Abstract
Interspecific polymorphisms of the 16S rRNA gene (rDNA) are widely used for species identification of mycobacteria. 16S rDNA sequences, however, do not vary greatly within a species, and they are either indistinguishable in some species, for example, in
Mycobacterium kansasii
and
M. gastri
, or highly similar, for example, in
M. malmoense
and
M. szulgai
. We determined 16S-23S rDNA internal transcribed spacer (ITS) sequences of 60 strains in the genus
Mycobacterium
representing 13 species (
M. avium
,
M. conspicuum
,
M. gastri
,
M. genavense
,
M. kansasii
,
M. malmoense
,
M. marinum
,
M. shimoidei
,
M. simiae
,
M. szulgai
,
M. triplex
,
M. ulcerans
, and
M. xenopi
). An alignment of these sequences together with additional sequences available in the EMBL database (for
M. intracellulare
,
M. phlei
,
M. smegmatis
, and
M. tuberculosis
) was established according to primary- and secondary-structure similarities. Comparative sequence analysis applying different treeing methods grouped the strains into species-specific clusters with low sequence divergence between strains belonging to the same species (0 to 2%). The ITS-based tree topology only partially correlated to that based on 16S rDNA, but the main branching orders were preserved, notably, the division of fast-growing from slowly growing mycobacteria, separate branching for
M. simiae
,
M. genavense
, and
M. triplex
, and distinct branches for
M. xenopi
and
M. shimoidei
. Comparisons of
M. gastri
with
M. kansasii
and
M. malmoense
with
M. szulgai
revealed ITS sequence similarities of 93 and 88%, respectively.
M. marinum
and
M. ulcerans
possessed identical ITS sequences. Our results show that ITS sequencing represents a supplement to 16S rRNA gene sequences for the differentiation of closely related species. Slowly growing mycobacteria show a high sequence variation in the ITS; this variation has the potential to be used for the development of probes as a rapid approach to mycobacterial identification.