ARTICLE IN PRESS Systematic and Applied Microbiology 33 (2010) 15–19
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Proposal to assign Aeromonas diversa sp. nov. as a novel species designation for Aeromonas group 501 ˜ ana-Galbis, Maribel Farfa´n, J. Gaspar Lore´n, M. Carmen Fuste´ n David Min Universitat de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain Facultat de Farmacia, Departament de Microbiologia i Parasitologia Sanitaries,
a r t i c l e in fo
abstract
Article history: Received 21 July 2009
The Aeromonas group 501, also named Aeromonas sp. HG13, is taxonomically close to A. schubertii. Results obtained in previous studies, including DNA–DNA hybridization and DNA fingerprinting, suggest that Aeromonas group 501 could constitute a different Aeromonas species. In this work we have performed a polyphasic study with the two strains comprising the Aeromonas sp. HG13 in order to propose a formal species name. They could be differentiated from A. schubertii by the indole and lysine decarboxylase tests and the utilization of L-lactate. Phenotypically, both strains were also easily separated from the other Aeromonas species. Sequence analysis of the 16S rRNA gene showed high sequence similarities ( 4 97%) between Aeromonas group 501 and all Aeromonas species. Nevertheless, sequence divergences of cpn60, dnaJ, gyrB and rpoD genes were higher than the intraspecific threshold values established for each gene (3.5%, 3.3%, 2.3% and 2.6%, respectively), while sequence divergences between strains CDC 2478-85T and CDC 2555-87 were low (0.6–1.1%). The DNA G +C content of the type strain was 62.2 mol%. Phenotypic and genotypic evidence strongly suggests that the Aeromonas group 501 is a novel species of the genus Aeromonas, for which the name Aeromonas diversa sp. nov. is proposed. The type strain is CDC 2478-85T (=CECT 4254T =ATCC 43946T = LMG 17321T). & 2009 Elsevier GmbH. All rights reserved.
Keywords: Aeromonas diversa sp. nov. Aeromonas group 501 Aeromonas sp. HG13 Housekeeping genes
Introduction Aeromonads inhabit a wide variety of ecological niches. They are primarily considered aquatic, even though they are frequently isolated from sediments, biofilms and foods, associated with aquatic animals and several species are primary or opportunistic pathogens in invertebrates and vertebrates including humans [16]. The taxonomy of the genus Aeromonas is complex due to the continuous description of novel species, new information about those previously described [8,10] and the rearrangement of strains and species described thus far [4,10,11,13,14]. The genus Aeromonas belongs to the family Aeromonadaceae within the Gammaproteobacteria, comprising the following species: A. allosaccharophila, A. bestiarum, A. bivalvium, A. caviae, A. encheleia, A. eucrenophila, A. fluvialis, A. hydrophila, A. jandaei, A. media, A. molluscorum, A. piscicola, A. popoffii, A. salmonicida, A. schubertii, A. simiae, A. sobria, A. tecta, A. trota and A. veronii[1,2,5,16,23]. Another species included in the genus, A. sharmana, has recently been questioned as a member of the genus Aeromonas on the basis
Abbreviations: FAFLP, fluorescence amplified fragment length polymorphism; HG, hybridization group n Corresponding author. Tel.: + 34 93 402 44 97; fax: + 34 93 402 44 98. E-mail address:
[email protected] (M. Carmen Fuste´). 0723-2020/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2009.11.002
of the 16S rRNA gene, cpn60, gyrB and rpoD sequences [18,24,28]. In addition, the taxonomic situation of A. aquariorum is also controversial since it is considered to be a later synonym of A. hydrophila subsp. dhakensis[12,19,20]. Although there have been many recent contributions to the taxonomy of the genus Aeromonas, there are still certain controversial taxa that need to be clarified. One of them is the Aeromonas group 501, constituted by two strains taxonomically close to A. schubertii. This group remains without a formal species name although it was described in 1988 [9] and proposed as DNA hybridization group 13 (HG13) in 1991 [3]. We have performed a polyphasic study with the two strains that constitute Aeromonas sp. HG13 (group 501), CDC 2478-85T ( = CECT 4254T = ATCC 43946T = LMG 17321T) and CDC 2555-87 (= CECT 5178= ATCC 700064) in order to propose a species name.
