Species identification of Chlamydia isolates by analyzing restriction fragment length polymorphism of the 16S-23S rRNA spacer region.

The genetic diversity of the 16S-23S rRNA spacer region of 12 Chlamydia pneumoniae isolates, 7 Chlamydia trachomatis isolates (human biovars: the trachoma serovars B and C, the urogenital serovars D, E, and F, and the lymphogranuloma venereum serovar L2; and a mouse biovar), 6 Chlamydia psittaci isolates (5 avian isolates and 1 feline isolate), and one Chlamydia pecorum isolate was studied. The 16S-23S rRNA spacer region was amplified by PCR and digested with the restriction enzymes MseI, PstI, AluI, BglII, NlaIV, and BclI. All 26 isolates could be amplified by using one genus-specific primer pair, yielding an amplicon with a size of 803 bp. The analytical sensitivity of the PCR assay was < or = 100 inclusion-forming units per reaction. Digestion with MseI or AluI made it possible to differentiate the four species from each other, the C. trachomatis human biovars from the mouse biovar, and the C. psittaci avian isolates from the feline isolate. The MseI profiles were more distinct than the AluI profiles. Phylogenetic analysis of the results for all enzymes combined supported the current classification of four Chlamydia species: C. pneumoniae, C. trachomatis, C. psittaci, and C. pecorum. Phylogenetic analysis also suggested subdivision of isolates of C. trachomatis and C. psittaci into subgroups that coincide with their host range. In conclusion, we have developed an easy and rapid method for species and subspecies identification of Chlamydia based on restriction fragment length polymorphism analysis of the 16S-23S rRNA spacer region.