Identification of Nontuberculous Mycobacteria by Sequence Analysis of the 16S Ribosomal RNA, the Heat-shock Protein 65 and the RNA Polymeraseβ-Subunit Genes

Abstract

The diagnosis of diseases caused by nontuberculous mycobacteria (NTM) is difficult, because NTM are prevalent in the environment such as soil and water, and because they have fastidious properties. In this study we investigated clinical isolates of NTM for their distribution pattern and accurate species identification. We selected presumptive NTM isolates negative for probe hybridization for M. tuberculosis complex, cultured in a third referral hospital from 21 January 2003 to 20 January 2004. Ninety seven-isolates were identified to the species level by direct sequencing of fragments of 16S rRNA, hsp65 and rpoB genes. A total of 120 isolates were studied for the distribution analysis. Frequently identified NTM species were M. avium (30.8%), M. intracellulare (23.3%) and M. abscessus (18.3%). Others were M. gordonae, M. senegalense, M. fortuitum, M. peregrinum, M. kansasii, M. terrae complex, M. lentiflavum, M. chelonae, and M. szulgai. Three M. tuberculosis complex (2.5%) were also identified among the presumptive NTM isolates. The identification rate by sequencing of 16S rRNA, rpoB, and hsp65 were 65%, 82% and 87%, respectively. The hsp65 or rpoB gene was more efficient than 16S rRNA for the identification of NTM by sequencing. Some NTM are increasingly considered to be the causative organisms in clinical diseases. Thus, direct sequencing could be adapted to routine work of clinical laboratories for accurate identification of NTM to the species level.