Materials and methods Strains CDC 2478-85 and CDC 2555-87 were obtained from the ˜ ola de Cultivos Tipo’’ as Aeromonas sp. CECT 4254 ‘‘Coleccio´n Espan and Aeromonas sp. CECT 5178. Strain CDC 2478-85 was isolated from the leg wound of a patient in New Orleans (LA, USA) and was originally described as Enteric Group 501 and distinguished from A. schubertii by DNA–DNA hybridization and phenotypical characterization [9]. Strain CDC 2555-87 was also isolated from
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a leg wound of a patient in New Orleans and was originally described as biochemically similar and highly related by DNA– DNA hybridization to strain CDC 2478-85 in an Addendum in Proof in the same paper [9]. The following physiological and biochemical tests were performed at 30 1C and determined as described previously [23]: Gram-staining, motility, glucose oxidation–fermentation, oxidase and catalase activity, nitrate reduction, susceptibility to O/129, swarming motility, indole production, production of a brown diffusible pigment, gas production from D-glucose, methyl red and Voges–Proskauer reactions, arginine dihydrolase and lysine and ornithine decarboxylase activity (Moeller’s method), ONPG, hydrogen sulphide production from cysteine and thiosulfate, growth on MacConkey agar, m-Aeromonas-selective agar base Havelaar and TCBS agar, salt tolerance, pH and temperature ranges for growth, hydrolysis of aesculin, arbutin, DNA, elastin, erythrocytes, gelatine, starch and xanthine, acid production from 17 carbohydrates, utilization of 32 substrates as sole carbon and energy sources and sensitivity to 18 antibiotics. The type strain of A. schubertii (CIP 103437T) was also included in this phenotypic study. Genomic DNA from the type strain of Aeromonas sp. HG13 was prepared using a modification of the procedure of Wilson [31] for the determination of DNA G +C content. The G +C content was determined by three independent analyses via the HPLC technique [21]. These analyses were performed by the BCCMTM/LMG Identification Service. DNA extraction, PCR amplification and sequencing of gyrB for the strains CDC 2478-85T and CDC 2555-87 and 16S rRNA, cpn60, dnaJ and rpoD genes for the strain CDC 2555-87 were performed using previously described methods [15,23,24,25,29]. Although the 16S rRNA gene sequence from the type strain CDC 2478-85T ( =CECT 4254T) is deposited in the GenBank (accession number U88663), it was also sequenced because Martı´nez-Murcia [17] found two inconsistencies in this sequence. Sequences of cpn60, dnaJ and rpoD genes from the strain CDC 2478-85T, some of them previously obtained by us, were taken from the GenBank. Sequences of 16S rRNA, cpn60, dnaJ, gyrB and rpoD genes from strains CDC 2478-85T and CDC 2555-87 were aligned independently with gene sequences of the type strains of all recognized
Aeromonas species (Table S1), except A. sharmana. Concatenated sequences of cpn60, dnaJ, gyrB and rpoD were also analyzed. Sequence alignment and phylogenetic analyses (maximum parsimony and neighbour-joining methods) were conducted by the MEGA software version 4.0 [30]. The topological robustness of the phylogenetic trees was evaluated by a bootstrap analysis through 1000 replicates.
