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1 Departamento de Microbiología y Genética, Edificio Departamental, Lab. 209, Campus Miguel de Unamuno, Universidad de Salamanca, Spain
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany
Correspondence
Martha E. Trujillo
mett{at}usal.es
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ABSTRACT |
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The GenBank accession number for the 16S rRNA gene sequence of strain WA101T is AJ534886.
A least-squares phylogenetic tree derived from analysis of 16S rRNA gene sequences of selected Mycobacterium species is available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Dailloux et al., 1999; Khan et al., 2002; Rhodes et al., 2003; Willumsen et al., 2001; Vuorio et al., 1999). Strain WA101T was isolated from pond water near a uranium mine in Spain. It was able to grow at 4 °C and is therefore considered to be psychrotolerant; further characterization using a polyphasic approach was used to resolve its taxonomic position. The results indicate that strain WA101T represents a novel Mycobacterium species.
Strain WA101T was isolated by direct plating of 100 µl pond water on soil extract agar (SEA) at pH 6·5. SEA plates were incubated for 2 weeks at 28 °C in the dark. The isolate was further purified by streaking onto glucose/yeast extract agar (GYEA) plates (Gordon & Mihm, 1962).
Colony morphology, pigment production and photoreactivity were determined on GYEA. Gram and acid–alcohol-fast stains were carried out on 3-day-old cultures as described by Doetsch (1981). Growth was also determined on Löwenstein–Jensen, modified Bennett's and nutrient agar media at 28 °C.
Genomic DNA extraction and enzymic amplification of the 16S rRNA gene sequence were carried out as described by Rivas et al. (2001, 2003). An almost complete sequence of the 16S rRNA gene was obtained and aligned against other Mycobacterium species in the EMBL/GenBank databases using CLUSTAL X (Thompson et al., 1997). Phylogenetic trees were constructed using the neighbour-joining, least-squares and maximum-parsimony methods. In all cases, bootstrap analysis was based on 1000 resamplings. The MEGA2 package (Kumar et al., 2001) was used for all analyses.
Strain WA101T was characterized using the API 50CH, API 20NE and API ZYM systems according to the manufacturer's instructions (bioMérieux). API 50CH strips were incubated for 9 days, whereas API ZYM and API 20NE strips were incubated for 48 h at 28 °C. Oxidase activity was recorded as described by Rivas et al. (2003). The ability to grow at various temperatures (0–45 °C) and different NaCl concentrations (1, 3, 5, 7, 10 %) was tested on GYEA plates. Growth on MacConkey agar (without crystal violet) was also tested. Catalase and other physiological characteristics were assessed as recommended by Lévy-Frébault & Portaels (1992).
Susceptibility to antibiotics was examined using penicillin (10 U), ampicillin (2 µg), oxytetracycline (30 µg), neomycin (5 µg), cloxacillin (1 µg), erythromycin (2 µg), cefuroxime (30 µg), ciprofloxacin (5 µg), polymyxin B (300 IU) and gentamicin (10 µg) discs (Becton Dickinson) and GYEA as the basal medium. Readings were taken after 5 and 10 days.
For whole-cell fatty acid and mycolic acid analyses, the strain was grown on Middlebrook 7H10 medium supplemented with glycerol and OADC (DSMZ medium 645) for 7 days at 35 °C.
Lipid analyses were carried out using TLC, GLC and HPLC. TLC analyses of mycolic acids were performed with whole cell methanolysates from freeze-dried cells as described by Springer et al. (1995). Fatty acid methyl esters were obtained from cells after saponification, methylation and extraction as described by Schröder et al. (1997). For mycolic acid analyses of cells by HPLC, about 40 mg biomass was scraped from Petri dishes and saponified in KOH. The free mycolic acids were extracted and transformed to their bromophenacyl esters as described by Butler et al. (1992) and Miller (1997). A low and a high molecular mass internal standard (Ribi Immunochem. Research) were added to the samples. HPLC was operated using SHERLOCK System software (MIDI Inc.) under the conditions described by Willumsen et al. (2001).
G+C content of the DNA was determined using the thermal denaturation procedure of Mandel & Marmur (1968).
