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1 Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan
2 Hiroshima Red Cross and Atomic Bomb Survivors Hospital, Hiroshima, Japan
3 Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany
Correspondence
Yuzuru Mikami
mikami{at}faculty.chiba-u.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Nocardia araoensis IFM 0575T and Nocardia pneumoniae IFM 0784T are AB108779 and AB108780, respectively.
Additional phenotypic characteristics and an expanded phylogenetic tree are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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) and has undergone a revolution of its taxonomy in recent years (Kiska et al., 2002; Roth et al., 2003). The genus Nocardia encompasses 31 recognized species at the time of writing, 20 of which have been described in the last 5 years.
Most Nocardia species are associated with human and animal infections that are difficult to diagnose because of their non-specific clinical manifestation and histological evidence (Gordon et al., 1974; Goodfellow, 1998; Boiron et al., 1992). Nocardiosis is a potentially life-threatening infection caused by several species of the genus Nocardia: Nocardia asteroides, Nocardia farcinica, Nocardia nova, Nocardia brasiliensis, Nocardia pseudobrasiliensis, Nocardia transvalensis and Nocardia otitidiscaviarum.
The two strains examined here (IFM 0575T and IFM 0784T), isolated from clinical specimens, were initially identified as N. asteroides based on morphological and biochemical characteristics. Strains IFM 0575T and IFM 0784T were respectively isolated from the sputum as well as bronchoalveolar lavage (BAL) of a 75-year-old Japanese male patient with non-tuberculous mycobacterial infection and the sputum of a 69-year-old Japanese male patient with a history of lung cancer, diabetes, emphysema and radiation pneumonitis. Based on phylogenetic criteria, the taxonomic position of the two strains was determined and revealed that both organisms are most closely related to Nocardia beijingensis. From phenotypic and phylogenetic characteristics, together with data regarding genomic DNA–DNA relatedness, we propose the inclusion of these strains within two novel Nocardia species, Nocardia araoensis sp. nov. and Nocardia pneumoniae sp. nov.
Strains IFM 0575T and IFM 0784T were initially isolated using Ogawa agar (Eiken). Strains IFM 0575T, IFM 0784T and Nocardia beijingensis IFM 10174T were then cultured on Mueller–Hinton II (MH II; Difco) agar slants with 1 % glucose and 1 % glycerol for 1 week at 30 °C. Strains IFM 0575T and IFM 0784T were cultured on MH II medium with 0·2 % glucose for 1 week at 30 °C for colonization. The isolated strains were cultured on brain heart infusion (BHI; Difco) broth with 0·1 % glucose and 0·1 % glycerol for 5 days at 30 °C for DNA sequencing. The bacterial strains were cultured on BHI broth with 2 % glucose and 2 % glycine for 3 days at 30 °C for DNA–DNA hybridization analysis. Strains were stained using a modified Kinyoun method (Chapin & Murray, 1999) using 0·5 % sulfuric acid for determination of acid–alcohol-fastness. Morphological observations under a scanning electron microscope (model S-5200; Hitachi) were made on cultures grown on MH II agar with 0·2 % glucose (Suzuki et al., 2000) at 30 °C for 7 days. For scanning electron microscope inspections, cultures were fixed in 2 % osmium tetroxide vapour in situ at 25 °C for 24 h, dehydrated with ethanol and t-butanol and then freeze-dried.
Decomposition of adenine, casein, hypoxanthine, tyrosine, urea and xanthine was examined using the methods of Gordon et al. (1974). Acid production from adonitol, arabinose, erythritol, galactose, glucose, inositol, maltose, mannose, rhamnose and sorbitol, utilization of citrate, adipic acid, acetamide and gluconate and growth at 37 and 45 °C were determined by the modified method of Poonwan et al. (1995). Decomposition of arbutin, elastin, aesculin and testosterone was examined using the methods of Goodfellow & Pirouz (1982). Utilization of nitrogen sources was tested using an N-Buiyon set (Eiken). Arylsulfatase activity was determined by the method of Kubica & Beam (1961). Isolated strains were tested for their ability to grow in MH II agar with 0·2 % glucose with each antibiotic TRIDISK (Eiken) at 32 °C for 2 or 3 days (Mikami & Yazawa, 1989).
