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Departament de Microbiologia i Parasitologia Sanitàries, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain
Correspondence
J. Gaspar Lorén
jgloren{at}ub.edu
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains 868ET and 665N are DQ504429 and DQ504430, respectively.
A table giving levels of DNA–DNA hybridization between strains 868ET and 665N and strains of phylogenetically related Aeromonas species is available as supplementary material in IJSEM Online.
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).
At the time of writing, 17 Aeromonas species and 20 DNA–DNA hybridization groups (HGs) have been described: Aeromonas hydrophila (HG1), A. bestiarum (HG2), A. salmonicida (HG3), A. caviae (HG4), A. media (HG5), A. eucrenophila (HG6), A. sobria (HG7), A. veronii bv. Sobria (HG8/10), A. jandaei (HG9), A. veronii bv. Veronii (HG10/8), Aeromonas sp. HG11, A. schubertii (HG12), Aeromonas sp. group 501 (HG13), A. trota (HG14), A. allosaccharophila (HG15), A. encheleia (HG16), A. popoffii (HG17), A. culicicola (HG18), A. simiae (HG19) and A. molluscorum (HG20) (Pidiyar et al., 2002; Harf-Monteil et al., 2004; Miñana-Galbis et al., 2004; Martin-Carnahan & Joseph, 2005). In addition to the continuous description of novel species, the complexity of Aeromonas taxonomy results from the isolation of motile and non-motile, mesophilic and psychrophilic, pigmented and non-pigmented strains within several Aeromonas species (Altwegg et al., 1990; Martin-Carnahan & Joseph, 2005), description of novel subspecies of A. hydrophila (Huys et al., 2002, 2003) and A. salmonicida (Pavan et al., 2000), taxonomic rearrangement of Aeromonas sp. HG11 (Huys et al., 1997) and A. culicicola (Huys et al., 2005) and extended descriptions of A. eucrenophila and A. encheleia (Huys et al., 1997) and A. jandaei (Esteve et al., 2003). In this context, several authors have proposed useful tables for the phenotypic differentiation of Aeromonas species based on a limited number of key tests (Carson et al., 2001; Miñana-Galbis et al., 2002, 2004; Valera & Esteve, 2002; Abbott et al., 2003; Martin-Carnahan & Joseph, 2005). Genotypic classification of aeromonads at the genus level is currently recommended based on DNA G+C content and for species delineation based on 16S rRNA gene sequence analyses and DNA–DNA reassociation experiments, although other genomic methods, such as DNA profiling or rpoD and gyrB gene sequencing, are useful for Aeromonas species discrimination (Stackebrandt et al., 2002; Soler et al., 2004; Martin-Carnahan & Joseph, 2005; Morandi et al., 2005).
In the present study, a polyphasic approach was used in order to determine the taxonomic position of two Aeromonas strains isolated from bivalve molluscs that clustered together as a separate phenon (phenon VII) but could not be identified at the species level in a previous phenotypic study of the genus Aeromonas (Miñana-Galbis et al., 2002). For this purpose, an extended phenotypic characterization, 16S rRNA gene sequencing, DNA G+C content determination, fluorescence amplified fragment length polymorphism (FAFLP) analysis and DNA–DNA hybridization experiments were performed for the two isolates. Based on the results obtained, these strains are considered to represent a novel species of the genus Aeromonas.
Strains 868ET and 665N were isolated from cockles (Cardium sp.) and razor shells (Ensis sp.), respectively, from retail markets in Barcelona (Spain). Isolation, growth and preservation of strains were performed as described previously (Miñana-Galbis et al., 2004).
Cell size, morphology and flagellar arrangement were determined by transmission electron microscopy (JEOL 1010). Cells were grown on trypticase soy agar (TSA) supplemented with 0.5 % (w/v) NaCl for 24 h at 25 °C and, following further suspension in MilliQ water, were examined by negative staining with 2 % (w/v) uranyl acetate.
Physiological and biochemical characterization experiments, unless stated otherwise, were performed at 25–30 °C and all media contained 1 % (w/v) NaCl. Tests for the following were determined as described previously (Miñana-Galbis et al., 2002): Gram-staining, motility, glucose oxidation–fermentation, oxidase and catalase activity, nitrate reduction, indole production, susceptibility to O/129, swarming motility, production of a brown diffusible pigment, gas production from D-glucose, methyl red and Voges–Proskauer reactions, ONPG, hydrogen sulfide production from cysteine and thiosulfate, growth on MacConkey agar and m-Aeromonas-selective agar base Havelaar, salt tolerance, pH and temperature ranges for growth, acid production from carbohydrates, hydrolysis of aesculin, arbutin, DNA, elastin, erythrocytes, starch, urea and xanthine, utilization of substrates as sole carbon and energy sources and sensitivity to antibiotics. Arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase activity (Moeller's method) and hydrolysis of gelatin by using tannic acid (1 %) as the gelatin-precipitating reagent were determined as described by Smibert & Krieg (1994).
