Genomic analysis of globally diverse Mycobacterium tuberculosis strains provides insights into the emergence and spread of multidrug resistance

Abstract

Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. Here we examined a data set of whole-genome sequences from 5,310 M. tuberculosis isolates from five continents. Despite the great diversity of these isolates with respect to geographical point of isolation, genetic background and drug resistance, the patterns for the emergence of drug resistance were conserved globally. We have identified harbinger mutations that often precede multidrug resistance. In particular, the katG mutation encoding p.Ser315Thr, which confers resistance to isoniazid, overwhelmingly arose before mutations that conferred rifampicin resistance across all of the lineages, geographical regions and time periods. Therefore, molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of polymorphisms that occur before the emergence of multidrug resistance, particularly katG p.Ser315Thr, into molecular diagnostics should enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.

Figure 1

A) Geographic distribution of M. tuberculosis isolates in our dataset by drug resistance pattern. This plot shows the distribution of the 5,310 M. tuberculosis isolates included in our dataset by drug resistance genotype (pie charts) and by 11 UN geographic subregions (coloring), and is not meant to indicate the overall global incidence of TB or drug resistance. There were no strains in our dataset from geographic regions colored grey. UN geographic subregions with fewer than 30 strains were excluded from this figure. Map modified from a blank map of UN geographical subregions. B) The overall proportion of drug-resistant strains identified among all 5,310 M. tuberculosis isolates in our dataset.

Figure 2

Across the globe, isoniazid resistance was overwhelmingly the first step towards drug resistance. Acquisition of a katG S315 mutation precedes all other resistance mutations for the majority of instances in which the order of acquisition can be disambiguated. We quantified the pairwise number of evolutions in which resistance to one drug preceded resistance to a second drug. Reported numbers represent the number of independent evolution events (not the number of strains) in which the drug resistance indicated by the row labeled “first resistance” was acquired before the drug resistance indicated by the column labeled “second resistance”. The shading color indicates the percentage of evolutionary events in which the “first resistance” clearly predates the “second resistance” for that drug pair. While inhA mutations can confer resistance to both isoniazid and ethionamide, we defined genotypic ethionamide resistance as mutations in only ethA to simplify the analysis and avoid double counting.

Figure 3

Sequential acquisition of drug resistance mutations reveals that isoniazid resistance-conferring mutations, specifically katG S315T, most often come first in sequential pairs. This figure includes data from 71 mutations conferring drug resistance with at least 10 occurrences in our dataset, which represent 93% of all drug resistance mutations in our dataset. Using PAUP analysis to assign specific mutation gains to individual nodes on the phylogeny, we tabulated all routes of drug resistance acquisition across the full strain phylogeny, examining only those nodes on the tree where drug resistance mutations arose (i.e. node 1 [mutation A] -> node 2 [mutations B and C] -> node 3 [mutation D]). We tabulated the number of times each pair of mutations arose sequentially at adjacent nodes (i.e. mutations A->B, A->C, B->D, and C->D). We removed node pairs that did not meet specific bootstrap and branch length criteria (see Methods). The ribbons in this figure depict the number of times that each pair of mutations arose sequentially at adjacent nodes across the entire dataset. The width of the ribbon at each end is proportional to the number of times mutation A arose before mutation B, or vice versa (i.e., a ribbon with a thick end at katG S315T and a thin end at rpoB S450L indicates that katG S315T mutation arose prior to rpoB S450L much more frequently than the opposite). Each ribbon is colored according to the mutation more often occurring first in each sequential pairing.

Figure 4

In all lineages and global regions, the katG mutation S315T occurs first, and few examples of the reverse ordering are observed. We separately recalculated phylogenies for isolates from patients in each of the 11 UN subregions and 5 lineages with greater than 30 representatives (see Methods). This figure depicts the pairwise ordering of the katG S315T mutation in relation to mutations conferring resistance to the other three XDR-definings drugs (rifampicin (R), kanamycin (K), and ofloxacin (O)), within each individual M. tuberculosis lineage and geographic region. The numbers in panel B do not necessarily add up to the numbers in panel A, as the analyses of regions and lineages were performed individually, which can affect the number of arisals. Grey shading indicates that there were not sufficient pairings. Data are not shown for the following regions and lineages, as there were insufficient pairings: West Africa, Southern Europe, Central Asia, Northern America, lineage 1, and M. bovis.

Figure 5

Non-rifampicin drug resistance often precedes the arisal of GeneXpert mutations. Data are shown here for the nodes at which a GeneXpert mutation arose. A) This plot shows the percentage of GeneXpert nodes where resistance to each of eight drugs unambiguously preceded the arisal of GeneXpert mutations. Drug resistances that appeared to arise coincident to the GeneXpert node were excluded from this representation. More than one additional resistance could precede a single GeneXpert node. No strains contained additional rifampicin mutations arising before GeneXpert mutations. The bars represent a lower bound on the percentage of GeneXpert mutations preceded by additional resistance mutations, as we were unable to disambiguate ordering for a substantial number of nodes where additional mutations arose at the same node (see Supplementary Figure 11). B) Percentage of nodes where resistance to one or more other drugs unambiguously preceded the arisal of GeneXpert mutations. 13% of GeneXpert arisal nodes unambiguously had no additional drug resistance mutations arising prior.

Figure 6

katG S315T is a commonly occurring mutation with very little resistance to other drugs arising prior to its occurrence. A) For each of the 16 “pre-MDR” mutations, the percentage of nodes where resistance to another drug unambiguously preceded the arisal of that mutation. These bars represent a lower bound on the percentage of nodes preceded by another resistance mutation, as we were unable to disambiguate ordering for a substantial number of nodes where additional mutations arose at the same node. B) The number of independent arisals for each of 16 “pre-MDR” (or harbinger) mutations. Since there are two embB M306I mutations, the nucleotide change at position 4,247,609 is also indicated for these two variants.