【作者】 陈军；

【导师】 徐顺清；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2011， 博士

【摘要】 结核病是严重危害人民群众健康的呼吸道传染病。在结核病防治过程中结核分枝杆菌耐药尤其是多重耐药结核病(MDR-TB)已成为有效控制结核病的重要障碍。氟喹诺酮类药物是治疗结核病的二线药,尤其是用于耐多药结核病的联合治疗的核心药物之一。MDR-TB的病人一旦对氟喹诺酮类耐药,病人的预后将更差。氟喹诺酮类药物主要作用于细菌的DNA旋转酶,使细菌DNA复制受阻,从而导致细菌死亡。DNA旋转酶由gyrA和gyrB基因编码。研究表明,细菌gyrA和/或gyrB基因上有一段核苷酸序列与喹诺酮耐药性密切相关,称为喹诺酮耐药决定区(QRDR)。QRDR碱基的改变引起相应的DNA旋转酶的改变,从而导致细菌耐药。1994年,Takiff HE首先克隆并测序了结核分枝杆菌的gyrA和gyrB基因,开始从分子水平研究结核分枝杆菌对氟喹诺酮类药物的耐药机制。在一些氟喹诺酮类药物耐药的结核分枝杆菌中,发现在gyrA基因的QRDR存在突变,突变主要集中在90、91、94位氨基酸,而gyrB基因的QRDR突变少见,但也在一些氟喹诺酮类药物耐药的结核分枝杆菌中未发现gyrA和gyrB基因的QRDR突变。其它的耐药机制如外排泵在临床结核分枝杆菌分离株中在氟喹诺酮耐药中的作用,需要进一步的研究。因此,为了解武汉地区结核分枝杆菌对氟喹诺酮的耐药情况,我们收集了一定数量的结核分枝杆菌,测定了这些菌株对主要抗结核药物的体外敏感性。对氧氟沙星耐药的结核分枝杆菌,测定了四种氟喹诺酮类药物最小抑菌浓度(MIC),了解新一代的氟喹诺酮能否用于治疗氧氟沙星耐药的结核分枝杆菌。采用DNA测序技术,检测氧氟沙星耐药和敏感结核杆菌菌株是否存在突变及突变类型,是否存在新的突变,探讨快速检测耐氟喹诺酮类药物结核杆菌的方法及结核杆菌对氟喹诺酮类药物耐药的分子机制,为进一步研究是否存在其它的耐药机制打下基础。第一部分结核分枝杆菌对抗结核药物的耐药性分析目的通过对武汉市结核病防治所检验科收集的1223株结核分枝杆菌药敏结果分析,了解初复治涂阳患者的结核分杆菌耐药情况,指导临床医生合理选择抗结核药物和治疗方案。方法收集2008年武汉市初治涂阳病人结核分枝杆菌菌株688株和武汉市结核病防治所检验科2008年至2009年两年间复治涂阳病人535株结核分枝杆菌的药敏数据进行分析。结果药敏试验结果显示:在1223株结核分枝杆菌分离株中,结核分枝杆菌的总耐药率为31.3%,其中,初始耐药率为18.0%,获得耐药率为48.4%。结核分枝杆菌的总耐多药率为12.7%,其中,初始耐多药率为4.5%,获得耐多药率为23.2%。对6种抗结核药物的耐药率:链霉素(SM) 18.7%、异烟肼(INH) 21.1%、利福平(RFP) 16.4%、乙胺丁醇(EMB) 8.3%、氧氟沙星(OFX) 7.6%、卡那霉素(KM) 0.9%,其中,在初治组,6种抗结核药物的耐药率:SM 12.3 %、INH 10.3 %、RFP 5.2%、EMB 2.5 %、OFX 3.6 %、KM 0.4 %;在复治组,对6种抗结核药物的耐药率:SM 26.9 %、INH 35.0%、RFP 30.7%、EMB 15.5%、OFX 12.9%、KM 1.5 %。在MDR-TB菌株中,OFX的耐药率为27.7%(43/155),KM的耐药率为5.2%(8/155)。结论复治涂阳病人的耐药率比较高,成为结核病预防控制的威胁,需根据药敏结果合理选择治疗方案。第二部分四种氟喹诺酮类药物对结核分枝杆菌的最小抑菌浓度的比较目的用刃天青显色法快速测定四种氟喹诺酮对结核分枝杆菌的最小抑菌浓度(MIC),探讨不同氟喹诺酮对结核分枝杆菌的作用和可能的交叉耐药性。方法用65株对氧氟沙星敏感的菌株(包括42株耐多药结核分枝杆菌(MDR-TB)菌株)和60株氧氟沙星耐药的菌株为试验菌株,在96孔酶标板中,用Middlebrook 7H9液体培养基将药物进行连续对倍稀释后,加入一定浓度菌液,用显色法测定最小抑菌浓度。结果氧氟沙星通过ROC曲线分析所确定的耐药折点浓度为2μg/ml,敏感度为98.3%,特异度为96.9%,准确度为97.6%。加替沙星和莫西沙星对结核分枝杆菌的MIC50和MIC90比氧氟沙星、环丙沙星要低4-8倍,具有比氧氟沙星和环丙沙星更好的抗结核分枝杆菌活性。结论新一代氟喹诺酮加替沙星和莫西沙星具有比第三代氟喹诺酮氧氟沙星和环丙沙星更低的最小抑菌浓度,更好的抗菌活性,有可能用于低度氧氟沙星耐药MDR-TB的治疗。第三部分gyrA/B基因突变与氟喹诺酮耐药的相关性研究目的通过检测gyrA和gyrB基因的喹诺酮耐药决定区(QRDR)序列突变,探讨武汉地区结核分枝杆菌的喹诺酮耐药突变类型及可能的耐药机制。方法共收集93株临床分离的结核分枝杆菌,以罗氏比例法为标准,通过PCR直接测序技术(PCR-DS)分析结核分枝杆菌中gyrA和gyrB基因突变情况。对未检测到突变的结核分枝杆菌,加入外排泵抑制剂利血平观察细菌最小抑菌浓度的改变。结果在61株氧氟沙星耐药的结核分枝杆菌中,54株(88.5%)在gyrA基因的喹诺酮耐药决定区均存在突变。突变类型有7种单位点突变(A90V,S91P,S91T,D94N, D94Y,D94G及D94A)和2种双位点突变(S91P&D94H,S91T&D94A)。在32株氧氟沙星敏感的MDR-TB菌株中,仅1株存在D94N突变,其余均未检测到突变。gyrB基因在全部菌株中均未检测到突变。7株无突变的结核分枝杆菌,有1株在加入外排泵抑制剂利血平后,最小抑菌浓度下降了8倍。氧氟沙星的MICs与gyrA基因94位不同氨基酸突变无统计学差异(F=0.40,P=0.755),而氧氟沙星的MICs与gyrA基因不同位点(90,91,94)的突变有统计学差异(F=11.70,P<0.0001)。结论gyrA基因在90,91和94位的突变是结核分枝杆菌中氟喹诺酮耐药的主要机制。外排泵可能在部分低度氧氟沙星耐药的结核分枝杆菌中起一定的作用。gyrA基因的91位突变可能与低水平氧氟沙星耐药相关。更多还原

【Abstract】 Tuberculosis is still a respiratory infectious disease that seriously threatens public health. In the practice of prevention from tuberculosis, drug resistance of mycobacterium tuberculosis, especially multidrug-resistant tuberculosis(MDR-TB) has become an important barrier to the effective control of tuberculosis. Fluoroquinolones are the pivotal second-line drugs , particularly in regimen for MDR-TB. Once MDR-TB patients are resistant to fluoroquinolones, they will have poor prognosis. DNA topoisomerases are a diverse set of essential enzymes responsible for maintaining chromosomes in an appropriate topological state. Fluoroquinolones inhibit