Phylogenetic Analysis of Prevalent Tuberculosis and Non-Tuberculosis Mycobacteria in Isfahan, Iran, Based on a 360 bp Sequence of the rpoB Gene

Abstract

Background: Taxonomic and phylogenetic studies of Mycobacterium species have been based around the 16sRNA gene for many years. However, due to the high strain similarity between species in the Mycobacterium genus (94.3 - 100), defining a valid phylogenetic tree is difficult; consequently, its use in estimating the boundaries between species is limited. The sequence of the rpoB gene makes it an appropriate gene for phylogenetic analysis, especially in bacteria with limited variation. Objectives: In the present study, a 360bp sequence of rpoB was used for precise classification of Mycobacterium strains isolated in Isfahan, Iran. Materials and Methods: From February to October 2013, 57 clinical and environmental isolates were collected, subcultured, and identified by phenotypic methods. After DNA extraction, a 360bp fragment was PCR-amplified and sequenced. The phylogenetic tree was constructed based on consensus sequence data, using MEGA5 software. Results: Slow and fast-growing groups of the Mycobacterium strains were clearly differentiated based on the constructed tree of 56 common Mycobacterium isolates. Each species with a unique title in the tree was identified; in total, 13 nods with a bootstrap value of over 50 were supported. Among the slow-growing group was Mycobacterium kansasii, with M. tuberculosis in a cluster with a bootstrap value of 98 and M. gordonae in another cluster with a bootstrap value of 90. In the fast-growing group, one cluster with a bootstrap value of 89 was defined, including all fast-growing members present in this study. Conclusions: The results suggest that only the application of the rpoB gene sequence is sufficient for taxonomic categorization and definition of a new Mycobacterium species, due to its high resolution power and proper variation in its sequence (85 - 100); the resulting tree has high validity.