Identification of Nocardia Species by Restriction Endonuclease Analysis of an Amplified Portion of the 16S rRNA Gene

Identification of clinical isolates of Nocardia to the species level is important for defining the spectrum of disease produced by each species and for predicting antimicrobial susceptibility. We evaluated the usefulness of PCR amplification of a portion of the Nocardia 16S rRNA gene and subsequent restriction endonuclease analysis (REA) for species identification. Unique restriction fragment length polymorphism (RFLP) patterns were found for Nocardia sp. type strains (except for the N. asteroides type strain) and representative isolates of the drug pattern types of Nocardia asteroides (except for N. asteroides drug pattern type IV, which gave inconsistent amplification). A variant RFLP pattern for Nocardia nova was also observed. Twenty-eight clinical isolates were evaluated both by traditional biochemical identification and by amplification and REA of portions of the 16S rRNA gene and the 65-kDa heat shock protein (HSP) gene. There was complete agreement among the three methods on identification of 24 of these isolates. One isolate gave a 16S rRNA RFLP pattern consistent with the biochemical identification but was not identifiable by its HSP gene RFLP patterns. Three isolates gave 16S rRNA RFLP patterns which were inconsistent with the identification obtained by both biochemical tests and HSP gene RFLP; sequence analysis suggested that two of these isolates may belong to undefined species. The PCR and REA technique described appears useful both for the identification of clinical isolates of Nocardia and for the detection of new or unusual species.