Sequence-Based Identification of Aerobic Actinomycetes

Abstract

We investigated the utility of 500-bp 16S rRNA gene sequencing for identifying clinically significant species of aerobic actinomycetes. A total of 28 reference strains and 71 clinical isolates that included members of the genera Streptomyces, Gordonia, and Tsukamurella and 10 taxa of Nocardia were studied. Methods of nonsequencing analyses included growth and biochemical analysis, PCR-restriction enzyme analysis of the 439-bp Telenti fragment of the 65 hsp gene, susceptibility testing, and, for selected isolates, high-performance liquid chromatography. Many of the isolates were included in prior taxonomic studies. Sequencing of Nocardia species revealed that members of the group were generally most closely related to the American Type Culture Collection (ATCC) type strains. However, the sequences of Nocardia transvalensis, N. otitidiscaviarum, and N. nova isolates were highly variable; and it is likely that each of these species contains multiple species. We propose that these three species be designated complexes until they are more taxonomically defined. The sequences of several taxa did not match any recognized species. Among other aerobic actinomycetes, each group most closely resembled the associated reference strain, but with some divergence. The study demonstrates the ability of partial 16S rRNA gene sequencing to identify members of the aerobic actinomycetes, but the study also shows that a high degree of sequence divergence exists within many species and that many taxa within the Nocardia spp. are unnamed at present. A major unresolved issue is the type strain of N. asteroides, as the present one (ATCC 19247), chosen before the availability of molecular analysis, does not represent any of the common taxa associated with clinical nocardiosis.

Identification of Pathogenic Nocardia Species by Reverse Line Blot Hybridization Targeting the 16S rRNA and 16S-23S rRNA Gene Spacer Regions

“…remains a challenge with the increasing recognition of novel species and taxonomic reassignment (5,23,25,27). The present study demonstrates the potential of combining a novel broad-range Nocardia PCR with probe hybridization technology in an RLB format to identify clinically relevant Nocardia species including newly described species.…”

“…However, the practical limitations of 16S rRNA gene sequencing have been realized (23). This method is often unable to distinguish between certain closely related Nocardia species due to insufficient interspecies polymorphisms within the 16S rRNA gene sequences (5,9,19).…”

Direct Identification of Mycobacteria in Primary Liquid Detection Media by Partial Sequencing of the 65-Kilodalton Heat Shock Protein Gene

“…We had encountered Tsukamurella species in our previous study, and to aid in their identification we had included sequences from six Tsukamurella species type strains in our most recent hsp65 sequence database. However, examination of the hsp65 sequence divergence for these species (unpublished data) indicated that, like the partial 16S rRNA gene sequence, hsp65 sequences may not be sufficiently divergent to report the results for isolates to the species level or that the current taxonomy is not adequate and we reported them only as Tsukamurella species, a cost-effective alternative to the use of our primary identification algorithm (15,22). Mixed cultures represented 3.3% (22 of 670) of the PLDM indicated to contain acid-fast bacilli.…”

Native Valve Endocarditis Due to Gordonia polyisoprenivorans : Case Report and Review of Literature of Bloodstream Infections Caused by Gordonia Species

“…This case illustrates that rapid commercial identification systems may provide inaccurate results, and identification to the genus level may be achieved by HPLC methods (which were not performed with this isolate); however, identification to the species level may be achieved only by molecular methods such as 16S rRNA gene sequencing (17) and, in our case, DNA reassociation. DNA reassociation studies were performed because of the uniqueness of this isolate in the Actinomycete Reference Laboratory.…”