For 16S rRNA gene sequencing and phylogenetic analysis, DNA was extracted by using a REALPURE genomic DNA extraction kit (RBMEG03; Durviz). Oligonucleotide primers used for PCR amplification and sequencing of the 16S rRNA gene were those described by Martínez-Murcia et al. (1999). DNA was subjected to PCR amplification in a total volume of 50 µl that contained 50 mM KCl, 15 mM Tris/HCl (pH 8.0), 1.5 mM MgCl2, 0.2 mM each dNTP (Amersham Biosciences), 1.25 U AmpliTaq Gold DNA polymerase (Applera) and 25 pmol each primer. The reaction mixtures were subjected to the following thermal cycling: an initial single step of 95 °C for 9 min, 35 cycles of 94 °C for 60 s, 51 °C for 30 s and 72 °C for 90 s and a final single step of 72 °C for 10 min. PCR products were purified by using Montage PCR centrifugal filter devices (Millipore) and prepared for sequencing by employing a BigDye Terminator v3.1 cycle sequencing kit (Applera). The amplified 16S rRNA genes were sequenced with an ABI PRISM 3730 DNA analyser by the Unitat de Genòmica of the Serveis Cientificotècnics of the Universitat de Barcelona.
The sequences obtained were aligned with 16S rRNA gene sequences of the type or reference strains of the Aeromonas DNA HGs by using the DNASTAR Lasergene software. Distances and clustering with the neighbour-joining and maximum-parsimony methods (pairwise deletion and Kimura two-parameter model) were determined by using MEGA version 2.1 (Kumar et al., 2001). Stability of the relationships was assessed by bootstrapping (1000 replicates).
DNA fingerprinting using FAFLP, which included DNA extraction and purification (Gevers et al., 2001), FAFLP fingerprinting, data processing and numerical analysis (Huys & Swings, 1999), was performed by the BCCMTM/LMG (Belgian Coordinated Collections of Microorganisms/Laboratorium voor Microbiologie from Universiteit Gent) Identification Service.
For DNA–DNA hybridization experiments and determination of DNA G+C content, genomic DNA of bacterial strains was prepared according to a modification of the procedure of Wilson (1987). The G+C content of each DNA sample was determined by three independent analyses via the HPLC technique (Mesbah et al., 1989). DNA–DNA hybridizations were performed in a minimum of four replicates at 47 °C according to a modification of the method described by Ezaki et al. (1989). These analyses were also performed by the BCCMTM/LMG Identification Service.
Strains 868ET and 665N were identified as belonging to the genus Aeromonas as they were Gram-negative, rod-shaped, motile by means of one polar flagellum, oxidase-positive, facultatively anaerobic, glucose-fermentative and resistant to O/129 and they grew in the absence of NaCl but not at 7 % (w/v) NaCl. Cells of the two isolates was coccoid to rod-shaped (0.3–1.0x0.5–2 µm). Their antibiotic-resistance pattern was similar to that of most recognized Aeromonas species (Kämpfer et al., 1999). The two strains were resistant to ampicillin and strain 665N was also resistant to cephalothin, whereas strain 868ET was not. These strains formed non-pigmented, circular colonies with a diameter of 3–4 mm on TSA when incubated at 25–30 °C. The growth temperature range was 4–37 °C, although strain 868ET was able to grow at 40.5 °C. Optimal growth occurred at 30–37 °C. The two isolates showed several differential phenotypic features with regard to other mesophilic Aeromonas species (Table 1). Three or more tests allowed differentiation of strains 665N and 868ET from all recognized Aeromonas species except A. caviae and A. media. A positive reaction in the lysine decarboxylase test allowed the separation of these strains from A. caviae and A. media. In addition, they could be differentiated from A. media based on the following tests: production of ONPG and brown pigment, fermentation of lactose and mannose and utilization of lactose, mannose and raffinose. In contrast to A. caviae, strains 665N and 868ET were able to use glycerol and to produce acid from it. These results allowed the phenotypic discrimination of strains 665N and 868ET from all recognized Aeromonas taxa.
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. The sequences of strains 665N and 868ET differed by 2 nt from each other and by 4 and 6 nt differences, respectively, with regard to that of A. popoffii (Table 2).
