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PLOS ONE  2012 

MLVA Based Classification of Mycobacterium tuberculosis Complex Lineages for a Robust Phylogeographic Snapshot of Its Worldwide Molecular Diversity

DOI: 10.1371/journal.pone.0041991

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Multiple-locus variable-number tandem repeat analysis (MLVA) is useful to establish transmission routes and sources of infections for various microorganisms including Mycobacterium tuberculosis complex (MTC). The recently released SITVITWEB database contains 12-loci Mycobacterial Interspersed Repetitive Units – Variable Number of Tandem DNA Repeats (MIRU-VNTR) profiles and spoligotype patterns for thousands of MTC strains; it uses MIRU International Types (MIT) and Spoligotype International Types (SIT) to designate clustered patterns worldwide. Considering existing doubts on the ability of spoligotyping alone to reveal exact phylogenetic relationships between MTC strains, we developed a MLVA based classification for MTC genotypic lineages. We studied 6 different subsets of MTC isolates encompassing 7793 strains worldwide. Minimum spanning trees (MST) were constructed to identify major lineages, and the most common representative located as a central node was taken as the prototype defining different phylogenetic groups. A total of 7 major lineages with their respective prototypes were identified: Indo-Oceanic/MIT57, East Asian and African Indian/MIT17, Euro American/MIT116, West African-I/MIT934, West African-II/MIT664, M. bovis/MIT49, M.canettii/MIT60. Further MST subdivision identified an additional 34 sublineage MIT prototypes. The phylogenetic relationships among the 37 newly defined MIRU-VNTR lineages were inferred using a classification algorithm based on a bayesian approach. This information was used to construct an updated phylogenetic and phylogeographic snapshot of worldwide MTC diversity studied both at the regional, sub-regional, and country level according to the United Nations specifications. We also looked for IS6110 insertional events that are known to modify the results of the spoligotyping in specific circumstances, and showed that a fair portion of convergence leading to the currently observed bias in phylogenetic classification of strains may be traced back to the presence of IS6110. These results shed new light on the evolutionary history of the pathogen in relation to the history of peopling and human migration.


