DIFFERENTIATION OF PHYLOGENETICALLY RELATED SLOWLY GROWING MYCOBACTERIA BASED ON 16S-23S RIBOSOMAL-RNA GENE INTERNAL TRANSCRIBED SPACER SEQUENCES

Interspecific polymorphisms of the 16S rRNA gene (rDNA) are widely use d for species identification of mycobacteria. 16S rDNA sequences, howe ver, do not vary greatly within a species, and they are either indisti nguishable in some species, for example, in Mycobacterium kansasii and M. gastri, or highly similar, for example, in M. malmoense and M. szu lgai. We determined 16S-23S rDNA internal transcribed spacer (ITS) seq uences of 60 strains in the genus Mycobacterium representing 13 specie s (M. avium, M. conspicuum, M. gastri, M. genavense, M. kansasii, M. m almoense, M. marinum, M. shimoidei, M. simiae, M. szulgai, M. triplex, M. ulcerans, and M. xenopi), An alignment of these sequences together ,vith additional sequences available in the EMBL database (for M. intr acellulare, M. phlei, M. smegmatis, and M. tuberculosis) was establish ed according to primary-and secondary-structure similarities. Comparat ive sequence analysis applying different treeing methods grouped the s trains into species-specific clusters with low sequence divergence bet ween strains belonging to the same species (a to 2%). The ITS-based tr ee topology only partially correlated to that based on 16S rDNA, but t he main branching orders were preserved, notably, the division of fast -growing from slowly growing mycobacteria, separate branching for M. s imiae, IM. genavense, and M. triplex, and distinct branches for M. xen opi and M. shimoidei. Comparisons of M. gastri with M. kansasii and M. malmoense with M. szulgai revealed ITS sequence similarities of 93 an d 88%, respectively. M. marinum and M. ulcerans possessed identical IT S sequences, Our results show that ITS sequencing represents a supplem ent to 16S rRNA gene sequences for the differentiation of closely rela ted species, Slowly growing mycobacteria show a high sequence variatio n in the ITS; this variation has the potential to be used for the deve lopment of probes as a rapid approach to mycobacterial identification.