Results and discussion We have recharacterized the Aeromonas group 501 obtaining identical results to the original description [9] and adding new phenotypical tests. Strains CDC 2478-85T and CDC 2555-87 showed identical phenotypic features, except in the case of Voges–Proskauer test which was positive for the strain CDC 247885T and negative for the CDC 2555-87. The Aeromonas group 501 was easily differentiated because it differed from each of the Aeromonas species in at least two of the 16 key tests shown in Table 1. Aeromonas HG13 could be differentiated from its closer Aeromonas species, A. schubertii, by the following key tests: indole, lysine decarboxylase and utilization of L-lactate (Table 1). Additionally, HG13 strains could be separated from A. schubertii CIP 103437T, because they showed b-hemolysis, and were negative for the ONPG test, fermentation of D-galactose and Dmannose and utilization of citrate, D-galactose, D-melezitose and D-xylose, and were resistant to cephalothin and cephoxitin. Therefore, phenotypically, the Aeromonas group 501 (HG13) shows enough differences from the other formally described Aeromonas to be considered a new species of this genus. Strains CDC 2478-85T and CDC 2555-87 showed identical sequences for the 16S rRNA gene (1504 nt). These results confirmed the same inconsistencies found by Martı´nez-Murcia [17] in the sequence U88663. Consequently, we propose that the new sequence obtained from the type strain of HG13 (GenBank accession number GQ365710) substitutes the one previously reported. 16S rRNA gene sequence similarity between Aeromonas sp. HG13 and other Aeromonas species ranged from 97.83% (31 nt differences) to 99.13% (13 nt differences), except in the case of A. schubertii, which showed a divergence of 99.67%
Table 1 Key tests for the phenotypic differentiation of strains CDC 2478-85T and CDC 2555-87 from other mesophilic Aeromonas species. Characteristic Production of Brown pigment Gas from D-glucose H2S from cysteine Indole Lysine decarboxylase Ornithine decarboxylase Voges–Proskauer reaction Acid from L-Arabinose D-Mannitol Sorbitol Sucrose Hydrolysis of Aesculin Arbutin Elastin Starch Utilization of L-lactate
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
– – – + – – V
– + + + + V –
– + + + + – +
– + + + + – +
– – – + + – –
– – – + – – –
– + + + – – –
– + + + – – –
– + – + – – –
– + + + + – +
– + + + + – +
+ – – + – – –
– – – – – – –
– + + + + – +
– + + + – – +
– + + + + – +
– – – – + – +
– – – – + – –
– + + + + – +
– + ND – V – V
– + + + + – –
– + + + + – +
– + V + + + +
– – – –
+ + – +
– + – +
+ + – +
+ + – +
+ + – +
– + – V
+ + – V
– – – +
V + – +
– + – V
+ + – +
+ + – +
– + V +
V + – –
+ + + +
– – – –
– – – +
– + – +
– + – –
– + – –
V + – +
– + – +
– – – + –
+ – – – V
+ + ND + –
+ + V + –
+ + – + +
+ + – + V
+ + – + –
+ + – + –
– ND – ND ND
+ + + + +
– – V + –
+ + – + +
+ + – – V
+ ND + + –
– – – + +
+ + + + –
– – – + +
– + – + ND
– – – + –
V ND ND ND –
– V – – +
– – – – –
+ + – + –
Taxa are identified as: 1, Strains CDC 2478-85T and CDC 2555-87 (data from this study); 2, A. allosaccharophila; 3, A. aquarioruma; 4, A. bestiarum; 5, A. bivalvium; 6, A. caviae; 7, A. encheleia; 8, A. eucrenophila; 9, A. fluvialis; 10, A. hydrophila; 11, A. jandaei; 12, A. media; 13, A. molluscorum; 14, A. piscicola; 15, A. popoffii; 16, A. salmonicida; 17, A. schubertii; 18, A. simiae; 19, A. sobria; 20, A. tecta; 21, A. trota; 22, A. veronii bv. sobria; 23, A. veronii bv. veronii. Abbreviations: + , 85–100% of strains positive;–, 0–15% of strains positive; V, 16–84% of strains positive; ND, no data available. Data taken from [1], [2], [5], [16] and [23], except those for strains CDC 2478-85T and CDC 2555-87. a A. aquariorum is considered to be a later synonym of A. hydrophila subsp. dhakensis[12,19,20].
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Fig. 1. Consensus neighbour-joining phylogenetic tree obtained from 16S rRNA gene sequences encompassing Aeromonas sp. HG13 and all type strains of the genus Aeromonas.
