The distribution of 20 variable regions resulting from insertion-deletion events in the genomes of the tubercle bacilli has been evaluated in a total of 100 strains of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium canettii, Mycobacterium microti, and Mycobacterium bovis. This approach showed that the majority of these polymorphisms did not occur independently in the different strains of the M. tuberculosis complex but, rather, resulted from ancient, irreversible genetic events in common progenitor strains. Based on the presence or absence of an M. tuberculosis specific deletion (TbD1), M. tuberculosis strains can be divided into ancestral and “modern” strains, the latter comprising representatives of major epidemics like the Beijing, Haarlem, and African M. tuberculosis clusters. Furthermore, successive loss of DNA, reflected by region of difference 9 and other subsequent deletions, was identified for an evolutionary lineage represented by M. africanum, M. microti, and M. bovis that diverged from the progenitor of the present M. tuberculosis strains before TbD1 occurred. These findings contradict the often-presented hypothesis that M. tuberculosis, the etiological agent of human tuberculosis evolved from M. bovis, the agent of bovine disease. M. canettii and ancestral M. tuberculosis strains lack none of these deleted regions, and, therefore, seem to be direct descendants of tubercle bacilli that existed before the M. africanum→M. bovis lineage separated from the M. tuberculosis lineage. This observation suggests that the common ancestor of the tubercle bacilli resembled M. tuberculosis or M. canettii and could well have been a human pathogen already.

https://www.pnas.org/content/pnas/99/6/3684.full.pdf

A new evolutionary scenario for the Mycobacterium tuberculosis complex

Molecular typing based on 12 loci containing variable numbers of tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTRs) has been adopted in combination with spoligotyping as the basis for large-scale, high-throughput genotyping of Mycobacterium tuberculosis. However, even the combination of these two methods is still less discriminatory than IS6110 fingerprinting. Here, we define an optimized set of MIRU-VNTR loci with a significantly higher discriminatory power. The resolution and the stability/robustness of 29 loci were analyzed, using a total of 824 tubercle bacillus isolates, including representatives of the main lineages identified worldwide so far. Five loci were excluded for lack of robustness and/or stability in serial isolates or isolates from epidemiologically linked patients. The use of the 24 remaining loci increased the number of types by 40%—and by 23% in combination with spoligotyping—among isolates from cosmopolitan origins, compared to those obtained with the original set of 12 loci. Consequently, the clustering rate was decreased by fourfold—by threefold in combination with spoligotyping—under the same conditions. A discriminatory subset of 15 loci with the highest evolutionary rates was then defined that concentrated 96% of the total resolution obtained with the full 24-locus set. Its predictive value for evaluating M. tuberculosis transmission was found to be equal to that of IS6110 restriction fragment length polymorphism typing, as shown in a companion population-based study. This 15-locus system is therefore proposed as the new standard for routine epidemiological discrimination of M. tuberculosis isolates and the 24-locus system as a high-resolution tool for phylogenetic studies.

Proposal for Standardization of Optimized Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing of Mycobacterium tuberculosis

Background
The Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database.

/pdf/mycobacterium-tuberculosis-complex-genetic-diversity-mining-25ve612xfv.pdf

Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology

The highly successful human pathogen Mycobacterium tuberculosis has an extremely low level of genetic variation, which suggests that the entire population resulted from clonal expansion following an evolutionary bottleneck around 35,000 y ago. Here, we show that this population constitutes just the visible tip of a much broader progenitor species, whose extant representatives are human isolates of tubercle bacilli from East Africa. In these isolates, we detected incongruence among gene phylogenies as well as mosaic gene sequences, whose individual elements are retrieved in classical M. tuberculosis. Therefore, despite its apparent homogeneity, the M. tuberculosis genome appears to be a composite assembly resulting from horizontal gene transfer events predating clonal expansion. The amount of synonymous nucleotide variation in housekeeping genes suggests that tubercle bacilli were contemporaneous with early hominids in East Africa, and have thus been coevolving with their human host much longer than previously thought. These results open novel perspectives for unraveling the molecular bases of M. tuberculosis evolutionary success.

Ancient Origin and Gene Mosaicism of the Progenitor of Mycobacterium

https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.0010005&type=printable

Ancient origin and gene mosaicism of the progenitor of Mycobacterium tuberculosis.

Large-scale genotyping of Mycobacterium tuberculosis is especially challenging, as the current typing methods are labor-intensive and the results are difficult to compare among laboratories. Here, automated typing based on variable-number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) in 12 mammalian minisatellite-like loci of M. tuberculosis is presented. This system combines analysis of multiplex PCRs on a fluorescence-based DNA analyzer with computerized automation of the genotyping. Analysis of a blinded reference set of 90 strains from 38 countries (K. Kremer et al., J. Clin. Microbiol. 37:2607-2618, 1999) demonstrated that it is 100% reproducible, sensitive, and specific for M. tuberculosis complex isolates, a performance that has not been achieved by any other typing method tested in the same conditions. MIRU-VNTRs can be used for analysis of the global genetic diversity of M. tuberculosis complex strains at different levels of evolutionary divergence. To fully exploit the portability of this typing system, a website was set up for the analysis of M. tuberculosis MIRU-VNTR genotypes via the Internet. This opens the way for global epidemiological surveillance of tuberculosis and should lead to novel insights into the evolutionary and population genetics of this major pathogen.

https://jcm.asm.org/content/jcm/39/10/3563.full.pdf

Automated High-Throughput Genotyping for Study of Global Epidemiology of Mycobacterium tuberculosis Based on Mycobacterial Interspersed Repetitive Units

Variable number tandem repeats (VNTRs) of elements named mycobacterial interspersed repetitive units (MIRUs) have previously been identified in 12 minisatellite loci of the Mycobacterium tuberculosis genome. These markers allow reliable high-throughput genotyping of M. tuberculosis and represent a portable approach to global molecular epidemiology of M. tuberculosis. To assess their temporal stability, we genotyped 123 serial isolates, separated by up to 6 years and belonging to a variety of distinct IS6110 restriction fragment length polymorphism (RFLP) families, from 56 patients who had positive sputum cultures. All 12 MIRU VNTR loci were completely identical within the groups of serial isolates in 55 out of 56 groups (98.2%), although 11 pairs of isolates from the same patients with conserved MIRU VNTRs displayed slightly different IS6110 RFLP profiles. In a single case, serial isolates with an unchanged IS6110 RFLP profile showed a change in 1 out of 12 MIRU VNTR loci. These results indicate that MIRU VNTRs are stable over time and therefore are suitable for reliable follow-up of patients chronically infected with tuberculosis over long periods. Moreover, they support MIRU VNTR genotyping as a powerful first-line method followed by subtyping by IS6110 RFLP to define ongoing transmission clusters.

Stability of Variable-Number Tandem Repeats of Mycobacterial Interspersed Repetitive Units from 12 Loci in Serial Isolates of Mycobacterium tuberculosis

Evgueni Savine

Papers

Proposal for Standardization of Optimized Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing of Mycobacterium tuberculosis

Automated High-Throughput Genotyping for Study of Global Epidemiology of Mycobacterium tuberculosis Based on Mycobacterial Interspersed Repetitive Units

Stability of Variable-Number Tandem Repeats of Mycobacterial Interspersed Repetitive Units from 12 Loci in Serial Isolates of Mycobacterium tuberculosis