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Institut für Medizinische Mikrobiologie und Immunologie der Universität Bonn, 53127 Bonn, Germany
Correspondence
A. F. Yassin
yassin{at}mibi03.meb.uni-bonn.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains IMMIB N-402T (=CCUG 50200T=CIP 108553T) and IMMIB N-403 (=CCUG 50201=CIP 108554) are AJ854057 and AJ854058, respectively.
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; Chun & Goodfellow, 1995; Rainey et al., 1995), in parallel with improved phenotypic approaches has greatly facilitated the discovery of novel species and has resolved the taxonomy of isolates that were collectively assigned to the Nocardia asteroides complex (Yassin et al., 2000b, 2001). The genus Nocardia has undergone considerable expansion in the past few years, and at the time of writing over 40 species are recognized. The vast majority of these species have been isolated from human clinical specimens (Hamid et al., 2001; Yassin et al., 2000a, b, 2001) and, to a lesser extent, from animal (Isik et al., 1999a) and soil (Isik et al., 1999b; Albuquerque de Barros et al., 2003) sources. In the present study, the taxonomic status of two isolates, IMMIB N-402T and IMMIB N-403, isolated from the sputa of a patient with a pulmonary infection, was determined using polyphasic taxonomic approaches. On the basis of the genotypic and phenotypic data reported here, the two isolates should be recognized as a novel species of the genus Nocardia for which the name Nocardia elegans sp. nov. is proposed.
Strains IMMIB N-402T and IMMIB N-403 were cultured on brain-heart infusion (Becton Dickinson) agar to determine their morphological properties. The strains were biochemically characterized by using tests for the hydrolysis of complex substrates, as described previously (Gordon, 1966, 1967; Gordon & Mihm, 1957), as well as tests to determine carbon-source utilization (according to Yassin et al., 1995). The presence of the isomeric form of diaminopimelic acid was tested by using the methods of Becker et al. (1964), while whole-cell sugars were determined by using the method of Lechevalier (1968). Lipids were extracted using acid methanolysis. Mycolic acids were detected by TLC as described by Minnikin et al. (1980); pyrolysis GC of the mycolates was performed according to Yassin et al. (1993a). Non-hydroxylated fatty acids were purified, identified and quantified by GC as described by Yassin (1988). Menaquinones were extracted, purified and identified according to Collins et al. (1977). Phospholipids were extracted, purified and identified as described previously (Yassin et al., 1993c).
DNA was isolated and purified as described previously (Yassin et al., 2000a). DNA–DNA hybridization studies were carried out by using the thermal renaturation method (Yassin et al., 1993b). Genomic DNA extraction, PCR-mediated amplification of 16S rRNA genes and the purification of PCR products were carried out using procedures described previously (Rainey et al., 1996). Purified PCR products were sequenced using a Taq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems) as described in the manufacturer's protocol. An Applied Biosystems 310 DNA genetic analyser was used for the electrophoresis of the sequence reaction products. The 16S rRNA gene sequences of strains IMMIB N-402T and IMMIB N-403, as well as those (retrieved from GenBank) of Nocardia species with validly published names, were added to the ARB database (Ludwig et al., 2004) and aligned using the integrated aligner software in the ARB package. The resulting alignment was corrected manually; evolutionary trees were inferred using maximum parsimony (Kluge & Farris, 1969), neighbour joining (Saitou & Nei, 1987) and maximum likelihood (Felsenstein, 1981). An evolutionary distance matrix was calculated using the corrections of Jukes & Cantor (1969). The tree topology was evaluated according to the results of the neighbour-joining and maximum-likelihood analyses. The robustness of the phyletic lines was evaluated by using bootstrap analyses (Felsenstein, 1985) of neighbour-joining datasets for 1000 resamplings. The phylogenetic analyses were carried out using the ARB package.