Whole-cell hydrolysates were analysed for diaminopimelic acid (DAP) isomers using TLC (Staneck & Roberts, 1974). Whole-cell sugars were prepared as described by Lechevalier & Lechevalier (1980) and analysed by TLC (Miyadoh, 2001). Mycolic acids were prepared as reported by Minnikin et al. (1980). Menaquinones were extracted from freeze-dried biomass (500 mg) and analysed as described by Chun & Goodfellow (1995). Strains were grown for fatty acid analysis for 7 days at 28 °C in Petri dishes on trypticase soy broth agar (DSMZ medium 535). Three to four inoculation loops of cell material were scraped from the plates and used for analysis. Fatty acid methyl esters were obtained from cells after saponification, methylation and extraction as described by Miller (1982). The fatty acid methyl ester mixtures were separated using a 5 % phenyl–methyl silicone capillary column (0·2 mmx25 m) and a gas chromatograph (model 5898A; Hewlett Packard) controlled by MIS software (Microbial ID). Peaks were automatically integrated and fatty acid names and percentages were determined using the MIS software package (Sasser, 1990). The following conditions were applied: carrier gas, ultrahigh-purity hydrogen; column head pressure, 60 kPa; injection volume, 2 µl; column split ratio, 100 : 1; septum purge, 5 ml min–1; column temperature, 170–270 °C at 5 °C min–1; injection port temperature, 250 °C; detector temperature, 300 °C.
Preparation of genomic DNA samples for sequencing was performed using the guanidine thiocyanate method of Kageyama et al. (2002).
16S rRNA genes were amplified and sequenced by PCR employing six prokaryotic 16S rRNA gene universal primers. PCR was performed with a DNA thermal cycler (TaKaRa) using 35 cycles consisting of denaturation at 94 °C for 60 s, primer annealing at 60 °C for 60 s and primer extension at 72 °C for 120 s. DNA sequences were determined with an automatic sequence analyser (ABI PRISM 3100; PE Applied Biosystems) using a dye terminator cycle sequencing kit (PE Applied Biosystems).
Species related to the new isolates were identified by performing a nucleotide sequence database search using BLAST. Sequence data of related species were retrieved from GenBank. Nucleotide substitution rates (Knuc values) were calculated (Kimura & Ohta, 1972) and phylogenetic trees were constructed by the neighbour-joining method (Saitou & Nei, 1987). The topology of the trees was evaluated by a bootstrap analysis of the sequence data using CLUSTAL W software (Thompson et al., 1994). Sequence similarity values were determined by visual comparison and manual calculation.
Preparation of genomic DNA samples for DNA–DNA hybridization was performed using a modified version of the method described by Saito & Miura (1983). DNA G+C content was estimated by HPLC (Tamaoka & Komagata, 1984) using the same DNA as for DNA–DNA hybridization. Levels of DNA–DNA relatedness were determined by the method of Ezaki et al. (1989) using photobiotin and microplates.
Comparison of nearly complete 16S rRNA gene sequences of isolated strains with the corresponding sequences of other Nocardia species shows that the two strains form a monophyletic clade with N. beijingensis in the neighbour-joining tree. Fig. 1 shows a phylogenetic tree of the two isolated strains and related Nocardia species (an extended tree including a larger selection of reference sequences is available as supplementary material in IJSEM Online). Sequence similarity values among the two strains and N. beijingensis were between 98·1 and 99·1 %, and a high bootstrap value (99 %) was obtained in the neighbour-joining analysis. However, DNA–DNA relatedness between the two isolated strains and N. beijingensis is <46 %, and that between strains IFM 0575T and IFM 0784T is 35 %. These values were well below the 70 % cut-off point for species classification as recommended by Wayne et al. (1987).
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; Goodfellow et al., 1999), i.e. they contained galactose and arabinose as characteristic whole-cell sugars in addition to meso-DAP as the dominant cell wall diamino acid. In addition, these strains contained mycolic acid that co-migrated (Rf value of about 0·47) with that extracted from Nocardia type strains. The major menaquinone was MK-8(H4
-cycl). Strains IFM 0575T and IFM 0784T were partially acid-fast. These two strains were also examined for phenotypic characterization. IFM 0575T decomposes urea and aesculin and reduces nitrate. Nocardia paucivorans and Nocardia veterana have the same characteristics, but IFM 0575T could be distinguished from these two species on the basis of utilization of glucose and rhamnose. IFM 0784T decomposes casein and utilizes citrate. N. brasiliensis and N. pseudobrasiliensis have the same characteristics, but IFM 0784T could be distinguished from these two species on the basis of decomposition of hypoxanthine. Table 1 shows phenotypic properties of the two isolated strains, N. asteroides and N. beijingensis. A comparison with further species of Nocardia is available as supplementary material in IJSEM Online. Fatty acid analysis by GLC revealed the expected pattern diagnostic for members of the genus Nocardia and related taxa, i.e. straight-chain saturated and unsaturated fatty acids together with a diagnostic percentage of tuberculostearic acid (10-methyl-octadecanoic acid). The major fatty acid for the two isolated strains, N. asteroides and N. beijingensis is 16 : 0. However, the second most abundant fatty acid varies: IFM 0575T, 18 : 1 cis-9; IFM 0784T, 16 : 1 cis-7; N. beijingensis, 18 : 0. Strains IFM 0575T and IFM 0784T could be separated from the closely related N. beijingensis IFM 10174T by quantitative differences in their fatty acid profiles (Table 2). Fig. 2 shows scanning electron micrographs (Hitachi) of strains IFM 0575T and IFM 0784T.
