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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 Yatsushiro Health Insurance General Hospital, 2-26 Yatsushiro, Kumamoto 866-0862, Japan
3 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany
Correspondence
Yuzuru Mikami
mikami{at}myco.pf.chiba-u.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains IFM 10311T, IFM 10312 and IFM 10313 and IFM 10084T are AB108775–AB108778, respectively.
An extended 16S rRNA gene-based phylogenetic tree and a comparison of properties of all Nocardia species are available as supplementary material in IJSEM Online.
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TOPABSTRACTMAIN TEXTREFERENCES |
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). This genus belongs to the mycolic acid-containing group of actinomycetes represented by the suborder Corynebacterineae (Chun et al., 1996; Kämpfer et al., 1999; Linos et al., 2002). Members of these genera can be distinguished from one another using a combination of biochemical, morphological and chemical characteristics (Goodfellow et al., 1999; Linos et al., 2002). The genus Nocardia currently encompasses 31 species with validly published names; 20 of these species have been described in the last 5 years. These species have been isolated from clinical samples and soils.
Nocardia asteroides is the major cause of nocardiosis; however, authors of many taxonomic studies (Schaal & Reuterberg, 1978; Orchard & Goodfellow, 1980) have considered N. asteroides as a heterogeneous species. Four strains that were similar to N. asteroides in their morphological and biochemical characteristics have been isolated in the present study. Further taxonomic studies revealed that the organisms consistently formed a distinct clade most closely associated with Nocardia farcinica. Comparative DNA–DNA relatedness data show that the four strains are distinguishable from the N. farcinica type strain and hence represent two novel species within the genus Nocardia. We propose the novel species designations Nocardia shimofusensis sp. nov. and Nocardia higoensis sp. nov.
Strains IFM 10311T, IFM 10312 and IFM 10313 were isolated by the method of Khan et al. (1997) from soils in the city of Choshi and in the village of Chyosei, respectively, in Japan in 1999. Strain IFM 10084T was isolated using Ogawa agar (Eiken) in 2001 from a 70-year-old Japanese female patient who had a history of autohepatitis and had received steroid therapy. She complained of chest pain, was hospitalized and her condition was diagnosed as pleurisy involving Nocardia species.
The four strains IFM 10311T, IFM 10312, IFM 10313, IFM 10084T and N. farcinica IFM 0284T were cultured on Mueller–Hinton medium II (MHII; Difco) slants with 1 % glucose and 1 % glycerol for 1 week at 30 °C. They were also cultured on MHII agar plates with 1 % glucose and 1 % glycerol for 1 week at 30 °C for colonization. The isolated strains were cultured in brain heart infusion broth (Difco) with 0·1 % glucose and 0·1 % glycerol for 5 days at 30 °C prior to DNA extraction, amplification and sequencing. Bacterial strains were cultured in brain heart infusion broth with 2 % glucose and 2 % glycine for 3 days at 30 °C for use in DNA–DNA hybridization experiments. Morphological observations under a scanning electron microscope (model S-5200; Hitachi) were made on cultures grown on MHII agar with 0·2 % glucose or humic acid-MOPS gellan gum medium (Suzuki et al., 2000) at 30 °C for 7–10 days.
Decomposition of adenine, casein, hypoxanthine, tyrosine, urea and xanthine was examined by 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 and growth at 37 and 45 °C were determined by using the modified method of Poonwan et al. (1995). The strains isolated were tested for their ability to grow on MHII 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 isomers using TLC (Staneck & Roberts, 1974). Whole-cell sugars were prepared as reported previously (Lechevalier & Lechevalier, 1980) and analysed by TLC (Miyadoh, 2001). Mycolic acids were prepared and analysed as described by Minnikin et al. (1980). Menaquinones were extracted from freeze-dried biomass (500 mg) and analysed as described by Chun & Goodfellow (1995). The strain was 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 the analyses. 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 package (Sasser, 1990). The following conditions were applied: carrier gas, ultra-high-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 (Kageyama et al., 2002).
16S rRNA genes were amplified and sequenced using a PCR employing six prokaryotic 16S rRNA universal primers. The PCR was performed in 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. PCR products were purified using a Centri-Sep column (Princeton Separations). DNA sequences were determined using an automatic sequence analyser (ABI PRISM 3100; PE Applied Biosystems) with 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 the BLAST programs (nucleotide–nucleotide BLAST: http://www.ncbi.nlm.nih.gov/BLAST). Sequences of related species were also retrieved from GenBank. Nucleotide substitution rates (Knuc values) were calculated (Kimura & Ohta, 1972) and phylogenetic trees were constructed using 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.
