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1 Research group on ‘Bacterial Opportunistic Pathogens and Environment’, Université de Lyon, F-69003 Lyon, France
2 Université Lyon 1 and CNRS, UMR 5557, Ecologie Microbienne, F-69622 Villeurbanne, France
3 Institut des Sciences Pharmaceutiques et Biologiques, F-69373 Lyon, France
4 Observatoire Français des Nocardioses, F-69373 Lyon, France
5 Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait
6 Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany
Correspondence
Verónica Rodríguez-Nava
veronica.Rodriguez{at}sante.univ-lyon1.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, hsp65 and sod gene sequences of strains N11 and N12T are DQ250024, DQ235689, DQ25025, DQ235688, DQ683744 and DQ683745, respectively.
Additional phylogenetic trees based on analysis of the 16S rRNA, hsp65 and sod gene sequences are available as supplementary figures with the online version of this paper.
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). This genus is a member of the family Nocardiaceae and the order Actinomycetales (Stackebrandt et al., 1997). During the last ten years, with the advent of molecular phylogenetic techniques, the genus Nocardia has undergone a taxonomic revolution (Roth et al., 2003). At the time of writing, 40 species have so far been isolated from human and animal samples and 23 species have been isolated from environmental samples (http://www.bacterio.cict.fr/n/nocardia.html). During a taxonomic re-evaluation of environmental and human strains deposited in the culture collection of the French Nocardiosis Observatory (OFN, Lyon, France), we comprehensively investigated the taxonomic position of two selected strains, OFN N11 and OFN N12T. An integrated approach was used based on molecular, chemotaxonomic and phenotypic methods.
The two novel strains were isolated in 1997 from soil from the Ahmadi oilfield which had been contaminated with crude oil during the Gulf war. The paraffin bait technique used to isolate these micro-organisms was first described by Kurup & Schmitt (1971). The novel strains were initially identified as Nocardia asteroides by using basic physiological methods (Khan et al., 1997). Following this initial identification, the isolates were sent to the OFN by the Microbiology Department of Kuwait University for accurate identification. The strains were maintained as glycerol suspensions (20 %, v/v) at –20 °C. Growth was achieved on Bennett's agar at 37 °C. On this agar, both isolates were able to grow at temperatures between 25 and 37 °C. The orange-coloured substrate mycelium was well-developed, with irregular branches penetrating the agar and bearing sparse white aerial hyphae. The cells were Gram-positive and acid-fast as determined by a modified Ziehl–Neelsen method (1 % acid decoloration).
Several molecular methods were used to characterize the two isolates. Their affiliation to the genus Nocardia was confirmed by genus-specific 16S rRNA gene amplification (Laurent et al., 1999). Hsp65 polymorphism analysis (Steingrube et al., 1997) yielded a restriction pattern (BstEII 320 and 80 bp, MspI 130, 120, 115 and 70 bp and HinfI 250 and 190 bp) that was identical to that found for Nocardia ignorata (Rodriguez-Nava et al., 2005).
A multigene sequencing approach was then applied. Part of the 16S rRNA gene (around 1322 nucleotides, corresponding to positions 46–1400 of the Escherichia coli numbering system) of strains OFN N11 and OFN N12T was amplified by using primers SQ1 (5'-AGAGTTTGATCMTGGCTCAG-3') and SQ6 (5'-CGGTGTGTACAAGGCCC-3') as described by Rodríguez-Nava et al. (2004). A partial hsp65 gene sequence (65 kDa heat-shock protein, 441 nucleotides corresponding to positions 396–836 of Mycobacterium tuberculosis CIP 105795) was amplified by using primers TB11 (5'-ACCAACGATGGTGTGTCCAT-3') and TB12 (5'-CTTGTCGAACCGCATACCCT-3') as described by Rodríguez-Nava et al. (2006) and a partial sod gene sequence (superoxide dismutase, 442 nucleotides corresponding to positions 82–523 of Nocardia farcinica IFM 10152) was obtained with primers Z205 (5'-ACGTTCACCACAGCAAGCACCA-3') and Z212 (5'-TCGGCCCAGTTCACGACGTT-3') for the two test strains and for ten Nocardia type strains with similar 16S rRNA genes (N. ignorata, Nocardia soli, Nocardia cummidelens, Nocardia salmonicida, Nocardia fluminea, N. asteroides, Nocardia brasiliensis, Nocardia uniformis, Nocardia cyriacigeorgica and Nocardia tenerifensis) as described by Devulder et al. (2005).
