Rifampin resistance, Beijing-W clade-single nucleotide polymorphism cluster group 2 phylogeny, and the Rv2629 191-C allele in Mycobacterium tuberculosis strains

Abstract

Rifampin resistance is a key prognostic marker for treatment success in tuberculosis patients. Recently, Wang et al. demonstrated that Rv2629 A191C mutations were present in 99.1% of rifampin-resistant and 0% of rifampin-susceptible clinical Mycobacterium tuberculosis isolates and that overexpression of the Rv2629 191C allele in Mycobacterium smegmatis produced an eightfold increase in rifampin resistance. These results suggested that Rv2629 could be a cause of rifampin resistance and a valuable target for rifampin resistance detection assays. We developed a molecular-beacon assay to study the association between Rv2629 191 alleles and rifampin resistance in 246 geographically and phylogenetically diverse clinical M. tuberculosis isolates. The 191C allele was present in 30/98 (30.6%) rifampin-resistant isolates and 25/148 (16.9%) rifampin-susceptible isolates and was more common in isolates from Asia. Phylogenetic analysis demonstrated complete overlap between the 191C allele and single nucleotide polymorphism cluster group 2 (SCG-2), a phylogenetic lineage that corresponds to the Beijing-W clade of M. tuberculosis. All 55 (100%) 191C isolates were SCG-2, while none of the 191 191A isolates were SCG-2 (P < 0.001). No association was found between the 191C allele and rifampin resistance in an analysis that included the SCG type (P = 1.0). Also, in contrast to the findings of Wang et al., we found that overexpression of either Rv2629 191 allele in M. smegmatis did not produce an increase in rifampin resistance. We conclude that the Rv2629 191C allele is not associated with rifampin resistance and that the allele cannot be used as a molecular target to detect rifampin resistance. The allele appears to be an excellent marker for the Beijing-W clade/SCG-2 phylogenetic group.

FIG. 1.

Phylogenetic distribution of M. tuberculosis isolates by SCG. (A) Distribution of rifampin-resistant isolates. (B) Distribution of rifampin-susceptible isolates. The circle sizes reflect the numbers of M. tuberculosis isolates in each SCG. ○, isolates containing the Rv2629 191C allele; •, isolates containing the Rv2629 191A allele.

FIG. 2.

Expression levels of Rv2629 mRNA in M. smegmatis strains. The mean ± 1 standard deviation of three real-time RT-PCR experiments is shown for each strain. The values were normalized to 16S mRNA levels. (A) Parental strain mc²155. (B) Vector-only control strain NJS31. (C) Rv2629 191A allele overexpression strain NJS30A. (D) Rv2629 191C allele overexpression strain NJS30C. *, fewer than 10 molecules of Rv2629 mRNA.