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J Clin Microbiol. Feb 2003; 41(2): 851–856.
PMCID: PMC149683

Phylogeny of the Genus Nocardia Based on Reassessed 16S rRNA Gene Sequences Reveals Underspeciation and Division of Strains Classified as Nocardia asteroides into Three Established Species and Two Unnamed Taxons


Conventional identification of Nocardia in the routine laboratory remains problematic due to a paucity of reliable phenotypic tests and due to the yet-unresolved taxonomy of strains classified as belonging to the species Nocardia asteroides, which comprises the type strain and isolates with drug pattern types II and VI. The 16S rRNA gene of 74 representative strains of the genus Nocardia, encompassing 25 established species, was sequenced in order to provide a molecular basis for accurate species identification and with the aim of reassessing the phylogeny of taxons assigned to the species N. asteroides. The result of this phylogenetic analysis confirms that the interspecies heterogeneity of closely related nocardial species can be considerably low (a sequence divergence of only 0.5% was found between N. paucivorans and N. brevicatena). We observed a sequence microheterogeneity (sequence divergence of fewer than five bases) in 8 of 11 species of which more than one strain in the species was studied. At least 10 taxons were found that merit description as new species. Strains previously classified as N. asteroides fell into five distinct phylogenetic groups: the type strain cluster (N. asteroides sensu strictu), N. abscessus, N. cyriacigeorgica, and two clusters closely related to N. carnea or N. flavorosea. The strains within the latter two groups probably represent new species, pending further genetic and phenotypic evaluation. Restricted phenotypic data revealed that N. abscessus, N. cyriacigeorgica, and the two Nocardia species taxons are equivalent to drug patterns I, VI, and II, respectively. In the future, these data will help in finding species-specific markers after adoption of a more precise nomenclature for isolates closely related to N. asteroides and unravel confusing phenotypic data obtained in the past for unresolved groups of strains that definitely belong to separate taxons from a phylogenetic point of view.

The genus Nocardia forms a group of 30 validly described species. Many of these can cause clinical diseases in humans and animals, including pulmonary, central nervous system, and cutaneous infections that are all diagnosed by culture and identification of the organisms (1, 10, 11). Identification to the genus level is well established based on chemotaxonomic characteristics, mainly on the presence of a major amount of meso-diaminopimelic acid, arabinose, galactose, and mycolic acids with 46 to 60 carbons in cell walls (13, 14). A variety of phenotypic characteristics for each Nocardia species can be found in the literature, but those of recently published species, such as N. abscessus and N. cyriacigeorgica (25, 26), have not been analyzed in collaborative numerical phenetic studies systematically in order to obtain a proposal of selected tests for routine identification of all medically relevant species (9, 13, 17, 19, 21). Studies of antimicrobial susceptibility revealed distinct drug pattern types within strains identified as N. asteroides, notably drug patterns I through VI (20). A few years later, Wallace et al. demonstrated that N. asteroides III and V strains are isolates of the species N. nova and N. farcinica (19, 21). In 1997 the same working group showed that N. asteroides IV represents an unknown taxon closely related to N. transvalensis, but this taxon has not yet been validly described as a new species (22). As the number of species is increasing, it is becoming evident that taxonomical complexity causes ambiguities in the interpretation of individually described phenotypic markers and that early identification strategies employed over the past 15 years have to be revised (9). Thus, albeit identification of Nocardia isolates to the species level is important for estimation of pathogenicity and prediction of antimicrobial susceptibility (and ultimately for prognosis), conventional species identification remains difficult, inaccurate, and time consuming. With the advent of molecular analyses, an identification method based on PCR-restriction fragment length polymorphism (RFLP) analysis of the 65-kDa heat shock protein gene was successfully introduced seven years ago (16, 23). Studies using this method have confirmed that the drug pattern types represent distinct taxons, but the taxonomic status of three of these, namely patterns I, II, and VI, still remained to be determined on the basis of 16S ribosomal DNA (rDNA) sequences (4, 5, 14). Although antimicrobial susceptibility was not specifically addressed by the authors who described N. abscessus as a new species, one of the strains identified as N. abscessus was N. asteroides ATCC 23824 (25), which is one of the known reference strains of drug pattern I (16). Recently a PCR-RFLP analysis of the 16S rDNA gene was described (5) which included N. asteroides strains belonging to all drug pattern types, but RFLP patterns were established for only six additional species. Available data on 16S rDNA sequences of Nocardia are incomplete, and 16S sequence-based identification is hampered by many faulty entries in the publicly accessible databases (18). This prompted us to reassess the 16S rDNA sequences of 74 representative strains (66 reference strains and 8 clinical isolates, including N. asteroides II and VI) encompassing 25 established species of the genus (Table (Table11).

