Pre-existing isoniazid resistance, but not the genotype of Mycobacterium tuberculosis drives rifampicin resistance codon preference in vitro.
Pre-existing isoniazid resistance, but not the genotype of Mycobacterium tuberculosis drives rifampicin resistance codon preference in vitro.
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DOI：
10.1371/journal.pone.0029108
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Anthony R
中科院分区：
文献类型：
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作者：
Bergval I;Kwok B;Schuitema A;Kremer K;van Soolingen D;Klatser P;Anthony R
突变发生的概率和突变持续的能力都会影响突变在种群中的分布。我们研究了这些因素的相互作用，以便在体外选择耐利福平（RIF）的结核分枝杆菌突变体。我们从异烟肼（INH）敏感和耐药的实验室菌株和临床分离株中鉴定了两组自发的体外rif耐药突变体，代表了不同的结核分枝杆菌基因型。第一个系列是从多个平行的1ml培养中选择的，第二个系列是从单个10ml培养中选择的。通过多重连接依赖探针扩增（Multiplex lig- dependent Probe Amplification， MLPA）或rpoB基因测序筛选rif抗性突变体。采用波动法测定所有菌株的RIF抗性突变率。最引人注目的观察结果是从10毫升培养物中选择的实验室生成的inh抗性突变体中向rpoB-S531L （TCG→TTG）突变转变（p<0.001）。所有测试菌株的突变率相似（1.33×10−8至2.49×10−7），但实验室产生的一种INH突变体的突变率为5.71×10−7，比INH敏感亲本菌株（5.46-7.44×10−8）高出10倍以上。不同基因型菌株之间的rpob突变谱没有明显的系统差异。在我们的耐inh实验室突变体中，rpoB-S531L的显著转变表明，耐药突变体的相对适应度可以显著影响（后续）在结核分枝杆菌群体中积累的突变的分布，至少在体外是这样。我们的结论是，在特定的遗传背景下，某些抗性突变特别容易传播。在临床分离株中对这些（组合）突变进行分子筛选可以快速识别这些特定的致病菌株。因此，我们建议对分离株进行耐药突变分布筛选，特别是在（多重）耐药结核病高度流行的地区。
Both the probability of a mutation occurring and the ability of the mutant to persist will influence the distribution of mutants that arise in a population. We studied the interaction of these factors for the in vitro selection of rifampicin (RIF)-resistant mutants of Mycobacterium tuberculosis. We characterised two series of spontaneous RIF-resistant in vitro mutants from isoniazid (INH)-sensitive and -resistant laboratory strains and clinical isolates, representing various M. tuberculosis genotypes. The first series were selected from multiple parallel 1 ml cultures and the second from single 10 ml cultures. RIF-resistant mutants were screened by Multiplex Ligation-dependent Probe Amplification (MLPA) or by sequencing the rpoB gene. For all strains the mutation rate for RIF resistance was determined with a fluctuation assay. The most striking observation was a shift towards rpoB-S531L (TCG→TTG) mutations in a panel of laboratory-generated INH-resistant mutants selected from the 10-ml cultures (p<0.001). All tested strains showed similar mutation rates (1.33×10−8 to 2.49×10−7) except one of the laboratory-generated INH mutants with a mutation rate measured at 5.71×10−7, more than 10 times higher than that of the INH susceptible parental strain (5.46–7.44×10−8). No significant, systematic difference in the spectrum of rpoB-mutations between strains of different genotypes was observed. The dramatic shift towards rpoB-S531L in our INH-resistant laboratory mutants suggests that the relative fitness of resistant mutants can dramatically impact the distribution of (subsequent) mutations that accumulate in a M. tuberculosis population, at least in vitro. We conclude that, against specific genetic backgrounds, certain resistance mutations are particularly likely to spread. Molecular screening for these (combinations of) mutations in clinical isolates could rapidly identify these particular pathogenic strains. We therefore recommend that isolates are screened for the distribution of resistance mutations, especially in regions that are highly endemic for (multi)drug resistant tuberculosis.
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