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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 Division of Respiratory Medicine, St Luke's International Hospital, 9-1 Akashicho, Chuo-ku, 104-8560, Tokyo, Japan
3 Discovery Research Laboratories, Tanabe Seiyaku Co. Ltd, 2-2-50 Kawagishi, Toda, Saitama 335-8505, Japan
4 Department of Laboratory Medicine, Chiba University Hospital, School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan
5 DSMZ – 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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An extended phylogenetic tree based on 16S rRNA gene sequences, the TLC mycolic acid patterns of strain IFM 0803T and a comparison of the physiological properties of 53 recognized Nocardia species are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). The primary treatment of nocardial infections in immunocompromised patients is by antibiotic therapy (Conville et al., 2004). The initial selection of antibiotic therapy should take account of the species involved as nocardial strains are known to show species-specific drug-susceptibility patterns (Mikami & Yazawa, 1989). The most prevalent species is considered to be Nocardia farcinica (Hitti & Wolff, 2005). However, it has been repeatedly reported that N. farcinica is resistant to most of the antibiotics that are typically used for the treatment of nocardial infections (Hitti & Wolff, 2005; Kageyama et al., 2004c). Recently, the whole genome of N. farcinica was sequenced and the study revealed that this organism has more sophisticated drug-resistance mechanisms than previously thought (Ishikawa et al., 2004). These data further suggest that nocardial species determination is necessary in order to choose the most appropriate therapeutic drug. Two bacterial strains, IFM 0803T and IFM 10383, isolated from clinical specimens in Japan, were initially identified in our laboratory as Nocardia asteroides from morphological and biochemical characteristics. However, 16S rRNA gene sequencing and DNA–DNA hybridization of the two novel strains showed that they belong to a distinct species. This study was performed to determine the taxonomic position of the two novel strains using a polyphasic taxonomic approach. Strains IFM 0803T and IFM 10383 were isolated from a bronchoalveolar lavage of a 43-year-old immunocompromised patient with lung abscess at Chiba University Hospital, Chiba, Japan, and from a 60-year-old immunocompromised patient diagnosed with pemphigus vulgaris at St Luke's International Hospital, Tokyo, Japan, respectively.
All nocardial strains used in this study were cultured on Muller Hinton II (MH II; Difco) agar slants with 1 % glucose and 1 % glycerol at 37 °C for 1 week. Colony morphology and micromorphological properties of strains IFM 0803T and IFM 10383 were observed by light and scanning electron microscopy according to previously described procedures (Kageyama et al., 2004b). Physiological and biochemical properties were examined using published procedures (Gordon et al., 1974; Poonwan et al., 1995). Arylsulfatase activity was determined using the method of Kubica & Beam (1961). The minimum inhibitory concentration (MIC) values for seven drugs, arbekacin, ampicillin, clarithromycin, cefetamet pivoxil cefotiam, erythromycin and a mixture of sulfamethoxazole–trimethoprim (152 : 8) were determined using the method described by Mikami & Yazawa (1989). Drug susceptibility tests were performed with TRIDISK (Eiken Chemical Co.) using the method described by Kageyama et al. (2004b). Strains were stained by a modified method (Chapin & Murray, 1999), using 0.5 % sulfuric acid for determination of acid–alcohol-fastness.
The 16S rRNA gene was amplified using PCR employing six prokaryotic 16S rRNA universal primers and sequenced following procedures described previously (Kageyama et al., 2004a). Species related to the novel isolates were identified by performing a nucleotide sequence database search using BLAST programs (nucleotide–nucleotide BLAST: http://www.ncbi.nlm.nih.gov/blast) from GenBank. 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). Tree topology was evaluated using a bootstrap analysis (based on 1000 resamplings) of sequence data using CLUSTAL W software (Thompson et al., 1994). Sequence similarity values were determined through visual comparison and manual calculation. Genomic DNA was prepared as described previously (Kageyama et al., 2004b, c). The DNA G+C composition was estimated by HPLC (Tamaoka & Komagata, 1984). Levels of DNA–DNA relatedness were determined using the method of Ezaki et al. (1989) using photobiotin and microplates.
Whole-cell hydrolysates were analysed for diaminopimelic acid (A2pm) isomers using TLC (Staneck & Roberts, 1974). Whole-cell sugars were prepared according to published protocols (Lechevalier & Lechevalier, 1980) and analysed using the TLC method (Miyadoh, 2001). Mycolic acids were prepared according to Minnikin et al. (1980). Menaquinones were analysed as described by Chun & Goodfellow (1995). Fatty acid methyl esters were prepared and analysed as described previously using the standard Microbial Identification System (MIDI) for automated GC analysis (Sasser, 1990; Kämpfer & Kroppenstedt, 1996).
