Sonia Borrell

Out-of-Africa migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans

Tuberculosis caused 20% of all human deaths in the Western world between the seventeenth and nineteenth centuries and remains a cause of high mortality in developing countries. In analogy to other crowd diseases, the origin of human tuberculosis has been associated with the Neolithic Demographic Transition, but recent studies point to a much earlier origin. We analyzed the whole genomes of 259 M. tuberculosis complex (MTBC) strains and used this data set to characterize global diversity and to reconstruct the evolutionary history of this pathogen. Coalescent analyses indicate that MTBC emerged about 70,000 years ago, accompanied migrations of anatomically modern humans out of Africa and expanded as a consequence of increases in human population density during the Neolithic period. This long coevolutionary history is consistent with MTBC displaying characteristics indicative of adaptation to both low and high host densities.

Whole-genome sequencing of rifampicin-resistant Mycobacterium tuberculosis strains identifies compensatory mutations in RNA polymerase genes

Epidemics of drug-resistant bacteria emerge worldwide, even as resistant strains frequently have reduced fitness compared to their drug-susceptible counterparts. Data from model systems suggest that the fitness cost of antimicrobial resistance can be reduced by compensatory mutations; however, there is limited evidence that compensatory evolution has any significant role in the success of drug-resistant bacteria in human populations. Here we describe a set of compensatory mutations in the RNA polymerase genes of rifampicin-resistant M. tuberculosis, the etiologic agent of human tuberculosis (TB). M. tuberculosis strains harboring these compensatory mutations showed a high competitive fitness in vitro. Moreover, these mutations were associated with high fitness in vivo, as determined by examining their relative clinical frequency across patient populations. Of note, in countries with the worlds highest incidence of multidrug-resistant (MDR) TB, more than 30% of MDR clinical isolates had this form of mutation. Our findings support a role for compensatory evolution in the global epidemics of MDR TB.

Acquired Resistance to Bedaquiline and Delamanid in Therapy for Tuberculosis

Treatment of multidrug-resistant Mycobacterium tuberculosis is a challenge. This letter describes the emergence of resistance to new therapies, bedaquiline and delamanid.

Putative Compensatory Mutations in the rpoC Gene of Rifampin-Resistant Mycobacterium tuberculosis Are Associated with Ongoing Transmission

ABSTRACT Rifampin resistance in clinical isolates of Mycobacterium tuberculosis arises primarily through the selection of bacterial variants harboring mutations in the 81-bp rifampin resistance-determining region of the rpoB gene. While these mutations were shown to infer a fitness cost in the absence of antibiotic pressure, compensatory mutations in rpoA and rpoC were identified which restore the fitness of rifampin-resistant bacteria carrying mutations in rpoB. To investigate the epidemiological relevance of these compensatory mutations, we analyzed 286 drug-resistant and 54 drug-susceptible clinical M. tuberculosis isolates from the Western Cape, South Africa, a high-incidence setting of multidrug-resistant tuberculosis. Sequencing of a portion of the RpoA-RpoC interaction region of the rpoC gene revealed that 23.5% of all rifampin-resistant isolates tested carried a nonsynonymous mutation in this region. These putative compensatory mutations in rpoC were associated with transmission, as 30.8% of all rifampin-resistant isolates with an IS6110 restriction fragment length polymorphism (RFLP) pattern belonging to a recognized RFLP cluster harbored putative rpoC mutations. Such mutations were present in only 9.4% of rifampin-resistant isolates with unique RFLP patterns (P < 0.01). Moreover, these putative compensatory mutations were associated with specific strain genotypes and the rpoB S531L rifampin resistance mutation. Among isolates harboring this rpoB mutation, 44.1% also harbored rpoC mutations, while only 4.1% of the isolates with other rpoB mutations exhibited mutations in rpoC (P < 0.001). Our study supports a role for rpoC mutations in the transmission of multidrug-resistant tuberculosis and illustrates how epistatic interactions between drug resistance-conferring mutations, compensatory mutations, and different strain genetic backgrounds might influence compensatory evolution in drug-resistant M. tuberculosis.

The heterogeneous evolution of multidrug-resistant Mycobacterium tuberculosis.

Recent surveillance data of multidrug-resistant tuberculosis (MDR-TB) reported the highest rates of resistance ever documented. As further amplification of resistance in MDR strains of Mycobacterium tuberculosis occurs, extensively drug-resistant (XDR) and totally drug-resistant (TDR) TB are beginning to emerge. Although for the most part, the epidemiological factors involved in the spread of MDR-TB are understood, insights into the bacterial drivers of MDR-TB have been gained only recently, largely owing to novel technologies and research in other organisms. Herein, we review recent findings on how bacterial factors, such as persistence, hypermutation, the complex interrelation between drug resistance and fitness, compensatory evolution, and epistasis affect the evolution of multidrug resistance in M. tuberculosis. Improved knowledge of these factors will help better predict the future trajectory of MDR-TB, and contribute to the development of new tools and strategies to combat this growing public health threat.

Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the “Beijing” sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive.

Strain diversity, epistasis and the evolution of drug resistance in Mycobacterium tuberculosis

Mycobacterium tuberculosis harbours little DNA sequence diversity compared with other bacteria. However, there is mounting evidence that strain-to-strain variation in this organism has been underestimated. We review our current understanding of the genetic diversity among M. tuberculosis clinical strains and discuss the relevance of this diversity for the ongoing global epidemics of drug-resistant tuberculosis. Based on findings in other bacteria, we propose that epistatic interactions between pre-existing differences in strain genetic background, acquired drug-resistance-conferring mutations and compensatory changes could play a role in the emergence and spread of drug-resistant M. tuberculosis.

Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages

Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.

Effect of mutation and genetic background on drug resistance in Mycobacterium tuberculosis.

ABSTRACT Bacterial factors may contribute to the global emergence and spread of drug-resistant tuberculosis (TB). Only a few studies have reported on the interactions between different bacterial factors. We studied drug-resistant Mycobacterium tuberculosis isolates from a nationwide study conducted from 2000 to 2008 in Switzerland. We determined quantitative drug resistance levels of first-line drugs by using Bactec MGIT-960 and drug resistance genotypes by sequencing the hot-spot regions of the relevant genes. We determined recent transmission by molecular methods and collected clinical data. Overall, we analyzed 158 isolates that were resistant to isoniazid, rifampin, or ethambutol, 48 (30.4%) of which were multidrug resistant. Among 154 isoniazid-resistant strains, katG mutations were associated with high-level and inhA promoter mutations with low-level drug resistance. Only katG(S315T) (65.6% of all isoniazid-resistant strains) and inhA promoter −15C/T (22.7%) were found in molecular clusters. M. tuberculosis lineage 2 (includes Beijing genotype) was associated with any drug resistance (adjusted odds ratio [OR], 3.0; 95% confidence interval [CI], 1.7 to 5.6; P < 0.0001). Lineage 1 was associated with inhA promoter −15C/T mutations (OR, 6.4; 95% CI, 2.0 to 20.7; P = 0.002). We found that the genetic strain background influences the level of isoniazid resistance conveyed by particular mutations (interaction tests of drug resistance mutations across all lineages; P < 0.0001). In conclusion, M. tuberculosis drug resistance mutations were associated with various levels of drug resistance and transmission, and M. tuberculosis lineages were associated with particular drug resistance-conferring mutations and phenotypic drug resistance. Our study also supports a role for epistatic interactions between different drug resistance mutations and strain genetic backgrounds in M. tuberculosis drug resistance.

“Pseudo-Beijing”: Evidence for Convergent Evolution in the Direct Repeat Region of Mycobacterium tuberculosis

Background Mycobacterium tuberculosis has a global population structure consisting of six main phylogenetic lineages associated with specific geographic regions and human populations. One particular M. tuberculosis genotype known as “Beijing” has repeatedly been associated with drug resistance and has been emerging in some parts of the world. “Beijing” strains are traditionally defined based on a characteristic spoligotyping pattern. We used three alternative genotyping techniques to revisit the phylogenetic classification of M. tuberculosis complex (MTBC) strains exhibiting the typical “Beijing” spoligotyping pattern. Methods and Findings MTBC strains were obtained from an ongoing molecular epidemiological study in Switzerland and Nepal. MTBC genotyping was performed based on SNPs, genomic deletions, and 24-loci MIRU-VNTR. We identified three MTBC strains from patients originating from Tibet, Portugal and Nepal which exhibited a spoligotyping patterns identical to the classical Beijing signature. However, based on three alternative molecular markers, these strains were assigned to Lineage 3 (also known as Delhi/CAS) rather than to Lineage 2 (also known as East-Asian lineage). Sequencing of the RD207 in one of these strains showed that the deletion responsible for this “Pseudo-Beijing” spoligotype was about 1,000 base pairs smaller than the usual deletion of RD207 in classical “Beijing” strains, which is consistent with an evolutionarily independent deletion event in the direct repeat (DR) region of MTBC. Conclusions We provide an example of convergent evolution in the DR locus of MTBC, and highlight the limitation of using spoligotypes for strain classification. Our results indicate that a proportion of “Beijing” strains may have been misclassified in the past. Markers that are more phylogenetically robust should be used when exploring strain-specific differences in experimental or clinical phenotypes.