Differentiation of phylogenetically related slowly growing mycobacteria bytheir gyrB sequences

The conventional methods for identifying mycobacterial species are based on their phenotypic characterization. Since some problematic species are slow growers, their taxonomy takes several weeks or months to identify. The rib osomal DNA (rDNA) sequence-based identification strategy has been adopted t o solve this problem. More recently, the gyrB sequences have been shown to be useful phylogenetic markers for the identification of species. We determ ined the gyrB sequences of 43 slowly growing strains belonging to 15 specie s in the genus Mycobacterium. The frequencies of base substitutions in the gyrB sequences were comparable to those in the 16S-23S rDNA internal transc ribed spacer (ITS) sequences. The ITS sequences of four species belonging t o the M. tuberculosis complex (M. tuberculosis, M. bovis, M. africanum, and M. microti) were 100% identical, while four synonymous substitutions were found in the gyrB sequences of these strains. Based on the differences foun d in the gyrB sequences, we developed PCR and PCR-restriction fragment leng th polymorphism methods to discriminate these species.