The Forest behind the Tree: Phylogenetic Exploration of a Dominant Mycobacterium tuberculosis Strain Lineage from a High Tuberculosis Burden Country

BACKGROUND: Genotyping of Mycobacterium tuberculosis isolates is a powerful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. Standardized PCR-based typing, based on 15 to 24 mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) loci combined with spoligotyping, has been shown to have adequate resolution power for tracing TB transmission and to be useful for predicting diverse strain lineages in European settings. Its informative value needs to be tested in high TB-burden countries, where the use of genotyping is often complicated by dominance of geographically specific, genetically homogeneous strain lineages. METHODOLOGY/PRINCIPAL FINDINGS: We tested this genotyping system for molecular epidemiological analysis of 369 M. tuberculosis isolates from 3 regions of Brazil, a high TB-burden country. Deligotyping, targeting 43 large sequence polymorphisms (LSPs), and the MIRU-VNTRplus identification database were used to assess phylogenetic predictions. High congruence between the different typing results consistently revealed the countrywide supremacy of the Latin-American-Mediterranean (LAM) lineage, comprised of three main branches. In addition to an already known RDRio branch, at least one other branch characterized by a phylogenetically informative LAM3 spoligo-signature seems to be globally distributed beyond Brazil. Nevertheless, by distinguishing 321 genotypes in this strain population, combined MIRU-VNTR typing and spoligotyping demonstrated the presence of multiple distinct clones. The use of 15 to 24 loci discriminated 21 to 25% more strains within the LAM lineage, compared to a restricted lineage-specific locus set suggested to be used after SNP analysis. Noteworthy, 23 of the 28 molecular clusters identified were exclusively composed of patient isolates from a same region, consistent with expected patterns of mostly local TB transmission. CONCLUSIONS/SIGNIFICANCE: Standard MIRU-VNTR typing combined with spoligotyping can reveal epidemiologically meaningful clonal diversity behind a dominant M. tuberculosis strain lineage in a high TB-burden country and is useful to explore international phylogenetical ramifications

Congruence analysis between MIRU-VNTR typing, deligotyping and spoligotyping.

<p>A selection of 137 isolates was used, representing the diversity of the different lineages and subgroups predicted based on MIRU-VNTR typing, spoligotyping and the MIRU-VNTR<i>Plus</i> database (see text, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018256#pone-0018256-t003" target="_blank">Table 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018256#pone-0018256-g001" target="_blank">Fig. 1</a>). A. A MIRU-VNTR-based dendrogram was generated using the neighbor-joining algorithm and rooted using a <i>M. prototuberculosis</i> C/D genotype (alias <i>M. canettii</i>) as outgroup. Solid coloured circles on tree nodes indicate MIRU-VNTR groupings that are monophyletic when compared to LSP-based (deligotyping) and/or (when deligotyping was not informative) spoligotyping-based groupings. Partially monophyletic groupings are indicated by coloured rings. B. MIRU-VNTR-based minimum spanning tree. The same isolates were used as in the neighbor-joining tree. Colours and grouping names correspond to those of panel A. Distances between circles are proportional to the number of allele differences between MIRU-VNTR genotypes; circle sizes are proportional to the numbers of isolates sharing an identical genotype. Del, deligotyping probe; RD, region of difference (reference LSP, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018256#pone-0018256-t001" target="_blank">Table 1</a>); spol, spoligotyping spacer; LAMu, unclassified LAM isolate. Color codes of phylogenetic groups (see text for further description): yellow, Indo-Oceanic (LSP)/EAI (spoligotyping); grey, S; light purple, X; red, Haarlem; blue, Brazil 1; dark purple; Brazil 2; pink, LAM II; khaki, LAM I; green, LAM III.</p

Discriminatory powers of different genotyping methods used.

<p>Discriminatory powers of different genotyping methods used.</p

Distribution of <i>M. tuberculosis</i> lineages in the study.

a<p>Nomenclatures corresponding to Gagneux et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018256#pone.0018256-Gagneux2" target="_blank">[30]</a>/Brudey et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018256#pone.0018256-Brudey1" target="_blank">[48]</a>, except for Brazil-1 and -2 named according to this study.</p

<i>M. tuberculosis</i> strain cluster distribution among three Brazilian regions.

<p>Molecular clusters are defined by isolates sharing identical 24-locus MIRU-VNTR genotypes and spoligotypes.</p

Genotypic diversity of <i>M. tuberculosis</i> isolates from 3 Brazilian regions.

<p>Colour-coded 24-locus MIRU-VNTR alleles and spoligotypes from 361 isolates are represented. A MIRU-VNTR-based dendrogram was generated using the neighbor-joining algorithm and rooted using a <i>M. prototuberculosis</i> C/D genotype (alias <i>M. canettii</i>) as outgroup. <i>M. tuberculosis</i> strain lineages and branches shown at the right were identified by analyzing the congruence of MIRU-VNTR typing and spoligotyping results within this collection and submitting the isolate genotypes to the MIRU-VNTR<i>Plus</i> identification database (see text). X gr1 and gr2 (groups 1 and 2) correspond to X del26/RD183 and Xdel29/RD193 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018256#pone-0018256-g003" target="_blank">Fig. 3</a>, respectively.</p

<i>M. tuberculosis strain</i> lineage distribution among three Brazilian regions.

<p>Lineage distributions result from the congruence analyses shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018256#pone-0018256-g001" target="_blank">Fig. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018256#pone-0018256-g003" target="_blank">3</a>. One isolate from Rio De Janeiro was of an unknown lineage/branch. Arrows indicate region-specific lineages. Br1 and Br2, Brazil 1 and 2, respectively.</p

Deligotyping genomic deletion targets and reference large sequence polymorphisms.

a<p>According to Gagneux et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018256#pone.0018256-Gagneux2" target="_blank">[30]</a>.</p