Identification of non-tuberculous mycobacteria isolated from clinical specimens by sequence analysis

Non-tuberculous mycobacteria (NTM), which was not accepted to cause disease in the past, has been observed to be isolated as an infection agent in increasing frequency in accordance with the rise in diseases and treatments that suppress immune system. Clinical syndromes that caused by NTM and their treatment protocols varies widely, therefore it is essential to be identified of NTM in species level. In our study, it is aimed to identify isolates in species level by DNA sequence analysis which is accepted as a gold standard in identification of NTM?s. 156 NTM isolates have been grown on LJ medium, which were isolated from the respiratory tract specimens in Central Laboratories of Dokuz Eylul University Hospital, between 2004 and 2010. Four hundred and forty-one -bp fragment of the hsp65 gene encoding the 65-kDa heat shock protein was amplified by polymerase chain reaction after DNA extraction. Hsp65 gene sequencing has been accomplished by service procurement. DNA sequences have been aligned in accordance with the hsp65 gene sequences of reference mycobacteria at Genbank. Aligned DNA sequences have been analyzed by BLAST software and 156 isolates have been identified in species level. Within isolates which were compared with reference sequences, 37 of them have been identified with 100% compatibility, whereas 111 of them have been identified with 99% compatibility, four of them with 98%, and remaining four isolates have been identified with 97%, 95%, 93% and 90% compatibilities. A total of the 156 isolates in the study have been identified by hsp65 sequence analysis. In the study, 15 different species have been detected 13 of which belong to NTM and 2 to Nocardia. The most frequently isolated NTM species was detected as M. abscessus; 69 of which were isolated (44,2%). The rest of the results were obtained as follows; 26 isolates for M. xenopi (16,7%), 17 isolates for M. peregrinum (10,9%), 13 isolates for M. fortuitum (8,3%), 6 isolates for M. intracellulare (3,8%), 5 isolates for M. simiae (3,2%), 5 isolates for M. lentiflavum (3,2%), 3 isolates for N. cyriacigeorgica (1,9%), 3 isolates for M. avium (1,9%), 2 isolates for N. abscessus (1,3%), 2 isolates for M. chelonae (1,3%), 2 isolates for M. bolletii (1,3%) and one isolate for M. senegalense, M. porcinum ve M. kansasii (0,6%) in order. As a result, hsp65 gene region?s partial DNA sequence analysis have been determined to be sufficient and succeeding at identification of NTM species.