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Journal of Clinical Microbiology, July 2004, p. 3248-3255, Vol. 42, No. 7
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.7.3248-3255.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

High Genetic Diversity Revealed by Variable-Number Tandem Repeat Genotyping and Analysis of hsp65 Gene Polymorphism in a Large Collection of "Mycobacterium canettii" Strains Indicates that the M. tuberculosis Complex Is a Recently Emerged Clone of "M. canettii"

Michel Fabre,1 Jean-Louis Koeck,2 Philippe Le Flèche,3 Fabrice Simon,4,{dagger} Vincent Hervé,1 Gilles Vergnaud,3 and Christine Pourcel5,6*

Laboratoire de Mycobactériologie, HIA Percy, 92140 Clamart,1 HIA Val de Grâce, 75005 Paris,2 Centre d'Etude du Bouchet, 91710 Vert le Petit,3 Génome, Polymorphisme et Minisatellites (GPMS), Institut de Génétique et Microbiologie, Université Paris XI, 91405 Orsay Cedex,5 Institut Pasteur, 75015 Paris, France,6 CHA Bouffard, Djibouti, Republic of Djibouti4

Received 2 February 2004/ Returned for modification 15 March 2004/ Accepted 6 April 2004

We have analyzed, using complementary molecular methods, the diversity of 43 strains of "Mycobacterium canettii" originating from the Republic of Djibouti, on the Horn of Africa, from 1998 to 2003. Genotyping by multiple-locus variable-number tandem repeat analysis shows that all the strains belong to a single but very distant group when compared to strains of the Mycobacterium tuberculosis complex (MTBC). Thirty-one strains cluster into one large group with little variability and five strains form another group, whereas the other seven are more diverged. In total, 14 genotypes are observed. The DR locus analysis reveals additional variability, some strains being devoid of a direct repeat locus and others having unique spacers. The hsp65 gene polymorphism was investigated by restriction enzyme analysis and sequencing of PCR amplicons. Four new single nucleotide polymorphisms were discovered. One strain was characterized by three nucleotide changes in 441 bp, creating new restriction enzyme polymorphisms. As no sequence variability was found for hsp65 in the whole MTBC, and as a single point mutation separates M. tuberculosis from the closest "M. canettii" strains, this diversity within "M. canettii" subspecies strongly suggests that it is the most probable source species of the MTBC rather than just another branch of the MTBC.

* Corresponding author. Present address: GPMS, Institut de Génétique et Microbiologie, Bat. 400, Université Paris XI, 91405 Orsay Cedex, France. Phone: (33) 1 69 15 30 01. Fax: (33) 1 69 15 66 78. E-mail: Christine.Pourcel{at}igmors.u-psud.fr.

{dagger} Present address: Service des maladies infectieuses et tropicales, HIA Laveran, 13013 Marseille, France.

Journal of Clinical Microbiology, July 2004, p. 3248-3255, Vol. 42, No. 7
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.7.3248-3255.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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