DNA was also isolated as described by Saito & Miura (1983) with modification for successive DNA base composition analysis as estimated by HPLC (Tamaoka & Komagata, 1984). Levels of DNA–DNA relatedness were determined by the method of Ezaki et al. (1989) using photobiotin and microplates.
The chemotaxonomic and morphological characteristics of these four isolates were consistent with their assignment to the genus Nocardia (Goodfellow, 1998; Goodfellow et al., 1999). All strains contained galactose and arabinose as characteristic whole-cell sugars in addition to meso-diaminopimelic acid as the dominant cell-wall diamino acid. In addition, the strains contained mycolic acid that co-migrated (Rf value approx. 0·47) with that extracted from Nocardia type strains. The major menaquinone was MK-8(H4
-cycl). Analysis of the fatty acids by GLC revealed the expected pattern that is diagnostic for members of the genus Nocardia and related taxa, i.e. straight-chain saturated and unsaturated fatty acids together with a diagnostic amount of tuberculostearic acid (10-methyl-branched octadecanoic acid) (Table 1).
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). It was clear from the phylogenetic tree (Fig. 1) that the four isolates formed a monophyletic clade loosely associated with N. farcinica. The sequence similarity values among IFM 10311T, IFM 10312 and IFM 10313 were >99·9 %, and these three isolates and IFM 10084T had 99·1–99·3 % identity; the sequence similarity values between these four strains and N. farcinica were <98·9 %. The distinction between these four strains and N. farcinica was supported by DNA–DNA relatedness data. The DNA relatedness levels among IFM 10311T, IFM 10312 and IFM 10313 were between 88 and 96 %. IFM 10311T, IFM 10312 and IFM 10313 showed DNA relatedness values of 17–32 % with IFM 10084T. The four isolates revealed DNA–DNA relatedness values of <12 % with the type strain of N. farcinica (Table 2). These values were well below the 70 % cut-off point for species classification as recommended by Wayne et al. (1987).
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). From the investigations described, it was clear that the strains could be distinguished from other Nocardia species with validly published names by a combination of phenotypic characteristics (see supplementary table in IJSEM Online). It was possible to differentiate Nocardia species from the strains isolated using a combination of the results of the utilization of sorbitol and citrate, susceptibility to fluorouracil, imipenem, tobramycin and kanamycin and growth at 45 °C.
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Description of Nocardia shimofusensis sp. nov.
Nocardia shimofusensis (shi.mo.fus.en'sis. N.L. fem. adj. shimofusensis pertaining to Shimofusa, a traditional geographical name for a northern part of Chiba Prefecture in Japan, the source of the isolates).
Aerobic, Gram-positive, partially acid-fast, non-motile actinomycetes that form an extensively branched substrate mycelium that fragments into rod-shaped elements (0·5–0·7x0·9–1·7 µm). Orange to reddish orange substrate mycelium carries white to reddish white aerial hyphae. No soluble pigment is produced. Colonies are 0·2–1·0 mm in diameter after 7 days at 30 °C on MHII medium with 0·2 % glucose. Arabinose, erythritol, galactose, glucose, inositol, maltose, mannose, rhamnose, sorbitol and citrate are not utilized. Adenine, casein, hypoxanthine, tyrosine and xanthine are not decomposed. Urea is decomposed. Does not grow at 45 °C. The G+C content of the DNA is 68–69 mol%.
The type strain, IFM 10311T (=NBRC 100134T=JCM 12122T=DSM 44733T), was isolated from soil at Choshi in Japan.
Description of Nocardia higoensis sp. nov.
Nocardia higoensis (hi.go.en'sis. N.L. fem. adj. higoensis pertaining to Higo, a traditional geographical name for Kumamoto Prefecture in Japan, the source of the type strain).
Aerobic, Gram-positive, partially acid-fast, non-motile actinomycetes that form an extensively branched substrate mycelium that fragments into rod-shaped elements (0·6–0·8x0·9–2·1 µm). Orange to reddish orange substrate mycelium carries white to reddish white aerial hyphae. Colonies without aerial hyphae may be produced, but they have a 16S rRNA gene sequence identical to that of the parent colonies (accession no. AB108778). No soluble pigment is produced. Colonies are 0·3–0·6 mm in diameter after 7 days at 30 °C on MHII medium with 0·2 % glucose. Arabinose, erythritol, galactose, glucose, inositol, maltose, mannose, rhamnose, sorbitol and citrate are not utilized. Adenine, casein, hypoxanthine, tyrosine and xanthine are not decomposed. Urea is decomposed. Grows at 45 °C. The G+C content of the DNA is 69 mol%.
The type strain, IFM 10084T (=NBRC 100133T=JCM 12121T=DSM 44732T), was isolated from a patient with lung nocardiosis.
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ACKNOWLEDGEMENTS |
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