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Journal of Clinical Microbiology, January 1998, p. 139-147, Vol. 36, No. 1
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Differentiation of Phylogenetically Related Slowly Growing Mycobacteria Based on 16S-23S rRNA Gene Internal Transcribed Spacer Sequences

Andreas Roth,1,* Marga Fischer,1 Mohamed E. Hamid,1,2 Sabine Michalke,1 Wolfgang Ludwig,3 and Harald Mauch1

Institut für Mikrobiologie und Immunologie, Krankenhaus Zehlendorf, 14109 Berlin,1 and Institut für Botanik und Mikrobiologie, Lehrstuhl für Mikrobiologie, Technische Universität München, 80290 Munich,3 Germany, and Faculty of Veterinary Science, University of Khartoum, Khartoum, Sudan2

Received 4 August 1997/Returned for modification 17 September 1997/Accepted 17 October 1997

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.

* Corresponding author. Mailing address: Institut für Mikrobiologie und Immunologie, Lungenklinik Heckeshorn-Zehlendorf, Zum Heckeshorn 33, D 14109 Berlin, Germany. Phone: 49-30-8002 2254. Fax: 49-30-8002 2299.

Journal of Clinical Microbiology, January 1998, p. 139-147, Vol. 36, No. 1
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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