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Description of Nocardia araoensis sp. nov.
Nocardia araoensis (a.ra.o.en'sis. N.L. fem. adj. araoensis referring to Arao city, where the bacterium was isolated).
Aerobic, Gram-positive, partially acid-fast, non-motile actinomycetes that form a branched substrate mycelium that fragments into rod-shaped elements (0·3–0·5x1·0–1·7 µm). Faint yellow substrate mycelium, and cotton-white to greyish white aerial mycelium. No soluble pigment is produced. Colony size is small; dimensions are 0·5–1·4 mm for 7 days at 30 °C on MH II medium with 0·2 % glucose. Acetate, citrate and glucose are utilized, but adonitol, arabinose, erythritol, galactose, inositol, maltose, mannose, rhamnose, sorbitol, adipic acid, acetamide and gluconate are not. Produces acid from glucose only. Adenine, casein, hypoxanthine, tyrosine, xanthine, aesculin, arbutin and elastin are not decomposed. Urea and testosterone are decomposed. Nitrogen sources are not utilized. No arylsulfatase activity. Grows at 45 °C. The DNA G+C content of the type strain is 69 mol%.
The type strain, IFM 0575T (=NBRC 100135T=JCM 12118T=DSM 44729T), was isolated from BAL of a male Japanese patient with non-tuberculosis mycobacterium infection.
Description of Nocardia pneumoniae sp. nov.
Nocardia pneumoniae (pneu.mo'ni.ae. N.L. gen. n. pneumoniae referring to the disease pneumonia).
Aerobic, Gram-positive, partially acid-fast, non-motile actinomycetes that form a branched substrate mycelium that fragments into rod-shaped elements (0·3–0·5x0·6–1·1 µm). Faint yellow substrate mycelium with white to greyish white aerial hyphae. No soluble pigment is produced. Dimensions of colonies are 2·0–3·0 mm for 7 days at 30 °C on MH II medium with 0·2 % glucose. Acetate, glucose, citrate and acetamide are utilized, but adonitol, arabinose, erythritol, galactose, inositol, maltose, mannose, rhamnose, sorbitol, adipic acid or gluconate are not. Produces acid from glucose only. Adenine, hypoxanthine, tyrosine, urea, xanthine and elastin are not decomposed. Casein, aesculin, arbutin and testosterone are decomposed. Nitrogen sources are utilized. Weak arylsulfatase activity. Grows at 45 °C. The DNA G+C content of the type strain is 68 mol%.
The type strain, IFM 0784T (=NBRC 100136T=JCM 12119T=DSM 44730T), was isolated from sputum of a male Japanese patient with a history of lung cancer, diabetes, emphysema and radiation pneumonia.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Chapin, K. C. & Murray, P. R. (1999). Stains. In Manual of Clinical Microbiology, pp. 1678. Edited by P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover & R. H. Yolken. Washington, DC: American Society for Microbiology.
Chun, J. & Goodfellow, M. (1995). A phylogenetic analysis of the genus Nocardia with 16S rRNA gene sequences. Int J Syst Bacteriol 45, 240–245.[CrossRef][Medline]
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.[CrossRef]
Goodfellow, M. (1998). Nocardia and related genera. In Topley and Wilson's Microbiology and Microbial Infections, 9th edn, vol. 2, Systematic Bacteriology, pp. 463–489. Edited by A. Balows & B. I. Duerden. London: Arnold.
Goodfellow, M. & Pirouz, T. (1982). Numerical classification of sporoactinomycetes containing meso-diaminopimelic acid in the cell wall. J Gen Microbiol 128, 503–527.[Medline]
Goodfellow, M., Isik, K. & Yates, E. (1999). Actinomycete systematics: an unfinished synthesis. Nova Acta Leopold 80 (312), 47–82.
Gordon, R. E., Barnett, D. A., Handerhan, J. E. & Pang, C. H.-N. (1974). Nocardia coeliaca, Nocardia autotrophica, and the nocardin strain. Int J Syst Bacteriol 24, 54–63.[CrossRef]
Kageyama, A., Sato, H., Nagata, M., Yazawa, K., Katsu, M., Mikami, Y., Kamei, K. & Nishimura, K. (2002). First human case of nocardiosis caused by Nocardia pseudobrasiliensis in Japan. Mycopathologia 156, 187–192.[Medline]
Kimura, M. & Ohta, T. (1972). On the stochastic model for estimation of mutation distance between homologous proteins. J Mol Evol 2, 87–90.[CrossRef][Medline]
Kinoshita, N., Homma, Y., Igarashi, M., Ikeno, S., Hori, M. & Hamada, M. (2001). Nocardia vinacea sp. nov. Actinomycetologica 15, 1–5.
