RAPID IDENTIFICATION OF CLINICALLY SIGNIFICANT SPECIES AND TAXA OF AEROBIC ACTINOMYCETES, INCLUDING ACTINOMADURA, GORDONA, NOCARDIA, RHODOCOCCUS, STREPTOMYCES, AND TSUKAMURELLA ISOLATES, BY DNA AMPLIFICATION AND RESTRICTION-ENDONUCLEASE ANALYSIS

A previously described PCR-restriction fragment length polymorphism (R FLP) identification schema for Nocardia that used an amplified 439-bp segment (amplicon) of the 65-kDa heat shock protein gene was evaluated for potential use with isolates of all clinically significant aerobic actinomycetes. The study included 28 reference (American Type Culture Collection) strains and 198 clinical isolates belonging to 20 taxonom ic groups. Of these 198 isolates, 188 could be differentiated by this PCR-RFLP method. Amplicons from all aerobic actinomycete isolates lack ed BstEII recognition sites, thereby distinguishing them from those of mycobacteria that contain one or more such sites. Of 29 restriction e ndonucleases, MspI plus HinfI produced RFLP patterns that differentiat ed 16 of the 20 taxa. A single RFLP pattern was observed for 15 of 20 taxa that included 65% of phenotypically clustered isolates. Multiple patterns were seen with Gordona bronchialis, Nocardia asteroides compl ex type VI, Nocardia otitidiscaviarum, Nocardia transvalensis, and Str eptomyces spp. Streptomyces RFLP patterns were the most heterogeneous (five patterns among 19 isolates), but exhibited a unique HinfI fragme nt of >320 bp. RFLP patterns that matched those from type strains of S treptomyces albus, Streptomyces griseus, or Streptomyces somaliensis w ere obtained from 14 of 19 Streptomyces isolates. Only 10 of 28 isolat es of N. otitidiscaviarum failed to yield satisfactory amplicons, whil e only 6 of 188 (3.2%) clinical isolates exhibited patterns that faile d to match one of the 21 defined RFLP patterns. These studies extended the feasibility of using PCR-RFLP analysis as a rapid method for the identification of all clinically significant species and taxa of aerob ic actinomycetes.