| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Sector 39A, Chandigarh 160 036, India
Correspondence
T. Chakrabarti
tapan{at}imtech.res.in
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
A transmission electron micrograph of a cell of strain GPTSA-6T is available as a supplementary figure in IJSEM Online.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). Aeromonads are autochthonous to aquatic environments worldwide and are the usual microbiota of fish, amphibians and other animals (Miñana-Galbis et al., 2004). At the time of writing, the family Aeromonadaceae is represented by four genera, Aeromonas, Tolumonas, Oceanimonas and Oceanisphaera (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Root). Of these genera, Aeromonas is the largest, containing 20 species plus 12 subspecies. Tolumonas, Oceanimonas and Oceanisphaera contain one, three and one species, respectively (http://www.bacterio.cict.fr/index.html). In the present communication, we report the taxonomic characterization of strain GPTSA-6T, and, on the basis of the polyphasic analysis, propose that it represents a novel species of the genus Aeromonas.
Strain GPTSA-6T was isolated from water sampled from a warm spring, using dilution plating on TSBA (tryptic soy broth plus 1.5 % agar; HiMedia) medium. All phenotypic characterizations were carried out according to standard methods (Cowan & Steel, 1965; Smibert & Krieg, 1994; Murray et al., 1994; Powers, 1995). Growth at various temperatures, pH values and NaCl concentrations was checked on basal TSBA medium. The cells of strain GPTSA-6T were found to be Gram-negative, motile, short rods. Transmission electron microscopy, performed as described previously (Pandey et al., 2002), demonstrated the presence of a single polar flagellum on each cell (see Supplementary Fig. S1 available in IJSEM Online). Detailed characteristics are given in the species description.
Antibiotic sensitivity was checked on Mueller–Hinton agar, using antibiotic-susceptibility discs (HiMedia) at the following antibiotic concentrations: ampicillin (10 µg), bacitracin (8 U), cephalothin (30 µg), chloramphenicol (30 µg), colistin (10 µg), erythromycin (15 µg), gentamicin (10 µg), kanamycin (30 µg), lincomycin (2 µg), meticillin (5 µg) neomycin (30 µg), nitrofurantoin (300 µg), norfloxacin (10 µg), novobiocin (30 µg), penicillin G (10 U), polymyxin B (300 U), rifampicin (2 µg), streptomycin (10 µg), sulfasomidine (300 µg) and tetracycline (3 µg). Susceptibility to the vibriostatic compound O/129 (150 µg) was checked on TSBA medium.
For cellular fatty acid analyses, the strain was grown on TSBA medium at 30 °C for 24 h. Extraction and analysis of the cellular fatty acids were performed according to the procedures for the SHERLOCK Microbial Identification system (MIDI) as described previously (Pandey et al., 2002). The fatty acid profile of strain GPTSA-6T showed a predominance of saturated and unsaturated unbranched fatty acids and included C16 : 0 (29.0 %), summed feature 3 (C16 : 1
7c and/or C15 : 0 iso 2-OH; 29.3 %), C18 : 17c (24.0 %), C12 : 0 (6.1 %), summed feature 2 (C14 : 0 3-OH and/or C16 : 1 iso I; 5.6 %) and C14 : 0 (4.7 %). An increased in the incubation time (to 48 h at 30 °C) did not seem to alter the overall fatty acid profile of the strain.
The genomic G+C content of the strain was determined spectrophotometrically as described previously (Saha et al., 2005). The G+C content of the strain was found to be 60.7 mol%.