Kiska, D. L., Hicks, K. & Pettit, D. J. (2002). Identification of medically relevant Nocardia species with an abbreviated battery of tests. J Clin Microbiol 40, 1346–1351.
Kubica, G. P. & Beam, R. E. (1961). The arylsulfatase activity of acid-fast bacilli. II. The differentiation of Mycobacterium avium from the unclassified group III nonphotochromogenic mycobacteria. Am Rev Resp Dis 83, 733–736.[Medline]
Kudo, T., Hatai, K. & Seino, A. (1988). Nocardia seriolae sp. nov. causing nocardiosis of cultured fish. Int J Syst Bacteriol 38, 173–178.[CrossRef]
Lechevalier, M. P. & Lechevalier, H. A. (1980). The chemotaxonomy of actinomycetes. In Actinomycete Taxonomy, pp. 227–291. Edited by A. Dietz & D. W. Thayer. Fairfax, VA: Society for Industrial Microbiology.
Maldonado, L., Hookey, J. V., Ward, A. C. & Goodfellow, M. (2000). The Nocardia salmonicida clade, including descriptions of Nocardia cummidelens sp. nov., Nocardia fluminea sp. nov. and Nocardia soli sp. nov. Antonie van Leeuwenhoek 78, 367–377.[CrossRef][Medline]
Mikami, Y. & Yazawa, K. (1989). Susceptibility patterns of pathogenic Nocardia to some selected antimicrobial agents and their usefulness in the identification work in a clinical laboratory. Bull JFCC 5, 89–95.
Miller, J. L. (1982). A single derivatization method for bacterial fatty acid methyl esters including hydroxy acids. J Clin Microbiol 16, 584–586.
Minnikin, D. E., Hutchinson, I. G., Caldicott, A. B. & Goodfellow, M. (1980). Thin-layer chromatography of methanolysates of mycolic acid-containing bacteria. J Chromatogr 188, 221–233.[CrossRef]
Miyadoh, M. (2001). Identification procedure at the genus level. In Identification Manual of Actinomycetes, pp. 9–19. Edited by S. Miyadoh, M. Hamada, K. Hotta, T. Kudo, A. Seino, K. Suzuki & A. Yokota. Tokyo: Business Center for Academic Societies Japan.
Poonwan, N., Kusum, M., Mikami, Y., Yazawa, K., Tanaka, Y., Gonoi, T., Hasegawa, S. & Konyama, K. (1995). Pathogenic Nocardia isolated from clinical specimens including those of AIDS patients in Thailand. Eur J Epidemiol 11, 507–512.[CrossRef][Medline]
Roth, A., Andrees, S., Kroppenstedt, R. M., Harmsen, D. & Mauch, H. (2003). Phylogeny of the genus Nocardia based on reassessed 16S rRNA gene sequences reveals underspeciation and division of strains classified as Nocardia asteroides into three established species and two unnamed taxons. J Clin Microbiol 41, 851–856.
Saito, H. & Miura, K. (1983). Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochem Biophys Acta 72, 619–629.[CrossRef]
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Sasser, M. (1990). Identification of Bacteria by Gas Chromatography of Cellular Fatty Acids. MIDI Technical Note 101. Newark, DE: MIDI Inc.
Stackebrandt, E., Rainey, F. A. & Ward-Rainey, N. L. (1997). Proposal for a new hierarchic classification system, Actinobacteria classis nov. Int J Syst Bacteriol 47, 479–491.[CrossRef]
Staneck, J. L. & Roberts, G. D. (1974). Simplified approach to identification of aerobic actinomycetes by thin-layer chromatography. Appl Microbiol 28, 226–231.[Medline]
Suzuki, S., Yamamoto, K., Okuda, T., Nishino, M., Nakanishi, N. & Komatsubara, S. (2000). Selective isolation and distribution of Actinomadura rugatobispora strains in soil. Actinomycetologica 14, 1–7.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Wang, L., Zhang, Y., Lu, Z., Shi, Y., Liu, Z., Maldonado, L. & Goodfellow, M. (2001). Nocardia beijingensis sp. nov., a novel isolate from soil. Int J Syst Evol Microbiol 51, 1783–1788.[Abstract]
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]
Yassin, A. F., Rainey, F. A. & Steiner, U. (2001). Nocardia ignorata sp. nov. Int J Syst Evol Microbiol 51, 2127–2131.[Abstract]
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