Contribution of rpoB mutations to development of rifamycin cross-resistance in Mycobacterium tuberculosis

Abstract

The contributions of 23 insertion, deletion, or missense mutations within an 81-bp fragment of rpoB, the gene encoding the beta-subunit of the DNA-dependent RNA polymerase of Mycobacterium tuberculosis, to the development of resistance to rifamycins (rifampin, rifabutin, rifapentine, and KRM-1648) in 29 rifampin-resistant clinical isolates were defined. Specific mutant rpoB alleles led to the development of cross-resistance to all rifamycins tested, while a subset of mutations were associated with resistance to rifampin and rifapentine but not to KRM-1648 or rifabutin. To further study the impact of specific rpoB mutant alleles on the development of rifamycin resistance, mutations were incorporated into the rpoB gene of M. tuberculosis H37Rv, contained on a mycobacterial shuttle plasmid, by in vitro mutagenesis. Recombinant M. tuberculosis clones containing plasmids with specific mutations in either codon 531 or 526 of rpoB exhibited high-level resistance to all rifamycins tested, whereas clones containing a plasmid with a mutation in codon 516 exhibited high-level resistance to rifampin and rifapentine but were susceptible to both rifabutin and KRM-1648. These results provided additional proof of the association of specific rpoB mutations with the development of rifamycin resistance and corroborate previous reports of the usefulness of rpoB genotyping for predicting rifamycin-resistant phenotypes.

FIG. 1

Nucleotide substitutions were incorporated into the rpoB gene of M. tuberculosis H37Rv contained on pLN-2, using mutagenic primers (Table 3) and long-fragment PCR–site-directed mutagenesis. (A) Signature nucleotides were incorporated into pLN-2 at codons 510 (CAG→CAA) and 511 (CTG→CTA) to yield the pLN-sig plasmid. (B) Mutant rpoB alleles were incorporated into pLN-sig as follows: 1) a GAC→GTC nucleotide substitution in codon 516 of pLN-sig to yield plasmid pAV, 2) a CAC→TAC nucleotide substitution in codon 526 of pLN-sig to yield plasmid pHT, and 3) a TCG→TTG nucleotide substitution in codon 531 of pLN-sig to yield plasmid pSL.