Amplification of the 16S rRNA gene from strain GPTSA-6T was done with primers 27f (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492r (5'-TACGGYTACCTTGTTACGACTT-3'). The amplification reaction and purification of amplicons were performed as described previously (Pandey et al., 2002). The amplified product was sequenced by the dideoxy chain terminator method, using the BigDye Terminator kit (Perkin-Elmer), followed by capillary electrophoresis on an ABI 310 Genetic Analyzer (Applied Biosystems). An almost-complete (1430 nt) 16S rRNA gene sequence of strain GPTSA-6T was used as the query to search for homologous sequences in the GenBank database. Sequence analysis revealed that its closest relative (99.23 % similarity) was an uncultured bacterial clone, A-8, reported during analysis of dissolved organic matter and a bacterial community involved in the degradation of an algal bloom (Kasuga et al., 2003). With regard to cultured bacteria, strain GPTSA-6T showed most similarity with Aeromonas sobria ATCC 43979T (95.13 %) followed by Aeromonas molluscorum 848TT (95.04 %), Aeromonas popoffii LMG 17541T (95.04 %), Aeromonas eucrenophila ATCC 2309T (94.92 %), Aeromonas encheleia CECT 4342T (94.90 %), Aeromonas salmonicida ATCC 33658T (94.85 %), Aeromonas veronii ATCC 35624T (94.85 %), Aeromonas caviae ATCC 15468T (94.84 %), Aeromonas bestiarum CIP 7430T (94.77 %), Aeromonas allosaccharophila CECT 4199T (94.77 %) and Aeromonas hydrophila ATCC 7966T (94.77 %). Sequence similarity with other species of the genus Aeromonas was less than 94.77 %. The strain showed much less sequence similarity with Tolumonas auensis DSM 9187T (91.95 %), Oceanimonas doudoroffii ATCC 27123T (90.70 %) and Oceanisphaera litoralis DSM 15406T (90.57 %), the type strains of the type species of the three other genera within the family Aeromonadaceae. Sequences from its closest uncultured relative and 22 type strains representing different species of the genera Aeromonas, Tolumonas, Oceanimonas and Oceanisphaera of the family Aeromonadaceae and of the genus Vibrio within the family Vibrionaceae were used for phylogenetic analysis. All these sequences were aligned by the CLUSTAL_X program (Thompson et al., 1997) and edited manually. Aligned sequences were analysed by the PHYLIP software package version 3.5c (Felsenstein, 1993). Pairwise evolutionary distances for the aligned sequences were computed using the DNADIST program with the Kimura two-parameter model (Kimura, 1980). To obtain a confidence value for the aligned sequence dataset, bootstrap analysis of 100 replications was done using SEQBOOT. A phylogenetic tree showing the relationship between GPTSA-6T and other reference strains was constructed using the neighbour-joining method (Saitou & Nei, 1987) and the UPGMA algorithm. Distance matrix data obtained from DNADIST were also used to construct a phylogenetic tree, by using KITSCH. Consensus trees for each of these methods were generated using CONSENSE from the PHYLIP package. Distance-based phylogenetic analysis was also performed with the TREECON software package (Van de Peer & De Wachter, 1997), using both Kimura (Kimura, 1980) and Jukes–Cantor (Jukes & Cantor, 1969) correction. Irrespective of the tree-generation software packages used, the overall tree topologies were similar in all cases. Phylogenetic analyses revealed that strain GPTSA-6T falls within the radiation of the family Aeromonadaceae (neighbour-joining analysis shown in Fig. 1). However, together with its closest uncultured bacterial relative, it formed a cluster that was well separated from the Aeromonas cluster and the Tolumonas–Oceanimonas–Oceanisphaera cluster with a very high bootstrap value.
|
95.13 %) strongly indicate that this strain represents a novel species. It is generally accepted that, when 16S rRNA gene sequence similarity is lower than 97 %, overall genomic relatedness is less than 70 % (Stackebrandt & Goebel, 1994), and these two criteria are considered crucial for prokaryotic species delineation. On the basis of phenotypic properties, strain GPTSA-6T could be differentiated from its five closest phylogenetic relatives belonging to the genus Aeromonas (Table 1). The type strains of Oceanimonas doudoroffii and Oceanisphaera litoralis are both strictly aerobic, negative for aesculin hydrolysis, can reduce nitrate to nitrite, can grow at 10 °C, tolerate 7 % NaCl, cannot grow in medium lacking NaCl, all of which contrast with the properties of strain GPTSA-6T, and they have G+C contents of 54 and 56.4 mol%, respectively (Baumann et al., 1983; Brown et al., 2001; Romanenko et al., 2003). Similarly, T. auensis differs from strain GPTSA-6T in being non-motile, in being unable to utilize cellobiose, D-lactose, maltose, D-mannitol, D-mannose and sucrose but being able to hydrolyse Tweens 40 and 80, and in having a comparatively low genomic G+C content (49 mol%) (Fischer-Romero et al., 1996). In terms of the fatty acid composition, fatty acids C13 : 0 iso, C15 : 0, C17 : 0, C17 : 18c, C15 : 0 iso, C16 : 0 iso, C17 : 0 iso, C17 : 19c iso and C16 : 19c alcohol, which are present in many members of the genus Aeromonas (Huys et al., 1994), were not detectable in strain GPTSA-6T. On the whole, GPTSA-6T is phylogenetically related to the members of the genus Aeromonas. Thus, on the basis of the results of our polyphasic approach, we conclude that strain GPTSA-6T represents a novel species within the genus Aeromonas, for which we propose the name Aeromonas sharmana sp. nov.