The PCR products were purified by using a Microspin Gel Extraction kit (Omega, Bio-tek) and sequenced on both strands by using the same primers, the Taq Dye Deoxy terminator cycle sequencing kit (Applied Biosystems) and a DNA sequencer (model 373A; Applied Biosystems). For phylogenetic analysis, the nucleotide sequences (excluding the primers) were aligned with the corresponding sequences of representative Nocardia species from the GenBank database and the BIBI database (Devulder et al., 2003) by using CLUSTAL_X (Thompson et al., 1997).
The phylogenetic trees were constructed with the MEGA (Kumar et al., 2004) and PHYLO_WIN (Galtier et al., 1996) software packages with three treeing algorithms, namely the maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Fitch, 1971) and neighbour-joining (Saitou & Nei, 1987) methods. The tree topology was determined by using 1000 bootstrapped datasets.
Strains OFN N12T and OFN N11 shared identical sequences for all three of the genes examined. The closest relative of strains OFN N12T and OFN N11 was N. ignorata DSM 44496T, showing 99.4 % sequence similarity (8 nucleotide differences out of 1322 nt) for the 16S rRNA gene (Fig. 1 and Supplementary Fig. S1 in IJSEM Online), 99.5 % sequence similarity (2 differences out of 401 nt, excluding the primers) for the hsp65 gene (see Supplementary Fig. S2 in IJSEM Online) and 98.5 % sequence similarity (6 differences out of 406 nt, excluding the primers) for the sod gene (see Supplementary Fig. S3 in IJSEM Online). Smaller sequence similarities were found with the other species of the genus Nocardia. On the basis of the 16S rRNA and hsp65 gene sequences, phylogenetic analysis showed that the two novel isolates matched the N. salmonicida subclade (N. cummidelens, N. fluminea, N. ignorata, N. salmonicida and N. soli) (Maldonado et al., 2000). In contrast, this specific subclade was not recognized when the sod gene sequences were analysed (see Supplementary Fig. S2). Regardless of which of the three genes was considered, N. ignorata was the closest species phylogenetically with bootstrap values of 98, 100 and 100, respectively, for the 16S rRNA, hsp65 and sod genes (See Supplementary Figs S1–S3). These results demonstrate that strains OFN N12T and OFN N11 form a distinct phyletic line within the genus Nocardia.
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. For strain OFN N12T, the cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. Analysis of whole-cell hydrolysates yielded arabinose, galactose, glucose and ribose.
Standard analyses of fatty acids, mycolic acids and isoprenoid quinones were performed by means of HPLC and were interpreted as described by Kroppenstedt with the standard Microbial Identification System (MIDI) for automated GC analysis (Kroppenstedt, 1982, 1985; Minnikin et al., 1975, 1984). The analyses revealed the presence of mycolic acids with a chain length of 52–58 carbon atoms (the principal mycolic acids have a chain length of 56 carbon atoms) as expected for members of the genus Nocardia (C50–C62). The fatty acid content was C18 : 0 (16.0 %), C18 : 1 (6.5 %), C17 : 0 (3.8 %), C15 : 0 (1.7 %), C17 : 1 (1.5 %), C14 : 0 (1.1 %), C15 : 1 (0.3 %) and C10 : 0 (0.1 %) and the main fatty acids were palmitic acid (C16 : 0, 27.2 %), palmitoleic acid (C16 : 1, 20.22 %) and tuberculostearic acid (10 methyl-branched C18 : 0, 19.8 %). The main menaquinone (74.2 %) was MK-8(H4cycl) and small amounts of MK-8(H2) (5.6 %) and MK-8(H4) (8 %) were also detected.
