Nocardia ninae sp. nov., isolated from a bronchial aspirate

A Gram-positive and acid-fast filamentous bacterium (OFN 02.72^T) was isolated from a bronchial aspirate from a 53-year-old patient. Chemotaxonomic data supported the affiliation of this organism to the genus Nocardia, and the phenotypic characteristics demonstrated that the strain differed from all previously described Nocardia species. Restriction enzyme analysis of the hsp65 gene (encoding the 65 kDa heat-shock protein) and sequencing of the 16S rRNA and hsp65 genes confirmed that this isolate is unique. The most closely related type strains were Nocardia alba YIM 30243^T (=DSM 44684^T) and Nocardia jejuensis N3-2^T (=JCM 13281^T) (with 16S rRNA gene sequence similarities of 98.3 and 97.2 %, respectively). On the basis of this polyphasic study, strain OFN 02.72^T represents a novel species within the genus Nocardia, for which the name Nocardia ninae sp. nov. is proposed. The type strain is OFN 02.72^T (=CIP 108955^T=DSM 44978^T).

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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and hsp65 gene sequences of strain OFN 02.72^T are DQ235687 and DQ250023, respectively.

Extended phylogenetic trees based on 16S rRNA and hsp65 gene sequences are available as supplementary figures in IJSEM Online.

Nocardia species are ubiquitous in the environment and are an increasingly recognized cause of suppurative and granulomatous opportunistic infections, especially in immunocompromised patients (McNeil & Brown, 1994). Accurate identification of the causative species is necessary for optimal treatment and for epidemiological purposes. The identification of novel bacterial species has long been based on epidemiological characteristics, the clinical disease spectrum and drug susceptibility, together with cultural, biochemical and chemotaxonomic characteristics (Boiron et al., 1993). Nocardia species are now classified using a range of genotypic and phenotypic data, and combinations of such characteristics have been used to describe many novel species (Maldonado et al., 2000; Li et al., 2004; Rodríguez-Nava et al., 2004), more than 30 having been described in the last decade. Here we describe a novel Nocardia species based on a single isolate obtained by bronchial aspiration of a patient. The isolate, strain OFN 02.72^T, was characterized by using phenotypic and genotypic methods, 16S rRNA and hsp65 gene sequence analyses and DNADNA hybridization.

Strain OFN 02.72^T was isolated from a 53-year-old patient admitted to Beaujon Hospital, Clichy, France, for an asthma attack. The patient was receiving long-term systemic steroid therapy. The isolate was co-cultured along with Haemophilus influenzae and Streptococcus pneumoniae. The patient had respiratory and inflammatory disorders (i.e. an elevated C-reactive protein level and hyperleukocytosis) but no fever or radiological evidence of abscesses. According to the attending physicians, the isolate was considered to be of uncertain clinical significance. After initial isolation on a blood-agar plate at 37 °C for 72 days, the isolate was subcultured on Bennett agar at 37 °C for 7 days and then maintained as a glycerol suspension (20 %, v/v) at 20 °C. It was examined for pigmentation, the production of aerial hyphae and morphological characteristics as described previously (Rodríguez-Nava et al., 2004). Acid-fastness was tested with a modified ZiehlNeelsen method (1 % acid decolorization) (Boiron et al., 1993). Growth at 25 and 37 °C was examined after cultivation for 2 weeks on Bennett agar.

Methods described by Boiron et al. (1993), Goodfellow (1992, 1998) and Goodfellow & Lechevalier (1989) were used to examine the following characteristics: (i) decomposition of adenine, casein, hypoxanthine, testosterone, tyrosine, uric acid and xanthine; (ii) utilization of L-arabinose, D-galactose, D-glucose, maltose, D-mannitol, mannose, raffinose, L-rhamnose, D-ribose, sucrose and sorbitol as sole carbon sources; and (iii) production of urease and catalase. β-Lactamase production was tested by using the chromogenic cephalosporin disc method (Cefinase; bioMérieux) as reported previously (Rodríguez-Nava et al., 2004). The diaminopimelic acid isomer was determined by TLC of whole-cell hydrolysates as described by Boiron et al. (1993). Standard procedures for the determination of fatty acids and mycolic acids were performed by the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany), using HPLC as described by Kroppenstedt (1985), with the standard Microbial Identification System (MIDI) for automated GC analysis, as described previously (Klatte et al., 1994). Isoprenoid quinones were extracted from freeze-dried biomass by using the small-scale procedure of Minnikin et al. (1975, 1984) and were then separated by HPLC and analysed as described previously (Kroppenstedt, 1982, 1985).

