DNA Gyrase Inhibition Assays Are Necessary To Demonstrate Fluoroquinolone Resistance Secondary to gyrB Mutations in Mycobacterium tuberculosis
DNA Gyrase Inhibition Assays Are Necessary To Demonstrate Fluoroquinolone Resistance Secondary to gyrB Mutations in Mycobacterium tuberculosis
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DOI：
10.1128/aac.00707-11
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发表时间：
2011-10-01
影响因子：
4.9
通讯作者：
Aubry, Alexandra
中科院分区：
文献类型：
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作者：
Pantel, Alix;Petrella, Stephanie;Aubry, Alexandra
结核分枝杆菌耐氟喹诺酮类药物（FQ）的主要机制是DNA回转酶(GyrA（2)GyrB(2)）的突变，特别是在GyrA中。然而，gyrB中未知突变的发现越来越频繁，这些突变与FQ耐药的关系尚不清楚。我们研究了结核分枝杆菌临床菌株中8个gyrB突变对FQ敏感性的影响，其中3个突变先前在FQ耐药菌株中被发现。为了阐明这些突变在FQ抗性中的作用，我们测量了FQ mic和FQ对DNA旋切酶的抑制作用。野生型GyrA、野生型GyrB和突变型GyrB亚基通过诱变从工程GyrB等位基因中产生，在大肠杆菌中过表达，纯化到均匀性，并用于重建高活性的gyrase复合物。测定莫西沙星、加替沙星、氧氟沙星、左氧氟沙星和依诺沙星的mic和DNA旋切酶抑制率。我们证明，最近在FQ耐药临床菌株中发现的或在法国分离的结核分枝杆菌菌株中发现的GyrB中的8个替换（D473N、P478A、R485H、S486F、A506G、A547V、G551R和G559A）与FQ耐药无关。这些结果强调，与表型FQ敏感性测试相反，DNA回转酶抑制试验是证明DNA回转酶突变在FQ抗性中的作用的唯一方法。因此，不应仅根据gyrB突变的存在就排除FQ在结核病患者治疗中的应用。
The main mechanism of fluoroquinolone (FQ) resistance in Mycobacterium tuberculosis is mutation in DNA gyrase (GyrA(2)GyrB(2)), especially in gyrA. However, the discovery of unknown mutations in gyrB whose implication in FQ resistance is unclear has become more frequent. We investigated the impact on FQ susceptibility of eight gyrB mutations in M. tuberculosis clinical strains, three of which were previously identified in an FQ-resistant strain. We measured FQ MICs and also DNA gyrase inhibition by FQs in order to clarify the role of these mutations in FQ resistance. Wild-type GyrA, wild-type GyrB, and mutant GyrB subunits produced from engineered gyrB alleles by mutagenesis were overexpressed in Escherichia coli, purified to homogeneity, and used to reconstitute highly active gyrase complexes. MICs and DNA gyrase inhibition were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin, and enoxacin. We demonstrated that the eight substitutions in GyrB (D473N, P478A, R485H, S486F, A506G, A547V, G551R, and G559A), recently identified in FQ-resistant clinical strains or encountered in M. tuberculosis strains isolated in France, are not implicated in FQ resistance. These results underline that, as opposed to phenotypic FQ susceptibility testing, the DNA gyrase inhibition assay is the only way to prove the role of a DNA gyrase mutation in FQ resistance. Therefore, the use of FQ in the treatment of tuberculosis (TB) patients should not be ruled out only on the basis of the presence of mutations in gyrB.