DNA gyrase(topoisomerase II) and topoisomerase IV, resulting in microbial death for the block of microbial replication.DNA gyrase is a tetrameric A2B2 protein. The A subunit carries the breakage-reunion active site, whereas the B subunit promotes ATP hydrolysis. Mycobacterium tuberculosis has respectively gyrA and gyrB genes encoding the A and B subunits. A conserved region, the quinolone-resistance-determining region(QRDR) of gyrA (320bp) and gyrB(375bp), has been found to be the most important area involved in the exhibition of fluoroquinolone resistance in Mycobacterium tuberculosis. Mutations within the QRDR of gyrA or gyrB gene will result in the resistance to fluoroquinolones. In 1994, Takiff HE for the first time cloned and sequenced the gyrA and gyrB genes and studied resistance mechanisms of fluoroquinolones. Mutations within the QRDR of gyrA have been identified in clinical and laboratory-selected isolates of Mycobacterium tuberculosis, largely clustered at codon 90,91,94,with Asp 94 being relatively frequent.For clinical isolates,gyrB mutations appear to be of much rarer occurrence. Among some fluoroquinolone-resistant Mycobacterium tuberculosis, there are no mutation in gyrA and gyrB genes. More researches are needed to identify new resistance mechanisms.In this study, clinical isolates of Mycobacterium tuberculosis were collected from Wuhan and the susceptibility to six antituberculosis drugs were determined. To understand the effect of newer fluoroquinolones on the ofloxacin-resistant Mycobacterium tuberculosis, the MICs of four fluoroquinolones were determined by the resazurin colorimetric assay among the ofloxacin-resistant Mycobacterium tuberculosis. Mutations within the QRDR of gyrA and gyrB genes were detected by DNA direct sequencing to identify the types and frequency of mutations.Part I Analysis of drug susceptibility testing results of 1223 MTB strains from patients with smear-positive sputaObjective To understand the drug resistance of mycobacterium tuberculosis in patients with smear positive, and provide reasonable regimens to doctors.Methods From Janurary 2008 to December 2009, 535 MTB isolates from previously treated patients with smear positive were collected and 688 MTB isolates from new cases with smear positive were also collected during resistance baseline survey in 2008 . The drug resistance data were analyzed by drug susceptibility testing.Results The drug susceptibility tests showed that the total resistance rate was 31.3%, the initial and the acquired resistance rate was 18.0% and 48.4%. The total MDR-TB resistance rate was 12.7%, the initial and the acquired MDR-TB resistance rate was 4.5% and 23.2%, respectively. The resistance rate of drug susceptibility testing of streptomycin, isoniazid, rifampicin, ethambutol, ofloxacin and kanamycin was18.7%, 21.1%, 19.4%, 8.3%, 7.6%, 0.9%, respectively. Among the new patients, the resistance rate of drug susceptibility testing of streptomycin, isoniazid, rifampicin, ethambutol , ofloxacin and kanamycin was 12.3 %, 10.3 %, 5.2%, 2.5 %, 3.6 %, 0.4 %, respectively. Among the previously treated patients, The resistance rate of drug susceptibility testing of streptomycin, isoniazid, rifampicin, ethambutol , ofloxacin and kanamycin was 26.9 %, 35.0%, 