结核分枝杆菌耐药性及耐药相关基因分子特征研究

发布时间：2018-04-24 10:32

本文选题：结核 + 复治肺结核患者　；参考：《中国疾病预防控制中心》2015年硕士论文

【摘要】：目的：基于专科医院数据,了解初复治肺结核患者的耐药谱及流行情况。阐明结核分枝杆菌耐药相关基因的突变频率和分子机制,并在多位点基因测序的基础上分析耐药相关基因的遗传多样性。方法：研究纳入2013年6月到2014年12月来自北京胸科医院的结核分枝杆菌临床分离株,培养后通过MGIT 960液体药敏实验对15种抗结核药物,包括异烟肼(Isoniazid, INH)、利福平(Rifampicin, RIF)、链霉素(Streptomycin, STR)、乙胺丁醇(Ethambuto, EMB)、卡那霉素(Kanamycin, KAN)、阿米卡星(Amikacin、AMK)、卷曲霉(Capreomycin, CPM)、氧氟沙星(Ofloxacin, OFX)、左氧氟沙星(Levofloxacin, LFX)、莫西沙星(Moxifloxacin、MFX)、对氨基水杨酸(Paza-aminosalicylate、PAS)、丙硫异烟胺(Protionamide、PTO)、利奈唑胺(Linezolid, LZD)、乙硫异烟胺(Ethionamide, ETH)、吡嗪酰胺(Pyrazinamide, PZA)进行耐药表型实验,并对耐药相关基因进行序列测定和分析。结果：本研究共纳入500例临床样本,菌种鉴定71例为NTM,12例分离培养污染,剩余417例结核分枝杆菌临床分离株进行四种一线抗结核药物的耐药表型检测,其总耐药率为47.2%(192/417),MDR耐药率为28.2%(120/417)。其中复治患者的总耐药率和MDR耐药率分别为66.3%(122/184)和47.3%(87/184),初治患者分别为32.2%(75/233)和14.2%(33/233),复治患者的总耐药率和MDR耐药率均显著高于初治患者(P0.001)。完成一线药敏后,筛选100例复治肺结核患者和50例初治肺结核患者样本进行后续11种抗结核药物的耐药表型实验(包括PZA、AMK、KAN、CPM、 OFX、LFX、MFX、PAS、PTO、LZD、ETO),有5例在药敏实验过程中发生污染,剩余初治患者样本48例,复治患者样本97例。其中PZA、AMK、 KAN、CPM、OFX、LFX和MFX对于复治患者的耐药率分别为34.0%(33/97)、13.4(13/97)、18.6%(18/97)、29.9%(29/97)、39.2%(38/97),33.0%(32/97)和35.1%(34/97),而初治患者的耐药率分别为12.5%(6/48)、0.0(0/48)、4.2%(2/48)、6.3%(3/48)、8.3%(4/48),12.5%(6/48)和12.5%(6/48)。复治患者对于这几种药物的耐药率显著高于初治患者(P0.05)。而PAS、PTO、LZD和ETO这4种药物在初、复治患者的耐药率之间无统计学差异。对已知15种药物耐药表型检测结果的结核分枝杆菌就耐药相关基因进行测序,结果显示对于INH表型耐药的菌株中katG的突变率为89.3%(50/56),以S315T突变为主；inhA的突变率为10.7%(6/56),以启动子区-15(C→T)突变为主。这两个位点可作为INH耐药的快速诊断的分子标记。oxyR-ahpC间区的突变率为7.1%,且所有突变位点均未在INH敏感的菌株中发现,提示该区间可能与INH耐药相关。katG的R463L位点与accD6的D229G位点的突变,在耐药和敏感株中突变比率相似,提示这两个位点可能与INH耐药无关。对于RIF, rpoB的突变率为85.9%,是RIF耐药主要机制。rpoB突变中以531和526为主要突变位点,可用于RIF耐药的快速诊断。对于STR,结核分枝杆菌rpsL和rrs的突变是链霉素耐药的主要分子机制。rpsL的突变率为56.1%,在STR耐药株中只发现K43R和K88R两个突变位点,该位点可用于STR的快速诊断。gidB的E92D+A205D联合突变出现在94.7%的结核分枝杆菌中,提示此突变可能与北京家族基因型相关。对于EMB,结核分枝杆菌EMB耐药发生与embB突变有关。研究中embB的突变率为50%,而306位点突变率可达到35%。该位点可用于EMB耐药的快速诊断的分子标记。结核分枝杆菌embB 297-497密码子区可能是EMB耐药决定区。二线抗结核药物包括喹诺酮类(Fluoroquinolone, FQNs)和注射类(Injectable Drugs, INJs)。对于FQNs,其耐药的发生主要与gyrA突变有关,本研究中gyrA的突变率达到77.8%,可用于FQNs耐药的快速诊断,其主要突变发生在是94和90位点。另外研究中显示gyrB可能与FQNS耐药没有相关性。对于INJs,结核分枝杆菌tlyA显示与CPM耐药相关,与AMK耐药无关。rrsA1401G和C1402T突变显示可能与AMK和CPM交叉耐药有关。对于PZA,在耐药菌株中pncA突变存在高度多样性且分布广泛,其突变的突变率为69.7%。提示与PZA耐药相关。对于PAS,其耐药发生可能与thyA突变有关。在本研究25例PAS耐药菌株中,thyA的突变率为12%,各突变位点都仅有1例发生,无法确定特异性耐药位点。对于ETH,其耐药相关基因ethA的突变率为34.2%。突变类型存在高度多样性,基本分布在1-1267碱基之间。通过多位点测序结果进行遗传多样性发现,MDR和XDR菌株dN/dS率大于1,显示耐药相关基因存在被正向选择驱动的进化。且MDR和XDR菌株的核苷酸多样性明显高于敏感株,不同组菌株间存在统计学差异(P0.05)。结论：专科医院就诊肺结核患者耐药率较高,且复治肺结核患者中耐药率明显高于初治患者。全面分析了常见抗结核药物的耐药相关基因的突变频率和分子特征,为基于分子生物学快速诊断技术的应用提供参考,并初步探讨了耐药相关基因的遗传多样性和选择强度。
[Abstract]:Objective: to understand the drug resistance spectrum and epidemic situation of the patients with primary and treatment pulmonary tuberculosis based on the data of specialized hospital. To clarify the mutation frequency and molecular mechanism of the resistance related genes of Mycobacterium tuberculosis, and to analyze the genetic diversity of the resistance related genes on the basis of the sequencing of multilocus genes. From the clinical isolates of Mycobacterium tuberculosis in Beijing Thoracic Hospital, 15 anti