Phylogenetic analysis of prevalent Mycobacterium species in Northeastern Iran based on hsp65 and tuf genes

Phylogenetic analysis of Mycobacterium species can provide valuable insights into their evolutionary relationships and help to identify species and strains. In this study, two genetic markers, including the heat shock protein 65 (hsp65) gene and the elongation factor (EF)-Tu (tuf) gene were used for phylogenetic analysis of Mycobacterium species. Clinical samples were collected from patients suspected of tuberculosis. Bacterial isolates were obtained from sputum samples and cultured on Löwenstein-Jensen medium. Thirty Mycobacterium isolates (acid-fast +, culture +), were included in our study. After DNA extraction, hsp65 (441 bp) and tuf (741 bp) genes were PCR-amplified and sequenced. The Neighbor-Joining method was employed to infer the evolutionary history of the isolates and the analyses were conducted with MEGA X software. The phylogenetic trees were validated using bootstrap analysis with 1000 replicates. Bootstrap values above 70 % considered indicative of well support for the branches. The phylogenetic trees revealed the overall natural relationships among Mycobacterium species. Our results demonstrated that the tuf gene provides superior resolution for identifying distinct mycobacterial species, closely aligning its phylogenetic profile with the hsp65 gene. However, neither of the markers was effective in distinguishing members of the Mycobacterium tuberculosis complex (MTBC). This study highlights the high discriminatory power of the tuf gene, recommending its use as a primary genomic marker for phylogenetic analysis.