Genomic DNA for hybridization studies was isolated using a French pressure cell (Thermo Spectronic) and purified by chromatography on hydroxyapatite as described by Cashion et al. (1997). DNA–DNA hybridization experiments (in 2x SSC buffer plus 10 % v/v DMSO at 67 °C) was performed between strain WA101T and Mycobacterium sphagni DSM 44076T as described by De Ley et al. (1970) and modified by Huß et al. (1983). Renaturation rates were calculated using the TRANSFER.BAS program (Jahnke, 1992).
Strain WA101T grew on GYEA as bright orange, smooth, entire and shiny scotochromogenic colonies. Good growth was also observed on Bennett's modified and nutrient agar media. Growth on Löwenstein–Jensen agar strips was discrete. Cells were rod-shaped, non-motile and did not produce aerial hyphae, capsules or spores; they stained Gram-positive and were acid–alcohol-fast. Diffusible pigments were not observed on the media tested.
A 16S rRNA gene sequence of 1517 nt obtained for isolate WA101T was compared to those of all Mycobacterium species available from the EMBL/GenBank databases. The sequence of WA101T was aligned with 60 sequences that corresponded to rapidly and slowly growing mycobacteria, and the closest similarity was found with the sequences of M. sphagni (98·6 %) and Mycobacterium parafortuitum (98·4 %); these values correspond to 19 and 20 nt differences, respectively. A phylogenetic tree based on the least-squares method and Kimura's two-parameter algorithm was constructed with the 60 sequences (data not shown). Fig. 1 shows the relationship of WA101T with its nearest phylogenetic relatives based on a subset of the data analysed. A more complete tree is provided as supplementary data in IJSEM Online. The same branch was recovered using the neighbour-joining and maximum-parsimony methods. The 16S rRNA gene sequence of isolate WA101T also contained the characteristic short helix in the hypervariable region 18 (nt 451–483, Escherichia coli numbering) (Kirschner et al., 1993); 7 and 8 nucleotide differences were found within the species-specific region V2 of helix 10 between WA101T and M. parafortuitum and M. sphagni, respectively.
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-, keto- and 
-carboxymycolates together with eicosanol (wax-ester mycolates). This pattern is common among mycobacteria, including M. sphagni (Minnikin et al., 1984; Lévy-Frébault & Portaels, 1992). GLC analyses of the fatty acids of strain WA101T revealed the expected pattern diagnostic for members of the genus Mycobacterium. It was composed of straight chain saturated and unsaturated fatty acids, including cis-9 octadecanoic acid (24·92 %), hexadecanoic acid (20·46 %), tuberculostearic acid (7·16 %) and cis-6 hexadecanoic acid (6·98 %); minor amounts of other fatty acids were also detected. The alcohols detected previously by TLC eluted together with the fatty acids and were identified as octadecanol (C18 : 0 alcohol) and eicosanol (C20 : 0 alcohol).
Analyses of mycolic acids by HPLC revealed a UV-HPLC chromatogram characteristic of Mycobacterium species, with widely separated, double-peak clusters; prominent peaks are seen in the early clusters that emerge prior to 5 min (Fig. 2). M. sphagni is also found in this group (Butler & Guthertz, 2001). WA101T can easily be separated from M. sphagni and other species of this group on the basis of quantitative differences. Isolate WA101T was also distinguished from Mycobacterium chubuense, Mycobacterium obuense and M. parafortuitum on the basis of HPLC mycolic acid chromatograms as the latter species present triple-peak clusters, with those in the early cluster emerging before 5 min (Butler & Guthertz, 2001).
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lists other tests used to differentiate WA101T from its closest phylogenetic neighbours. Other metabolic properties and antibiotic susceptibilities observed for strain WA101T are given under the species description.
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DNA–DNA hybridization experiments revealed a low level of relatedness between strain WA101T and M. sphagni DSM 44076T. The value of 22·7 % obtained falls below the threshold value (70 %) recommended by Wayne et al. (1987) for distinguishing between separate species. Based on the overall results presented it is proposed that strain WA101T represents a novel species of Mycobacterium, for which the name Mycobacterium psychrotolerans sp. nov. is proposed.
Description of Mycobacterium psychrotolerans sp. nov.
Mycobacterium psychrotolerans (psy.chro.tol'er.ans. Gr. adj. psychros cold; L. pres. part. tolerans tolerating; N.L. part. adj. psychrotolerans cold-tolerating).
