Definition of the Beijing/W lineage of Mycobacterium tuberculosis on the basis of genetic markers

Abstract

Mycobacterium tuberculosis Beijing genotype strains are highly prevalent in Asian countries and in the territory of the former Soviet Union. They are increasingly reported in other areas of the world and are frequently associated with tuberculosis outbreaks and drug resistance. Beijing genotype strains, including W strains, have been characterized by their highly similar multicopy IS6110 restriction fragment length polymorphism (RFLP) patterns, deletion of spacers 1 to 34 in the direct repeat region (Beijing spoligotype), and insertion of IS6110 in the genomic dnaA-dnaN locus. In this study the suitability and comparability of these three genetic markers to identify members of the Beijing lineage were evaluated. In a well-characterized collection of 1,020 M. tuberculosis isolates representative of the IS6110 RFLP genotypes found in The Netherlands, strains of two clades had spoligotypes characteristic of the Beijing lineage. A set of 19 Beijing reference RFLP patterns was selected to retrieve all Beijing strains from the Dutch database. These reference patterns gave a sensitivity of 98.1% and a specificity of 99.7% for identifying Beijing strains (defined by spoligotyping) in an international database of 1,084 strains. The usefulness of the reference patterns was also assessed with large DNA fingerprint databases in two other European countries and for identification strains from the W lineage found in the United States. A standardized definition for the identification of M. tuberculosis strains belonging to the Beijing/W lineage, as described in this work, will facilitate further studies on the spread and characterization of this widespread genotype family of M. tuberculosis strains.

FIG.1.

For both panels A and B, the dendrogram (left) reflects the similarity of the IS6110 RFLP patterns. The IS6110 RFLP patterns (middle) are shown, with the PvuII restriction fragments depicted from high (left) to low (right) molecular weight. The spoligo patterns (right) are shown from spacer 1 to spacer 43 (left to right). Related groups of DNA fingerprints are marked A or B, followed by a number, to the right of the spoligo patterns. These groups mark the isolates that belong to certain branches of the dendrogram. Group A3 represents clade 47, and group B3 represents clade 61. For some branches in the dendrograms, the similarity between the branches is indicated as a percentage at the left of the dendrogram. The clade 47 (group A3) isolates and all except one of the isolates showing IS6110 RFLP patterns with 60% or more similarity to clade 47 patterns (groups A1, A2, A4, and A5) belong to the Beijing genotype family, as determined by spoligotyping and region A RFLP. The strain at the bottom of the dendrogram in panel A is the only strain that has an IS6110 RFLP pattern that exhibits >60% similarity to clade 47 patterns and that has spoligo and region A RFLP patterns not characteristic of Beijing strains. The clade 61 isolates (group B3) belong to the Beijing lineage, as determined by spoligotyping and region A RFLP. In addition, six of the strains in the branches neighboring clade 61 are of the Beijing lineage. Note that many of the Beijing isolates in this figure show a false-positive weak hybridization to spacer 28; this was due to the quality of the spoligotype membranes of batch 03052001.

FIG. 2.

IS6110 RFLP patterns of the 19 M. tuberculosis Beijing reference strains and of M. tuberculosis reference strain Mt14323. The molecular sizes of the IS6110-containing PvuII restriction fragments of Mt14323 are shown in kilobases. Two strains are from the PHRI collection and are represented here as NLA000200230 (TN14439N4) and NLA000300957 (TN11265LB).