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"

Abstract

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.

FIG. 1.

MLVA cluster analysis. Clustering analysis was done using the categorical and unweighted pair group method with arithmetic averages options. From left to right, the columns designate the strains names, the genotype number (geno), the result obtained for the PCR amplification of part of the DR locus using “canettii-specific” spacers 73 to 76 or 80 to 83 (24), and the hsp65 profile. The asterisks indicate that sequencing data have been produced. The hsp65 fragment has been sequenced in at least one strain from each genotype (SNPs 2 and 3 [Fig. 5] are not detected by PCR-restriction fragment length polymorphism analysis). Strains H37Rv and CIPT060 are underlined.

FIG. 2.

Minimum spanning tree analysis and comparison with the other approaches. A minimum spanning tree was constructed using the genotyping data provided in Table 1. The parameters used for the construction of the tree authorized the introduction of hypothetical (missing) links which appear as open circles (data predicted by the software as intermediates, not present in the available collection). The genotype numbers listed in Fig. 1 are indicated in the circles. The three group A genotypes comprising 31 strains are shown in blue. The two closely related strains, including the “M. canettii” strain CIPT060 (genotype 12), and in which four additional spacers in the DR locus have been found are shown in yellow. Genotypes with the hsp65 SNP 3 variant are presented in green. The exceptional strain percy65 (genotype 14; hsp65 SNPs 1, 2, and 5) is colored in red. The two groups of strains devoid of DR regions are circled. Strain percy79 (genotype 4, absence of the “M. canettii”-specific set of spacers, presence of new spacers) is indicated. Dotted lines are used to link genotypes separated by seven or more differences in the MLVA genotype.

FIG. 3.

Structure of the DR locus. PCR using primers Mcan80For and Mcan83Rev (A) or Mcan73For and Mcan76Rev (B) on 24 smooth strains and H37Rv. The 100-bp ruler is used as size marker (lanes 1, 11, 19, and 28). No amplicon is observed in H37Rv (lane 2) and in seven smooth strains (lanes 9, 12, 21, 23, 24, 25, and 27). (B) Strains percy32 and CIPT060 have four additional new spacers as shown by the presence of a 505-bp amplicon and by sequencing.

FIG. 4.

Cloning of new spacers from strain percy79. (A) PCR amplification was performed using primers DRa and DRb on 16 “M. canettii” strains. Amplification product from strain percy79 was purified and cloned into the pGEM-T Easy vector (B), and inserts were analyzed by PCR amplification (C). Lanes 1, 10, and 19 in panels A and C contain 100-bp ruler DNA size markers.

FIG. 5.

PCR-restriction fragment length polymorphism analysis of the hsp65 locus. (A) hsp65 PRA patterns of representative strains from the MTBC after digestion with HhaI (left) or DdeI (right). The representative “M. canettii” strain CIPT060 shows a different pattern with HhaI as expected (lane 7) but not with DdeI (lane 14). (B) Fifteen smooth strains and H37Rv upon digestion of the 441-bp amplicon with HhaI. (C) Same as panel B, using DdeI instead of HhaI. A 100-bp ruler is used as size marker. Strain percy65 has a unique digestion pattern as exemplified. (D) Multiple alignments using the CLUSTALW algorithm of the 180-bp portion of the hsp65 gene in H37Rv and smooth strains showing five single nucleotide changes, numbered 1 to 5. The SNP described by Goh et al. (8) is SNP 4.