A smooth tubercle bacillus from Ethiopia phylogenetically close to the Mycobacterium tuberculosis complex

Abstract

The Mycobacterium tuberculosis complex (MTBC) includes several human- and animal-adapted pathogens. It is thought to have originated in East Africa from a recombinogenic Mycobacterium canettii-like ancestral pool. Here, we describe the discovery of a clinical tuberculosis strain isolated in Ethiopia that shares archetypal phenotypic and genomic features of M. canettii strains, but represents a phylogenetic branch much closer to the MTBC clade than to the M. canettii strains. Analysis of genomic traces of horizontal gene transfer in this isolate and previously identified M. canettii strains indicates a persistent albeit decreased recombinogenic lifestyle near the emergence of the MTBC. Our findings support that the MTBC emergence from its putative free-living M. canettii-like progenitor is evolutionarily very recent, and suggest the existence of a continuum of further extant derivatives from ancestral stages, close to the root of the MTBC, along the Great Rift Valley.

Fig. 1. Schematic workflow showing main steps of sequencing and post-sequencing analyses of the study.

The hybrid strategy combining Illumina and Oxford Nanopore sequencing used to obtain the complete assembly and functional annotation of the genome of M. canettii ET1291, and to perform comparative genomics analyses with previously known M. canettii and MTBC strains, are represented in the center. Illumina whole-genome sequencing data were also used to perform SNP-based phylogenetic reconstructions and to determine the ratio of recombination versus mutation in the strain dataset. Results of targeted next-generation sequencing using the Deeplex Myc-TB assay, identifying ET1291 as M. canettii, are represented on the circular map on the right. Information on hsp65 best match-based and phylogenetic SNP-based identification of M. canettii is shown inside the circle, along with information on spoligotype (without any spacer detected). Target gene regions in the map are grouped within sectors according to the anti-tuberculous drug resistance with which they are associated. Sectors in red (for pyrazinamide (PZA) here, as typically expected for M. canettii) and green indicate targets in which resistance-associated mutations or either no mutation or only mutations not associated with resistance (shown in gray) are detected, respectively. Blue sectors refer to regions where as yet uncharacterized mutations are detected (see ref. for further details on the Deeplex map). gDNA, genomic DNA; MTBC, M. tuberculosis complex, MCAN, M. canettii; MAP, M. tuberculosis (MTB)-associated phylotype. Abbreviations of anti-tuberculous drugs on the Deeplex map: RIF: rifampicin, INH: isoniazid, PZA: pyrazinamide, EMB: ethambutol, SM: streptomycin, FQ: Fluoroquinolones, KAN: kanamycin, AMI: amikacin, CAP: capreomycin, ETH: ethionamide, LIN: linezolid, BDQ: bedaquiline, CFZ: clofazimine. Figure created using BioRender.com by Arash Ghodousi with license to publish.

Fig. 2. Whole-genome alignment-based phylogeny and recombinogenic versus clonal structure of ET1291, other M. canettii and MTBC strains.

a Maximum likelihood phylogenetic tree including 121 genomes before accounting for recombination. Bootstrap support values are shown only for values below 100%; the value is not shown for branches with 100% bootstrap support, which represent the majority of the long branches as expected. b Maximum likelihood phylogenetic tree including the same genomes after correction for recombination. Branches are colored according to the estimated ratios of recombination versus mutation (r/m). r/m values are only shown for branches longer than 6e-5 substitutions/site, because of inherent stochasticity in signal to noise ratios in the shortest branches. The intermediate phylogenetic position of ET1291 between the MTBC and the other, currently known M. canettii strains is indicated by a red arrow. c ClonalFrameML analysis of the 121 genomes, showing the extent and segments of recombination mapped on the branches of ET1291 and other M. canettii strains and the branch leading to the common ancestor of the MTBC. Note that the red dotted lines indicate two branches that are ancestral to the progenitor of the MTBC and to the progenitor of the MTBC and ET1291, respectively, and are thus not part of the MTBC clade. MTBC, M. tuberculosis complex; MCAN, M. canettii. ET1291 indicated by a red arrow.

Fig. 3. Colony morphology and architecture of the pks5 genome region in ET1291, M. canettii STB-A, M. tuberculosis H37Rv.

a Similar smooth colony morphotypes shared by ET1291 and M. canettii STB-A, contrasting with the rough colony morphotype of M. tuberculosis H37Rv. b Aligned genome segments showing the dual polyketide synthase-encoding pks5 gene configuration with an intervening pap gene shared by ET1291 and M. canettii STB-A (and all other M. canettii strains), instead of the single pks5 gene found in M. tuberculosis H37Rv (and all other MTBC members). Colors show the correspondence between gene orthologues among ET1291, M. canettii STB-A, and M. tuberculosis H37Rv.

Fig. 4. Presence of two prophage-like regions in ET1291 unlike in other M. canettii strains.

a PhiRv1 prophage region. b PhiRv2 prophage region. These two prophage-like regions of ~9–10 kb found are located in the same genomic locations as the PhiRv1 and PhiRv2 prophage-like regions of MTBC strains. Percent identities are shown for genes with at least 70% identity with an orthologue in M. tuberculosis H37Rv. Only a fraction of these phage gene sequences show substantial identity (76–96%, lower than the 99–100% seen for the core genes) with the MTBC PhiRv1 and PhiRv2 genes, suggesting that these prophages were acquired independently from those in the MTBC.

Fig. 5. CRISPR-Cas systems in ET1291, STB-G, STB-I, and M. tuberculosis H37Rv.

Percentages of identities are indicated with arrows between cas2, cas1e, cas6/cse3/casE, cas5/casD and cas7/cse4/casC genes of ET1291, and STB-G/I. CRISPR repeats sequences (shown above the respective CRISPR arrays) are identical between ET1291, STB-G, and STB-I (as shown as same black vertical bars in CRISPR arrays), while CRISPR spacers are strain-specific (as shown by different colors interspaced between vertical bars among respective CRISPR arrays). Colors show the correspondence between gene orthologues among ET1291, STB strains, and M. tuberculosis H37Rv.