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The discriminatory power of  gyrB  DNA sequence polymorphisms for differentiation of the species of the Mycobacterium tuberculosis  complex (MTBC) was evaluated by sequencing and restriction fragment length polymorphism (RFLP) analysis of a 1,020-bp fragment amplified from clinical isolates of  M. tuberculosis ,  Mycobacterium bovis  (pyrazinamide [PZA] resistant as well as PZA susceptible),  Mycobacterium africanum  subtypes I and II, and  Mycobacterium microti  types vole and llama. We found sequence polymorphisms in four regions described previously and at one additional position. These differences in the  gyrB  sequences allow an accurate discrimination of  M. bovis ,  M. microti , and M. africanum  subtype I. The PZA-susceptible subtypes of M. bovis  shared the  M. bovis -specific substitution at position 756 with the PZA-resistant strains, but can be unambiguously differentiated by a characteristic substitution at position 1311. As a drawback,  M. tuberculosis  and  M. africanum  subtype II showed an identical  gyrB sequence that facilitates discrimination from the other species, but not from each other. A PCR-RFLP technique applying three restriction enzymes could be shown to be a rapid and easy-to-perform tool for the differentiation of the members of the MTBC. Based on these results, we present a clear diagnostic algorithm for the differentiation of species of the MTBC.

Differentiation of Clinical Mycobacterium tuberculosis Complex Isolates by gyrB DNA Sequence Polymorphism Analysis