Genotyping of Mycobacterium avium complex organisms using multispacer sequence typing

Mycobacterium avium complex (MAC) currently comprises eight species of environmental and animal-associated, slowly-growing mycobacteria: Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium colombiense, Mycobacterium arosiense, Mycobacterium bouchedurhonense, Mycobacterium marseillense and Mycobacterium timonense. In humans, MAC organisms are responsible for opportunistic infections whose unique epidemiology remains poorly understood, in part due to the lack of a genotyping method applicable to all eight MAC species. In this study we developed multispacer sequence typing (MST), a sequencing-based method, for the genotyping of MAC organisms. An alignment of the genome sequence of M. avium subsp. hominissuis strain 104 and M. avium subsp. paratuberculosis strain K-10 revealed 621 intergenic spacers <1000 bp. From these, 16 spacers were selected that ranged from 300 to 800 bp and contained a number of variable bases, <50 within each of the 16 spacers. Four spacers were successfully PCR-amplified and sequenced in 11 reference strains. Combining the sequence of these four spacers in 106 MAC organisms, including 83 M. avium, 11 M. intracellulare, six M. chimaera, two M. colombiense and one each of M. arosiense, M. bouchedurhonense, M. marseillense and M. timonense, yielded a total of 45 spacer types, with an index of discrimination of 0.94. Each spacer type was specific for a species and certain spacer types were specific for subspecies of M. avium. MST is a new method for genotyping of organisms belonging to any one of the eight MAC species tested in this study.