Polyphasic taxonomic analysis establishes Mycobacterium indicus pranii as a distinct species

Abstract

Background: Mycobacterium indicus pranii (MIP), popularly known as Mw, is a cultivable, non-pathogenic organism, which, based on its growth and metabolic properties, is classified in Runyon Group IV along with M. fortuitum, M. smegmatis and M. vaccae. The novelty of this bacterium was accredited to its immunological ability to undergo antigen driven blast transformation of leukocytes and delayed hypersensitivity skin test in leprosy patients, a disease endemic in the Indian sub-continent. Consequently, MIP has been extensively evaluated for its biochemical and immunological properties leading to its usage as an immunomodulator in leprosy and tuberculosis patients. However, owing to advances in sequencing and culture techniques, the citing of new strains with almost 100% similarity in the sequences of marker genes like 16S rRNA, has compromised the identity of MIP as a novel species. Hence, to define its precise taxonomic position, we have carried out polyphasic taxonomic studies on MIP that integrate its phenotypic, chemotaxonomic and molecular phylogenetic attributes.

Methodology/principal findings: The comparative analysis of 16S rRNA sequence of MIP by using BLAST algorithm at NCBI (nr database) revealed a similarity of > or =99% with M. intracellulare, M. arosiense, M. chimaera, M. seoulense, M. avium subsp. hominissuis, M. avium subsp. paratuberculosis and M. bohemicum. Further analysis with other widely used markers like rpoB and hsp65 could resolve the phylogenetic relationship between MIP and other closely related mycobacteria apart from M. intracellulare and M. chimaera, which shares > or =99% similarity with corresponding MIP orthologues. Molecular phylogenetic analysis, based on the concatenation of candidate orthologues of 16S rRNA, hsp65 and rpoB, also substantiated its distinctiveness from all the related organisms used in the analysis excluding M. intracellulare and M. chimaera with which it exhibited a close proximity. This necessitated further analysis of MIP with more sensitive and segregating parameters to ascertain its precise taxonomic position as a new species. The analysis of MIP and its comparison with other mycobacterial reference strains based on cellular and biochemical features, growth characteristics and chemotaxonomic studies like FAME profiling confirmed that MIP is uniquely endowed with diverse metabolic attributes that effectively distinguishes it from all the closely related mycobacteria including M. intracellulare and M. chimaera.

Conclusion: The results presented in this study coupled with the non-pathogenic nature and different biochemical and immunomodulatory properties of MIP affirm it as a distinct species belonging to M. avium complex (MAC). It is further proposed to use an earlier suggested name Mycobacterium indicus pranii for this newly established mycobacterial species. This study also exemplifies the growing need for a uniform, consensus based broader polyphasic frame work for the purpose of taxonomy and speciation, particularly in the genus Mycobacterium.

Figure 1. Growth rate analysis of MIP:

MIP was cultured in MB7H9-ADC medium at 30°C and 37°C. The A_600nm of liquid culture of MIP was plotted against time to analyze the pattern of MIP growth. Growth was monitored by measuring the change in the value of A_600nm over time. Each experiment was performed with replicates and error bars for each time point are shown. A typical growth curve with three distinct phases was generated with culture becoming saturated in 8 to 10 days.

Figure 2. Phylogenetic positioning of MIP:

The Phylogenetic positioning of MIP with respect to other members of genus Mycobacterium was performed by making concatenated tree of 16S rRNA, rpoB and hsp65 involving members of MAC and other environmental isolates, which are close to MIP. The alignment was carried out using clustal x ver 1.81 and a phylogenetic tree rooted to M. malmoense was constructed using Neighbor joining (NJ) method with 1,000 bootstrap iterations .