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.The genotyping of Mycobacterium tuberculosis isolates contributes to tuberculosis (TB) control by, e.g., indicating possible epidemiological links between TB patients, detecting (un)suspected outbreaks and laboratory cross-contamination, and distinguishing exogenous reinfection from endogenous reactivation in relapse cases. For these purposes, IS6110 restriction fragment length polymorphism (RFLP) typing (48) has been used as the gold standard method for more than a decade. However, this method is labor-intensive, requires weeks for culturing the isolates and subsequent DNA purification, and suffers from problems of interpretability and portability of the complex banding patterns. In addition, it provides insufficient discrimination among isolates with low (Ͻ6) IS6110 copy numbers, a problem that is only partly overcome by using PCR-based spoligotyping as a secondary method (6).Genotyping based on variable numbers of tandem repeats (VNTRs) of different classes of interspersed genetic elements named mycobacterial interspersed repetitive units (MIRUs) (12,25,32,36,40,43,44) is increasingly used to solve these problems. This method relies on PCR amplification of multiple loci using primers specific for the flanking regions of each repeat locus and on the determination of the sizes of the amplicons...
Mycobacterium tuberculosis remains a major cause of morbidity and mortality worldwide. Studies have reported human pathogens to have geographically structured population genetics, some of which have been linked to ancient human migrations. However, no study has addressed the potential evolutionary consequences of such longstanding human-pathogen associations. Here, we demonstrate that the global population structure of M. tuberculosis is defined by six phylogeographical lineages, each associated with specific, sympatric human populations. In an urban cosmopolitan environment, mycobacterial lineages were much more likely to spread in sympatric than in allopatric patient populations. Tuberculosis cases that did occur in allopatric hosts disproportionately involved high-risk individuals with impaired host resistance. These observations suggest that mycobacterial lineages are adapted to particular human populations. If confirmed, our findings have important implications for tuberculosis control and vaccine development.coevolution ͉ deletions ͉ lineage ͉ polymorphism ͉ population
Mycobacterium avium subsp. paratuberculosis, the etiological agent of paratuberculosis, affects a wide range of domestic ruminants and has been suggested to be involved in Crohn's disease in humans. Most available methods for identifying and differentiating strains of this difficult species are technically demanding and have limited discriminatory power. Here, we report the identification of novel PCR-based typing markers consisting of variable-number tandem repeats (VNTRs) of genetic elements called mycobacterial interspersed repetitive units (MIRUs). Eight markers were applied to 183 M. avium subsp. paratuberculosis isolates from bovine, caprine, ovine, cervine, leporine, and human origins from 10 different countries and to 82 human isolates of the closely related species M. avium from France. Among the M. avium subsp. paratuberculosis isolates, 21 patterns were found by MIRU-VNTR typing, with a discriminatory index of 0.751. The predominant R01 IS900 restriction fragment length polymorphism type, comprising 131 isolates, was divided into 15 MIRU-VNTR types. Among the 82 M. avium isolates, the eight MIRU-VNTR loci distinguished 30 types, none of which was shared by M. avium subsp. paratuberculosis isolates, resulting in a discriminatory index of 0.889. Our results suggest that MIRU-VNTR typing is a fast typing method that, in combination with other methods, might prove to be optimal for PCR-based molecular epidemiological studies of M. avium/M. avium subsp. paratuberculosis pathogens. In addition, presumably identical M. avium subsp. paratuberculosis 316F vaccine strains originating from the Weybridge laboratory and from different commercial batches from Mérial actually differed by one or both typing methods. These results indicate a substantial degree of genetic drift among different vaccine preparations, which has important implications for prophylactic approaches.
We give an update on the worldwide spoligotype database, which now contains 3,319 spoligotype patterns of Mycobacterium tuberculosis in 47 countries, with 259 shared types, i.e., identical spoligotypes shared by two or more patient isolates. The 259 shared types contained a total of 2,779 (84%) of all the isolates. Seven major genetic groups represented 37% of all clustered isolates. Two types (119 and 137) were found almost exclusively in the USA and accounted for 9% of clustered isolates. The remaining 1,517 isolates were scattered into 252 different spoligotypes. This database constitutes a tool for pattern comparison of M. tuberculosis clinical isolates for global epidemiologic studies and phylogenetic purposes.
The contribution of horizontal gene transfer (HGT) to the evolution of Mycobacterium tuberculosis -- the main causal agent of tuberculosis in humans -- and closely related members of the M. tuberculosis complex remains poorly understood. Using a combination of genome-wide parametric analyses, we have identified 48 M. tuberculosis chromosomal regions with atypical characteristics, potentially due to HGT. These specific regions account for 4.5% of the genome (199 kb) and include 256 genes. Many display features typical of the genomic islands found in other bacteria, including residual material from mobile genetic elements, flanking direct repeats, insertion in the vicinity of tRNA sequences, and genes with putative or documented virulence functions. Southern blotting analysis of nine of these 48 regions confirmed their presence in "Mycobacterium prototuberculosis," the ancestral species of the M. tuberculosis complex. Finally, our results strongly suggest that the ancestor of the tubercle bacilli was an environmental bacillus that exchanged genetic material with other bacterial species, including Proteobacteria in particular, present in its surroundings. This study describes a rational approach to searching for mycobacterial virulence genes, and highlights the importance of dissecting gene transfer networks to improve our understanding of mycobacterial pathogenicity and evolution.
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