[1]  Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, et al. (1997) Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol 35: 907–914.
[2]  Mazars E, Lesjean S, Banuls AL, Gilbert M, Vincent V, et al. (2001) High-resolution minisatellite-based typing as a portable approach to global analysis of Mycobacterium tuberculosis molecular epidemiology. Proc Natl Acad Sci U S A 98: 1901–1906.
[3]  Supply P, Lesjean S, Savine E, Kremer K, van Soolingen D, et al. (2001) Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units. J Clin Microbiol 39: 3563–3571.
[4]  Supply P, Allix C, Lesjean S, Cardoso-Oelemann M, Rüsch-Gerdes S, et al. (2006) Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis. J Clin Microbiol 44: 4498–4510.
[5]  Demay C, Liens B, Burguière T, Hill V, Couvin D, et al. (2012) SITVITWEB – a publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology. Infect Genet Evol 12: 755–766.
[6]  Brudey K, Driscoll JR, Rigouts L, Prodinger WM, Gori A, et al. (2006) Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology. BMC Microbiol 6: 23.
[7]  Rastogi N, Sola C (2007) Molecular evolution of the Mycobacterium tuberculosis complex. In: Palomino JC, Leao S, Ritacco V, editors. Tuberculosis 2007: from basic science to patient care. 53–91. Amedeo Online Textbooks:, Accessed 15 March 2012.
[8]  Sreevatsan S, Pan X, Stockbauer KE, Connell ND, Kreiswirth BN, et al. (1997) Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination. Proc Natl Acad Sci U S A 94: 9869–9874.
[9]  Brosch R, Gordon SV, Marmiesse M, Brodin P, Buchrieser C, et al. (2002) A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc Natl Acad Sci U S A 99: 3684–3689.
[10]  Fenner L, Malla B, Ninet B, Dubuis O, Stucki D, et al. (2011) “Pseudo-Beijing”: evidence for convergent evolution in the direct repeat region of Mycobacterium tuberculosis. PLoS One 6: e24737.
[11]  Comas I, Homolka S, Niemann S, Gagneux S (2009) Genotyping of genetically monomorphic bacteria: DNA sequencing in Mycobacterium tuberculosis highlights the limitations of current methodologies. PLoS One 4: e7815.
[12]  Allix-Béguec C, Harmsen D, Weniger T, Supply P, Niemann S (2008) 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. J Clin Microbiol. 46: 2692–2699.
[13]  Kato-Maeda M, Gagneux S, Flores LL, Kim EY, Small PM, et al. (2011) Strain classification of Mycobacterium tuberculosis: congruence between large sequence polymorphisms and spoligotypes. Int J Tuberc Lung Dis 15: 131–133.
[14]  Abadia E, Zhang J, dos Vultos T, Ritacco V, Kremer K, et al. (2010) Resolving lineage assignation on Mycobacterium tuberculosis clinical isolates classified by spoligotyping with a new high-throughput 3R SNPs based method. Infect Genet Evol 10: 1066–1074.
[15]  van der Zanden AG, Kremer K, Schouls LM, Caimi K, Cataldi A, et al. (2002) Improvement of differentiation and interpretability of spoligotyping for Mycobacterium tuberculosis complex isolates by introduction of new spacer oligonucleotides. J Clin Microbiol. 40: 4628–4639.
[16]  Brudey K, Gutierrez MC, Vincent V, Parsons LM, Salfinger M, et al. (2004) Mycobacterium africanum genotyping using novel spacer oligonucleotides in the direct repeat locus. J Clin Microbiol 42: 5053–5057.
[17]  van Embden JD, van Gorkom T, Kremer K, Jansen R, van Der Zeijst BA, et al. (2000) Genetic variation and evolutionary origin of the direct repeat locus of Mycobacterium tuberculosis complex bacteria. J Bacteriol 182: 2393–2401.
[18]  Filliol I, Sola C, Rastogi N (2000) Detection of a previously unamplified spacer within the DR locus of Mycobacterium tuberculosis: epidemiological implications. J Clin Microbiol. 38: 1231–1234.
[19]  Legrand E, Filliol I, Sola C, Rastogi N (2001) Use of spoligotyping to study the evolution of the direct repeat locus by IS6110 transposition in Mycobacterium tuberculosis. J Clin Microbiol 39: 1595–1599.
[20]  Roberts DE, Ascherman D, Kleckner N (1991) IS10 promotes adjacent deletions at low frequency. Genetics 128: 37–43.
[21]  Fang Z, Forbes KJ (1997) A Mycobacterium tuberculosis IS6110 preferential locus (ipl) for insertion into the genome. J Clin Microbiol 35: 479–481.
[22]  Gagneux S, DeRiemer K, Van T, Kato-Maeda M, de Jong BC, et al. (2006) Variable host-pathogen compatibility in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 103: 2869–2873.
[23]  Gagneux S, Small PM (2007) Global phylogeography of Mycobacterium tuberculosis and implications for tuberculosis product development. Lancet Infect Dis 7: 328–337.
[24]  Lazzarini LC, Huard RC, Boechat NL, Gomes HM, Oelemann MC, et al. (2007) Discovery of a novel Mycobacterium tuberculosis lineage that is a major cause of tuberculosis in Rio de Janeiro, Brazil. J Clin Microbiol 45: 3891–3902.
