郑州地区耐多药结核分枝杆菌gyrA和gyrB基因突变特征及对氟喹诺酮类药物耐药研究

Correlation between phenotypic resistance to fluoroquinolones and gyrA and gyrB gene mutations in multidrug-resistant Mycobacterium tuberculosis in Zhengzhou,China

目的分析郑州地区耐多药(MDR)结核分枝杆菌(MTB)临床分离株中gyrA和gyrB基因突变特征及其对氟喹诺酮类(FQs)药物耐药的特点。方法收集2015年5月至2016年12月河南省胸科医院收治的256例肺结核患者的痰标本,进行MTB固体罗氏培养、液体分枝杆菌培养,MTB培养阳性者行比例法药物敏感性试验,采用聚合酶链式反应扩增MDR MTB的gyrA和gyrB基因,对扩增产物进行测序和序列比对分析。应用耐药基因判断MDR MTB对FQs药物耐药的敏感性和特异性。结果 256例患者中MTB培养阳性215例,其中非MDR肺结核患者137例,MDR肺结核患者78例。78株MDR MTB临床分离株对氧氟沙星(Ofx)、0.5 mg·L^(-1)莫西沙星(Mfx 0.5)及2.0 mg·L^(-1)莫西沙星(Mfx 2.0)的耐药率分别为55.1%(43/78)、55.1%(43/78)、32.1%(25/78);137株非MDR MTB临床分离株对Ofx、Mfx 0.5及Mfx 2.0的耐药率分别为7.3%(10/137)、5.8%(8/137)、0.7%(1/137);非MDR MTB临床分离株对Ofx、Mfx0.5、Mfx2.0的耐药率显著低于MDR MTB临床分离株(χ~2=61.21、66.73、45.87,P<0.001)。gyrA基因、gyrB基因在Ofx耐药株中的突变率分别为90.7%(39/43)、11.6%(5/45);gyrA基因、gyrB基因在Mfx 0.5耐药株中的突变率分别为88.4%(38/43)、11.6%(5/43);gyrA基因、gyrB基因在Mfx 2.0耐药株中的突变率分别为100.0%(25/25)、12.0%(3/25)。以FQs药物敏感性试验结果为金标准,gyrA基因突变判断MDR MTB对Ofx、Mfx 0.5、Mfx 2.0耐药的敏感性分别为90.7%、88.4%、58.1%,特异性分别为88.6%、85.7%、100.0%,gyrB基因突变判断MDR MTB对Ofx、Mfx 0.5、Mfx 2.0耐药的敏感性分别为62.5%、62.5%、37.5%,特异性分别为45.7%、45.7%、68.6%,检测gyrA基因突变判断MDR MTB对FQs药物耐药的特异性高于gyrB基因突变(χ~2=17.86、15.44、13.92,P<0.001),检测gyrA基因突变判断MDR MTB对Ofx耐药的敏感性高于gyrB基因突变(χ~2=4.53,P<0.05)。结论郑州地区MDR MTB对FQs耐药率较高,耐药的主要原因是gyrA基因突变,最常见的gyrA基因突变位于94位点。

Objective To analyze the characteristics of gyrA and gyrB gene mutations and the phenotypic resistance to fluoroquinolones（FQs） in multi-drug resistant（MDR） Mycobacterium tuberculosis （MTB） isolates in Zhengzhou,China.Methods The sputum samples of 256 patients with pulmonary tuberculosis were collected in Henan Provincial Chest Hospital from May 2015 to December 2016.The sputum samples were performed with MTB solid Roche culture method and liquid culture method.The samples with positive MTB were performed with proportional drug sensitivity test.The gyrA and gyrB genes of MDR MTB were amplified by PCR,and the amplified products were sequenced and analyzed.The sensitivity and specificity of MDR MTB to FQs were determined by Drug resistance genes.Results In the 256 patients,MTB culture was positive in 215 patients.Among 215 MTB positive patients,137 cases were non-MDR pulmonary tuberculosis patients and 78 cases were MDR pulmonary tuberculosis patients.The resistance rates of 78 MDR MTB isolates to ofloxacin（Ofx）,0.5 mg·L -1 moxifloxacin（Mfx 0.5） and 2.0 mg·L -1 moxifloxacin（Mfx 2.0） were 55.1%（43/78）,55.1%（43/78） and 32.1%（25/78） respectively.The resistance rates of 137 non-MDR MTB isolates to Ofx,Mfx 0.5 and Mfx 2.0 were 7.3%（10/137）,5.8%（8/137） and 0.7%（1/137） respectively.The resistance rates of non-MDR MTB isolates to Ofx,Mfx 0.5 and Mfx 2.0 were significantly lower than those of MDR MTB isolates（ χ^2=61.21,66.73,45.87; P 〈0.001）.The mutation rate of gyrA and gyrB gene was 90.7%（39/43） and 11.6%（5/45） in Ofx resistant strains.The mutation rate of gyrA gene was 88.4%（38/43） in Mfx 0.5 resistant strains,and the mutation rate of gyrB gene was 11.6%（5/43） in Mfx 0.5 resistant strains.The mutation rates of gyrA and gyrB genes were 100.0%（25/25） and 12.0%（3/25） in Mfx 2.0 resistant strains.The sensitivity and specificity of MDR-MTB to FQs were determined by the drug-resistance gene based on the results of FQs phenotypic drug sensitivity test.The sensitivity of gyrA gene mutation in estimating the resistance of MDR MTB to Ofx,Mfx 0.5 and Mfx 2.0 was 90.7%,88.4% and 58.1% respectively;and the specificity was 88.6%,85.7% and 100.0% respectively.The sensitivity of gyrB gene mutation in estimating the resistance of MDR MTB to Ofx,Mfx 0.5,Mfx 2.0 was 62.5%,62.5 and 37.5%;and the specificity was 45.7%,45.7% and 68.6%.The specificity of judging resistance of MDR MTB to FQs drug by gyrA gene mutation was better than that by gyrB gene mutation（ χ^2=17.86,15.44,13.92; P 〈0.001）.The sensitivity of judging resistance of MDR MTB to Ofx drug resistance by gyrA gene mutation was higher than that by gyrB gene mutation（ χ^2=4.53, P 〈0.05）.Conclusion The resistance rate of MDR MTB to fluoroquinolones is high in Zhengzhou,China.The main reason of drug resistance is gyrA gene mutation,and the most common gyrA gene mutation is located at 94 site.