Source of 74 Nocardia strains used, the accession numbers of 51 complete 16S rDNA sequences submitted, and the genotypes (sequence microheterogeneities) found in 8 of 25 established species

The nearly complete 16S rDNA gene sequence (1,494 bp) of 63 strains and a partial sequence (at least 600 bp) at the 5′ end of the gene of 11 isolates were determined using standard universal primers for PCR and sequencing (3). Sequences were obtained by standard procedures of cycle sequencing and fluorescence-based analysis of both the forward and the reverse strands with the ABI 310 genetic analyzer (Perkin-Elmer Applied Biosystems). The sequences were aligned manually, and a phylogenetic tree was inferred using the neighbor-joining method applied to distances corrected for multiple hits and for unequal transition and transversion rates by Kimura's two-parameter model (8, 15) with the aid of the TREECON for Windows software (version 3.1b; University of Antwerp, Antwerp, Belgium). In an effort to determine the status and to clarify the phenetic integrity of defined clusters in the phylogenetic tree generated, 42 isolates unable to decompose casein, hypoxanthine, tyrosine, and xanthine from the group of strains classified as N. asteroides together with strains of N. abscessus, N. carnea, N. flavorosea, N. nova, and N. paucivorans were submitted to a limited number of biochemical tests: growth at 45°C (9); urea hydrolysis (25); assimilation of carbohydrate substrates in the API ID 32C (BioMerieux, Marcy-l'Etoile, France), the latter according to a method described previously (12); and diagnostic susceptibility patterns by disk diffusion tests (2, 20). The strains studied were susceptible when zone diameters (in millimeters) of the disk diffusion test for piperacillin (100 μg), ampicillin (10 μg), erythromycin (15 μg), imipenem (10 μg), ciprofloxacin (5 μg), cefotaxime (30 μg), and tobramycin (10 μg) were larger than 20, 25, 30, 15, 20, 25, and 20, respectively. Drug patterns were determined according to the method of Wallace et al. (20) as follows: type I, susceptible to ampicillin, carbenicillin, and cephalosporins but resistant to ciprofloxacin (more than half of the strains are resistant to imipenem); type II, same as type I, susceptible to ciprofloxacin; type III, susceptible to ampicillin but resistant to carbenicillin, and susceptible to erythromycin (N. nova); type V, resistant to penicillins and cephalosporins but susceptible to ciprofloxacin (N. farcinica); type VI, resistant to penicillins and ciprofloxacin but susceptible to broad-spectrum cephalosporins. Strains belonging to drug pattern type IV show resistance to amikacin and are able to hydrolyze hypoxanthine (22) and were not included in our phenotypic analysis. The sequences determined in this study have been deposited in the GenBank database under the accession numbers shown in Table Table11 and will also be available in the near future for similarity searches in a database which is currently being expanded in the RIDOM project (7, 18).