30.7%, 15.5%, 12.9%, 1.5%, respectively. The ofloxacin and kanamycin resistance rate was 27.7%(43/144) and 5.2%(8/155) among the MDR-TB strains.Conclusion The resistance rate is higher among previously treated patients, suggesting that treatment regimen should be changed according to the result of drug susceptibility testing.The high prevalence of drug resistance has been a major challenge for TB control.Part II Comparison of MICs of four fluoroquinolones for Mycobaterium tuberculosis by resazurin colometric assay.Objective To understand effects and cross-resistance of fluoroquinolones on mycobacterium tuberculosis, four fluoroquinolones’MICs for Mycobaterium tuberculosis were determined by resazurin colorimetric assay.Methods MICs of 65 ofloxacin-susceptible Mycobacterium tuberculosis strains and 60 ofloxacin-resistant clinical isolates were detected by resazurin colorimetric assay. The proportion method on L-J medium was used as a gold standard. Breakpoint concentration of ofloxacin were determined by ROC curve analysis.Results The best breakpoint value of drug susceptibility testing of Ofloxacin were 2μg/ml, sensitivity was 98.3%, specificity was 96.9% and accuracy was 97.6%. MIC50 and MIC90 of gatifloxacin and moxifloxacin for Mycobaterium tuberculosis were 4-8 times lower than ofloxacin and ciprofloxacin.Conclusion Newer fluoroquinolones such as moxifloxacin, gatifloxacin may be used to treat low degree ofloxacin-resistant MDR-TB. Part III Association of Mutations of gyrA/B and fluoroquinolone resistance in Mycobacterium tuberculosis.Objective The aim of this study was to observe gyrA and gyrB genes mutations and resistance mechnisms of ofloxacin-resistant Mycobacterium tuberculosis(MTB) isolates from Wuhan, China.Methods A total of 93 MTB clinical strains were originally isolated from patients with pulmonary tuberculosis. The phenotype of susceptibility of each strain was determined by the proportion method and the QRDR in gyrA and gyrB genes were sequenced with DNA direct sequencing technique. The MICs of ofloxacin and ciprofloxacin were determined at the presence of the efflux pump inhibitor reserpine.Results 54 of 61 (88.5%) ofloxacin-resistant MTB clinical isolates had mutations in the QRDR of gyrA gene. The mutation patterns involved seven patterns of single codon mutation ( A90V,S91P,S91T,D94N, D94Y,D94G and D94A) and two patterns of double codons mutation (S91P with D94H, S91P with D94A ). No mutation in gyrB gene was detected among all MTB strains. Among seven MTB strains without gyrA and gyrB genes, the MICs of ofloxacin and ciprofloxacin in one MTB strain decreased 8-fold at the presence of the efflux pump inhibitor reserpine. No differences were detected among strains with different amino acid mutations at codon 94 in the QRDR of gyrA gene(F=0.40,P=0.755). The MICs of all ofloxacin-resistant strains showed significant difference among strains with mutions at condons 90,91 or 94 in gyrA gene(F=11.70,P<0.0001).Conclusion Mutations of gyrA codons 90, 91 and 94 constitute the primary mechanism of FQ resistance among Mycobacterium tuberculosis. Efflux pump system may play a role in some low level ofloxacin-resistant MTB strains. Mutations at codon 91 in gyrA gene may relate to low level resistance of ofloxacin.更多还原