Horng Yu-Tze, Jeng Wen-Yih, Chen Yih-Yuan, Liu Che-Hung, Dou Horng-Yunn, Lee Jen-Jyh, Chang Kai-Chih, Chien Chih-Ching, Soo Po-Chi
Department of Laboratory Medicine and Biotechnology, Tzu Chi University, College of Medicine, Hualien, Taiwan, Republic of China.
Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan, Republic of China Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei, Taiwan, Republic of China.
Antimicrob Agents Chemother. 2015 Mar;59(3):1542-8. doi: 10.1128/AAC.04374-14. Epub 2014 Dec 22.
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.
大多数结核分枝杆菌耐利福平菌株与rpoB基因中一个81bp的利福平耐药决定区（RRDR）的突变有关。然而，如果仅针对该区域，RRDR以外发生突变的耐利福平菌株将无法被检测到。在本研究中，对51株耐利福平临床分离株进行测序分析，这些分离株包含RRDR的1681bp长DNA片段，其中47株在RRDR内有突变，3株在RRDR内外均有突变，只有1株在RRDR外有一个单错义突变（Arg548His）。对携带突变rpoB（Arg548His）的重组耻垢分枝杆菌和结核分枝杆菌分离株进行药敏试验，结果显示与对照菌株相比，利福平的最低抑菌浓度（MIC）有所增加。对Arg548His突变型RpoB-DNA复合物进行建模显示，His548侧链形成的氢键结构比Arg548更稳定，降低了RpoB耐利福平簇II区域的灵活性，这表明RpoB Arg548His突变体不能有效地与利福平相互作用，从而导致细菌对该药物产生耐药性。这是关于RpoB第548位密码子突变与结核病耐利福平之间关系的首次报道。本文描述了rpoB基因新的突变谱，这将有助于全面了解利福平耐药模式，并有助于开发一种用于简单快速药敏试验的有用工具。
