Differentiation of Mycobacterium species by analysis of the heat-shock protein 65 gene (hsp65)

1 Department of Microbiology, College of Medicine, Seoul National University, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Korea 2 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-600, Korea 3 Department of Laboratory Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul 138-600, Korea 4 The Korean Institute of Tuberculosis, The Korean National Tuberculosis Association, Seoul 137-140, Korea 5 Department of Microbiology, College of Medicine, Konkuk University, Chungju 380-230, Korea 6 Institute of Hansen's Disease, The Catholic University Medical College, Seoul 137-7014, Korea Correspondence Bum-Joon Kim kbumjoon{at}snu.ac.kr The nucleotide sequences (604 bp) of partial heat-shock protein genes ( hsp65 ) from 161 Mycobacterium strains containing 56 reference Mycobacterium species and 105 clinical isolates were determined and compared. hsp65 sequence analysis showed a higher degree of divergence between Mycobacterium species than did 16S rRNA gene analysis. Generally, the topology of the phylogenetic tree based on the hsp65 DNA sequences was similar to that of the 16S rRNA gene, thus revealing natural relationships among Mycobacterium species. When a direct sequencing protocol targeting 422 bp sequences was applied to 70 non-tuberculous mycobacterium (NTM) clinical isolates, all NTMs were clearly identified. In addition, an Xho I PCR restriction fragment length polymorphism analysis method for the differentiation of Mycobacterium tuberculosis complex from NTM strains was developed during this study. The results obtained suggest that 604 bp hsp65 sequences are useful for the phylogenetic analysis and species identification of mycobacteria. Abbreviations: NTM, non-tuberculous mycobacterium; PRA, PCR-restriction fragment analysis The GenBank/EMBL/DDBJ accession numbers for the partial hsp65 sequences determined in this study are AF057449 –AF057493. Details of reference strains, including GenBank accession numbers, trees comparing the hsp65 and 16S rRNA genes of the same sample of reference strains and details of a protocol to distinguish M. tuberculosis complex and NTM isolates are available as supplementary material in IJSEM Online.