(5 nt differences). A Jukes–Cantor neighbour-joining tree is shown in Fig.1, dendrograms generated by neighbour-joining (Jukes–Cantor, Kimura-two-parameter and Tamura–Nei models) and maximum parsimony methods were almost identical (data not shown). The DNA G+ C content of the type strain was 62.2 mol%, which agrees with the range described for the genus Aeromonas (57– 63 mol%) [16]. In the description of A. schubertii[9], the strain CDC 2478-85T later considered as Aeromonas HG 13 was included in the DNA hybridization experiments but it was not considered as belonging to this species due to its level of divergence (70% at 60 1C and 61% at 75 1C, with a variation of 5%). Other DNA hybridization determinations have shown a low DNA relatedness ( o30%) between the type strain of HG13 and some type strains of Aeromonas species [3,6,7,22,26]. Hickman-Brenner et al. [9] also carried out DNA hybridizations with the other strain of the Aeromonas group 501 (strain CDC 2555-87), which was only closely related to strain CDC 2478-85T (87% at 60 1C, 86% at 75 1C, with a divergence of 1%). These results suggested that the Aeromonas group 501 could constitute a different Aeromonas species [9,17]. The type strain of HG13 was also included in a previous analysis of the FAFLP [22,23]. Cluster analysis of FAFLP fingerprints revealed that the FAFLP pattern of this strain is unique and with a very low genotypic relatedness to other members of the genus Aeromonas ( o10%), including A. schubertii. These results
allow a clear genotypic discrimination of Aeromonas group 501 from other Aeromonas species. Sequences of cpn60, dnaJ, gyrB and rpoD genes were analyzed independently and concatenated. In all cases, phylogenetic trees generated by neighbour-joining (Jukes–Cantor, Kimura-two-parameter and Tamura–Nei models) and maximum parsimony methods were similar. As shown in the phylogenetic tree obtained from concatenated sequences (Fig. 2), strains CDC 2478-85T and CDC 2555-87 (HG13) clustered together in a separate phylogenetic line close to A. schubertii (100% bootstrap). These results were similar to other phylogenetic analyses [15,24,27]. Previous studies [24,25,29] have established the following intraspecific threshold values based on divergences of gene sequences: 3.5% for cpn60, 3.3% for dnaJ, 2.3% for gyrB and 2.6% for rpoD. Sequence divergence between strains CDC 2478-85T and CDC 2555-87 was 1.1% for cpn60 and dnaJ, 0.6% for gyrB and 1% for rpoD, indicating that both strains belonged to the same species. Sequence divergence between both HG13 strains and the type strains of all Aeromonas species were higher than the threshold values. Similarly, the Aeromonas group 501 showed divergences of 5.6% for cpn60, 4.9–5.0% for dnaJ, 2.8–3.1% for gyrB and 4.5–4.8% for rpoD with respect to A. schubertii. Nevertheless, divergences observed when comparing with other Aeromonas species were even higher: 410% for cpn60, 47.5% for dnaJ, 49.5% for gyrB and 411.5% for rpoD, confirming that the Aeromonas group 501 represents a novel Aeromonas species.
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Fig. 2. Consensus neighbour-joining phylogenetic tree obtained from cpn60-dnaJ-gyrB-rpoD concatenated sequences encompassing Aeromonas sp. HG13 and all type strains of the genus Aeromonas.
Based on the results of phenotypic characterization, DNA–DNA hybridization, FAFLP analysis and sequence analyses from 16S rRNA, cpn60, dnaJ, gyrB and rpoD genes, we propose to assign Aeromonas diversa sp. nov. as a novel species designation for strains CDC 2478-85T( =CECT 4254T) and CDC 2555-87 ( = CECT 5178), named Aeromonas group 501 or Aeromonas sp. HG13.
