DNA amplification and restriction endonuclease analysis for differentiation of 12 species and taxa of Nocardia, including recognition of four new taxa within the Nocardia asteroides complex

Nineteen reference and 156 clinical strains of the genus Nocardia belonging to 12 taxonomic groups were studied for restriction fragment length polymorphism (RFLP) by using an amplified 439-bp segment of the 65-kDa heat shock protein gene. Of 30 restriction endonucleases, digestion with MspI and then digestion with BsaHI produced RFLP band patterns which separated all 12 groups except N. asteroides type IV from 6 of 12 N. transvalensis isolates and N. carnea from the N. asteroides type VI isolates. Commonly encountered species such as N. nova, N. farcinica, N. brasiliensis sensu stricto, and N. otitidiscaviarum were easily separated. Each taxon resulted in a single RFLP band pattern that included > or = 96% of all biochemically grouped isolates for 9 of 12 taxa with MspI and for 8 of 12 taxa with BsaHI. With the use of both patterns, only 6 of 175 (3.4%) isolates failed to fit the biochemically defined group patterns. These studies provide the first evidence for the separate identities of four antibiogram-defined (but currently unnamed) groups within the N. asteroides complex (types I, II, IV, and VI) and the presence of two subgroups within N. transvalensis. They also provide genotypic evidence for the separate identities of N. nova and N. farcinica. The lack of BstEII recognition sites in amplicons obtained from nocardiae provides a simple and rapid method for the differentiation of nocardiae from mycobacteria. DNA amplification with RFLP analysis is the first rapid method that distinguishes all clinically significant taxa and recognized species within the genus Nocardia.