Identification of a contaminating Mycobacterium tuberculosis strain with a transposition of an IS6110 insertion element resulting in an altered spoligotype

Abstract

Molecular fingerprinting with the IS6110 insertion sequence is useful for tracking transmission of Mycobacterium tuberculosis within a population or confirming specimen contamination in the laboratory or through instrumentation. Secondary typing with other molecular methods yields additional information as to the relatedness of strains with similar IS6110 fingerprints. Isolated, relatively rare, random events within the M. tuberculosis genome alter molecular fingerprinting patterns with any of the methods; therefore, strains which are different by two or more typing methods are usually not considered to be closely related. In this report, we describe two strains of M. tuberculosis, obtained from the same bronchoscope 2 days apart, that demonstrated unique molecular fingerprinting patterns by two different typing methods. They were closely linked through the bronchoscope by a traditional epidemiologic investigation. Genetic analysis of the two strains revealed that a single event, the transposition of an IS6110 insertion sequence in one of the strains, accounted for both the differences in the IS6110 pattern and the apparent deletion of a spacer in the spoligotype. This finding shows that a single event can change the molecular fingerprint of a strain in two different molecular typing systems, and thus, molecular typing cannot be the only means used to track transmission of this organism through a population. Traditional epidemiologic techniques are a necessary complement to molecular fingerprinting so that radical changes within the fingerprint pattern can be identified.

FIG. 1

Molecular typing of the strains. (A) Spoligotype image of isolates from patient no. 1 (P1) and patient no. 2 (P2). The arrow indicates spacer 9, which was missing in the P2 isolate. (B) IS6110 RFLP fragment digested with PvuII. An arrow indicates the additional 2.5-kb band in the P2 isolate. (C) IS6110 RFLP fragment digested with SacI. The additional band is indicated by the arrow.

FIG. 2

PCR products of isolates from patients no. 1 and 2 obtained with a forward primer corresponding to spacer 12 (DR7) and a reverse primer corresponding to a sequence downstream of spacer 1 (U16B7). The predicted size of the band from H37Rv was 1009 bp, which matches the size of the band of the isolate from patient 1.

FIG. 3

Sequence indicating a portion of the direct repeat region and the IS6110 insertion site of the 2.5-kb PCR fragment from patient no. 2's isolate. Primers used are indicated by bold type, with the name in bold on the 5′ end. Direct repeats are indicated by a single underline, with the spacer number indicated 3′ of the direct repeat just before that number's spacer. The three duplicated nucleotides are indicated by a double underline. The boxed numbers indicate the IS6110 sequences that were deleted.

FIG. 4

A common spoligotype (bottom) compared with spoligotypes of patient no. 2's isolate. The spoligotype of patient no. 2's strain using DRa and DRb showed a deletion of spacer 9; using only INS1 (an IS6110-specific primer directed toward the right end of the insertion sequence) and DRa (which is biotin labeled and hybridizes to the direct repeat region with the 3′ end toward the lower numbered spacer) showed that the IS6110 adjacent spacers 9 and 25 were amplified.