Association of non-tuberculous mycobacteria with Mycobacterium leprae in environment of leprosy endemic regions in India

Non-tuberculous mycobacteria (NTM) are environmental mycobacteria found ubiquitously in nature. The present study was conducted to find out the presence of various species of NTM in leprosy endemic region along with Mycobacterium (M) leprae. Water and wet soil samples from the periphery of ponds used by the community were collected from districts of Purulia of West Bengal and Champa of Chhattisgarh, India. Samples were processed and decontaminated followed by culturing on Lowenstein Jensen (LJ) media. Polymerase chain reaction (PCR) was performed using 16S rRNA gene target of mycobacteria and species was confirmed by sequencing method. Indirect immune-fluorescent staining of M. leprae from soil was performed using M. leprae-PGL-1 rabbit polyclonal antibody. The phylogenetic tree was constructed by using MEGA-X software. From 380 soil samples 86 NTM were isolated, out of which 34(40%) isolates were rapid growing mycobacteria (RGM) and 52(60%) isolates were slow growing mycobacteria (SGM). Seventy-seven NTM isolates were obtained from 250 water samples, out of which 35(45%) were RGM and 42(55%) were SGM. Amongst all the RGM, we isolated M. porcinum, M. psychrotolerans, M. alsenase, M. arabiense and M. asiaticum from Indian environmental samples. M. fortuitum was the most commonly isolated species of all RGM. Out of all SGM, M. holsaticum, M. yongonense, M. seoulense, M. szulgai, M. europaeum, M. simiae and M. chimaera were isolated for the first time from Indian environment. M. intracellulare was the commonest of all isolated SGM. Presence of M. leprae was confirmed by indirect immunofluorescent microcopy and PCR method from the same environmental samples. Phylogenetic tree was showing a close association between these NTMs and M. leprae in these samples. Several NTM species of pathogenic and nonpathogenic in nature along with M. leprae were isolated from soil and pond water samples from leprosy endemic regions and these might be playing a role in causing disease and maintaining leprosy endemicity in India.