Reply to '"Mycobacterium indicus pranii" is a strain of Mycobacterium intracellulare': "M. indicus pranii" is a distinct strain, not derived from M. intracellulare, and is an organism at an evolutionary transition point between a fast grower and slow grower.

REPLY

Alexander and Turenne in their letter (1) almost completely reiterate their previous comments on our earlier article, which was published in Nucleic Acids Research (2), to which we replied; their letter and the rebuttal were subsequently published (18, 19). In our earlier rebuttal, we clearly addressed all these issues and left no scope for revisiting them, and hence we are quite surprised that they did not even cite our original paper, their comments on this paper, or our earlier response in their latest letter. We are once again responding to their comments with a hope that there will be no further room for discussion on these issues.

They state that the name “Mycobacterium indicus pranii” does not figure in the “List of Prokaryotic Names with Standing in Nomenclature” and that the designation M. indicus pranii does not conform to the binomial naming convention. This opinion is, in our view, irrelevant and nonsustainable. While most bacteria follow a binomial naming system, there are several examples of bacteria being named differently. M. avium paratuberculosis, from the mycobacterial family itself, is one such example. The fact that the name M. indicus pranii has three words does not take away its distinct morphological, biochemical, and genomic identity.

Alexander’s and Turenne’s claim that they are unaware of any comparison of M. indicus pranii with a comprehensive panel of M. intracellulare or M. avium complex (MAC) reflects their ignorance (deliberate?) of published literature. We state once again that M. indicus pranii is very different from all known members of the MAC, including M. intracellulare, in various respects that include colony type, growth pattern, biochemical features, chemotaxonomic features, etc. (Table 1). M. indicus pranii is a fast grower (6 to 8 days) compared to M. tuberculosis (>3 weeks) and members of the MAC complex (including M. intracellulare) (>2 weeks). M. indicus pranii shares several other biochemical characteristics usually associated with a rapid grower (like M. smegmatis and M. vaccae), such as the absence of pigmentation and the presence of nitrate reduction, aryl sulfatase (14 days), and catalase, to name a few. Fatty acid methyl ester (FAME) analysis is a highly sensitive indicator of uniqueness of a species and is used to define the precise taxonomic position (3). Our earlier comprehensive FAME analysis (4) and comparison with profiles in the Microbial Identification System (3) showed the presence of a unique metabolic machinery in M. indicus pranii very different from that in M. intracellulare and the rest of the organisms in the FAME database. Pathway analyses confirmed the presence of unique KEGG pathways in M. indicus pranii compared to M. intracellulare and M. avium, notably the lipopolysaccharide biosynthesis pathway (KEGG identifier mid00540) and nitrotoluene degradation pathway (KEGG identifier mid00633). The lipopolysaccharide biosynthesis pathway is involved in the synthesis of lipopolysaccharides (also known as lipoglycans), which elicit strong immune responses. The nitrotoluene degradation pathway is found in soil-dwelling bacteria, is associated with metabolism of 2,4,6-trinitrotoluene under aerobic conditions, and has been reported to be present in Mycobacterium sp. strain HL 4-NT-1, isolated from polynitroaromatic compound-rich soil (5).

TABLE 1 

Comparative analyses of M. indicus pranii with M. intracellulare

Feature (reference[s])	Result for:
	M. indicus pranii	M. intracellulare
Growth patterna (4, 16, 17)
Growth rate	Rapid (6–8 days)	Slow (>2 weeks)
Growth at 45°C	Yes	No
Pigmentation	No pigment	Scotochromogenic
Colony type	Smooth	Variable
Intracellular growth	No	Yes
Biochemical featuresa (4, 16, 17)
Nitrate reduction	+	–
Aryl sulfatase (14 days)	+	–
Catalase semiquantitative	+	–
Chemotaxonomic featuresb
Fatty acid fraction	Higher	Lower
18:1ω7c	Present	Absent
20:0 fatty acid	Absent	Present
18:1ω9c	Lower	Higher
16:1ω10c	Absent	Present
Unique metabolic genes and pathways (KEGG)
Unique genes	Present	Absent
UniProt: J9WDV5	Present	Absent
UniProt: J9WLX6	Present	Absent
UniProt: J9WHE4	Present	Absent
Unique nitrotoluene degradation pathway (KEGG identifier eco00633)	Present	Absent

Saini et al. (4), Saxena et al. (16), Katoch (17).

