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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 Clinical Laboratory Chiba University Hospital, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8673, Japan
3 Department of Laboratory Medicine, Chiba University School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8673, Japan
4 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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Published online ahead of print on 15 April 2005 as DOI 10.1099/ijs.0.63282-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of IFM 10032T and IFM 10148 are AB122026 and AB122027, respectively.
Detailed physiological properties of strains IFM 10032T and IFM 10148 and representative Gordonia species are given in supplementary tables in IJSEM Online.
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Gordonia includes around 20 species (Arenskotter et al., 2005), among which Gordonia terrae has been considered to be a cause of primary cutaneous infection, and Gordonia aichiensis, Gordonia bronchialis and Gordonia rubropertincta have been isolated from sputum specimens of patients with pulmonary disease. Gordonia sputi was also isolated from the sputa of patients with chronic pulmonary diseases (Klatte et al., 1994). Therefore, most of the Gordonia species described hitherto have been considered as opportunistic pathogens in humans (Tsukamura, 1971, 1978, 1982). However, most of the species of Gordonia recently described were isolated from the environment, and have been reported to play important roles in bioremediation or the biodegradation of pollutants (Linos et al., 1999, 2002; Maldonado et al., 2003; Kim et al., 2000, 2003; Xue et al., 2003; Yoon et al., 2000).
Two hundred and thirty-five strains of pathogenic actinomycetes were isolated from clinical specimens in Japan from 2000 to 2003 and referred to our research centre for identification. Of the 235 isolates, 15 strains were found to belong to Gordonia species. From these 15 isolates, strains IFM 10032T and IFM 10148 were chosen for identification. Based on 16S rRNA gene sequence analysis these two strains are most closely related to G. aichiensis, G. sputi and ‘Gordonia jacobaea’ (de Miguel et al., 2000). The proposed type strain of ‘G. jacobaea’, MV-1 (=IFM 10484), was also obtained and used for comparative purposes. Comparative DNA–DNA relatedness data showed that strains IFM 10032T and IFM 10148 belong to the same species, but are distinct from G. aichiensis, G. sputi and ‘G. jacobaea’. Based on our phenotypic and genotypic data, the clinical isolates represent a novel species within the genus Gordonia, for which the name Gordonia otitidis sp. nov. is proposed.
Strains IFM 10032T and IFM 10148 were isolated in 2000 from ear discharge of a 28-year-old Japanese female patient with external otitis, and in 2002 from pleural fluid of a 60-year-old Japanese male patient with bronchitis, respectively. Both strains were isolated on blood agar plates at 37 °C for 7 days.
Strains IFM 10032T, IFM 10148, G. aichiensis IFMO473T, G. sputi IFM 0477T and ‘G. jacobaea’ IFM 10484 were cultured on Muller–Hinton II (MH II; Difco) agar slants with 1 % glucose and 1 % glycerol for 1 week at 27 °C. For extraction of DNA and sequencing, bacterial strains were cultured in a 1 % glucose containing brain heart infusion (BHI; Difco) broth. Morphological observations under a scanning electron microscope (model S-5200; Hitachi) were made on cultures grown on BHI agar.
Carbon source utilization tests were performed as described by Takeuchi & Hatano (1998). Biochemical characterization of the isolates was performed using the Biolog GP2 MicroPlate assay (Biolog). A modified Ziehl–Nielsen method was used for acid-fast staining test. Whole-cell hydrolysates were analysed for diaminopimelic acid (DAP) isomers using TLC (Staneck & Roberts, 1974). Whole-cell sugars were prepared as detailed by Lechevalier & Lechevalier (1980) and were analysed by a TLC method (Miyadoh, 2001). Fatty acid methyl esters and mycolic acid trimethylsilyl ethers were prepared and analysed as described by Klatte et al. (1994) using the standard Microbial Identification System (MIDI Inc.) for automated GC analyses (Sasser, 1990; Kämpfer & Kroppenstedt, 1996). Menaquinones were extracted from freeze-dried biomass and analysed as described by Chun & Goodfellow (1995).
Preparation of genomic DNA samples for sequencing was performed using the guanidine thiocyanate method of Kageyama et al. (2004a, b).
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 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 the BigDye terminator cycle sequencing ready reaction kit (PE Applied Biosystems). The 16S rRNA gene sequences were analysed and species related to the isolates were identified by performing a BLAST sequence database search. Nucleotide 200 substitution rates (Knuc values) were calculated (Kimura & Ohta, 1972). Phylogenetic trees were constructed by the neighbour-joining method (Saitou & Nei, 1987), and the topology of the trees was evaluated by a bootstrap analysis of the sequence data using CLUSTAL W software (Thompson et al., 1994).
