Differentiation of clinical Mycobacterium tuberculosis complex isolates bygyrB DNA sequence polymorphism analysis

The discriminatory power of gyrB DNA sequence polymorphisms for differentia tion of the species of the Mycobacterium tuberculosis complex (MTBC) was ev aluated by sequencing and restriction fragment length polymorphism (RFLP) a nalysis of a 1,020-bp fragment amplified from clinical isolates of M. tuber culosis, Mycobacterium bovis (pyrazinamide [PZA] resistant as well as PZA s usceptible), Mycobacterium africanum subtypes I and II, and Mycobacterium m icroti types vole and llama, We found sequence polymorphisms in four region s described previously and at one additional position. These differences in the gyrB sequences allow an accurate discrimination of M, bovis, M. microt i, and M. africanum subtype I, The PZA-susceptible subtypes of M. bovis sha red the M. bovis-specific substitution at position 756 with the PZA-resista nt strains, but can be unambiguously differentiated by a characteristic sub stitution at position 1311. As a drawback, M. tuberculosis and M. africanum subtype II showed an identical gyrB sequence that facilitates discriminati on from the other species, but not from each other, A PCR-RFLP technique ap plying three restriction enzymes could be shown to be a rapid and easy-to-p erform tool for the differentiation of the members of the MTBC, Based on th ese results, we present a clear diagnostic algorithm for the differentiatio n of species of the MTBC.