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Molecular analysis of codon 548 in the rpoB gene involved in Mycobacterium tuberculosis resistance to rifampin.

ABSTRACT: Most Mycobacterium tuberculosis rifampin-resistant strains have been associated with mutations in an 81-bp rifampin resistance-determining region (RRDR) in the gene rpoB. However, if this region alone were targeted, rifampin-resistant strains with mutations outside the RRDR would not be detected. In this study, among 51 rifampin-resistant clinical isolates analyzed by sequencing 1,681-bp-long DNA fragments containing the RRDR, 47 isolates contained mutations within the RRDR, three isolates contained mutations both within and outside the RRDR, and only one isolate had a single missense mutation (Arg548His) located outside the RRDR. A drug susceptibility test of recombinant Mycobacterium smegmatis and M. tuberculosis isolates carrying mutated rpoB (Arg548His) showed an increased MIC for rifampin compared to that of the control strains. Modeling of the Arg548His mutant RpoB-DNA complex revealed that the His548 side chain formed a more stable hydrogen bond structure than did Arg548, reducing the flexibility of the rifampin-resistant cluster II region of RpoB, suggesting that the RpoB Arg548His mutant does not effectively interact with rifampin and results in bacterial resistance to the drug. This is the first report on the relationship between the mutation in codon 548 of RpoB and rifampin resistance in tuberculosis. The novel mutational profile of the rpoB gene described here will contribute to the comprehensive understanding of rifampin resistance patterns and to the development of a useful tool for simple and rapid drug susceptibility tests.

SUBMITTER: Horng YT

PROVIDER: S-EPMC4325785 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Molecular analysis of codon 548 in the rpoB gene involved in Mycobacterium tuberculosis resistance to rifampin.

Horng Yu-Tze YT Jeng Wen-Yih WY Chen Yih-Yuan YY Liu Che-Hung CH Dou Horng-Yunn HY Lee Jen-Jyh JJ Chang Kai-Chih KC Chien Chih-Ching CC Soo Po-Chi PC

Antimicrobial agents and chemotherapy 20141222 3

Most Mycobacterium tuberculosis rifampin-resistant strains have been associated with mutations in an 81-bp rifampin resistance-determining region (RRDR) in the gene rpoB. However, if this region alone were targeted, rifampin-resistant strains with mutations outside the RRDR would not be detected. In this study, among 51 rifampin-resistant clinical isolates analyzed by sequencing 1,681-bp-long DNA fragments containing the RRDR, 47 isolates contained mutations within the RRDR, three isolates conta ...[more]

PMID: 25534743

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Project description:We aimed to study the distribution and contribution of mutations in the rpoB whole gene in rifampin-resistant/rifabutin-resistant (RIF(r)/Rfb(r)) (or RIF/Rfb cross-resistant) clinical Mycobacterium tuberculosis isolates. One standard M. tuberculosis strain (H37Rv) and 392 other clinical M. tuberculosis isolates mainly from Guangdong Province of China whose susceptibilities to rifampin (RIF), rifabutin (Rfb), streptomycin (SM), ethambutol (EMB), and isoniazid (INH) were previously determined were subjected to DNA sequencing of their rpoB whole genes. H37Rv and the 30 drug-susceptible clinical isolates had no mutations in rpoB whole genes. In 43 rifampin-resistant/rifabutin-susceptible (RIF(r)/Rfb(s)) isolates, the most frequent mutation codons were 516 (62.80%), 526 (14.0%), and 533 (6.98%), but codon 531 had no mutation. Twenty-one of the 43 isolates (48.84%) had single mutations of H526L, H526S, D516V, D516Y, and D516F. In 319 RIF(r)/Rfb(r) isolates, the most frequent mutation codons were 531 (73.7%) and 526 (18.8%); the mutation frequency for codon 516 was 2.5%, and that for codon 533 was only 0.31%. A total of 82.8% (264/319) of them had single mutations of S531L, S531W, H526D, H526Y, H526R, Q513K, Q513P, Q510H, V176F, P206(T)R, Y314(T)C, and H323(T)Y (the superscript T indicates M. tuberculosis numbering; the remaining codons use the E. coli numbering), among which V176F, P206(T)R, Y314(T)C, and H323(T)Y were located in the beginning of rpoB, and all of them were present in 1.9% (6/319) of RIF(r)/Rfb(r) isolates. The multiple mutations in RIF(r)/Rfb(r) isolates and in RIF(r)/Rfb(s) isolates were also different from each other either in mutation positions or in types of mutation combinations. In conclusion, the mutations of rpoB in RIF-R/Rfb(s) and in RIF-R/Rfb-R isolates differ significantly from each other not only in the most frequent mutation codons (516, 531, and 533) but also in the most frequent single mutations (S531L, H526L, D516V, D516Y, and D516F), and the beginning of rpoB may confer a RIF/Rfb cross-resistance phenotype in M. tuberculosis. Molecular assays for identifying RIF/Rfb cross-resistance in M. tuberculosis might be improved in terms of accuracy by including this region, in addition to the rifampin resistance determination region.

Dataset Information

Molecular analysis of codon 548 in the rpoB gene involved in Mycobacterium tuberculosis resistance to rifampin.

Publications

Molecular analysis of codon 548 in the rpoB gene involved in Mycobacterium tuberculosis resistance to rifampin.

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