Philip Supply

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

ABSTRACT 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.

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

ABSTRACT Large-scale genotyping of Mycobacterium tuberculosisis 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 ofM. 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 ofM. 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.

Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome.

Mycobacterial interspersed repetitive units (MIRUs) are 40–100 bp DNA elements often found as tandem repeats and dispersed in intergenic regions of the Mycobacterium tuberculosis complex genomes. The M. tuberculosis H37Rv chromosome contains 41 MIRU loci. After polymerase chain reaction (PCR) and sequence analyses of these loci in 31 M. tuberculosis complex strains, 12 of them were found to display variations in tandem repeat copy numbers and, in most cases, sequence variations between repeat units as well. These features are reminiscent of those of certain human variable minisatellites. Of the 12 variable loci, only one was found to vary among genealogically distant BCG substrains, suggesting that these interspersed bacterial minisatellite‐like structures evolve slowly in mycobacterial populations.

Ancient Origin and Gene Mosaicism of the Progenitor of Mycobacterium tuberculosis

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.

Origin, Spread and Demography of the Mycobacterium tuberculosis Complex

The evolutionary timing and spread of the Mycobacterium tuberculosis complex (MTBC), one of the most successful groups of bacterial pathogens, remains largely unknown. Here, using mycobacterial tandem repeat sequences as genetic markers, we show that the MTBC consists of two independent clades, one composed exclusively of M. tuberculosis lineages from humans and the other composed of both animal and human isolates. The latter also likely derived from a human pathogenic lineage, supporting the hypothesis of an original human host. Using Bayesian statistics and experimental data on the variability of the mycobacterial markers in infected patients, we estimated the age of the MTBC at 40,000 years, coinciding with the expansion of “modern” human populations out of Africa. Furthermore, coalescence analysis revealed a strong and recent demographic expansion in almost all M. tuberculosis lineages, which coincides with the human population explosion over the last two centuries. These findings thus unveil the dynamic dimension of the association between human host and pathogen populations.

Evaluation and Strategy for Use of MIRU-VNTRplus, a Multifunctional Database for Online Analysis of Genotyping Data and Phylogenetic Identification of Mycobacterium tuberculosis Complex Isolates

ABSTRACT Because of its portable data, discriminatory power, and recently proposed standardization, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing has become a major method for the epidemiological tracking of Mycobacterium tuberculosis complex (MTBC) clones. However, no public MIRU-VNTR database based on well-characterized reference strains has been available hitherto for easy strain identification. Therefore, a collection of 186 reference strains representing the primary MTBC lineages was used to build a database, which is freely accessible at http://www.MIRU-VNTRplus.org . The geographical origin and the drug susceptibility profile of each strain were stored together with comprehensive genetic lineage information, including the 24-locus MIRU-VNTR profile, the spoligotyping pattern, the single-nucleotide- and large-sequence-polymorphism profiles, and the IS6110 restriction fragment length polymorphism fingerprint. Thanks to flexible import functions, a single or multiple user strains can be analyzed, e.g., for lineage identification with or without the use of reference strains, by best-match or tree-based analyses with single or combined marker data sets. The results can easily be exported. In the present study, we evaluated the database consistency and various analysis parameters both by testing the reference collection against itself and by using an external population-based data set comprising 629 different strains. Under the optimal conditions found, lineage predictions based on typing by 24-locus MIRU-VNTR analysis optionally combined with spoligotyping were verified in >99% of the cases. On the basis of this evaluation, a user strategy was defined, which consisted of best-match analysis followed, if necessary, by tree-based analysis. The MIRU-VNTRplus database is a powerful tool for high-resolution clonal identification and has little equivalent in terms of functionalities among the bacterial genotyping databases available so far.

Whole Genome Sequencing versus Traditional Genotyping for Investigation of a Mycobacterium tuberculosis Outbreak: A Longitudinal Molecular Epidemiological Study

In an outbreak investigation of Mycobacterium tuberculosis comparing whole genome sequencing (WGS) with traditional genotyping, Stefan Niemann and colleagues found that classical genotyping falsely clustered some strains, and WGS better reflected contact tracing.

MIRU-VNTRplus: a web tool for polyphasic genotyping of Mycobacterium tuberculosis complex bacteria

Harmonized typing of bacteria and easy identification of locally or internationally circulating clones are essential for epidemiological surveillance and disease control. For Mycobacterium tuberculosis complex (MTBC) species, multi-locus variable number tandem repeat analysis (MLVA) targeting mycobacterial interspersed repetitive units (MIRU) has been internationally adopted as the new standard, portable, reproducible and discriminatory typing method. However, no specialized bioinformatics web tools are available for analysing MLVA data in combination with other, complementary typing data. Therefore, we have developed the web application MIRU-VNTRplus (http://www.miru-vntrplus.org). This freely accessible service allows users to analyse genotyping data of their strains alone or in comparison with a reference database of strains representing the major MTBC lineages. Analysis and comparisons of genotypes can be based on MLVA-, spoligotype-, large sequence polymorphism and single nucleotide polymorphism data, or on a weighted combination of these markers. Tools for data exploration include search for similar strains, creation of phylogenetic and minimum spanning trees and mapping of geographic information. To facilitate scientific communication, an expanding genotype nomenclature (MLVA MtbC15-9 type) that can be queried via a web- or a SOAP-interface has been implemented. An extensive documentation guides users through all application functions.

Identification of novel intergenic repetitive units in a mycobacterial two‐component system operon

Mycobacterial interspersed repetitive units (MIRUs), a novel class of repeated sequences, were identified within the intercistronic region of an operon coding for a mycobacterial two‐component system, named senX3‐regX3. Southern blot analysis and homology searches revealed the presence of several homologous sequences in intergenic regions dispersed throughout the genomes of Mycobacterium bovis BCG, Mycobacterium tuberculosis and Mycobacterium leprae. These could be grouped into three major families, containing elements of 77–101 bp, 46–53 bp and 58–101 bp. Based on the available mycobacterial sequences, the total number of MIRUs is estimated to be about 40–50 per genome. Similar to previously identified small repetitive sequences, the MIRUs of the two‐component operon are transcribed on a polycistronic mRNA. Unlike previously identified small repetitive sequences, however, MIRUs do not contain dyad symmetries, comprise small open reading frames (ORFs) whose extremities overlap those of the contiguous ORFs and are oriented in the same translational direction as those of the adjacent genes. Analyses of the sequences at the insertion sites suggest that MIRUs disseminate by transposition into DTGA sites involved in translational coupling in polycistronic operons.

Evolutionary history and global spread of the Mycobacterium tuberculosis Beijing lineage

Mycobacterium tuberculosis strains of the Beijing lineage are globally distributed and are associated with the massive spread of multidrug-resistant (MDR) tuberculosis in Eurasia. Here we reconstructed the biogeographical structure and evolutionary history of this lineage by genetic analysis of 4,987 isolates from 99 countries and whole-genome sequencing of 110 representative isolates. We show that this lineage initially originated in the Far East, from where it radiated worldwide in several waves. We detected successive increases in population size for this pathogen over the last 200 years, practically coinciding with the Industrial Revolution, the First World War and HIV epidemics. Two MDR clones of this lineage started to spread throughout central Asia and Russia concomitantly with the collapse of the public health system in the former Soviet Union. Mutations identified in genes putatively under positive selection and associated with virulence might have favored the expansion of the most successful branches of the lineage.