Gram-positive, acid-fast, non-spore-forming, non-motile short rods. Smooth, entire, bright orange, scotochromogenic colonies appear after 2 days in GYEA, Bennett's and nutrient agars. Growth on Löwenstein–Jensen agar is moderate; no growth occurs on MacConkey agar. Grows at 4–37 °C and tolerates 7 % NaCl. Shows a positive reaction for nitrate reduction, acid phosphatase, 2-naphthyl-butyrate (esterase), 2-naphthyl-caprylate (lipase), 2-naphthyl-myristate, cysteine arylamidase, leucine arylamidase, valine arylamidase, arginine dehydrolase, trypsin, phosphohydrolase, -glucosidase and 
-glucosidase; urea reaction is weak. Negative for -chymotrypsin, -fucosidase, -galactosidase, -galactosidase, -glucuronidase, N-acetyl--glucosaminidase and -mannosidase. Adonitol, aesculin, erythritol, D-fructose, gluconate, D-glucose, N-acetylglucosamine, glycerol, inositol, malate, mannitol, D-mannose, sucrose, sorbitol, trehalose and D-xylose are used as sole carbon sources. Does not use amygdalin, D-arabinose, L-arabinose, arbutin, cellobiose, dulcitol, D-fucose, L-fucose, galactose, -gentiobiose, glycogen, inulin, lactose, maltose, melezitose, melibiose, D-raffinose, rhamnose, ribose, salicin, L-sorbose, starch, D-tagatose, D-turanose, L-xylose or methyl -xyloside. Resistant to ampicillin, cefuroxime, cloxacillin, erythromycin, penicillin and polymyxin. Sensitive to ciprofloxacin, gentamicin, neomycin and oxytetracycline. TLC of mycolic acid methanolysates reveals -mycolates, keto-mycolates and -carboxymycolates plus 2-eicosanol (wax-ester mycolates). The mycolic acid HPLC elution profile is unique and can be used for differentiation from the closely related species Mycobacterium aichiense, Mycobacterium gilvum, M. chlorophenolicum, M. parafortuitum, M. sphagni and other mycobacteria. The G+C composition of the DNA of the type strain is 64 mol%.
The type strain, WA101T (=DSM 44697T=LMG 21953T), was isolated from a pond in Salamanca, Spain.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Butler, W. R., Thibert, L. & Kilburn, J. O. (1992). Identification of Mycobacterium avium complex strains and some similar species by high-performance liquid chromatography. J Clin Microbiol 30, 2698–2704.
Cashion, P., Holder-Franklin, M. A., McCully, J. & Franklin, M. (1977). A rapid method for the base ratio determination of bacterial DNA. Anal Biochem 81, 461–466.[CrossRef][Medline]
Collins, C. H., Grange, J. M. & Yates, M. D. (1984). A review: mycobacteria in water. J Appl Bacteriol 57, 193–211.[Medline]
Dailloux, M., Laurain, C., Weber, M. & Hartemann, P. H. (1999). Water and nontuberculous mycobacteria. Water Res 33, 2219–2228.[CrossRef]
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[Medline]
Doetsch, R. N. (1981). Determinative methods of light microscopy. In Manual of Methods for General Bacteriology, pp. 21–33. Edited by P. Gerdhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg & G. B. Phillips. Washington, DC: American Society for Microbiology.
Gordon, R. E. & Mihm, J. M. (1962). Identification of Nocardia caviae (Erikson) nov. comb. Ann N Y Acad Sci 98, 628–636.[CrossRef]
Häggblom, M. M., Nohynek, L. J., Palleroni, N. J., Kronqvist, K., Nurmiaho-Lassila, E.-L., Salkinoja-Salonen, M. S., Klatte, S. & Kroppenstedt, R. M. (1994). Transfer of polychlorophenol-degrading Rhodococcus chlorophenolicus (Apajalahti et al. 1986) to the genus Mycobacterium as Mycobacterium chlorophenolicum comb. nov. Int J Syst Bacteriol 44, 485–493.[CrossRef][Medline]
Huß, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the spectrophotometric determination of DNA hybridisation from renaturation rates. Syst Appl Microbiol 4, 184–192.