Nocardia-specific 16S rRNA gene amplification (Laurent et al., 1999) and hsp65 gene polymorphism restriction analysis were also used (Steingrube et al., 1997; Rodríguez-Nava et al., 2006). An almost-complete 16S rRNA gene sequence (1321 nt, corresponding to positions 461400 of the Escherichia coli numbering system) and a partial hsp65 gene sequence (440 nt) were obtained. For phylogenetic analysis, these sequences were aligned with the corresponding sequences of all Nocardia species (from the GenBank database) by using the multiple sequence alignment program CLUSTAL W (Thompson et al., 1994). Using Phylo_win software (Galtier et al., 1996), evolutionary trees were inferred from three treeing algorithms, the maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Kluge & Farris, 1969) and neighbour-joining (Saitou & Nei, 1987) methods. The robustness of the trees was assessed by bootstrap resampling (1000 replicates each). The degree of genomic relatedness between strain OFN 02.72^T and Nocardia alba DSM 44684^T (taxonomically, the most closely related species on the basis of 16S rRNA gene sequence similarity) was determined by DNADNA hybridization, as described initially by De Ley et al. (1970) and modified by Huß et al. (1983). DNA was isolated by using a French pressure cell and was purified by chromatography on hydroxyapatite as described by Cashion et al. (1977).

The morphological and chemotaxonomic characteristics obtained for strain OFN 02.72^T were consistent and support its assignment to the genus Nocardia. The isolate formed small, orange colonies on Bennett-agar plates. No diffusible pigmentation was observed. The bacteria were Gram-positive and produced branched hyphae that tended to fragment into rod-like and coccoid elements. The filaments were acid-fast in the modified acid-fast staining method.

Whole-cell hydrolysates of strain OFN 02.72^T contained meso-diaminopimelic acid as the only peptidoglycan diamino acid. Analysis of the cell wall composition revealed mycolic acids with a chain length of 5258 carbon atoms. This pattern was compatible with that of Nocardia species (C₅₀C₆₂) (McNeil & Brown, 1994). The main fatty acids were C_{16 : 0} (38.29 %), C_{16 : 1} (23.90 %), C_{18 : 0} (2.09 %) and tuberculostearic acid (10.76 %). The main menaquinone was MK-8(H₄, cycl.) (constituting 68 % of the total menaquinone content). Smaller amounts of MK-8(H₂) (9 %) and MK-8(H₄) (23 %) were also detected. All of these chemotaxonomic properties are typical of members of the genus Nocardia (Goodfellow, 1998; Minnikin et al., 1975).

Detailed results from the physiological tests are shown in Table 1. The phenotypic properties, particularly the decomposition of complex substrates, were sufficiently atypical to distinguish the isolate from all Nocardia species with validly published names.

Table 1. Physiological characteristics of strain OFN 02.72T and related type strains of the genus Nocardia Strains: 1, strain OFN 02.72T; 2, N. alba DSM 44684T; 3, N. jejuensis JCM 13281T; 4, Nocardia fluminea DSM 44489T; 5, Nocardia ignorata DSM 44496T; 6, Nocardia soli DSM 44488T; 7, Nocardia cummidelens DSM 44490T; 8, Nocardia asteroides ATCC 19247T; 9, Nocardia farcinica DSM 43578T; 10, Nocardia brasiliensis ATCC 19296T; 11, Nocardia pseudobrasiliensis DSM 44290T. Data for reference strains were taken from Isik et al. (1999) and Maldonado et al. (2000). All of the strains were negative for the decomposition of xanthine (at 0.4 %, w/v). +, Positive; , negative; W, weak. ND, no data available.

