Detection of a previously unamplified spacer within the DR locus of Mycobacterium tuberculosis: epidemiological implications

Abstract

Spoligotyping, a method based on the variability of distribution of the 43 inter-direct repeat (DR) spacers of Mycobacterium tuberculosis and Mycobacterium bovis BCG, is useful to study the molecular epidemiology of bovine and human tuberculosis. Recently, a major family of M. tuberculosis clinical isolates named the Haarlem family, which did not contain spacers 31 and 33 to 36, was reported in a multicenter study. Independently, a data bank containing all the published spoligotypes showed that the two most prevalent spoligotypes in the world differed only by the presence or absence of spacer 31. A careful analysis of the DR locus sequence led us to hypothesize that spacer 31 may not have been amplified in some isolates with the primer sets DRa and DRb currently used for spoligotyping. Consequently, a modified spoligotyping method based on different combinations of the 36-bp DR and IS6110 primers was devised that was able to discriminate between the left and the right parts of the DR locus and demonstrated the presence of the previously unamplified spacer 31 for some of the clinical isolates. By analogy, we suggest that a single-spacer difference in some epidemiologically linked cases of tuberculosis may simply arise due to the insertion of an extra copy of IS6110 within the DR locus, leading to its asymmetrical disruption and subsequent lack of the DRa or DRb targets. The influence of the IS6110 preferential insertion sites within the DR locus on spoligotyping results should be further investigated.

FIG. 1

Schematic representation of the DR locus of M. tuberculosis with an enlargement of the two IS6110 insertion sites. The top of the figure shows the general organization of the DR locus, whereas the bottom of the figure is an enlargement showing the flanking regions of IS6110 insertions. In the bottom part, the left-hand copy of IS6110 is inserted exactly in the middle of the DRr sequence between spacers 24 and 25, splitting the DRr into two equal 18-bp fragments which serve as PCR targets for primers DRa and DRb and give a positive hybridization signal for spacers 24 and 25. On the other hand, an asymmetrical disruption of the DRr sequence due to the insertion of a second copy of IS6110 in the right-hand side of the DR locus between spacers 31 and 25 results in two unequal parts in such a way that only an 8-bp fragment of the DRa target is conserved toward spacer 31, not enough for successful amplification and subsequent detection of this spacer.

FIG. 2

Correlation between spoligotypes and PvuII-DR RFLP results showing a link between the absence of spacer 31, the presence of a third DRr band, and the consequent presence of a second copy of IS6110 inserted within the DR locus.

FIG. 3

Schematic representation of results obtained by L-R spoligotyping of M. tuberculosis H37Rv and by the routine spoligotyping method of representative clinical isolates that lacked spacer 31. The primer sets A, B, C, and D have been detailed in the text and in Table 1.