Phylogenomics of Mycobacterium africanum reveals a new lineage and a complex evolutionary history

Abstract

Human tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC). The MTBC comprises several human-adapted lineages known as M. tuberculosis sensu stricto, as well as two lineages (L5 and L6) traditionally referred to as Mycobacterium africanum. Strains of L5 and L6 are largely limited to West Africa for reasons unknown, and little is known of their genomic diversity, phylogeography and evolution. Here, we analysed the genomes of 350 L5 and 320 L6 strains, isolated from patients from 21 African countries, plus 5 related genomes that had not been classified into any of the known MTBC lineages. Our population genomic and phylogeographical analyses showed that the unclassified genomes belonged to a new group that we propose to name MTBC lineage 9 (L9). While the most likely ancestral distribution of L9 was predicted to be East Africa, the most likely ancestral distribution for both L5 and L6 was the Eastern part of West Africa. Moreover, we found important differences between L5 and L6 strains with respect to their phylogeographical substructure and genetic diversity. Finally, we could not confirm the previous association of drug-resistance markers with lineage and sublineages. Instead, our results indicate that the association of drug resistance with lineage is most likely driven by sample bias or geography. In conclusion, our study sheds new light onto the genomic diversity and evolutionary history of M. africanum, and highlights the need to consider the particularities of each MTBC lineage for understanding the ecology and epidemiology of TB in Africa and globally.

Keywords: Mycobacterium africanum; Mycobacterium tuberculosis; diversity; evolution; genome; mycobacteria.

Fig. 1.

Maximum-likelihood phylogeny of 424 M . africanum genomes analysed together with animal-associated genomes used as references. Support bootstrap values are indicated at the nodes. The scale bar indicates the number of nucleotide substitutions per site. Nodes are coloured according to country or origin, and the shape of the node indicates susceptible or drug resistance based on absence or presence at least one of the drug-resistance mutations indicated in Table S8.

Fig. 2.

Maximum-likelihood phylogeny of 5 unclassified genomes analysed together with a dataset of 249 MTBC genomes used as references. The five unclassified genomes are coloured in light green and tagged as L9. Animal-associated clades A1 to A4 are indicated and coloured in black. Support bootstrap values are indicated at the deepest nodes. The scale bar indicates the number of nucleotide substitutions per site.

Fig. 3.

PCA based on genomic variable SNPs. The PCA was conducted separately for L5 (a) and L6 (b). Colours indicate different sublineages and grey indicates genomes with no sublineage assigned ‘nolin’.

Fig. 4.

Phylogeographical structure in L5 and L6. Linear axis plot between the genomic phylogeny and the geographical origin of the genomes for L5 (a) and L6 (c), with minimum crossing between each leaf node in the phylogeny and its geographical distribution. Histograms show the number of crossing for each inclination of the axis, and the red lines indicate the number of crossings expected by chance for L5 (b) and L6 (d).

Fig. 5.

Geographical ancestral distributions of L5, L6 and L9. (a) Ancestral area reconstruction by the Bayesian binary model onto the maximum-likelihood phylogeny. Circles represent the probabilities of ancestral ranges, and the most likely ancestral areas are indicated by their corresponding colour codes. (b) The four geographical areas considered in this analysis are coloured in the map, the most likely ancestral areas for each lineage are shown as stars, and movements of strains inferred from phylogeny indicated as arrows. The map was created using Mapchart (https://mapchart.net/africa.html).

Fig. 6.

Nucleotide diversity (π). Comparison of pairwise nucleotide diversity (π) between L5 and L6 across gene categories