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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 National Institute of Health, Department of Medical Sciences, Nonthaburi, Thailand
Correspondence
Yuzuru Mikami
mikami{at}myco.pf.chiba-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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The DDBJ accession numbers for the 16S rDNA sequences of Nocardia asiatica IFM 0245T, IFM 0263, IFM 0425, IFM 0731 and IFM 0860 are respectively AB092566–AB092570.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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contains several species, such as Nocardia asteroides, Nocardia farcinica, Nocardia nova, Nocardia brasiliensis, Nocardia pseudobrasiliensis, Nocardia transvalensis and Nocardia otitidiscaviarum, that are major pathogens that cause clinical disease in humans. Nocardiosis, which occurs most frequently in immunocompromised patients, is usually caused by the N. asteroides group, N. asteroides sensu stricto, N. farcinica and N. nova (Poonwan et al., 1995; Javaly et al., 1992). Cutaneous nocardiosis is usually caused by N. brasiliensis and N. otitidiscaviarum. Recently, several novel pathogenic species of Nocardia have been described (Wang et al., 2001; Yassin et al., 2001a). Nocardia africana was also described in 2001 (Hamid et al., 2001), having been isolated from sputum samples taken from patients with pulmonary diseases in Sudan. To date, it has been isolated only in that country. The five strains were also isolated in restricted areas. The strains described here have been isolated only in Japan and Thailand, i.e. in Asia. In these cases, it appears that some agents of nocardiosis may be restricted in their geographical distribution.
The genus Nocardia has been redefined (Goodfellow, 1992, 1998), and current methods of identification of Nocardia species now include not only analysis of microscopic and colony morphology and biological and chemotaxonomic characteristics, but also phylogenetic analyses. In recent years, many novel species of the genus, for example Nocardia beijingensis (Wang et al., 2001), Nocardia cyriacigeorgica (Yassin et al., 2001a), Nocardia ignorata (Yassin et al., 2001b) and Nocardia vinacea (Kinoshita et al., 2001), have been proposed.
In this paper, we report on the morphological, physiological and biochemical characteristics, analysis of cell compositions, DNA–DNA hybridization and the 16S rRNA gene sequence of five Nocardia isolates in comparison with those of reference strains of Nocardia. On the basis of the characteristics presented, these isolates represent a novel taxon within the genus Nocardia, for which we propose the name Nocardia asiatica sp. nov.
Strain IFM 0245T was isolated in 1985 from the sputum of a 41-year-old female patient with nocardiosis. Strain IFM 0263 was isolated in 1986 from a granuloma from a 68-year-old male patient with nocardiosis. These two strains were isolated in Japan. Strain IFM 0425 was isolated in 1994 from trans-tracheal aspirate, and strains IFM 0731 and IFM 0860 were respectively isolated in 1997 and 1999 from sputum; these three strains were isolated in Thailand. Clinical histories of the Thai patients were not publicly available. Strains IFM 0245T, IFM 0263, IFM 0425, IFM 0731, IFM 0860, N. beijingensis and N. brasiliensis were cultured on Mueller–Hinton II agar slants with 1 % glucose and 1 % glycerol for 1 week at 27 °C. For extraction of DNA and sequencing, bacterial strains were cultured on brain heart infusion (BHI; Difco) broth for 4 days at 32 °C. Bacterial strains were cultured on BHI broth with 2 % glucose and 2 % glycine for 3 days at 32 °C for DNA–DNA hybridization. Morphological observations under a scanning electron microscope (model S-5200; Hitachi) were made on cultures grown on humic acid/MOPS/gellan gum medium (HMG; Suzuki et al., 2000) at 30 °C for 10 days. For inspections using a scanning electron microscope (Hitachi), the cultures were fixed in 2 % osmium tetroxide vapour in situ at 25 °C for 24 h, dehydrated with ethanol and tert-butanol and then freeze-dried. Each specimen was coated with osmium using an osmium plasma coater OPC 80N (NLE Nippon Laser & Electronics Laboratory).
Decomposition of adenine, casein, hypoxanthine, tyrosine, urea and xanthine was examined by using the methods of Gordon et al. (1974). Acid production from carbohydrates, utilization of organic acids and growth temperature were determined by using the modified method of Poonwan et al. (1995). Decomposition of arbutin, elastin, aesculin and testosterone were examined by 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), Kubica & Rigdon (1961) and Kubica & Vestal (1961). Isolated strains were tested for their ability to grow in Mueller–Hinton II agar supplied 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 isomers using TLC (Staneck & Roberts, 1974). Whole-cell sugars were prepared as reported by Lechevalier & Lechevalier (1980) and analysed by using the TLC method (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).
A 1 ml volume of broth culture was centrifuged at 12 000 r.p.m. for 10 min. The pellet was resuspended in 200 µl TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8·0), 250 µl GPT reagent (6 M guanidine thiocyanate dissolved in 50 mM Tris/HCl, pH 8·3) and 450 µl Tris-buffered phenol (pH 8·0). The tube was placed in a boiling-water bath for 15 min and extracted with 250 µl chloroform/isoamyl alcohol (24 : 1, v/v). After 10 min centrifugation at 12 000 r.p.m., the aqueous phase (
500 µl) was transferred to a fresh tube, mixed with 500 µl 100 % 2-propanol and 50 µl 3 M sodium acetate and then centrifuged at 12 000 r.p.m. for 15 min at 4 °C before the supernatant was decanted. Traces of GPT reagent were removed by the addition of 500 µl ice-cold 70 % ethanol to the nucleic acid pellet and a further centrifugation at 12 000 r.p.m. for 5 min at 4 °C. The ethanol was then removed and the pellet dried under a vacuum for 20 min. The pellet was finally resuspended in 50 µl TE buffer.