Jahnke, K. D. (1992). Basic computer program for evaluation of spectroscopic renaturation data from GILFORD System 2600 spectrometer on a PC/XT/AT type personal computer. J Microbiol Methods 15, 61–73.
Kazda, J. (1980). Mycobacterium sphagni sp. nov. Int J Syst Bacteriol 30, 77–81.[CrossRef]
Khan, A. A., Kim, S.-J., Paine, D. D. & Cerniglia, C. E. (2002). Classification of a polycyclic aromatic hydrocarbon-metabolizing bacterium, Mycobacterium sp. strain PYR-1, as Mycobacterium vanbaalenii sp. nov. Int J Syst Evol Microbiol 52, 1997–2002.[Abstract]
Kirschner, P., Springer, B., Vogel, U., Meier, A., Wrede, A., Kiekenbeck, M., Bange, F.-C. & Böttger, E. C. (1993). Genotypic identification of mycobacteria by nucleotide acid sequence determination: report of a 2-year experience in a clinical laboratory. J Clin Microbiol 31, 2882–2889.
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). MEGA 2: molecular evolutionary genetics analysis software. Bioinformatics 17, 1244–1245.
Lévy-Frébault, V. V. & Portaels, F. (1992). Proposed minimal standards for the genus Mycobacterium and for description of new slowly growing Mycobacterium species. Int J Syst Bacteriol 42, 315–323.[CrossRef][Medline]
Mandel, M. & Marmur, J. (1968). Use of ultraviolet absorbance temperature profile for determining the guanine plus cytosine content of DNA. Methods Enzymol 12B, 195–206.
Miller, J. L. (1997). Sherlock Mycobacteria Identification by High Performance Liquid Chromatography. A Training Manual. Newark, DE: MIDI, Inc.
Minnikin, D. E., Minnikin, S. M., Parlett, J. H., Goodfellow, M. & Magnusson, M. (1984). Mycolic acid patterns of some species of Mycobacterium. Arch Microbiol 139, 241–256.[CrossRef]
Rhodes, M. W., Kator, H., Kotob, S. & 7 other authors (2003). Mycobacterium shottsii sp. nov., a slowly growing species isolated from Chesapeake Bay striped bass (Morone saxatilis). Int J Syst Evol Microbiol 53, 421–424.
Rivas, R., Velázquez, E., Valverde, A., Mateos, P. F. & Martínez-Molina, E. (2001). A two primers random amplified polymorphic DNA procedure to obtain polymerase chain reaction fingerprints of bacterial species. Electrophoresis 22, 1086–1089.[CrossRef][Medline]
Rivas, R., Sánchez, M., Trujillo, M. E., Zurdo-Piñeiro, J. L., Mateos, P. F., Martínez-Molina, E. & Velázquez, E. (2003). Xylanimonas cellulosilytica gen. nov., sp. nov., a xylanolytic bacterium isolated from a decayed tree (Ulmus nigra). Int J Syst Evol Microbiol 53, 99–103.
Schröder, K.-H., Naumann, L., Kroppenstedt, R. M. & Reischl, U. (1997). Mycobacterium hassiacum sp. nov., a new rapidly growing thermophilic mycobacterium. Int J Syst Bacteriol 47, 86–91.[CrossRef][Medline]
Springer, B., Tortoli, E., Richter, I. & 7 other authors (1995). Mycobacterium conspicuum sp. nov., a new species isolated from patients with disseminated infections. J Clin Microbiol 33, 2805–2811.[Abstract]
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 24, 4876–4882.
Vuorio, R., Andersson, M. A., Rainey, F. A., Kroppenstedt, R. M., Kämpfer, P., Busse, H.-J., Viljanen, M. & Salkinoja-Salonen, M. (1999). A new rapidly growing mycobacterial species, Mycobacterium murale sp. nov., isolated from the indoor walls of a children's day care centre. Int J Syst Bacteriol 49, 25–35.[CrossRef][Medline]
Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.[CrossRef]
Willumsen, P., Karlson, U., Stackebrandt, E. & Kroppenstedt, R. M. (2001). Mycobacterium frederiksbergense sp. nov., a novel polycyclic aromatic hydrocarbon-degrading Mycobacterium species. Int J Syst Evol Microbiol 51, 1715–1722.[Abstract]
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