Molecular identification and phylogenetic relationships of clinical Nocardia isolates.

Species identification of Nocardia is difficult because of a complex and rapidly evolving taxonomy. In this study, gene sequencing (16S rRNA, gyrB, secA1, hsp65, rpoB), single 16S rRNA gene sequence phylogenetic analysis, and MALDI-TOF analysis were used to accurately identify 46 clinical Nocardia isolates to the species level. This provided a basis for establishing a routine method of multi-locus sequence analysis (MLSA) for molecular identification of Nocardia species. Genetic polymorphism analysis showed that MLSA was a powerfully discriminating method compared with the 16S rRNA gene to identify clinical Nocardia isolates. However, five-locus (gyrB-16S rRNA-secA1-hsp65-rpoB) MLSA led to misidentifications of all of the five Nocardia abscessus, which were confirmed by digital DNA-DNA hybridization (DDH) analysis. Interestingly, four strains identified as Nocardia beijingensis by a 16S rRNA gene phylogenetic tree may be novel species as suggested by DDH studies. For the purpose of achieving both accuracy and discrimination, the data of MLSA were reanalyzed. A three-locus MLSA with concatenated gyrB-16S rRNA-secA1 sequences was used to construct the phylogenetic tree with high accuracy and powerful discrimination. Therefore, a routine method of MLSA was developed to identify clinical Nocardia species.