Description of Aeromonas diversa sp. nov. Aeromonas diversa (di’ver.sa. L. fem. adj. diversa different, distinct) Cells are Gram-negative, straight, motile rods. Optimal growth occurs at 30–37 1C but growth occurs at up to 40.5 1C but not at 4 1C. Oxidase- and catalase-positive, reduces nitrate to nitrite, produces indole from tryptophan and is resistant to vibriostatic agent O/129 (150 mg). Positive for glucose oxidation–fermentation, arginine dihydrolase and methyl red tests. Negative for lysine decarboxylase, ornithine decarboxylase and ONPG tests. Strain CDC 2478-85T is positive for Voges–Proskauer test, whereas strain CDC 2555-87 is not. Brown diffusible pigment, swarming, gas from D-glucose and hydrogen sulphide from cysteine and thiosulfate are not produced. Grows on MacConkey agar and mAeromonas-selective agar base Havelaar but not on TCBS agar. Able to grow at pH 9 0 and 0–3% NaCl but not at pH 4.5 or 6% NaCl. Hydrolyses DNA, gelatin and starch, but not aesculin, arbutin, elastin or xanthine. Produces b-hemolysis and acid from dextrin and D-trehalose, but not from L-arabinose, arbutin,
D-cellobiose, D-galactose,
lactose, D-mannitol, D-mannose, D-melibiose, D-raffinose, L-rhamnose, salicin, sorbitol, D-sucrose or Dxylose. The following substrates are used as sole carbon and energy sources: acetate, N-acetylglucosamine, L-arginine, D-fructose, D-glucose, L-histidine, maltose, starch and D-trehalose. Does not use adonitol, aesculin, L-arabinose, r-arbutin, D-cellobiose, citrate, dulcitol, D-galactose, inositol, inulin, L-lactate, lactose, Dmannose, D-mannitol, D-melezitose, D-melibiose, D-raffinose, Lrhamnose, salicin, sorbitol, L-sorbose, D-sucrose or D-xylose. Resistant to ampicillin (10 mg), cephalothin (30 mg), cefoxitin (30 mg), penicillin G (10 mg), streptomycin (10 mg) and tobramycin (10 mg), shows intermediate sensitivity to amikacin (30 mg), amoxycillin/clavulanic acid (30 mg) and erythromycin (15 mg) and is sensitive to ceftriaxone (30 mg), ciprofloxacin (5 mg), colistin (50 mg), gentamicin (10 mg), imipenem (10 mg), polymyxin B (300 U), tetracycline (30 mg), ticarcillin (75 mg) and trimethoprim/sulfamethoxazole (1.25/23.75 mg). A phenotypic comparison with other related Aeromonas is provided in Table 1. The DNA G+ C content is 62.2 mol%. The type strain is CDC 2478-85T (= CECT 4254T =ATCC 43946T = LMG 17321T). The GenBank accession numbers for the 16S rRNA gene and gyrB sequences of A. diversa strains CDC 2478-85T and CDC 2555-87, and for the rpoD, cpn60 and dnaJ sequences of A. diversa strain CDC 2555-87 are GQ365710, GU062400, GQ365711, GU062401, GQ365712, GQ365713 and GQ365714, respectively (GenBank accession numbers of cpn60, dnaJ, gyrB and rpoD sequences used in this study are shown in Table S1, which are available online).
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Acknowledgements ¨ and Marina Diana for help and the We thank Mariana S. Guel Serveis Cientificote cnics of the Universitat de Barcelona (Unitat de Genomica) for technical assistance. We would like to thank Drs. A.J. Martı´nez-Murcia and M.J. Figueras for kindly providing type strains of A. aquariorum, A. fluvialis, A. piscicola and A. tecta. This work has been supported by Project CGL2008–03281/BOS from the Ministerio de Ciencia e Innovacio´n, Spain.
[13]
[14]
[15]
[16]
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.syapm.2009.11.002.