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; Huys et al., 2003; Miñana-Galbis et al., 2004). This comparison revealed that isolates 665N and 868ET clustered together with a similarity of 78 % but did not cluster within any of the 20 Aeromonas DNA HGs currently described. A dendrogram of the cluster analysis of FAFLP fingerprints is shown in Fig. 2. These results indicated that strains 665N and 868ET probably belonged to the same species and represented a novel Aeromonas species.
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). Levels of DNA–DNA relatedness were determined between the type strain of the presumptively novel Aeromonas species (868ET) and the type or reference strains of the phylogenetically most closely related Aeromonas HGs (Fig. 1
) and between strains 868ET and 665N. Strains 868ET and 665N showed a level of DNA–DNA relatedness of 76 %, above the level of 70 % that is accepted as the limit for species delineation (Wayne et al., 1987). Strain 868ET clearly showed <70 % DNA reassociation (30–44 %) with any of the type or reference Aeromonas strains analysed (see Supplementary Table S1 in IJSEM Online). Based on the results of DNA–DNA hybridization, phenotypic characterization, FAFLP analysis and 16S rRNA gene sequencing, we consider that strains 868ET and 665N represent a single novel species of the genus Aeromonas, for which the name Aeromonas bivalvium sp. nov. is proposed.
Description of Aeromonas bivalvium sp. nov.
Aeromonas bivalvium (bi.val'vi.um. N.L. pl. neut. n. Bivalvia scientific name of a class of molluscs; N.L. neut. gen. pl. n. bivalvium of bivalves of the class Bivalvia).
Cells are Gram-negative, straight, motile coccoids/rods with a polar flagellum, 0.5–2.0 µm long and 0.3–1.0 µm wide. Colonies on TSA are 3–4 mm in diameter, opaque, circular and beige in colour after 48 h at 25 °C. Optimal growth occurs at 30–37 °C but growth occurs at 4–37 °C and strain 868ET grows at up to 40.5 °C. Oxidase- and catalase-positive, reduces nitrate to nitrite, produces indole from tryptophan and is resistant to the vibriostatic agent O/129 (150 µg). Positive for glucose oxidation–fermentation, lysine decarboxylase, methyl red and ONPG tests. Negative for arginine dihydrolase, ornithine decarboxylase and Voges–Proskauer tests. Negative for brown diffusible pigment, swarming, and production of gas from D-glucose and hydrogen sulfide from cysteine and thiosulfate. Grows on MacConkey agar and m-Aeromonas-selective agar base Havelaar. Able to grow at pH 9.0 and 0–3 % NaCl, grows weakly at 6 % NaCl but does not grow at pH 4.5 or 7 % NaCl. Hydrolyses arbutin, DNA, aesculin, gelatin and starch, but not elastin, erythrocytes, urea or xanthine. Produces acid from L-arabinose, arbutin, dextrin, D-galactose, glycerol, D-mannitol, salicin, sucrose and D-trehalose but not from lactose, D-mannose, D-melibiose, D-raffinose, L-rhamnose, sorbitol or D-xylose. The following substrates are used as sole carbon and energy sources: acetate, N-acetylglucosamine, aesculin, L-arabinose, p-arbutin, L-arginine, D-cellobiose, citrate, D-fructose, D-galactose, D-glucose, glycerol, L-histidine, L-lactate, maltose, D-mannitol, salicin, starch, sucrose and D-trehalose. Does not use adonitol, dulcitol, inositol, lactose, D-mannose, D-melezitose, D-melibiose, D-raffinose, L-rhamnose, sorbitol, L-sorbose or D-xylose. Resistant to amoxycillin/clavulanic acid (30 µg), ampicillin (10 µg), erythromycin (15 µg) and penicillin G (10 µg), shows intermediate sensitivity to cefuroxime (30 µg) and streptomycin (10 µg) and is sensitive to amikacin (30 µg), ceftriaxone (30 µg), ciprofloxacin (5 µg), colistin (50 µg), gentamicin (10 µg), imipenem (10 µg), polymyxin B (300 U), tetracycline (30 µg), tobramycin (10 µg) and trimethoprim/sulfamethoxazole (1.25/23.75 µg). Strain 665N is resistant to cephalothin (30 µg), cefoxitin (30 µg) and ticarcillin (75 µg), whereas strain 868ET is not. The DNA G+C content is 62.3 mol% for strain 665N and 62.6 mol% for strain 868ET.
The type strain, 868ET (=CECT 7113T=LMG 23376T), was isolated from cockles (Cardium sp.) obtained from a retail market in Barcelona (Spain). Strain 665N (=CECT 7112=LMG 23377) is a second strain of the species.
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ACKNOWLEDGEMENTS |
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