The two novel isolates were also characterized phenotypically. The methods used for biochemical tests have been described previously (Boiron et al., 1993; Goodfellow, 1992, 1998). The ability to grow on various carbon sources was tested according to the methods of Goodfellow & Lechevalier, 1989 and Boiron et al., 1993 and by using the API 50 CH (bioMérieux) system according to the manufacturer's instructions. The API 50 CH strips were utilized for only the type strain of N. ignorata and strains OFN N11 and OFN N12T. Inocula were standardized to obtain a final concentration of 1x104–1x105 c.f.u. and were then suspended in API 50 CHB broth (bioMérieux). Tests were read daily, with a final reading taken at 4 days. Incubation beyond 4 days gave weak reactions in many wells that were interpreted as non-specific. Only the assimilation results were considered for the API 50 CH system as oxidation and fermentation reactions were weak and difficult to interpret. Sugar assimilations in the API 50 CH system were read as positive when heavy growth in the wells obscured the underlying stripes. These results showed that strains OFN N11 and OFN N12T could be distinguished from the type strain of the closest phylogenetic species, N. ignorata, by their growth on media containing glycerol, raffinose and sucrose as the sole carbon sources by using conventional methods and the API 50CH system. Some other biochemical properties useful for identifying and differentiating strain OFN N12T and the type strains of other species of the genus Nocardia are presented in Table 1 and the species description.
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, with modifications as described by Huß et al. (1983) and Escara & Hutton (1980). DNA was isolated by using a French pressure cell and was purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). Renaturation rates were computed with the TRANSFER.BAS program (Jahnke & Bahnweg, 1986; Jahnke, 1992). The DNA–DNA relatedness of strain OFN N12T and N. ignorata DSM 44496T was low (26 %) and well below the 70 % cut-off for species delineation as recommended by Wayne et al. (1987), confirming that strain OFN N12T and N. ignorata are not related at the species level. On the basis of the genotypic and phenotypic data described above, it is clear that strains OFN N11 and OFN N12T represent a novel Nocardia species. The name Nocardia coubleae sp. nov. is proposed and isolate OFN N12T (=CIP 108996T=DSM 44960T) is designated as the type strain.
The results obtained in this study confirm the utility of this multigenic approach (16S rRNA, hsp65, and sod gene sequencing) for the description of Nocardia coubleae sp. nov. and for the description of other novel species belonging to the genus Nocardia in the future.
Description of Nocardia coubleae sp. nov.
Nocardia coubleae (cou'ble.ae. N.L. gen. n. coubleae of Couble, named after Andrée Couble, in recognition of her contribution to the French Nocardiosis Observatory, Lyon, France).
Cells are Gram-positive, acid–alcohol-fast and strictly aerobic. Colonies on Bennett's agar are rough, 1–2 mm in diameter and include an orange to white substrate mycelium bearing sparse to abundant white aerial hyphae. Grows at 25 °C and 37 °C (optimum at 37 °C) and is able to utilize D-glucose, glycerol, raffinose and sucrose as sole carbon sources. Testosterone is decomposed, but adenine, casein, hypoxanthine, tyrosine, uric acid and xanthine are not decomposed. The most abundant fatty acids are palmitic acid (C16 : 0), 27.2 % and tuberculostearic acid, 19.8 %. Mycolic acids are 52–58 carbon atoms long. The cell-wall chemotype is IV. The main menaquinone is MK-8(H4cycl).
The type strain, strain OFN N12T (=DSM 44960T=CIP 108996T), was isolated from Kuwaiti soil.
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ACKNOWLEDGEMENTS |
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