|
Gram-negative, facultatively anaerobic, mesophilic, motile, short rods occurring in singly or in pairs. Single polar flagellum. Colonies on TSBA after 36 h growth are round, convex with slightly irregular margins, opaque and whitish in colour without the production of any diffusible pigment. Cells are 1–2 µm long and 0.4–0.5 µm wide. Oxidase-positive and very weakly positive for catalase. Grows at temperatures between 15 and 42 °C, at pH 5.7–8.0 and can tolerate 2 % NaCl. Does not produce indole, gas from glucose, H2S, gelatinase, phenylalanine deaminase, DNase, arginine dihydrolase, lysine decarboxylase or ornithine decarboxylase. Hydrolyses starch, aesculin and ONPG but not casein, urea, fat or Tweens 20, 40 and 80. Does not reduce nitrate to nitrite. Produces acid from arbutin, D-amygdalin, cellobiose, fructose, D-glucose, D-galactose, inulin, D-maltose, D-mannitol, D-mannose, salicin, sucrose and trehalose but not from adonitol, L-arabinose, D-arabinose, L-arabitol, dulcitol, glycerol, myo-inositol, D-melibiose, D-melezitose, D-raffinose, L-rhamnose, D-ribose, D-sorbitol, L-sorbose, xylitol, D-xylose and L-xylose. Utilizes L-arabinose, D-cellobiose, D-fructose, D-glucose, D-galactose, D-lactose, D-mannose, D-maltose, sucrose and D-xylose (weakly) but not D-arabinose, L-arabitol, arbutin, D-amygdalin, adonitol, dulcitol, glycerol, melezitose, L-rhamnose, D-ribose, D-sorbitol, L-sorbose, acetate, citrate, fumarate, glutarate, malate, propionate or succinate as sole carbon sources. The type strain is resistant to the vibriostatic compound O/129, lincomycin and methicillin but is susceptible to ampicillin, bacitracin, cephalothin, chloramphenicol, colistin, erythromycin, gentamicin, kanamycin, neomycin, nitrofurantoin, novobiocin, polymyxin B, penicillin G, rifampicin, streptomycin, sulfasomidine and tetracycline. The major whole-cell fatty acids are C16 : 0 (29.0 %), summed feature 3 (C16 : 17c and/or C15 : 0 iso 2-OH; 29.3 %), C18 : 17c (24.0 %), C12 : 0 (6.1 %), summed feature 2 (C14 : 0 3-OH and/or C16 : 1 iso I; 5.6 %) and C14 : 0 (4.7 %). The genomic DNA G+C content is 60.7 mol%.
The type strain, GPTSA-6T (=MTCC 7090T=DSM 17445T), was isolated from a water sample from a warm spring in Assam, India.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Brown, G. R., Sutcliffe, I. C. & Cummings, S. P. (2001). Reclassification of [Pseudomonas] doudoroffii (Baumann et al. 1983) into the genus Oceanimonas gen. nov. as Oceanimonas doudoroffii comb. nov., and description of a phenol-degrading bacterium from estuarine water as Oceanimonas baumannii sp. nov. Int J Syst Evol Microbiol 51, 67–72.[Abstract]
Colwell, R. R., MacDonell, M. T. & De Ley, J. (1986). Proposal to recognize the family Aeromonadaceae fam. nov. Int J Syst Bacteriol 36, 473–477.
Cowan, S. T. & Steel, K. J. (1965). Manual for the Identification of Medical Bacteria. London: Cambridge University Press.