[25]  Cruciani F, Trombetta B, Sellitto D, Massaia A, Destro-Bisol G, et al. (2010) Human Y chromosome haplogroup R-V88: a paternal genetic record of early mid Holocene trans-Saharan connections and the spread of Chadic languages. Eur J Hum Genet 18: 800–807.
[26]  Asgharzadeh M, Kafil HS, Roudsary AA, Hanifi GR, et al. (2011) Tuberculosis transmission in Northwest of Iran: using MIRU-VNTR, ETR-VNTR and IS6110-RFLP methods. Infect Genet Evol 11: 124–131.
[27]  Viegas SO, Machado A, Groenheit R, Ghebremichael S, Pennhag A, et al. (2010) Molecular diversity of Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in Mozambique. BMC Microbiol 10: 195.
[28]  Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574.
[29]  Alexander DC, Jones JR, Liu J (2003) A rifampin-hypersensitive mutant reveals differences between strains of Mycobacterium smegmatis and presence of a novel transposon, IS1623. Antimicrob Agents Chemother 47: 3208–3213.
[30]  Caimi K, Romano MI, Alito A, Zumarraga M, Bigi F, et al. (2001) Sequence analysis of the direct repeat region in Mycobacterium bovis. J Clin Microbiol 39: 1067–1072.
[31]  Schürch AC, Kremer K, Kiers A, Boeree MJ, Siezen RJ, et al. (2011) Preferential deletion events in the direct repeat locus of Mycobacterium tuberculosis. J Clin Microbiol 49: 1318–1322.
[32]  Lari N, Rindi L, Garzelli C (2001) Identification of one insertion site of IS6110 in Mycobacterium tuberculosis H37Ra and analysis of the RvD2 deletion in M. tuberculosis clinical isolates. J Med Microbiol. 50: 805–811.
[33]  Sampson SL, Warren RM, Richardson M, Victor TC, Jordaan AM, et al. (2003) IS6110-mediated deletion polymorphism in the direct repeat region of clinical isolates of Mycobacterium tuberculosis. J Bacteriol 185: 2856–2866.
[34]  Brodin P, Eiglmeier K, Marmiesse M, Billault A, Garnier T, et al. (2002) Bacterial artificial chromosome-based comparative genomic analysis identifies Mycobacterium microti as a natural ESAT-6 deletion mutant. Infect Immun 70: 5568–5578.
[35]  Rao KR, Kauser F, Srinivas S, Zanetti S, Sechi LA, et al. (2005) Analysis of genomic downsizing on the basis of region-of-difference polymorphism profiling of Mycobacterium tuberculosis patient isolates reveals geographic partitioning. J Clin Microbiol 43: 5978–5982.
[36]  Tsolaki AG, Gagneux S, Pym AS, Goguet de la Salmoniere YO, Kreiswirth BN, et al. (2005) Genomic deletions classify the Beijing/W strains as a distinct genetic lineage of Mycobacterium tuberculosis. J Clin Microbiol 43: 3185–3191.
[37]  Fang Z, Morrison N, Watt B, Doig C, Forbes KJ (1998) IS6110 transposition and evolutionary scenario of the direct repeat locus in a group of closely related Mycobacterium tuberculosis strains. J Bacteriol 180: 2102–2109.
[38]  Jansen R, Embden JD, Gaastra W, Schouls LM (2002) Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol 43: 1565–1575.
[39]  Renfrew C (2010) Archaeogenetics – towards a ‘new synthesis’? Curr Biol 20: R162–165.
[40]  Underhill PA, Kivisild T (2007) Use of y chromosome and mitochondrial DNA population structure in tracing human migrations. Annu Rev Genet 41: 539–564.
[41]  Kayser M (2010) The human genetic history of Oceania: near and remote views of dispersal. Curr Biol 20: R194–201.
[42]  Majumder PP (2010) The human genetic history of South Asia. Curr Biol 20: R184–187.
[43]  Mokrousov I, Ly HM, Otten T, Lan NN, Vyshnevskyi B, et al. (2005) Origin and primary dispersal of the Mycobacterium tuberculosis Beijing genotype: clues from human phylogeography. Genome Res 15: 1357–1364.
[44]  Myres NM, Rootsi S, Lin AA, J?rve M, King RJ, et al. (2011) A major Y-chromosome haplogroup R1b Holocene era founder effect in Central and Western Europe. Eur J Hum Genet 19: 95–101.
[45]  Sola C, Ferdinand S, Sechi LA, Zanetti S, Martial D, et al. (2005) Mycobacterium tuberculosis molecular evolution in western Mediterranean Islands of Sicily and Sardinia. Infect Genet Evol 5: 145–156.
[46]  Gibson AL, Huard RC, Gey van Pittius NC, Lazzarini LC, Driscoll J, et al. (2008) Application of sensitive and specific molecular methods to uncover global dissemination of the major RDRio Sublineage of the Latin American-Mediterranean Mycobacterium tuberculosis spoligotype family. J Clin Microbiol 46: 1259–1267.
[47]  Mokrousov I, Valcheva V, Sovhozova N, Aldashev A, Rastogi N, et al. (2009) Penitentiary population of Mycobacterium tuberculosis in Kyrgyzstan: exceptionally high prevalence of the Beijing genotype and its Russia-specific subtype. Infect Genet Evol 9: 1400–1405.
[48]  Filliol I, Motiwala AS, Cavatore M, Qi W, Hazbón MH, Bobadilla del Valle M, et al. (2006) Global phylogeny of Mycobacterium tuberculosis based on single nucleotide polymorphism (SNP) analysis: insights into tuberculosis evolution, phylogenetic accuracy of other DNA fingerprinting systems, and recommendations for a minimal standard SNP set. J Bacteriol 188: 759–772.


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