[17]
[18]
References
[19]
[1] Alperi, A., Martı´nez-Murcia, A.J., Monera, A., Saavedra, M.J., Figueras, M.J. Aeromonas fluvialis sp. nov., isolated from Spanish river, Int. J. Syst. Evol. Microbiol., in press doi: 10.1099/ijs.0.011643-0. [2] Beaz-Hidalgo, R., Alperi, A., Figueras, M.J., Romalde, J.L., 2009. Aeromonas piscicola sp. nov., isolated from diseased fish. Syst. Appl. Microbiol. 32, 471– 479. [3] Carnahan, A.M., Behram, S., Joseph, S.W., 1991. Aerokey II: a flexible key for identifying clinical Aeromonas species. J. Clin. Microbiol. 29, 2843–2849. [4] Collins, M.D., Martı´nez-Murcia, A.J., Cai, J., 1993. Aeromonas enteropelogenes and Aeromonas ichthiosmia are identical to Aeromonas trota and Aeromonas veronii, respectively, as revealed by small-subunit rRNA sequence analysis. Int. J. Syst. Bacteriol. 43, 855–856. ¨ [5] Demarta, A., Kupfer, M., Riegel, P., Harf-Monteil, C., Tonolla, M., Peduzzi, R., Monera, A., Saavedra, M.J., Martı´nez-Murcia, A., 2008. Aeromonas tecta sp. nov., isolated from clinical and environmental sources. Syst. Appl. Microbiol. 31, 278–286. [6] Esteve, C., Gutie´rrez, M.C., Ventosa, A., 1995. DNA relatedness among Aeromonas allosaccharophila strains and DNA hybridization groups of the genus Aeromonas. Int. J. Syst. Bacteriol. 45, 390–391. [7] Esteve, C., Gutie´rrez, M.C., Ventosa, A., 1995. Aeromonas encheleia sp. nov., isolated from European eels. Int. J. Syst. Bacteriol. 45, 462–466. [8] C. Esteve, L. Valera, C. Gutie´rrez, A. Ventosa, Taxonomic study of sucrosepositive Aeromonas jandaei-like isolates from faeces, water and eels: emendation of A. jandaei Carnahan et al. 1992, Int. J. Syst. Evol. Microbiol. 53 (2003) 1411–1419. [9] Hickman-Brenner, F.W., Fanning, G.R., Arduino, M.J., Brenner, D.J., Farmer III, J.J., 1988. Aeromonas schubertii, a new mannitol-negative species found in human clinical specimens. J. Clin. Microbiol. 26, 1561–1564. ¨ [10] Huys, G., Kampfer, P., Altwegg, M., Coopman, R., Janssen, P., Gillis, M., Kersters, K., 1997. Inclusion of Aeromonas DNA hybridization group 11 in Aeromonas encheleia and extended descriptions of the species Aeromonas eucrenophila and A. encheleia. Int. J. Syst. Bacteriol. 47, 1157–1164. ¨ [11] Huys, G., Kampfer, P., Swings, J., 2001. New DNA–DNA hybridization and phenotypic data on the species Aeromonas ichthiosmia and Aeromonas allosaccharophila: A. ichthiosmia Schubert et al. 1990 is a later synonym of A. veronii Hickman-Brenner et al. 1987. Syst. Appl. Microbiol. 24, 177–182. ¨ ¨ [12] Huys, G., Kampfer, P., Albert, M.J., Kuhn, I., Denys, R., Swings, J., 2002. Aeromonas hydrophila subsp. dhakensis subsp. nov., isolated from children with diarrhoea in Bangladesh, and extended description of Aeromonas
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27] [28] [29]
[30]
[31]
19
hydrophila subsp. hydrophila (Chester 1901) Stanier 1943 (approved lists 1980). Int. J. Syst. Evol. Microbiol. 52, 705–712. Huys, G., Denys, R., Swings, J., 2002. DNA–DNA reassociation and phenotypic data indicate synonymy between Aeromonas enteropelogenes Schubert et al. 1990 and Aeromonas trota Carnahan et al. 1991. Int. J. Syst. Evol. Microbiol. 52, 1969–1972. Huys, G., Cnockaert, M., Swings, J., 2005. Aeromonas culicicola Pidiyar et al. 2002 is a later subjective synonym of Aeromonas veronii Hickman-Brenner et al. 1987. Syst. Appl. Microbiol. 