Strains IMMIB N-402T and IMMIB N-403 have morphological properties consistent with their assignment to the genus Nocardia. They are aerobic organisms that form hyphae that are Gram-positive and slightly acid–alcohol-fast. The vegetative hyphae are orange in colour, well developed, with irregular branches penetrating the agar and bearing white aerial hyphae. At a late stage of growth the hyphae fragment into rod-shaped elements characteristic of nocardiae. The physiological properties of isolates IMMIB N-402T and IMMIB N-403 are cited in detail later, in the description of Nocardia elegans sp. nov. Biochemical differences between the isolates and some Nocardia species with validly published names (i.e. those examined in this study) are shown in Table 1.
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). One-dimensional TLC of whole-cell acid methanolysates of strain IMMIB N-402T and strain IMMIB N-403 revealed the presence of two lipid spots, for each organism, with different flow rates on the chromatogram. The lower spots correspond to mycolic acids, as identified by their RF value (0·47), and the higher ones correspond to the non-hydroxylated fatty acids. Pyrolysis GC of the purified mycolic acid methyl esters from isolates IMMIB N-402T and IMMIB N-403 released fatty acid methyl esters of C16 : 0 (33·2 % of total cleavage products of the mycolates from strain IMMIB N-402T), C18 : 1 (27·8 %) and C18 : 0 (39·0 %) as pyrolysis cleavage products. GC analyses of the non-hydroxylated fatty acid methyl esters revealed the presence of tetradecanoate (0·26 % of total fatty acids from isolate IMMIB N-402T), pentadecanoate (0·52 %), cis-hexadecenoate (0·89 %), hexadecanoate (30·98 %), 10-methylhexadecanoate (0·1 %), heptadecanoate (1·86 %), 10-methylheptadecanoate (0·39 %), octadecenoate (3·85 %), octadecanoate (18·15 %), tuberculostearic acid (10-methyl octadecanoate, 38·91 %), nonadecanoate (0·14 %), eicosenoate (0·95 %), eicosanoate (1·4 %), heneicosenoate (0·61 %) and heneicosanoate (1·0 %) as the major cellular fatty acid methyl esters. Polar lipid analysis showed that strains IMMIB N-402T and IMMIB N-403 contain phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and diphosphatidylglycerol as the characteristic phospholipids (i.e. phospholipid type PII sensu Lechevalier et al., 1977). Mass-spectral analysis of the respiratory quinones showed that isolate IMMIB N-402T and isolate IMMIB N-403 possess hexahydrogenated menaquinones with eight isoprene units in which the two terminal isoprene moieties are cyclized. The main component corresponds to MK-8(H6) and the minor component corresponds to 2,3-epoxy-MK-8(H6). These chemotaxonomic similarities to Nocardia species with validly published names are supported by the high levels of 16S rRNA gene sequence similarity observed between isolates IMMIB N-402T and IMMIB N-403 and members of the genus Nocardia.
To ascertain the phylogenetic position of strains IMMIB N-402Tand IMMIB N-403, their almost-complete 16S rRNA gene sequences [1483 nt; 96·1 % of the Escherichia coli sequence (Brosius et al., 1978)] were determined in this study and subjected to a comparative analysis. The 16S rRNA gene sequence comparison clearly shows that strains IMMIB N-402T and IMMIB N-403 are members of the family Nocardiaceae (Stackebrandt et al., 1997). The high values for the 16S rRNA gene sequence similarities to other previously described members of the genus Nocardia (95·5–99·6 %) support the addition of strains IMMIB N-402T and IMMIB N-403 to this genus. There were significantly lower levels of similarity with other Actinomycetales taxa (data not shown). The highest sequence similarities were shown with Nocardia nova, Nocardia vaccinii, Nocardia cerradoensis, Nocardia veterana and Nocardia africana (98·3, 98·5, 98·7, 99·2 and 99·6 % sequence similarity, respectively). A tree constructed using the neighbour-joining method, depicting the phylogenetic placement of strains IMMIB N-402T and IMMIB N-403 within a subset of the genus Nocardia, is shown in Fig. 1. It is evident from the tree that the two isolates represent a distinct subline within the genus Nocardia that is associated with N. africana, N. veterana, N. cerradoensis and N. vaccinii. These results suggest that strains IMMIB N-402T and IMMIB N-403 belong to a genetically distinct Nocardia species showing close relatedness with N. africana, N. veterana, N. vaccinii and N. cerradoensis. These sequence similarities are too high to allow the definition of a novel species, since values below 97 % and/or genomic DNA reassociation values below 70 % are considered necessary for the establishment of a novel bacterial species (Stackebrandt & Goebel, 1994). In view of the high levels of 16S rRNA sequence similarities between isolates IMMIB N-402T and IMMIB N-403 and some Nocardia species, chromosomal DNA–DNA hybridization studies were performed to establish whether strains IMMIB N-402T and IMMIB N-403 represent distinct species. Strains IMMIB N-402T and IMMIB N-403 displayed low levels of DNA–DNA reassociation with the type strains of N. africana SD 769T (30·2 and 33·6 %, respectively) and N. veterana DSM 44445T (33·6 % and 27·5 %, respectively), results which are below the cut-off point recommended by Wayne et al. (1987) for the circumscription of bacterial genomic species, and confirm the separation of isolates IMMIB N-402T and IMMIB N-403 from their nearest phylogenetic neighbours.
