Novel diagnostic algorithm for identification of mycobacteria using genus-specific amplification of the 16S-23S rRNA gene spacer and restriction endonucleases

A novel genus-specific PCR for mycobacteria with simple identification to t he species level by restriction fragment length polymorphism (RFLP) was est ablished using the 16S-23S ribosomal RNA gene (rDNA) spacer as a target. Pa nspecificity of primers was demonstrated on the genus level by testing 811 bacterial strains (122 species in 37 genera from 286 reference strains and 525 clinical isolates). All mycobacterial isolates (678 strains among 48 de fined species and 5 indeterminate taxons) were amplified by the new primers . Among nonmycobacterial isolates, only Gordonia terrae was amplified. The RFLP scheme devised involves estimation of variable PCR product sizes toget her with HaeIII and CfoI restriction analysis. It yielded 58 HaeIII pattern s, of which 49 (84%) were unique on the species level. Hence, HaeIII. diges tion together with CfoI results was sufficient for correct identification o f 39 of 54 mycobacterial taxons and one of three or four of seven RFLP geno types found in Mycobacterium intracellulare and Mycobacterium kansasii, res pectively. Following a clearly laid out diagnostic algorithm, the remaining unidentified organisms fell into five clusters of closely related species (i.e., the Mycobacterium avium complex or Mycobacterium chelonae-Mycobacter ium abscessus) that were successfully separated using additional enzymes (T aqI, MspI, DdeI, or AvaII). Thus, next to slowly growing mycobacteria, all rapidly growing species studied, including M. abscessus, M. chelonae, Mycob acterium farcinogenes, Mycobacterium fortuitum, Mycobacterium peregrinum, a nd Mycobacterium senegalense (with a very high 16S rDNA sequence similarity ) were correctly identified. A high intraspecies sequence stability and the good discriminative power of patterns indicate that this method is very su itable for rapid and cost-effective identification of a wide variety of myc obacterial species without the need for sequencing. Phylogenetically, space r sequence data stand in good agreement with 16S rDNA sequencing results, a s was shown by including strains with unsettled taxonomy. Since this approa ch recognized significant subspecific genotypes while identification of a b road spectrum of mycobacteria rested on identification of one specific RFLP pattern within a species, this method can be used by both reference (or re search) and routine laboratories.