Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase beta subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas

Abstract

Automated DNA sequencing was used to characterize mutations associated with rifampin resistance in a 69-bp region of the gene, rpoB, encoding the P subunit of RNA polymerase in Mycobacterium tuberculosis. The data confirmed that greater than 90% of rifampin-resistant strains have sequence alterations in this region and showed that most are missense mutations. The analysis also identified several mutant rpoB alleles not previously associated with resistant organisms and one short region of rpoB that had an unusually high frequency of insertions and deletions. Although many strains with an identical IS6110 restriction fragment length polymorphism pattern have the same variant rpoB allele, some do not, a result that suggests the occurrence of evolutionary divergence at the clone level.

Antimicrobial agent resistance in mycobacteria: molecular genetic insights

“…In addition, one strain each had an insertion and deletion of one amino acid in this region. As described by other investigators (58,77,78,158), most amino acid substitutions affected His-526 or Ser-531. A summary of rpoB mutations associated with RIF resistance is presented in Fig.…”

“…In an effort to directly determine the role of rpoB mutations in conferring RIF resistance in mycobacteria, Miller et al (103) cloned and sequenced the entire M. tuberculosis rpoB gene and then used sophisticated genetic techniques to conclusively show that one common mutant M. tuberculosis rpoB allele conferred RIF resistance to M. smegmatis (103). The results of this experiment contribute to evidence that the rpoB mutations identified by Telenti et al (158), Kapur et al (77,78), and others (106,170) directly confer RIF resistance to M. tuberculosis. It remains unknown if RIF-resistant M. tuberculosis isolates exhibit pleiotropic phenotypes (including, for example, alterations in growth characteristics or virulence), as described for E. coli rpoB mutants (75).…”

“…The positions of insertions and deletions are illustrated above the wild-type sequence, and missense mutations are recorded below. The codon numbering system initially described by Telenti detected mutations had not been previously described (77,78,158). No mutations were found in 27 RIF-susceptible strains examined.…”

The evolving story of Mycobacterium tuberculosis clade members detected in fish

“…The gene for 65-kDa heat shock protein (hsp65) is useful for differentiation of most mycobacterial species (Telenti, Marchesi, Balz, Bally, Böttger & Bodmer 1993;Devallois, Goh & Rastogi 1997;Devulder et al 2005), with the exception of those within the M. tuberculosis complex and subspecies of both M. avium and M. fortuitum (McNabb, Eisler, Adie, Amos, Rodrigues, Stephens, Black & Isaac-Renton 2004). The hsp65 gene is known to be more variable within the Mycobacterium genus than the gene encoding 16S rRNA (Kapur, Li, Iordanescu, Hamrick, Wanger, Kreiswirth & Musser 1994). By combining sequence analysis of both the 16S rRNA gene and the protein-encoding hsp65 gene (Ucko et al 2002;Ucko & Colorni 2005), it was possible to differentiate between strains of piscine and clinical M. marinum.…”

Use of a high-density DNA probe array for detecting mutations involved in rifampicin resistance in Mycobacterium tuberculosis

“…Several molecular assays have been developed to screen the rpoB gene for Rif R mutations, including single-strand conformation polymorphism analysis, heteroduplex and mismatch analyses, dot spot, DNA sequencing, and other assays [8][9][10][11][12][13]. A commercial test, the Inno-LiPa assay, is available for detection of the four commonest mutations in clinical isolates (D516V, H526Y, H526D, S531L) [14,15].…”