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). More critical examples are N. veterana and N. africana, the 16S rRNA gene sequences of which are 99·3 % similar, whereas the total labelled genomic DNA of the two species exhibited only 28·8 % homology. Similar situations were found for Nocardia brevicatena and Nocardia paucivorans, which have a 16S rRNA similarity value of 99·6 % but 61·9 % DNA–DNA relatedness (Yassin et al., 2000a), and for Nocardia carnea and Nocardia flavorosea, which have a 16S rRNA similarity value of 99·2 % but 5·0 % DNA–DNA relatedness (Chun et al., 1998). Given the 1·4, 1·5 and 1·8 % sequence divergence between isolates IMMIB N-402T and IMMIB N-403 and their closest relatives, N. cerradoensis, N. vaccinii and N. nova, respectively, and the biochemical tests (Table 1
) differentiating isolates IMMIB N-402T and IMMIB N-403 from the latter three species, it is reasonable to define a novel species. The chemical (and morphological) data clearly indicate that the isolates belong to the genus Nocardia. The 16S rRNA gene sequencing, the DNA–DNA pairing and the biochemical results indicate that the two isolates are similar and represent a novel species when compared with the type strains of Nocardia species with validly published names. Thus, on the basis of the results of the polyphasic taxonomic study reported here, we consider that isolates IMMIB N-402T and IMMIB N-403 represent one species and merit classification as a novel species of the genus Nocardia, for which the name Nocardia elegans sp. nov. is proposed, with IMMIB N-402T as the type strain.
Description of Nocardia elegans sp. nov.
Nocardia elegans (e'le.gans. L. adj. elegans fastidious – with respect to utilization of nutrients).
The hyphae are Gram-positive and partially acid–alcohol-fast. Vegetative hyphae are orange in colour, well developed with irregular branches penetrating the agar and bear white aerial hyphae. At a late stage of growth the hyphae fragment into rod-shaped elements. Grows at temperatures in the range 22–42 °C. The organism possesses the salient chemotaxonomic characteristics of the genus Nocardia. Its mycolic acids are cleaved, upon pyrolysis, releasing fatty acids C16 : 0, C18 : 1 and C18 : 0 with C18 : 0 as the major cleavage product. Hydrolyses aesculin, testosterone and urea but not adenine, casein, elastin, gelatin, guanine, hypoxanthine, tyrosine or xanthine. Assimilates acetate and glucose as carbon sources, but not adonitol, adipate, iso-amyl alcohol, arabinose, 2,3-butanediol, cellobiose, citrate, meso-erythritol, galactose, gluconate, m-hydroxybenzoate, p-hydroxybenzoate, myo-inositol, lactate, lactose, maltose, mannitol, melezitose, 1,2-propanediol, raffinose, rhamnose, sorbitol, sucrose, trehalose or xylose. Does not utilize acetamide, arginine, gelatin, ornithine, proline or serine as simultaneous carbon and nitrogen sources.
The type strain of Nocardia elegans is strain IMMIB N-402T (=CCUG 50200T=CIP 108553T).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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