Use of PCR-restriction enzyme pattern analysis and sequencing database for hsp65 gene-based identification of Nocardia species

Abstract

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.

FIG. 1.

Steingrube's decision tree for the identification of Nocardia species by hsp65 PRA (29), based on reference strains. The previously tested species are indicated by arrows. ^a, reclassified as N. abscessus by Roth et al. (25); ^b, reclassified as a Nocardia sp. by Roth et al. (25); *, results from the work of Steingrube et al. (29).

FIG. 2.

Phylogenetic tree based on 16S rRNA sequencing of collection strains belonging to the genus Nocardia. The tree was constructed by using the neighbor-joining method, based on a 569-nucleotide stretch. Bootstrap values are expressed as a percentage of 1,000 replications. The scale bar represents 0.02 substitutions per nucleotide position.

FIG. 3.

Phylogenetic tree based upon hsp65 sequencing of collection strains belonging to the genus Nocardia. The tree was constructed by using the neighbor-joining method, based on a 401-nucleotide stretch. Bootstrap values are expressed as a percentage of 1,000 replications. The scale bar represents 0.02 substitutions per nucleotide position.