结核分枝杆菌喹诺酮类耐药基因的研究

发布时间：2018-06-12 08:13

本文选题：gyrA基因 + gyrB基因　；参考：《广州医学院》2010年硕士论文

【摘要】：研究背景与目的结核病是一种对人类影响严重的传染性疾病,2006年全球有180万人死于结核病,920万人成为新发结核病患者。该病需要较长时间的药物联合治疗才能治愈,但目前能用于该病治疗的药物有限,而且随着耐药结核病特别是耐多药结核病的出现,结核病控制工作和公共健康安全正遭受越来越严峻的挑战。作为广谱的抗生素,喹诺酮类药物逐渐被用于结核病的治疗,并有可能作为一线药物使用。不幸的是,随着该类药物的广泛使用及不同程度的滥用和误用,对该类药物耐受的结核分枝杆菌(MTB)开始出现并逐渐增多。现有的研究已经证实gyrA基因和gyrB基因的突变是MTB喹诺酮类耐药的主要分子机制,但各研究者所发现的突变位点及突变频率尚存在差异,因此需要进一步的研究来深入分析MTB的喹诺酮类耐药机制。本研究通过检测耐喹诺酮类结核分枝杆菌临床菌株gyrA基因和gyrB基因的突变类型和突变频率,分析突变类型与耐药水平之间的关系,阐述gyrA基因和gyrB基因突变在结核分枝杆菌喹诺酮类耐药中作用。方法从肺结核患者的痰液中分离出67株MTB临床菌株,采用绝对浓度法测定MTB临床菌株对左氧氟沙星(LVX)的敏感性,并应用聚合酶链反应(PCR)和DNA直接测序(DS)技术检测其gyrA基因和gyrB基因的突变情况。结果 1. 64株MTB临床LVX耐药菌株有47株gyrA基因序列发生突变,突变率为73.4%。其中42株低浓度耐药菌株有27株存在突变,突变率为64.3%;22株高浓度耐药菌株有20株发生突变,突变率为90.9%。47株gyrA基因突变菌株中,其中45株为单位点突变,另2株为双位点突变。单位点突变发生在70位、89位、90位、91位和94位,双位点突变发生在90位+94位,94位为突变频率最高的位点,占总例数的63.8%。具体的突变类型包括H70R、D89N、A90V、S91A、D94G、D94A、D94N等7种单位点突变和1种A90V+D94A双位点突变。其中,H70R、D89N单位点突变和A90V+D94A双位点突变均只导致高浓度耐药,而其它突变类型即可致低浓度耐药也可引起高浓度耐药。 2. 64株LVX耐药MTB临床菌株中,仅有1株发生gyrB基因突变,为Thr511Asn突变。结论 gryA基因和gyrB基因的突变是MTB喹诺酮类耐药的主要分子机制,其不同的突变类型可导致不同程度的耐药水平改变。但是,gryA基因和gyrB基因的突变并不能解释所有MTB喹诺酮类耐药现象。因此,有必要对其它耐药机制(如药物外排泵)进行研究。
[Abstract]:Research background and purpose
Tuberculosis is an infectious disease seriously affected by human beings. In 2006, 1 million 800 thousand people worldwide died of tuberculosis and 9 million 200 thousand were newly diagnosed with tuberculosis. The disease needed a long time of combined treatment to cure, but the drugs used for the treatment of the disease were limited and with drug-resistant TB, especially MDR - TB. As a broad-spectrum antibiotic, quinolones are gradually used for the treatment of tuberculosis and may be used as a first-line drug. Unfortunately, with the widespread use of such drugs and the misuse and misuse of different degrees, tolerance to this type of drugs is unfortunately tolerated. Mycobacterium tuberculosis (MTB) begins to appear and increases gradually. Existing studies have confirmed that the mutation of gyrA and gyrB genes is the main molecular mechanism of MTB quinolone resistance, but the mutation sites and frequency of mutation found by the researchers are still different, so a further study is needed to analyze the resistance of MTB to quinolones. Mechanism.
In this study, the relationship between mutation type and drug resistance level was analyzed by detecting the mutation type and mutation frequency of gyrA and gyrB gene in clinical strains resistant to quinolone Mycobacterium tuberculosis, and the use of gyrA and gyrB gene mutations in quinolone resistance of Mycobacterium tuberculosis was described.
Method
67 MTB clinical strains were isolated from the sputum of the patients with pulmonary tuberculosis. The sensitivity of MTB clinical strains to levofloxacin (LVX) was determined by absolute concentration, and the mutation of the gyrA gene and gyrB gene was detected by polymerase chain reaction (PCR) and DNA direct sequencing (DS).
Result
There were 47 strains of gyrA gene sequence mutation in 1.64 MTB clinical LVX resistant strains. The mutation rate was 73.4%. of which 42 strains of low concentration resistant strains were mutated, the mutation rate was 64.3%, 20 strains of high concentration resistant strains of 22 strains were mutated, the mutation rate was 90.9%.47 strain gyrA mutant strain, 45 of them were unit point mutation and 2 was double. Site mutation. Unit point mutation occurred in 70, 89, 90, 91 and 94. The double site mutation occurred at 90 +94 sites and 94 was the highest mutation frequency. The 63.8%. specific mutation types included 7 unit point mutations, such as H70R, D89N, A90V, S91A, D94G, D94A, D94N, and 1 A90V+D94A double site mutations. Point mutation and A90V+D94A double site mutation only lead to high concentration of resistance, while other mutant types can cause low concentration resistance and high concentration resistance.
Of the 2.64 LVX resistant MTB clinical isolates, only 1 of them had gyrB gene mutation, which was Thr511Asn mutation.
conclusion
Mutations in the gryA and gyrB genes are the main molecular mechanisms of the resistance to MTB quinolones, and the different mutations can lead to varying levels of resistance to varying degrees. However, mutations in the gryA and gyrB genes do not explain all MTB quinolones resistance. Therefore, it is necessary to study other mechanisms of drug resistance, such as the drug efflux pump. Study.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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