SPECIES IDENTIFICATION OF CHLAMYDIA ISOLATES BY ANALYZING RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM OF THE 16S-23S RIBOSOMAL-RNA SPACER REGION

The genetic diversity of the 16S-23S rRNA spacer region of 12 Chlamydi a 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 Ch lamydia psittaci isolates (5 avian isolates and 1 feline isolate), and one Chlamydia pecorum isolate was studied. The 16S-23S rRNA spacer re gion was amplified by PCR and digested with the restriction enzymes Ms eI, PstI, AluI, BglII, NlaIV, and BclI, All 26 isolates could be ampli fied by using one genus-specific primer pair, yielding an amplicon wit h a size of 803 bp. The analytical sensitivity of the PCR assay was le ss than or equal to 100 inclusion-forming units per reaction. Digestio n with MseI or AluI made it possible to differentiate the four species from each other, the C. trachomatis human biovars from the mouse biov ar, and the C. psittaci avian isolates from the feline isolate. The Ms eI profiles were more distinct than the AluI profiles, Phylogenetic an alysis of the results for all enzymes combined supported the current c lassification of four Chlamydia species: C. pneumoniae, C. trachomatis , C. psittaci, and C. pecorum. Phylogenetic analysis also suggested su bdivision 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.