Genomic Insights Into the Mycobacterium kansasii Complex: An Update

Abstract

Only very recently, has it been proposed that the hitherto existing Mycobacterium kansasii subtypes (I-VI) should be elevated, each, to a species rank. Consequently, the former M. kansasii subtypes have been denominated as Mycobacterium kansasii (former type I), Mycobacterium persicum (II), Mycobacterium pseudokansasii (III), Mycobacterium innocens (V), and Mycobacterium attenuatum (VI). The present work extends the recently published findings by using a three-pronged computational strategy, based on the alignment fraction-average nucleotide identity, genome-to-genome distance, and core-genome phylogeny, yet essentially independent and much larger sample, and thus delivers a more refined and complete picture of the M. kansasii complex. Furthermore, five canonical taxonomic markers were used, i.e., 16S rRNA, hsp65, rpoB, and tuf genes, as well as the 16S-23S rRNA intergenic spacer region (ITS). The three major methods produced highly concordant results, corroborating the view that each M. kansasii subtype does represent a distinct species. This work not only consolidates the position of five of the currently erected species, but also provides a description of the sixth one, i.e., Mycobacterium ostraviense sp. nov. to replace the former subtype IV. By showing a close genetic relatedness, a monophyletic origin, and overlapping phenotypes, our findings support the recognition of the M. kansasii complex (MKC), accommodating all M. kansasii-derived species and Mycobacterium gastri. None of the most commonly used taxonomic markers was shown to accurately distinguish all the MKC species. Likewise, no species-specific phenotypic characteristics were found allowing for species differentiation within the complex, except the non-photochromogenicity of M. gastri. To distinguish, most reliably, between the MKC species, and between M. kansasii and M. persicum in particular, whole-genome-based approaches should be applied. In the absence of clear differences in the distribution of the virulence-associated region of difference 1 genes among the M. kansasii-derived species, the pathogenic potential of each of these species can only be speculatively assessed based on their prevalence among the clinically relevant population. Large-scale molecular epidemiological studies are needed to provide a better understanding of the clinical significance and pathobiology of the MKC species. The results of the in vitro drug susceptibility profiling emphasize the priority of rifampicin administration in the treatment of MKC-induced infections, while undermining the use of ethambutol, due to a high resistance to this drug.

Keywords: Mycobacterium kansasii complex; Mycobacterium ostraviense sp. nov.; non-tuberculous mycobacteria (NTM); taxonomy; whole genome sequencing.

Figure 1

Application of the Microbial Species Identifier (MiSI) method in classifying Mycobacterium kansasii subtype I-VI strains. Separation of different species into different cliques is depicted. Each color represents a species, while each circle—a genome of that species. Within cliques corresponding to M. persicum (formerly subtype II), M. pseudokansasii (III), M. innocens (V), and M. attenuatum (VI), genomes, which had been analyzed previously and first assigned to newly proposed species are shown in other than red color. Within the M. tuberculosis (MTB) complex clique, genomes of M. tuberculosis, M. africanum, M. bovis, M. caprae, and M. microti are depicted in yellow, orange, green, blue, and gray, respectively.

Figure 2

Maximum-likelihood phylogenetic tree based on the amino acid alignment of concatenated single-copy orthologous genes. Bar, number of amino acid substitutions per amino acid site. Node supports were computed using the Shimodaira-Hasegawa test.

Figure 3

Phylogenetic tree based on 16S rRNA gene sequences, constructed using the Neighbor-Joining method. The bootstrap values were calculated from 1,000 replications. Bootstrap values are given at nodes. Strains of M. kansasii type IV (241/15, 1010001458) and M. gastri are shown boxed in solid and dashed lines, respectively. Strains of M. kansasii type I, as per WGS-based analysis, that clustered either within type I or II, upon single-gene or concatenated gene phylogenies are shown in bold. GenBank accession numbers for the sequences are parenthesized. Bar, 0.01 substitutions per nucleotide position.

Figure 4

Phylogenetic tree based on ITS sequences constructed using the Neighbor-Joining method. The bootstrap values were calculated from 1,000 replications. Bootstrap values are given at nodes. Strains of M. kansasii type I, as per WGS-based analysis, that clustered either within type I or II, upon single-gene or concatenated gene phylogenies are shown in bold. GenBank accession numbers for the sequences are parenthesized. Bar, 0.01 substitutions per nucleotide position.

Figure 5

Phylogenetic tree based on hsp65 gene sequences constructed using the Neighbor-Joining method. The bootstrap values were calculated from 1,000 replications. Bootstrap values are given at nodes. Strains of M. kansasii type I, as per WGS-based analysis, that clustered either within type I or II, upon single-gene or concatenated gene phylogenies are shown in bold. GenBank accession numbers for the sequences are parenthesized. Bar, 0.01 substitutions per nucleotide position.

Figure 6

Phylogenetic tree based on tuf gene sequences constructed using the Neighbor-Joining method. The bootstrap values were calculated from 1,000 replications. Bootstrap values are given at nodes. Strains of M. kansasii type I, as per WGS-based analysis, that clustered either within type I or II, upon single-gene or concatenated gene phylogenies are shown in bold. GenBank accession numbers for the sequences are parenthesized. Bar, 0.01 substitutions per nucleotide position.

Figure 7

Phylogenetic tree based on rpoB sequences constructed using the Neighbor-Joining method. The bootstrap values were calculated from 1,000 replications. Bootstrap values are given at nodes. Strains of M. kansasii type I, as per WGS-based analysis, that clustered either within type I or II, upon single-gene or concatenated gene phylogenies are shown in bold. GenBank accession numbers for the sequences are parenthesized. Bar, 0.01 substitutions per nucleotide position.

Figure 8

Phylogenetic tree based on concatenated 16S rRNA, hsp65, and rpoB gene sequences constructed using the Neighbor-Joining method. The bootstrap values were calculated from 1,000 replications. Bootstrap values are given at nodes. Strains of M. kansasii type I, as per WGS-based analysis, that clustered either within type I or II, upon single-gene or concatenated gene phylogenies are shown in bold. GenBank accession numbers for the sequences are parenthesized. Bar, 0.01 substitutions per nucleotide position.