Development of amplified 16S ribosomal DNA restriction analysis for identification of Actinomyces species and comparison with pyrolysis-mass spectrometry and conventional biochemical tests

Identification of Actinomyces spp. by conventional phenotypic methods is no toriously difficult and unreliable. Recently, the application of chemotaxon omic and molecular methods has clarified the taxonomy of the group and has led to the recognition of several new species. A practical and discriminato ry identification method is now needed for routine identification of clinic al isolates. Amplified 16S ribosomal DNA restriction analysis (ARDRA) was a pplied to reference strains (n = 27) and clinical isolates (n = 36) of Acti nomyces spp. and other gram-positive rods. Clinical strains were identified initially to the species level by conventional biochemical tests. However, given the low degree of confidence in conventional methods, the findings o btained by ARDRA were also compared with those obtained by pyrolysis-mass s pectrometry. The ARDRA profiles generated by the combination of HaeIII and HpaII endonuclease digestion differentiated all reference strains to the sp ecies or subspecies level. The profiles correlated well with the findings o btained by pyrolysis-mass spectrometry and by conventional tests and enable d the identification of 31 of 36 clinical isolates to the species level. AR DRA was shown to be a simple, rapid, cost-effective, and highly discriminat ory method for routine identification of Actinomyces spp. of clinical origi n.