Reverse line blot hybridization and DNA sequencing studies of the 16S-23S rRNA gene intergenic spacer regions of five emerging pathogenic Nocardia species.

The objective of this study was to examine DNA sequence polymorphisms in the 16S-23S rRNA gene intergenic spacer (ITS) regions of five emerging pathogenic Nocardia species: Nocardia beijingensis, Nocardia blacklockiae, Nocardia thailandica, Nocardia elegans and Nocardia vinacea. A set of six isolates belonging to the species of interest and 135 isolates belonging to other Nocardia species was studied. A PCR-based reverse line blot (RLB) hybridization assay incorporating species- or intraspecies ITS rRNA gene operon-specific probes was then developed for species identification. Substantial intraspecies sequence variation among different ITS operons was identified. Four sequence types of N. thailandica, eight sequence types of N. beijingensis (four types for each of two strains) and five sequence types of N. blacklockiae, N. elegans and N. vinacea were found. The results represent the first evidence of ITS sequence heterogeneity in emerging species of Nocardia. By incorporating species/operon-specific probes into a RLB assay, unique RLB patterns were identified for each of the species and every sequence type. The PCR/RLB assay demonstrated high specificity and showed promise in both the identification and genotyping of Nocardia species. More detailed studies of the polymorphism within the ITS locus may further advance our capacity to reliably identify and subtype medically important Nocardia species.