For sequencing of 16S rRNA genes, 16S rDNA was amplified and sequenced using a PCR and the following universal prokaryotic 16S rDNA primers: 8F (5'-AGAGTTTGATCCTGGCTCAG-3') and 691R (5'-ACCGCTACACCAGGA-3'), 520F (5'-CAGCAGCCGCGGTAATAC-3') and 1100R (5'-GGGTTGCGCTGTTG-3') and 926F (5'-AAACTCAAAGGAATTGACGG-3') and 1542R (5'-ACAAAGGAGGTGATC-3'). PCR was performed with a DNA thermal cycler (TaKaRa) using 35 cycles 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. The PCR products were purified with a Centri-Sep column (Princeton Separations). DNA sequences were determined with an automatic sequence analyser (ABI PRISM 3100; PE Applied Biosystems) using the BigDye Terminator cycle sequencing ready reaction kit (PE Applied Biosystems).
A BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST/) against various sequence databases was used to identify strains related to strain IFM 0245T. Sequence data for related species were retrieved from GenBank. Nucleotide substitution rates (Knuc values) were calculated (Kimura & Ota, 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 (Thompson et al., 1994). Sequence similarity values were calculated manually.
DNA was isolated as described by Saito & Miura (1983), with modifications. DNA base compositions were estimated by HPLC (Tamaoka & Komagata, 1984) and levels of DNA–DNA relatedness were determined by the method of Ezaki et al. (1989), using photobiotin and microplates.
Almost complete 16S rDNA sequences were determined for the five isolated strains; these sequences have been deposited in the DDBJ database. A database search demonstrated that the isolated strains belong to the suborder Corynebacterineae and the family Nocardiaceae (Stackebrandt et al., 1997). It is clear from the phylogenetic tree (Fig. 1) that the five isolates belong to the same cluster and are loosely associated with N. beijingensis. Nocardia sp. DSM 43301 and Nocardia sp. DSM 43396 are unnamed strains of the genus Nocardia (Roth et al., 2003); the BLAST search showed that these two strains are also closely related to the five isolated strains. Sequence similarity values among the five strains were 99·24–99·93 %. This is a high degree of similarity, but strain IFM 0245T has a 16S rDNA sequence that is slightly different from that of the other four isolates. On the other hand, sequence similarity values between these five strains and N. beijingensis were 98·49–98·66 %.
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; Goodfellow et al., 1999). All of them contain galactose and arabinose as characteristic whole-cell sugars in addition to meso-diaminopimelic acid as the wall diamino acid; all contain two kinds of mycolic acid that co-migrated (Rf values of approximately 0·47 and 0·91) with those extracted from marker Nocardia strains. These types of mycolic acid were seen only in Nocardia abscessus, N. asiatica and N. beijingensis. More than 90 % of the menaquinones were MK-8(H4
-cycl).
In contrast to the chemotaxonomic similarities between these five strains and other species of the genus Nocardia, the results of physiological tests showed clear differences between them (Table 1). Table 1 lists all Nocardia species with the phenotypic properties that distinguish the present five strains from the type strains of Nocardia species. Fig. 2 shows scanning electron micrographs (Hitachi) of strain IFM 0245T.
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). Therefore, these five strains were confirmed as belonging to the same novel Nocardia species.
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Description of Nocardia asiatica sp. nov.
Nocardia asiatica (a.si.a'ti.ca. L. gen. masc. n. asiatica of Asia, the source of the isolates).
Aerobic, Gram-positive, partially acid-fast, non-motile actinomycetes forming a beige substrate. Substrate mycelia are fragmented. Aerial mycelia are straight to flexuous with a mean spore number of 2–20. Spores are cylindrical (0·3–0·5x1·5–1·7 µm). Colonies on BHI agar and on most other commonly used media, such as Sabouraud's dextrose agar, are very small compared with those of N. brasiliensis and N. asteroides type strains. The reverse side of colonies is yellowish. Colonies are 0·3–1·0 mm in diameter after 7 days at 30 °C on Mueller–Hinton II medium with 0·2 % glucose. Glucose, acetate, rhamnose and citrate are utilized, but adipic acid, adonitol, arabinose, arbutin, aesculin, erythritol, galactose, inositol, mannose and sorbitol are not. Adenine, casein, hypoxanthine, testosterone and xanthine are not hydrolysed. The type strain is a clinical isolate. It grows at 37 °C, but not at 45 °C; nitrate is reduced to nitrite. It does not have arylsulfatase activity. The G+C content of the DNA is 68·4–69·9 mol%.
The type strain is strain IFM 0245T (=NBRC 100129T= JCM 11892T=DSM 44668T). The five strains described were isolated from Japan and Thailand.
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ACKNOWLEDGEMENTS |
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