The phylogenetic analysis presented in Fig. Fig.11 revealed the following results. First, the interspecies heterogeneity of closely related nocardial species is considerably low. The highest 16S rDNA similarity value between two established species, namely N. paucivorans and N. brevicatena (both have a DNA-DNA relatedness value of less than 70% [24]), was as high as 99.5%. This value, based on sequences devoid of ambiguities, corresponds to only seven nucleotide differences and is much lower than previously discussed (6, 24). In contrast, intraspecies heterogeneity (arbitrarily defined as a nucleotide difference of fewer than five bases) occurred frequently: in 8 of 11 species of which more than one strain in a species was studied. These results stand in agreement with recent studies (5, 6), and it is interesting that most of these were species of medical importance, e.g., N. nova (Table (Table2).2). This highlights the need for high-quality sequences in comprehensive databases for accurate molecular identification. Second, our results provide clear evidence that the species in the genus are strongly undercounted. Under the assumption that strains DSM 43254, 43576, 43263, and 43253 represent one taxon, we can state that 10 taxons shown as Nocardia species in the tree were found that merit description as new species in light of their significant low level of sequence similarity to their next relative (values between 98 and 99.4%). Third, strains previously classified as N. asteroides fell into five distinct phylogenetic groups: (A) the type strain cluster, (B) N. abscessus, (C) N. cyriacigeorgica, (D) strains Nocardia species DSM 43261 and 43260, and (E) four additional Nocardia species DSM strains that clustered within the N. carnea/N. flavorosea clade. As stated above, the strains found in these last two groups probably represent new species and need to be reclassified. Delineation of a species in group E will have to implement DNA-DNA hybridization, because the strains in group E show a high level of similarity to N. flavorosea. Phenotypic data revealed that the clusters of strains in groups B and C are equivalent to drug patterns I and VI. In contrast to this, the drug pattern type II appears to reflect a phenotypic feature found in different phylogenetic groups, since all strains in both groups D and E and two N. carnea strains (genotype II) had this antibiogram pattern type (Table (Table2).2). No reference strain for the drug pattern type II is available (5, 16), but the sequence of strain N-565 drug pattern type II (5) submitted to the GenBank database (accession no. AF163818) is identical to our sequence obtained from strain DSM 43576, belonging to group E. The API ID 32C method was time consuming because a long incubation period of at least 14 days was required. Moreover, the reaction profiles were difficult to interpret because they showed intraspecies variability and overlap between species. Thus, this method appears to be of little use for the identification of Nocardia strains that are closely related to N. asteroides. A selection of a few of these reactions (e.g., utilization of gluconate for N. cyriacigeorgica) is shown in Table Table2.2. A surprising and yet to us unknown finding was that the combination of only three tests (growth at 45°C, urea hydrolysis, and the susceptibility tests) gave a rather good discriminatory power between most of the species shown in Table Table2.2. Of importance, 10 strains assigned to the species N. cyriacigeorgica (25), including the reference strain ATCC 14759 for the antibiogram type VI, revealed a very consistent phenotype that enables us to delineate an authentic Nocardia species. Ultimately, a larger number of strains will have to be tested in a polyphasic, more extensive taxonomical approach to see if this holds true (this taxon is one of the species most frequently isolated in clinical specimens [20]). The closely related species N. carnea and the Nocardia species within the N. carnea/N. flavorosea clade can be confused on the sole basis of the phenetic markers applied in this study, which are shown in Table Table22 (what may be the case for some N. asteroides sensu stricto strains and N. abscessus too). This means that the drug patterns I and II are not species-specific markers per se and that further distinguishing phenotypic characters have to be determined.

FIG. 1.
Phylogenetic tree generated by using 63 complete 16S rDNA sequences (1,494 bp) determined in this study from 25 established Nocardia species and 14 Nocardia species (N. sp.) strains. The sequences of four species not determined in this study were derived ...
Genotypes and phenotypic characteristics of 42 Nocardia strains differentiating species frequently isolated in clinical specimens and closely related species that are unable to decompose casein, hypoxanthine, tyrosine, or xanthine

Molecular identification of bacteria using 16S rDNA sequencing provides three primary advantages over phenotypic identification: rapid turn-around time, improved accuracy, and taxonomical meaningfulness (7). This study provides the molecular basis for accurate species identification of Nocardia and proposes how we should retaxonomize taxons so far assigned to the species N. asteroides. The data presented indicate the phylogenetic position of 30 established species (except for N. coeliaca, the only species not included in this study, for which there is also no 16S rDNA available in the database) and 10 unclassified taxons in the genus. This will aid in finding adequate additional discriminating phenotypic or chemotaxonomic markers for conventional identification of all members in this genus and ultimately will unravel confusing data obtained on the ground of unresolved groups of strains that clearly belong—from a phylogenetic standpoint—to different taxons.


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