Nocardia identification required laborious and time-consuming phenotypic and chemotaxonomic methods until molecular methods were developed in the mid-1990s. Here we reassessed the capacity of PCR-restriction enzyme pattern analysis (PRA) of the hsp65 gene to differentiate Nocardia species, including 36 new species. Our results confirm that hsp65 PRA must no longer be used for Nocardia species identification, as many species have the same restriction pattern. We then compared sequencing-based strategies using an hsp65 database and a 16S rRNA database and found that the hsp65 region contained sufficient polymorphisms for comprehensive Nocardia species identification.

Phylogenetic analyses of 56 type species of Nocardia were conducted using the partial nucleotide sequences of the gyrase B-encoding gene (gyrB). The interspecies similarities of the gyrB gene for the 56 type species were 82.4-99.9 %, which corresponded to 270-2 nt differences in the partial gene sequences of approximately 1200 nt. In comparison with phylogenetic relationships, gyrB gene sequence information was generally consistent with that of 16S rRNA gene sequences with minor exceptions. However, the degree of divergence of the gyrB gene sequences was approximately 3.6 times greater than those of the 16S rRNA gene, suggesting a higher discriminative power of gyrB sequence information compared with 16S rRNA gene sequences for Nocardia species. The Nocardia type species were clustered based on gyrB sequence similarity values of 93.5 % and above. Among the 56 type species, 38 were distributed in 13 clusters, each comprising 2 to 7 species. The remaining 18 species were classified into an independent cluster, in which the similarity between each species and the other 55 Nocardia species was less than 93.5 %. Among the eight mycolic acid-containing actinomycete genera in the suborder Corynebacterineae, Nocardia was clearly differentiated from the other genera, such as Rhodococcus, by gyrB gene analyses (similarity values of gyrB sequences for Nocardia and Rhodococcus were 75-85 %), indicating that the gyrB gene is a useful alternative to the 16S rRNA gene for the determination of phylogenetic relationships between the genus Nocardia and the seven other actinomycete genera.