Saini et al. (4).

The name M. indicus pranii has been accepted in scientific literature, and this new species has been deposited in the DSMZ, Germany (DSM45239T), and MTCC, India (MTCC 9506T), per well-established international guidelines. This saprophytic bacterium, with a much bigger genome than that of M. intracellulare, lacks the mce operon (required for invasion), is commercially available for therapeutic intervention against leprosy, and is currently undergoing large-scale clinical trials against many dreaded infections and diseases such as cancer, HIV, anal warts, tuberculosis, etc. (reference 6 and references cited therein).

We stand by our key conclusion (2, 7) that M. indicus pranii is an ancestor of the M. avium complex. The evidence presented by Alexander and Turenne in support of their hypothesis that M. indicus pranii is a strain of M. intracellulare lack scientific rigor and scrutiny and deserve to be trashed. They assert that phenotypic results can be misleading and hence have suggested genomic sequence comparison for drawing definitive conclusions, primarily based on >99% similarity between M. indicus pranii and M. intracellulare at the level of DNA sequences of hsp70, gyrA, dnaJ, and 16S rRNA genes. It is surprising that they are unaware of the fact that most members of the mycobacterial family that have biomedical importance display >99.95% similarity (8). Such similarities are often misleading, as they do not represent the full genomic picture, and accordingly, mycobacteria, despite having almost 100% similarity in these marker genes, have been assigned different species status (9–11). For example, with respect to the 16S rRNA gene, M. kansasii and M. gastri share 100% identity and M. malmoense and M. szulgai share 99.9% identity, as do M. microti and M. bovis. Similarly for other markers, despite identity to the extent of 100%, distinct species status has been given by taking into account the difference in ecological niches, host preferences, etc. (12). Therefore, drawing evolutionary evidence based on such assumptions of marker gene identities is erroneous (10) since candidate genes do not represent the entire genome complexity. Alexander and Turenne mention a complex genetic event as an illustration to support their claim that M. indicus pranii is derived from M. intracellulare. We respond that this single genetic event could not form the basis of an argument that M. indicus pranii is not a different species. The inversion, gain in transposons, is highly unlikely during evolution of a strain under environmental conditions. Furthermore, mycobacterium evolution involves genome reduction (13); hence, M. indicus pranii with a much bigger genome is very unlikely to be derived from M. intracellulare just because of the acquisitions of the transposons and the inversion events. On the contrary, it can be argued that the predecessor of M. indicus pranii witnessed selective massive gene acquisitions as a prelude to a soil-water interface habitat that evolved into a parasitic lifestyle (2). Genomic analysis (2) of M. indicus pranii demonstrated the presence of genetic features required for its unique lifestyle. They include 24 sigma factors (a very large number); the abundance of cytochrome P450 genes, genes involved in aerobic metabolism of phenol during degradation of plant substrates; machinery for biodegradation of cyanide and for thiocyanate degradation (absent in pathogenic mycobacteria); the presence of a complete hydrogenases enzyme complex, etc.

In summary, the array of features, including growth, biochemical and chemotaxonomic characteristics, and metabolic genes and pathways, and finally the complete genomic analyses of M. indicus pranii constitute the comprehensive basis of our assertion that M. indicus pranii occupies a unique phylogenetic place, making it the immediate predecessor of opportunistic mycobacterial species represented by the M. avium complex. We believe that the progenitor status of M. indicus pranii lends support to the idea of a shared aquatic past between saprophytic and pathogenic mycobacteria (14, 15) and provides new insights on “evolutionary habitat diversification and advent of pathogenic attributes in mycobacterium” (2).