tuberculosis drugs, including isoniazid (Isoniazid, INH), Li Fuping (Rifampicin, RIF), Streptomycin, STR, ethambutol (Ethambuto, EMB), kanamycin (Kanamycin, KAN), and Amikacin (Amikacin,), were tested through the MGIT 960 liquid drug sensitivity test. Capreomycin (CPM), ofloxacin (Ofloxacin, OFX), Levofloxacin, LFX, moxifloxacin (Moxifloxacin, MFX), p-aminosalicylic acid (Paza-aminosalicylate, PAS), propylene thioisoamine (Protionamide, PTO), linezolid, ethyl thiazinamide, and pyrazinamide. A total of 500 clinical samples were included in this study. In this study, 71 cases were identified as NTM, 12 cases were isolated and cultured, and the remaining 417 Mycobacterium tuberculosis clinical isolates were tested for the drug resistant phenotype of four first-line anti tuberculosis drugs, the total resistance rate was 47.2% (192/417), M The drug resistance rate of DR was 28.2% (120/417). The total resistance rate and MDR resistance rate of the retreated patients were 66.3% (122/184) and 47.3% (87/184) respectively. The initial treatment patients were 32.2% (75/233) and 14.2% (33/233) respectively. The total drug resistance rate and the MDR resistance rate of the retreated patients were significantly higher than those of the first treated patients (P0.001). After completing the first-line drug sensitivity, 100 cases of retreated pulmonary tuberculosis patients were screened. PZA, AMK, KAN, CPM, OFX, LFX, MFX, PAS, PTO, LZD, ETO) were carried out in 50 cases of first treated tuberculosis patients (including AMK, OFX, LFX, MFX, PAS, PTO, LZD, ETO). There were 5 cases in the drug sensitivity test, 48 cases of residual treatment and 97 cases of retreated patients. The resistance rates were 34% (33/97), 13.4 (13/97), 18.6% (18/97), 29.9% (29/97), 39.2% (38/97), 33% (32/97) and 35.1% (34/97), while the drug resistance rates of the first treated patients were 12.5% (6/48), 0 (0/48), 4.2% (2/48), 6.3% (3/48), 8.3% (4/48), 12.5% (4/48), and 12.5%. The drug resistance rate of these retreated patients was significantly higher than that of the first treated patients. .05). There was no statistical difference between the 4 drugs of PAS, PTO, LZD and ETO. The resistance related genes of Mycobacterium tuberculosis with known 15 drug resistance phenotypes were sequenced. The results showed that the mutation rate of katG in the strains resistant to INH phenotypes was 89.3% (50/56), and inhA was dominated by S315T mutation; inhA. The mutation rate was 10.7% (6/56) and the main mutation was -15 (C to T) in the promoter region. The two loci could be used as a molecular marker for the rapid diagnosis of INH resistance. The mutation rate was 7.1%, and all the mutation sites were not found in the INH sensitive strain, suggesting that the interval could be associated with R463L loci of.KatG associated with INH resistance and accD6 D229G. Point mutation is similar in resistance and sensitive strain, suggesting that these two loci may not be related to INH resistance. For RIF, the mutation rate of rpoB is 85.9%, and the main mechanism of RIF resistance is 531 and 526 as