CLONING AND NUCLEOTIDE-SEQUENCE OF MYCOBACTERIUM-TUBERCULOSIS GYRA AND GYRB GENES AND DETECTION OF QUINOLONE RESISTANCE MUTATIONS
CLONING AND NUCLEOTIDE-SEQUENCE OF MYCOBACTERIUM-TUBERCULOSIS GYRA AND GYRB GENES AND DETECTION OF QUINOLONE RESISTANCE MUTATIONS
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DOI：
10.1128/aac.38.4.773
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发表时间：
1994-04-01
影响因子：
4.9
通讯作者：
TELENTI, A
中科院分区：
文献类型：
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作者：
TAKIFF, HE;SALAZAR, L;TELENTI, A
随着耐多药结核分枝杆菌的出现，氟喹诺酮类药物作为抗结核药物的研究日益受到重视。目的：研究稻瘟病菌对氟喹的耐药频率及耐药机制。在结核病中，我们克隆并测序了野生型gyrA和gyrB基因，它们分别编码DNA促旋酶的A和B亚基; DNA促旋酶是FQs的主要靶标。在此基础上，我们对参考菌株（n = 4）和临床分离株（n = 55）的gyrA和gyrB基因进行了DNA扩增测序和单链构象多态性分析，以确定其可能的喹诺酮类耐药区域。在环丙沙星MIC>2 μ g/ml的所有分离株（n = 14）中鉴定出与其他耐药细菌中描述的类似的gyrA密码子突变。此外，我们还选择了牛分枝杆菌BCG和M.结核病Erdman和H37 ra。在2 μ g/ml的环丙沙星浓度下，自发抗性突变体以1/10（7）至10（8）的频率产生，但在更高的环丙沙星浓度下没有选择出初级抗性菌落。对那些第一步突变体进行重新铺板，这些突变体选择具有高水平抗性的突变体，其具有与临床耐药分离株中发现的gyrA突变相似的gyrA突变。gyrA和gyrB序列信息将有助于分析对靶向旋转酶的药物的耐药机制和实施快速评估临床M的FQ敏感性的策略。结核病分离株。
The emergence of multidrug-resistant strains of Mycobacterium tuberculosis has resulted in increased interest in the fluoroquinolones (FQs) as antituberculosis agents. To investigate the frequency and mechanisms of FQ resistance in M. tuberculosis, we cloned and sequenced the wild-type gyrA and gyrB genes, which encode the A and B subunits of the DNA gyrase, respectively; DNA gyrase is the main target of the FQs. On the basis of the sequence information, we performed DNA amplification for sequencing and single-strand conformation polymorphism analysis to examine the presumed quinolone resistance regions of gyrA and gyrB from reference strains (n = 4) and clinical isolates (n = 55). Mutations in codons of gyrA analogous to those described in other FQ-resistant bacteria were identified in all isolates (n = 14) for which the ciprofloxacin MIC was >2 mug/ml. In addition, we selected ciprofloxacin-resistant mutants of Mycobacterium bovis BCG and M. tuberculosis Erdman and H37ra. Spontaneously resistant mutants developed at a frequency of 1 in 10(7) to 10(8) at ciprofloxacin concentrations of 2 mug/ml, but no primary resistant colonies were selected at higher ciprofloxacin concentrations. Replating of those first-step mutants selected for mutants with high levels of resistance which harbored gyrA mutations similar to those found among clinical FQ-resistant isolates. The gyrA and gyrB sequence information will facilitate analysis of the mechanisms of resistance to drugs which target the gyrase and the implementation of rapid strategies for the estimation of FQ susceptibility in clinical M. tuberculosis isolates.