementary Fig. S4). As a control a correct276
identification of E. coli CECT 433 was obtained with profiles always above log score277
2.400. Strain S1.2 with its own profile had a mean log score of 2.744. Mean log scores278
with A. popoffii, A. salmonicida, and A. bestiarum were 2.348, 2.324 and 2.234279
respectively.280
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Based on the results of the phylogenetic analyses using gyrB, rpoD, recA and dnaJ282
genes, DNA-DNA hybridization, MALDI-TOF-MS analysis and phenotyping283
characterization, it is evident that the strains isolated from diseased fish represent a284
single novel species of the genus Aeromonas, for which the name A. piscicola sp. nov.285
is proposed with the strain S1.2T (=CECT 7443T= LMG 24783T) as the type strain.286
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Description of Aeromonas piscicola sp. nov.288
pis. ci. co´ la, piscicola (pis. ci´co. la L.n. piscis fish; L. suff.- cola dweller; M. L. n.289
piscicola, fish dweller).290
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Cells are Gram-negative, motile rods with a polar flagellum, 1.5-2.0 !m long and 0.6-292
1.0 !m wide (Fig. 3). Growth occurs at 4-37ºC and at 0-3% NaCl (w/v). Optimum293
growth temperature is 25-30ºC. Oxidase and catalase positive, reduce nitrates to nitrites294
and are resistant to the vibriostatic agent O/129 (150 !g). No brown diffusible pigment295
is produced. Chemo-organotrophic, with both oxidative and fermentative metabolism.296
Positive for ADH, LDC, indol, VP and !-galactosidase test. All strains hydrolyse297
gelatin, elastin, esculin, starch, Tween 80 and DNA. Produce gas from D-glucose, H2S298
from cysteine and are !-hemolytic in sheep blood agar. Negative for ODC and MR299
tests. All strains utilize sucrose and mannitol as an energy source but not m-inositol, L-300
lactate or raffinose. Acid is produced from glycerol, D-ribose, D-galactose, D-glucose,301
D-f ruct ose, D-m anno se, D-ma nnit ol, meth yl-"D-glucopyranoside, N-302
acetylglucosamine, arbutin, aesculin, salicin, D-maltose, D-sucrose, D-trehalose, starch,303
glycogen and potassium gluconate. Does not produce acid from erythritol, D-arabinose,304
D-xylose, L-xylose, D-adonitol, methyl-"D-xylopyranoside, L-sorbose, dulcitol,305
inositol, methyl-"D-mannopyranoside, amygdalin, D-cellobiose, D-lactose, D-306
melibiose, inulin, D-melezitose, D-raffinose, xylitol, gentiobiose, D-turanose, D-lyxose,307
D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium 2-ketogluconate and308
potassium 5-ketogluconate. Type strain utilizes D-cellobiose and does not produce acid309
from L-arabinose and L-rhamnose. All strains are resistant to ampicillin (10!g),310
amoxicillin (25!g), amoxycilin + clavulanic acid (30!g), tricarcillin (75!g), cephalotin311
(30!g), erythromycin (5!g) and penicillin (10!g). All strains show susceptibility dose312
dependent to streptomycin (10!g) and sensitive to gentamicin (10!g), norfloxacin313
(10!g), trimethoprim + sulfamethoxazole (25!g) and chloramphenicol (10!g).314
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The type strain is S1.2T, was isolated in 2005 from wild diseased salmon, (Salmo salar)316
in Galicia (Spain).317
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The GenBank accession numbers for the 16S rRNA gene sequences of Aeromonas319
piscicola strains S1.2T, EO-0505, TC1 and TI 1.1 are FM999971, FM999970,320
FM999972 and FM999973. The GenBank accession numbers for the rpoD, gyrB, dnaJ321
and recA gene sequences of strains S1.2T, EO-0505, TC1 and TI 1.1 are FM999069,322
FM999963, FM999949, FM999941, FM998645, FM999962, FM999948, FM999939,323
FM999068, FM999964, FM999950, FM999940, FM999070, FM999965, FM999951,324
FM999942 respectively. (GenBank accession numbers for the rest of species sequenced325
in the study are in Fig. 1, S1, S2 and S3 available on line).326
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Figure Legends.494
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Fig.1. Unrooted neighbour-joining phylogenetic tree derived from partial 16S rRNA496
gene sequences showing the corresponding relationships of Aeromonas piscicola sp.497
nov. to known species of Aeromonas. The phylogenetic tree was constructed by using498
1338 nt. Numbers shown next to each node indicate bootstrap values (percentages of499
1000 replicates).500
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Fig.2. Unrooted neighbour-joining phylogenetic tree derived from MLSA (16S rRNA,502
rpoD, gyrB, dnaJ and recA genes) showing the corresponding relationships of503
Aeromonas piscicola sp. nov. to all other Aeromonas species described. The504
phylogenetic tree was constructed by using 4321 nt. Numbers shown next to each node505
indicate bootstrap values (percentages of 1000 replicates).506
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Fig.3. Transmission electron micrograph of Aeromonas piscicola sp. nov strain.508
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Fig.S1. Unrooted maximum likelihood phylogenetic tree derived from partial 16S631
rRNA gene sequences showing the corresponding relationships of Aeromonas piscicola632
sp. nov. to known species of Aeromonas. The phylogenetic tree was constructed by633
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Fig.S2. Unrooted neighbour-joining phylogenetic tree derived from rpoD (A) and gyrB638
(B) gene sequences showing the corresponding relationships of Aeromonas piscicola sp.639
nov. to known species of Aeromonas. The phylogenetic tree was constructed by using640
743 nt and 894 nt respectively Numbers shown next to each node indicate bootstrap641
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Fig.S3. Unrooted neighbour-joining phylogenetic tree derived from dnaJ (A) and recA648
(B) gene sequences showing the corresponding relationships of Aeromonas piscicola sp.649
nov. to known species of Aeromonas. The phylogenetic tree was constructed by using650
781 nt and 559 nt respectively. Numbers shown next to each node indicate bootstrap651
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Fig.S3. Unrooted maximum likelihood phylogenetic tree derived from concatenated658
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Fig. S4. MALDI-TOF-MS spectra of (a) A. piscicola S1.2T, (b) A. bestiarum CECT674
6277T, (c) A. salmonicida CECT 894T, (d) A. popoffii CECT 5176T in the mass range675
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