Preparation of genomic samples for DNA–DNA hybridization was performed using a modified method of that described by Saito & Miura (1983). 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.
Whole-cell hydrolysates of strains IFM 10032T and IFM 10148 contained meso-DAP as the only diamino acid of the peptidoglycan and arabinose plus galactose as major whole-cell sugars. The fatty acid pattern of strain IFM 10032T comprised straight-chain saturated and unsaturated fatty acids plus tuberculostearic acid (Table 1). Although this pattern is roughly the same in all gordoniae, there are qualitative and quantitative species-specific and often intraspecific differences (Klatte et al., 1994) (Table 1). The predominant menaquinone is MK-9(H2), but substantial amounts of MK-8(H2) are also present. The G+C contents of the DNA of strains IFM 10032T and IFM 10148 are 64·9 and 65·2 mol%, respectively.
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; Stackebrandt et al., 1988; Klatte et al., 1994).
Strains IFM 10032T and IFM 10148 produced short elementary branching hyphae that disintegrated into rod- and cocci-like elements. They showed the typical rod–coccus growth cycle that is usually found among strains of the genus Gordonia and related taxa (Goodfellow, 1992).
The mycolic acid pattern of strain IFM 10032T is similar to those of G. aichiensis, G. sputi, Gordonia desulfuricans, ‘G. jacobaea’ and Gordonia polyisoprenivorans, which possess similar sized mycolic acids of chain length of about C58–C64 and with the principal mycolic acid C62 comprising about 50 % (54 % for IFM 10032T) of the total (Table 2).
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The phylogenetic tree shown in Fig. 1 based on the 16S rRNA gene sequences shows that strains IFM 10032T and IFM 10148 cluster with G. aichiensis IFM 0473T, G. sputi IFM 0477T and ‘G. jacobaea’ IFM 10484.
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for the delineation of separate species.
Results of physiological tests obtained from growth on agar plates revealed that strains IFM 10032T and IFM 10148 were able to utilize 18 out of 32 carbon sources. Strains IFM 10032T and IFM 10148 gave identical responses in carbon utilization tests (Table 3). Some carbon utilization patterns useful for identifying and differentiating strains IFM 10032T and type strains of other species of the genus Gordonia are given in Table 3 and under the species description below. The utilization pattern clearly showed that IFM 10032T could be differentiated from G. aichiensis IFM 0473T, G. sputi IFM 0477T and ‘G. jacobaea’ IFM 10484.
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(see also Supplementary Table S2), and also confirmed that IFM 10032T could be differentiated from G. sputi IFM 0477T, G. aichiensis IFM 0473T and ‘G. jacobaea’ IFM 10484.
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). Based on the source of isolation, from a patient with external otitis, the name Gordonia otitidis sp. nov. is proposed. Because the name ‘G. jacobaea’ has not been validly published in IJSEM in accordance with Rule 27 of the Bacteriological Code, the present studies included characterization of the proposed type strain of ‘G. jacobaea’, MV-1 (=IFM 10484=DSM 44880=JCM 12643).
Description of Gordonia otitidis sp. nov.
Gordonia otitidis (o.ti'ti.dis. N.L. gen. n. otitidis of otitis, inflammation of the ear).
Aerobic, Gram-positive, partially acid-fast, nitrate-reducing, non-motile actinomycetes that form rod/coccus-like elements (0·6–0·8x2·5–3·1 µm). Colonies are white, changing with time to apricot to pale orange. Colonies of the type strain are rough with irregular margins. Utilizes sucrose, D-turanose, D-arabitol, N-acetyl-D-glucosamine, 4-aminobutyrate, 2-hydroxyvalerate, 2-oxoglutarate, pimelate, benzoate, 4-hydroxybenzoate, phenylacetate, quinate, L-alanine, L-leucine, L-proline, L-serine, L-valine, putrescine and acetamide. Does not utilize D-galactose, L-rhamnose, D-ribose, i-inositol, glucarate, gluconate, caprate, succinate, 3-hydroxybenzoate, citrate, L-aspartate or tyramine. Whole-cell hydrolysates contain meso-DAP, arabinose and galactose (cell-wall chemotype IV sensu Lechevalier & Lechevalier, 1980). Predominant menaquinone is MK-9(H2); a small amount of MK-8(H2) is also present. Principal mycolic acids have a chain length (58–64 carbons) equivalent to those of other Gordonia species. Physiological properties are given in Supplementary Table S1 available in IJSEM Online. The type strain is a clinical isolate. The G+C content of the DNA is 64·9–65·2 %.
The type strain, IFM 10032T (=DSM 44809T=JCM 12355T=NBRC 100426T), was isolated from ear discharge of a patient with external otitis, Japan.
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ACKNOWLEDGEMENTS |
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