28, 604–609. ¨ Kupfer, M., Kuhnert, P., Korczak, B.M., Peduzzi, R., Demarta, A., 2006. Genetic relationships of Aeromonas strains inferred from 16 S rRNA, gyrB and rpoB gene sequences. Int. J. Syst. Evol. Microbiol. 56, 2743–2751. Martin-Carnahan, A., Joseph, S.W., 2005. Genus I. Aeromonas Stanier 1943, 213AL. In: second ed. In: Garrity, G.M., Brenner, D.J., Krieg, N.R., Staley, J.T. (Eds.), Bergey’s Manual of Systematic Bacteriology, vol. 2. Springer, New York, pp. 557–578. Martı´nez-Murcia, A.J., 1999. Phylogenetic positions of Aeromonas encheleia, Aeromonas popoffii, Aeromonas DNA hybridization group 11 and Aeromonas group 501. Int. J. Syst. Bacteriol. 49, 1403–1408. Martı´nez-Murcia, A.J., Figueras, M.J., Saavedra, M.J., Stackebrandt, E., 2007. The recently proposed species Aeromonas sharmana sp. nov., isolate GPTSA6T, is not a member of the genus Aeromonas. Int. Microbiol. 10, 61–64. Martı´nez-Murcia, A.J., Saavedra, M.J., Mota, V.R., Maier, T., Stackebrandt, E., Cousin, S., 2008. Aeromonas aquariorum sp. nov., isolated from aquaria of ornamental fish. Int. J. Syst. Evol. Microbiol. 58, 1169–1175. Martı´nez-Murcia, A., Monera, A., Alperi, A., Figueras, M.J., Saavedra, M.J., 2009. Phylogenetic evidence suggests that strains of Aeromonas hydrophila subsp. dhakensis belong to the species Aeromonas aquariorum sp. nov. Curr. Microbiol. 58, 76–80. Mesbah, M., Premachandran, U., Whitman, W.B., 1989. Precise measurement of the G+ C content of deoxyribonucleic acid by high-performance liquid chromatography. Int. J. Syst. Bacteriol. 39, 159–167. ˜ ana-Galbis, D., Farfa´n, M., Fuste´, M.C., Lore´n, J.G., 2004. Aeromonas Min molluscorum sp. nov., isolated from bivalve molluscs. Int. J. Syst. Evol. Microbiol. 54, 2073–2078. ˜ ana-Galbis, D., Farfa´n, M., Fuste´, M.C., Lore´n, J.G., 2007. Aeromonas Min bivalvium sp. nov., isolated from bivalve molluscs. Int. J. Syst. Evol. Microbiol. 57, 582–587. ˜ ana-Galbis, D., Urbizu-Serrano, A., Farfa´n, M., Fuste´, M.C., Lore´n, J.G., Min 2009. Phylogenetic analysis and identification of Aeromonas species based on sequencing of the cpn60 universal target. Int. J. Syst. Evol. Microbiol. 59, 1976–1983. Nhung, P.H., Hata, H., Ohkusu, K., Noda, M., Shah, M.M., Goto, K., Ezaki, T., 2007. Use of the novel phylogenetic marker dnaJ and DNA–DNA hybridization to clarify interrelationships within the genus Aeromonas. Int. J. Syst. Evol. Microbiol. 57, 1232–1237. Pidiyar, V., Kaznowski, A., Narayan, N.B., Patole, M., Shouche, Y.S., 2002. Aeromonas culicicola sp. nov., from the midgut of Culex quinquefasciatus. Int. J. Syst. Evol. Microbiol. 52, 1723–1728. Saavedra, M.J., Figueras, M.J., Martı´nez-Murcia, A.J., 2006. Updated phylogeny of the genus Aeromonas. Int. J. Syst. Evol. Microbiol. 56, 2481–2487. Saha, P., Chakrabarti, T., 2006. Aeromonas sharmana sp. nov., isolated from a warm spring. Int. J. Syst. Evol. Microbiol. 56, 1905–1909. ˜ ez, M.A., Chaco´n, M.R., Aguilera-Arreola, M.G., Catala´n, V., Soler, L., Ya´n Figueras, M.J., Martı´nez-Murcia, A.J., 2004. Phylogenetic analysis of the genus Aeromonas based on two housekeeping genes. Int. J. Syst. Evol. Microbiol. 54, 1511–1519. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599. Wilson, K., 1987. Preparation of genomic DNA from bacteria. In: Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K. (Eds.), Current Protocols in Molecular Biology. Greene Publishing and WileyInterscience, New York, pp. 2.4.1–2.4.5.