Felsenstein, J. (1993). PHYLIP (phylogeny inference package), version 3.5c. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, USA.
Fischer-Romero, C., Tindall, B. J. & Jüttiner, F. (1996). Tolumonas auensis gen. nov., sp. nov., a toluene-producing bacterium from anoxic sediments of a freshwater lake. Int J Syst Bacteriol 46, 183–188.
Harf-Monteil, C., Le Flèche, A., Riegel, P., Prévost, G., Bermond, D., Grimont, P. A. D. & Monteil, H. (2004). Aeromonas simiae sp. nov., isolated from monkey faeces. Int J Syst Evol Microbiol 54, 481–485.
Holt, J. G., Krieg, N. R., Sneath, P. H. A., Staley, J. T. & Williams, S. T. (1994). Bergey's Manual of Determinative Bacteriology, 9th edn. Baltimore: Williams & Wilkins.
Huys, G., Vancanneyt, M., Coopman, R., Janssen, P., Falsen, E., Altwegg, M. & Kersters, K. (1994). Cellular fatty acid composition as chemotaxonomic marker for the differentiation of phenospecies and hybridization groups in the genus Aeromonas. Int J Syst Bacteriol 44, 651–658.
Huys, G., Kämpfer, P., Altwegg, M. & 7 other authors (1997). Aeromonas popoffii sp. nov., a mesophilic bacterium isolated from drinking water production plants and reservoirs. Int J Syst Bacteriol 47, 1165–1171.
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
Kasuga, I., Nakajima, F. & Furumai, H. (2003). Analysis of dissolved organic matter and bacterial community in degradation of algal bloom by EEMS and PCR-DGGE. J Jpn Soc Water Environ 26, 171–174 (in Japanese).
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]
Miñana-Galbis, D., Farfán, M., Fusté, C. & Lorén, J. G. (2004). Aeromonas molluscorum sp. nov., isolated from bivalve molluscs. Int J Syst Evol Microbiol 54, 2073–2078.
Murray, R. G. E., Doetsch, R. N. & Robinow, C. F. (1994). Determinative and cytological light microscopy. In Methods for General and Molecular Bacteriology, pp. 21–41. Edited by P. Gerhard, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.
Pandey, K. K., Mayilraj, S. & Chakrabarti, T. (2002). Pseudomonas indica sp. nov., a novel butane-utilizing species. Int J Syst Evol Microbiol 52, 1559–1567.[Abstract]
Popoff, M. (1984). Genus III Aeromonas Kluvyer and Van Niel 1936, 398AL. In Bergey's Manual of Systematic Bacteriology, vol. 1, pp. 545–548. Edited by N. R. Krieg & J. G. Holt. Baltimore: Williams & Wilkins.
Powers, E. M. (1995). Efficacy of the Ryu nonstaining KOH technique for rapidly determining gram reactions of food-borne and waterborne bacteria and yeast. Appl Environ Microbiol 61, 3756–3758.[Abstract]
Romanenko, L. A., Schumann, P., Zhukova, N. V., Rohde, M., Mikhailov, V. V. & Stackebrandt, E. (2003). Oceanisphaera litoralis gen. nov., sp. nov., a novel halophilic bacterium from marine bottom sediments. Int J Syst Evol Microbiol 53, 1885–1888.
Saha, P., Krishnamurthi, S., Mayilraj, S., Prasad, G. S., Bora, T. C. & Chakrabarti, T. (2005). Aquimonas voraii gen. nov., sp. nov., a novel gammaproteobacterium isolated from a warm spring of Assam, India. Int J Syst Evol Microbiol 55, 1491–1495.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for General and Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Van de Peer, Y. & De Wachter, R. (1997). Construction of evolutionary distance trees with TREECON for Windows: accounting for variation in nucleotide substitution rate among sites. Comput Appl Biosci 13, 227–230.
This article has been cited by other articles:
|
|
 |
|
  A. J. Martinez-Murcia, M. J. Saavedra, V. R. Mota, T. Maier, E. Stackebrandt, and S. Cousin Aeromonas aquariorum sp. nov., isolated from aquaria of ornamental fish Int J Syst Evol Microbiol, May 1, 2008; 58(5): 1169 - 1175. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
