Isolation of non- tuberculosis mycobacterial strains from suspected pulmonary tuberculosis patients by 16S-23S rRNA inter transcribed spacer PCR – RFLP

Abstract

Pulmonary infections due to Non-Tuberculosis Mycobacteria (NTM) has emerged as an increasingly prevalent clinical entity in the past two to three decades. The prevalence of tuberculosis has reduced in developed countries, but infections due to NTM are on rising, whereas in developing countries like India, tuberculosis is still a significant health problem. This study was undertaken to investigate the overall burden and diversity of NTM among clinical pulmonary isolates from a south Indian population. This study was conducted from March 2017 to December 2017, to identify species of Non- tuberculous mycobacteria from specimens suspected with pulmonary tuberculosis. We applied conventional biochemical test like growth on the LJ medium containing PNBA (para-nitrobenzoic acid) for differentiation of NTM and M.tuberculosis and PCR- RFLP of the 16S-23S rRNA ITS gene for species identification. A 221 bp fragment of ITS gene was amplified and the amplification product was digested by two restriction enzymes, BstEII and HaeIII. Digested products were analyzed using polyacrylamide gel electrophoresis. Out of 1138 positive cultures 96 were identified as Non- tuberculous mycobacteria. Of the 96 Non-tuberculous mycobacteria 59 were from pulmonary sites. Among the 59 NTM, 74.5% (44/59) were rapidly growing mycobacteria (RGM) and 15 (25.4%) were slow growing mycobacteria (SGM). The most predominant NTM species among RGM was Mycobacterium abscessus followed by Mycobacterium simiae, Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium gordonae. Regular documentation of NTM isolated from clinical specimens and reporting their antibiogram is essential to be aware of the clinical spectrum of disease associated and preferred treatment option.