the main mutation site, which can be used for the rapid diagnosis of RIF resistance. For STR, the mutation of Mycobacterium tuberculosis rpsL and RRS is the chain. The mutation rate of.RpsL, the main molecular mechanism of mycophenin resistance, was 56.1%. Only the two mutation sites of K43R and K88R were found in the STR resistant strains. This site could be used for the E92D+A205D joint mutation of STR in the rapid diagnosis of.GidB in 94.7% Mycobacterium tuberculosis, suggesting that the mutation may be related to the genotype of the Beijing family. For EMB, the Mycobacterium tuberculosis is a member of the Mycobacterium tuberculosis. EMB resistance is associated with embB mutation. The mutation rate of embB in the study is 50%, and the mutation rate of the 306 loci can reach 35%., a molecular marker for rapid diagnosis of EMB resistance. The embB 297-497 codon area of Mycobacterium tuberculosis may be the EMB resistant region. The second line anti tuberculosis drugs include quinolones (Fluoroquinolone, FQNs), and Injection class (Injectable Drugs, INJs). For FQNs, the occurrence of drug resistance is mainly related to gyrA mutation. In this study, the mutation rate of gyrA is 77.8%, which can be used for the rapid diagnosis of FQNs resistance. The main mutation occurs at the 94 and 90 loci. In addition, the study shows that gyrB may not be associated with FQNS resistance. INJs, tlyA Mycobacterium tuberculosis It was found to be associated with CPM resistance..rrsA1401G and C1402T mutations associated with AMK resistance were associated with AMK and CPM cross resistance. For PZA, there was a high diversity and wide distribution of pncA mutations in resistant strains. The mutation rate of the mutant was associated with PZA resistance. For PAS, the occurrence of drug resistance may be associated with thyA mutations. In the study of 25 PAS resistant strains, the mutation rate of thyA was 12%, and only 1 of the mutation sites were occurring, and the specific resistance loci were not determined. For ETH, the mutation rate of the drug resistance related gene ethA was highly diverse in the 34.2%. mutation type and was basically distributed among the 1-1267 bases. Genetic diversity was carried out by the results of multipoint sequencing. The dN/dS rate of MDR and XDR was more than 1, indicating that the resistance related genes were driven by positive selection, and the nucleotide diversity of MDR and XDR strains was significantly higher than that of the sensitive strains, and there was a statistical difference between the different groups (P0.05). Conclusion: the drug resistance rate of the patients with pulmonary tuberculosis in the hospital was higher and the drug resistance rate was retreated in the patients with pulmonary tuberculosis. The mutation frequency and molecular characteristics of resistance related genes of common anti tuberculosis drugs were analyzed, and the genetic diversity and selection intensity of resistance related genes were preliminarily discussed.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R52;R446.5

【相似文献】