The Nocardia-specific 16S rRNA gene PCR produced a positive result, and the hsp65 polymorphism restriction analysis yielded a restriction pattern (BstEII, 440 bp; MspI, 70, 110 and 265 bp; HinfI, 60, 130 and 250 bp) distinct from those of all Nocardia species (Rodríguez-Nava et al., 2006). Comparison of the almost-complete 16S rRNA sequence of strain OFN 02.72^T with those of the type strains of all species of the genus Nocardia with validly published names (i.e. sequences extracted from GenBank) indicated that the isolate was affiliated to this genus. The evolutionary trees inferred from the three treeing algorithms (maximum likelihood, maximum parsimony and neighbour joining) were in agreement: the various branches were supported in all three methods. On the basis of these sequences, the most closely related type strains were N. alba YIM 30243^T (=DSM 44684^T) (Li et al., 2004) and Nocardia jejeunesis N3-2^T (=JCM 13281^T) (Lee, 2006). The phylogenetic tree based on the 16S rRNA gene sequence (Fig. 1 and Supplementary Fig. S1 available in IJSEM Online) showed that the isolate differed markedly from all other Nocardia species. N. jejuensis is the most closely related species according to the phylogenetic tree, but the bootstrap percentage was low (47 %; i.e. below 50 %). This shows clearly that the two species are not actually phylogenetically related.

(40K): Fig. 1. Phylogenetic tree, based on 16S rRNA gene sequences, showing the relationships of strain OFN 02.72T and the 10 most closely related species belonging to the genus Nocardia. This tree was constructed by using the neighbour-joining method and was based on a comparison of 1321 nt. The tree was rooted by using Streptomyces somaliensis DSM 40738T as the outgroup (not shown). Bootstrap values were expressed as percentages of 1000 replications. The branching is supported by the results from the three algorithms used. Bar, 0.01 substitutions per nucleotide position. The full tree from which this area is taken is available as Supplementary Fig. S1 in IJSEM Online.

The degree of 16S rRNA gene sequence similarity between strain OFN 02.72^T and N. jejuensis N3-2^T was only 97.2 % (corresponding to 36 nucleotide differences among 1321 nucleotide positions), whereas it was 98.3 % (corresponding to 22 nucleotide differences among 1321 nucleotide positions) between strain OFN 02.72^T and N. alba YIM 30243^T. These data were confirmed by the levels of hsp65 gene sequence similarity: strain OFN 02.72^T was closer to N. alba DSM 44684^T (94.2 % similarity) than to N. jejuensis JCM 13281^T (only 90.6 % similarity). On the basis of phylogenetic analysis using hsp65 gene sequences, OFN 02.72^T was clearly separated from N. alba DSM 44684^T, from N. jejuensis JCM 13281^T and from all other Nocardia species (Fig. 2 and Supplementary Fig. S2). The level of DNADNA relatedness between strain OFN 02.72^T and N. alba DSM 44684^T (<20 %) was well below the 70 % cut-off recommended for the circumscription of bacterial genomic species (Wayne et al., 1987).

(30K): Fig. 2. Phylogenetic tree, based on hsp65 gene sequences, showing the relationships of strain OFN 02.72T and the eight most closely related species belonging to the genus Nocardia. This tree was constructed by using the neighbour-joining method and was based on a comparison of 440 nt. The tree was rooted by using Streptomyces somaliensis DSM 40738T as the outgroup (not shown). Bootstrap values were expressed as percentages of 1000 replications. The branching is supported by the results from the three algorithms used. Bar, 0.01 substitutions per nucleotide position. The full tree from which this area is taken is available as Supplementary Fig. S2 in IJSEM Online.

This polyphasic taxonomic study clearly showed that the isolate warrants classification as a novel species within the genus Nocardia; therefore, the name Nocardia ninae sp. nov. is proposed for strain OFN 02.72^T.

Description of Nocardia ninae sp. nov.
Nocardia ninae (ni'nae. N.L. fem. gen. n. ninae of Nina, the first name of the patient from whom the type strain was isolated).

Aerobic, Gram-positive, acidalcohol-fast (modified ZiehlNeelsen method), non-motile actinomycete that forms a branched, orange substrate mycelium carrying sparse to moderate amounts of white aerial hyphae. Colonies on Bennett agar are rough and 13 mm in diameter. Distinguishing phenotypic properties are reported in Table 1.

The type strain, OFN 02.72^T (=CIP 108955^T=DSM 44978^T), was isolated from a human respiratory sample.