Comparison of the almost-complete 16S rRNA gene sequences of strains IFM 0803T and IFM 10383 with those of representatives of genera classified in the suborder Corynebacterineae showed that they contained all the signature nucleotides that are expected for members of the family Nocardiaceae (Stackebrandt et al., 1997) and the genus Nocardia (Chun & Goodfellow, 1995). Highest sequence similarities were found with Nocardia beijingensis, Nocardia arthritidis, Nocardia abscessus, Nocardia brevicatena, Nocardia paucivorans and Nocardia xishanensis, with sequence similarities of 98.3, 98.9, 98.6, 98.4, 98.3 and 98.9 %, respectively. Gene sequence similarity between strains IFM 0803T and IFM 10383 was 99.7 %. A tree constructed using the neighbour-joining method depicting the phylogenetic placement of strains IFM 0803T and IFM 10383 within a subset of the genus Nocardia is shown in Fig. 1. This tree shows that the two novel strains form a monophyletic clade that is loosely associated with N. xishanensis JCM 12160T. Sequence similarity values between strains IFM 0803T and IFM 10383 and N. xishanensis were 98.6 % (1367/1389) and 98.9 % (1396/1412).
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Chemotaxonomic and morphological characteristics of strains IFM 0803T and IFM 10383 were consistent with their assignment to the genus Nocardia (Goodfellow, 1998; Goodfellow et al., 1999). Both contain galactose and arabinose as characteristic whole-cell sugars and meso-A2pm as the diagnostic cell-wall diamino acid (chemotype IV sensu Lechevalier & Lechevalier, 1980). The major menaquinone was MK-8(H4
-cycl). The mycolic acid major chain length ranged from C48 to C56 for strains IFM 0803T and IFM 10383. TLC separation of whole-cell trans esterification extracts of strains IFM 0803T and IFM 10383 revealed two or three spots (see Supplementary Fig. S1 in IJSEM Online). The lower spot co-migrated with nocardia-mycolic acids. The second spot, an unknown lipid, migrated to a position between nocardia-mycolic acid and non-hydroxylated fatty acids and the RF value of the higher spot (third spot) correlated with non-hydroxylated fatty acids. The higher spot of strain IFM 0803T co-migrated with extracts from N. xishanensis JCM 12160T (Zhang et al., 2004). Our TLC analysis revealed the presence of the higher spot of mycolic acid in a few species of Nocardia, such as N. abscessus DSM 44432T, N. beijingensis JCM 10666T and Nocardia asiatica IFM 0245T (Poonwan et al., 2005). Analyses of fatty acids of strains IFM 0803T and IFM 10383 by GLC revealed the diagnostic pattern of members of the genus Nocardia and related taxa: straight-chain saturated and unsaturated fatty acids, together with a diagnostic amount of tuberculostearic acid.
Phenotypic characteristics of the two novel strains were also examined and compared with those of recognized species of the genus Nocardia. The novel strains were able to decompose casein, but N. xishanensis JCM 12160T was not. N. xishanensis utilizes adipic acid, but IFM 0803T and IFM 10383 do not. Citrate is used by the novel strains, but not by N. xishanensis JCM 12160T. Based on these phenotypic characteristics, strains IFM 0803T and IFM 10383 can be distinguished from N. xishanensis JCM 12160T. The two novel strains could also be distinguished from N. xishanensis JCM 12160T on the basis of drug susceptibility tests. The differential features of the phenotypes are summarized in Table 1. A comparison of phenotypic characteristics between the two novel strains and additional species of Nocardia is available as Supplementary Table S1 in IJSEM Online. Both phenotypic and genotypic data show that the two novel isolates represent a novel species within the genus Nocardia, for which the name Nocardia exalbida sp. nov. is proposed.
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Aerobic, Gram-positive, partially acid-fast, non-motile actinomycetes that form an extensively branched substrate mycelium that fragments into rod-shaped elements (0.4–0.8x0.8–1.7 µm). Orange–tan to tan colonies with abundant white to off-white aerial mycelium. No soluble pigment is produced. Colonies are 1.0–2.5 mm in diameter after 7 days at 30 °C on MH II medium with 0.2 % glucose. Casein is decomposed, but adenine, arbutin, elastin, hypoxanthine, tyrosine and xanthine are not decomposed. Urea is decomposed. Glucose is utilized, but arabinose, erythritol, galactose, inositol, maltose, mannose, rhamnose and sorbitol are not. Citrate and testosterone are utilized, but adipic acid, acetamide and gluconate are not. No growth at 45 °C. The G+C content of the DNA is 68 mol%. Susceptible to imipenem and tobramycin (growth inhibition zone around a paper disc with 2.5 µg drug per disc), moderately susceptible to kanamycin (10 µg disc), but not susceptible to 5-fluorouracil (30 µg disc).
The type strain, IFM 0803T (=NBRC 100660T=DSM 44883T=JCM 12667T), was isolated from the bronchoalveolar lavage of a patient with lung nocardiosis in Japan.
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ACKNOWLEDGEMENTS |
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