Silent nucleotide polymorphisms and a phylogeny for Mycobacterium tuberculosis

Abstract

Much remains unknown of the phylogeny and evolution of Mycobacterium tuberculosis, an organism that kills 2 million people annually. Using a population-based approach that analyzes multiple loci around the chromosome, we demonstrate that neutral genetic variation in genes associated with antimicrobial drug resistance has sufficient variation to construct a robust phylogenetic tree for M. tuberculosis. The data describe a clonal population with a minimum of four distinct M. tuberculosis lineages, closely related to M. bovis. The lineages are strongly geographically associated. Nucleotide substitutions proven to cause drug resistance are distributed throughout the tree, whereas nonsynonymous base substitutions unrelated to drug resistance have a restricted distribution. The phylogenetic structure is concordant with all the previously described genotypic and phenotypic groupings of M. tuberculosis strains and provides a unifying framework for both epidemiologic and evolutionary analysis of M. tuberculosis populations.

Figure 2

Relationship between the 37 silent single nucleotide polymorphisms, the synonymous sequence types, and major lineages.

Figure 1

Unifying phylogeny for Mycobacterium tuberculosis. A) Maximum parsimony tree of M. tuberculosis and M. bovis based on 37 silent single-nucleotide polymorphisms in 225 isolates. Synonymous sequence types (SST) are marked 1–35. The frequency of each SST is marked in parentheses. The nodes of the major lineages are highlighted: lineage I (cyan), lineage II (red), lineage III (blue), lineage IV (yellow), and M. bovis (green). The colors correspond to those in Figure 2. Note both M. africanum Type I isolates sequenced were SST 1. B) Schematic representation of the genetic groups 1, 2, and 3 defined by the katG-gyrA scheme. C) Schematic representation of the presence or absence of the tuberculosis specific region of difference, TbD1. D) Schematic representation of the strain families Beijing, Haarlem, Africa, Delhi, East Africa-India (EA-I), and Latin America-Mediterranean (LA-M), previously described by IS6110 restriction fragment length polymorphism typing and spoligotyping, demonstrating concordance with the phylogenetic tree. E) Spoligotyping patterns for representative isolates of each lineage demonstrating lack of probe hybridization at spacers 1–34 in lineage I, 33–36 in lineage II, 4–7 and 23–24 in lineage III, 29–32 and 34 in lineage IV, and 39–43 in M. bovis.

Figure 3

Relationship between Mycobacterium tuberculosis synonymous sequence type (SST), and lineage (left hand column), spoligotype pattern (middle column), and IS6110 restriction fragment length polymorphism.