New design of multilocus sequence analysis of rpoB, ssrA, tuf, atpE, ku, and dnaK for identification of Mycobacterium species.

BACKGROUND: Differentiating Mycobacterium tuberculosis (MTB) from nontuberculous mycobacteria (NTM) is very important in the treatment process of patients. According to the American Thoracic Society guideline (ATS), NTM clinical isolates should be identified at the species level proper treatment and patient management. This study aimed to identify NTM clinical isolates by evaluationg rpoB, ssrA, tuf, atpE, ku, and dnaK genes, and use multilocus sequence analysis (MLSA) to concatenate the six genes. METHODS: Ninety-six Mycobacterium isolates, including 86 NTM and 10 MTB isolates, from all the patients referred to the certain TB Reference Centres were included. All isolates were evaluated by PCR amplification of rpoB, ssrA, tuf, ku, atpE, and dnaK genes and MLSA. RESULTS: Out of 96 isolates, 91 (94.8%), 87 (90.6%), 72 (75%), 84 (87.5%) and 79 (82.3%) were differentiated to the species level by rpoB, tuf, ssrA, dnaK and atpE genes, respectively. The ku gene was able to identify 69 (80.2%) isolates of the 86 NTM isolates to the species level. We could identify 100% of the isolates to the species level by MLSA. CONCLUSIONS: None of the PCR targets used in this study were able to completely differentiate all species. The MLSA technique used to concatenate the six genes could increase the identification of clinical Mycobacterium isolates and all 16 species were well-differentiated.