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. 2016 Jan 11;10(1):e0004332.
doi: 10.1371/journal.pntd.0004332. eCollection 2016 Jan.

Whole Genome Sequencing of Mycobacterium Africanum Strains From Mali Provides Insights Into the Mechanisms of Geographic Restriction

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Free PMC article

Whole Genome Sequencing of Mycobacterium Africanum Strains From Mali Provides Insights Into the Mechanisms of Geographic Restriction

Kathryn Winglee et al. PLoS Negl Trop Dis. .
Free PMC article

Abstract

Background: Mycobacterium africanum, made up of lineages 5 and 6 within the Mycobacterium tuberculosis complex (MTC), causes up to half of all tuberculosis cases in West Africa, but is rarely found outside of this region. The reasons for this geographical restriction remain unknown. Possible reasons include a geographically restricted animal reservoir, a unique preference for hosts of West African ethnicity, and an inability to compete with other lineages outside of West Africa. These latter two hypotheses could be caused by loss of fitness or altered interactions with the host immune system.

Methodology/principal findings: We sequenced 92 MTC clinical isolates from Mali, including two lineage 5 and 24 lineage 6 strains. Our genome sequencing assembly, alignment, phylogeny and average nucleotide identity analyses enabled us to identify features that typify lineages 5 and 6 and made clear that these lineages do not constitute a distinct species within the MTC. We found that in Mali, lineage 6 and lineage 4 strains have similar levels of diversity and evolve drug resistance through similar mechanisms. In the process, we identified a putative novel streptomycin resistance mutation. In addition, we found evidence of person-to-person transmission of lineage 6 isolates and showed that lineage 6 is not enriched for mutations in virulence-associated genes.

Conclusions: This is the largest collection of lineage 5 and 6 whole genome sequences to date, and our assembly and alignment data provide valuable insights into what distinguishes these lineages from other MTC lineages. Lineages 5 and 6 do not appear to be geographically restricted due to an inability to transmit between West African hosts or to an elevated number of mutations in virulence-associated genes. However, lineage-specific mutations, such as mutations in cell wall structure, secretion systems and cofactor biosynthesis, provide alternative mechanisms that may lead to host specificity.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. M. africanum and M. tuberculosis drug resistance is genetically similar.
A) SNP-based phylogenetic tree of 92 newly sequenced strains from Mali (the Mali Collection), constructed using FastTree [55]. B) Groups differing by 10, 20, 30, or 50 SNPs are connected with black bars, as calculated in Cohen et al. [25]. C) Comparison of genotypic and phenotypic DST (drug susceptibility testing). Genotypic drug resistance was calculated using a list of mutations known to confer drug resistance (S2A Table) [59, 60]. D) Drug resistance category (mono-DR, poly-DR, MDR, or pre-XDR) based on genotype. E) Digital spoligotype clade and F) digital spoligotype international type (SIT), colored by lineage. F) The SIT is blank if the SITVITWEB database [45] did not contain a SIT for that strain’s digital spoligotype pattern.
Fig 2
Fig 2. Phylogenetic tree of 92 newly sequenced strains from Mali, together with 45 additional strains with whole-genome assemblies (the Assembly Collection).
Nodes, lineages, and newly-sequenced Mali strains are indicated. All key nodes separating the major lineages had bootstrap values of 100%, except for the node separating M. tuberculosis lineage 1 and M. africanum lineage 5, which had a bootstrap value of 83%. Letters indicate key nodes analyzed in detail: (A) lineage 6, (B) the clade including M. bovis and lineage 6, (C) lineage 5, and (D) the clade including lineages 5, 6 and M. bovis.
Fig 3
Fig 3. Average nucleotide identity (ANI) analysis indicates M. africanum and M. tuberculosis are not separate species.
A) ANI values when comparing M. africanum and M. tuberculosis do not cross the ANI species threshold of 94–95%. In fact, this comparison shows that the distribution of M. africanum/M. tuberculosis comparisons (red) overlaps that of inter-lineage M. tuberculosis comparisons (purple), indicating that M. africanum should be considered another lineage of M. tuberculosis. B) Similarly, ANI values when comparing M. bovis and M. tuberculosis also overlap with inter-lineage M. tuberculosis, and indicate that M. bovis should also be considered another lineage of M. tuberculosis. C) ANI values comparing M. africanum and M. bovis (pink) also overlap inter-lineage M. tuberculosis comparisons (green).
Fig 4
Fig 4. Diversity in Mali lineage 4 and lineage 6 strains.
ANI values for comparisons (A) within all Mali lineage 4 isolates and (B) within all lineage 6 isolates. Blue lines indicate mean ± standard deviation. The means of these two groups was not significantly different using the Mann-Whitney test.
Fig 5
Fig 5. Percentage of lineage-specific mutations in virulence associated genes.
A) Percentage of lineage-specific mutations in coding sequences of the genes in each category. Sassetti virulence genes are genes that were identified in [66] as being required for virulence in mice. Sassetti essential and slow growth genes were identified by Sassetti et al. under in vitro conditions using TraSH [65]. Rengarajan macrophage genes were identified by Rengarajan et al. as being required for growth in macrophages [67]. Comas antigen genes were genes identified by Comas et al. as containing T cell epitopes [4]. The color of the bar indicates type of mutation. B) Percentage of lineage-specific pseudogenes falling into the above defined categories. Missing categories had no pseudogenes in any lineage. Lineage is indicated by the number below each bar, while ‘af’ indicates mutations found in both lineages 5 and 6 (both M. africanum lineages).

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