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Journal of Clinical Microbiology, November 2004, p. 5058-5064, Vol. 42, No. 11
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.11.5058-5064.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Characterization of Ancestral Mycobacterium tuberculosis by Multiple Genetic Markers and Proposal of Genotyping Strategy

Yong-Jiang Sun,1* Ann S. G. Lee,2 Sze Ta Ng,1 Sindhu Ravindran,1 Kristin Kremer,3 Richard Bellamy,1 Sin-Yew Wong,1 Dick van Soolingen,3 Philip Supply,4 and Nicholas I. Paton1

Department of Infectious Diseases, Tan Tock Seng Hospital,1 Division of Medical Sciences, National Cancer Centre, Singapore,2 Diagnostic Laboratory for Infectious Diseases and Perinatal Screening, National Institute for Public Health and the Environment, Bilthoven, The Netherlands,3 Laboratoire des Mécanismes Moléculaires de la Pathogenèse Bactérienne, Institut Pasteur de Lille, Lille, France4

Received 24 March 2004/ Returned for modification 28 June 2004/ Accepted 5 July 2004

Sixty-eight ancestral Mycobacterium tuberculosis isolates were previously identified by using the tuberculosis-specific deletion 1 (TbD1) PCR and mycobacterial interspersed-repetitive-unit-variable-number tandem repeat (MIRU-VNTR) typing (Y. J. Sun, R. Bellamy, A. S. G. Lee, S. T. Ng, S. Ravindran, S.-Y. Wong, C. Locht, P. Supply, and N. I. Paton, J. Clin. Microbiol. 42:1986-1993, 2004). These TbD1+ ancestral isolates were further characterized and typed in this study by IS6110 restriction fragment length polymorphism (RFLP) typing, VNTR typing using exact tandem repeats (VNTR-ETR), and spoligotyping of the direct-repeat region. To our knowledge, this is the first characterization of this genogroup by multiple genetic markers based on a fairly large sample size. In this genogroup, all spoligotypes were characterized by the absence of spacers 29 to 32 and 34. In addition, VNTR-ETR typing could add further resolution to the clustered isolates identified by MIRU-VNTR, and the combination of MIRU-VNTR and VNTR-ETR, called MIRU-ETR, showed the highest discriminatory power for these strains compared to IS6110 RFLP typing and spoligotyping alone. However, MIRU-ETR appeared to still cluster some probably epidemiologically unrelated strains, as judged by IS6110 RFLP divergence. Therefore, a typing strategy based on stepwise combination of MIRU-ETR and IS6110 RFLP is proposed to achieve maximal discrimination for unrelated TbD1+ strains. This typing strategy may be useful in areas where TbD1+ ancestral strains are prevalent.

* Corresponding author. Mailing address: Infectious Disease Research Laboratory, Level 2, TTSH Medical Centre, Tan Tock Seng Hospital, 11 Jalan Tan Tock Seng, Singapore 308433. Phone: 65-6357-8959. Fax: 65-6256-7023. E-mail: Yong_Jiang_Sun{at}ttsh.com.sg.

Journal of Clinical Microbiology, November 2004, p. 5058-5064, Vol. 42, No. 11
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.11.5058-5064.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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