北京地区结核分枝杆菌耐药分子特点及结核分枝杆菌耐喹诺酮类药物泵机制的初步研究

【作者】 孙勇；

【导师】 许绍发；

【作者基本信息】 北京市结核病胸部肿瘤研究所 ， 免疫学， 2012， 博士

【摘要】 目的研究北京地区结核分枝杆菌临床分离株katG、inhA、oxyR、ahpC、ndh、dfrA、kasA、inhA启动子区、oxyR-ahpC基因间区、rpoB、rpsL、rrs、gyrA、gyrB、tlyA和thyA基因基因突变特点，探讨结核分枝杆菌对异烟肼、利福平、链霉素、乙胺丁醇、喹诺酮类、阿米卡星、卷曲霉素和对氨基水杨酸钠的耐药分子机制。方法对303例异烟肼耐药菌株和61例异烟肼敏感菌株、195例利福平耐药菌株和169例利福平敏感菌株、360例链霉素耐药菌株和53例链霉素敏感菌株、266例乙胺丁醇耐药菌株和32例乙胺丁醇敏感菌株、118例喹诺酮耐药菌株和128例喹诺酮敏感菌株、10例阿米卡星耐药菌株、25例卷曲霉素耐药菌株、15例阿米卡星和卷曲霉素交叉耐药菌株及30例双敏感菌株、31例对氨基水杨酸钠耐药菌株和65例对氨基水杨酸钠敏感菌株临床分离株相关基因进行PCR扩增及DNA测序分析。结果①303例耐异烟肼结核分枝杆菌有84.8%katG基因发生突变，97.4%突变集中在katG基因85-463密码子区，其中S315T位点最多占55.4%，而R463L位点在敏感菌株中突变达到80.3%高于耐药菌株62.7%(P<0.05)；inhA启动子区18.8%的点突变发生在-15(C→T)高于敏感菌株的4.9%(P<0.05)；在oxyR-ahpC间区突变菌株，异烟肼耐药菌株比例是9.6%高于敏感菌株的1.6%(P<0.05)。②在195例利福平耐药菌株中，168例（86.2%）rpoB基因发生突变，突变主要集中在531（41%）、526（22.5%）、516（9.7%）三个位点，同时在27例多位点突变的菌株中，25例（92.6%）为利福平耐药菌株。③360例链霉素耐药菌株中，分别有71.7%rpsL基因和11.6%rrs基因发生突变，其中rpsL基因K43R和K88R位点突变比例分别是60.6%和11.1%，而rrs基因A1401G位点，其在耐药中比例（2.2%）少于敏感菌株（9.4%），两组间差异有统计学上意义（P<0.05）。④266例乙胺丁醇耐药菌株中，共检测到45.1%的embB基因突变菌株，98.3%突变发生在319-497密码子区，其中突变最多的位点是306位点（26.1%）。⑤在118例喹诺酮类耐药菌株中，共检测到74.6%的菌株gyrA基因突变，最常见是94位点（49.9%）和90位点（19.5%）⑥从tlyA基因突变结果显示，阿米卡星和敏感菌株均未发现突变株；卷曲霉素单耐药菌株有6株突变（24%），在阿米卡星和卷曲霉素交叉耐药菌株中有1例同义突变；A1401G在阿米卡星、卷曲霉素、阿米卡星和卷曲霉素交叉耐药菌株中突变比例10%、8%、13.3%；C1402T在阿米卡星、阿米卡星和卷曲霉素交叉耐药中各1例。⑦在31例对氨基水杨酸钠耐药菌株中，71%thyA基因发生突变，以第19位碱基C缺失和第168位碱基C缺失突变为主，在耐药菌株中的突变率分别是54.8%、25.8%，其敏感菌株中突变率也达了35.5%、12.3%，耐药与敏感相比两个位点突变差异均没有统计学意义（P>0.05）。结论本项研究进一步明确了北京地区结核分枝杆菌8种抗结核药物与相关基因之间的关系，有助于北京地区流行分子特点的研究及建立快速、特异、准确的药物敏感性分子诊断的方法。目的筛选左氧氟沙星（LFX）耐药和敏感的临床分离菌株，分析外排泵系统在LFX耐药的作用，并初步筛选LFX类药物的外排泵基因。方法采用刃天青的药敏检测方法，分析加入三种泵抑制剂利血平（Res）、羰基氰氯苯腙（CCCP）和维拉帕米(Ver)前后最低抑菌浓度（MIC）的变化，并通过荧光定量PCR的方法检测药物刺激组和药物未刺激组对编码13种外排泵基因表达量的影响。结果筛选得到59例LFX耐药菌株和4例敏感菌株。在应用泵抑制剂之前，耐药菌株MIC大于2mg/l（包含2mg/l）的菌株共52株，MIC小于2mg/l菌株共7株；在应用泵抑制剂Res之后，耐药菌株MIC大于2mg/l菌株共43株，MIC小于2mg/l菌株共16株，应用泵抑制剂前后差异有统计学意义(X2=4.374，P<0.05)，MIC值降低2-16倍；在应用泵抑制剂CCCP之后，59株耐药菌株MIC值全部小于2mg/l，应用泵抑制剂前后差异有统计学意义(X2=970，P<0.05)，MIC值降低2-64倍；在应用泵抑制剂Ver之后，耐药菌株MIC大于2mg/l共菌株31株，MIC小于2mg/l菌株共28株，耐药率下降明显，应用泵抑制剂前后差异有统计学意义(X2=17.913，P<0.05)， MIC值降低2-64倍；同时泵抑制剂对4例敏感菌株外排泵系统也有不同程度的抑制作用，其中抑制剂CCCP和Ver作用最明显：应用泵抑制剂后仅有1株MIC值为2mg/l，而应用泵抑制剂Res后外排泵作用不明显。对59例LFX耐药菌株和4例LFX敏感的菌株gyrA和gyrB基因进行检测，45株（95.8%，45/47）突变中MIC值大于2mg/l，仅两株D94G突变菌株MIC值小于2mg/l。此外发现有12株耐药不突变的菌株，而63株结核分枝杆菌gyrB基因均未发现突变。12株耐药不突变的菌株MIC值结果显示，在应用CCCP泵抑制剂后，12株耐药菌株MIC值降低2-16倍；在应用Ver泵抑制剂，10株耐药菌株MIC值降低2-4倍，仅451和575菌株MIC值没有发生变化；在应用Res泵抑制剂后，729、777、896、838、529、642、325菌株MIC值降低2-4倍以上，其他5株在应用泵抑制剂后MIC值没有变化。在LFX药物刺激后，结果显示药物处理后有10种泵基因水平下降，其中Rv1348和Rv2686c下降比较明显，其基因水平分别是药物处理前的0.52和0.42倍。有三个泵基因水平有所上升，分别是Rv2846c基因水平是药物处理前的1.21倍；Rv3239c基因水平是药物处理前的1.17倍；Rv2936基因水平是药物处理前的1.06倍。结论靶基因gyrA突变是喹诺酮的耐药的主要机制，而且其突变与喹诺酮高水平耐药相关。分枝杆菌的外排泵系统参与了喹诺酮耐药的发生，而泵抑制剂对外排泵系统有抑制作用，其中CCCP作用最明显。潜在的外排泵基因Rv2846c、Rv3239c和Rv2936在耐药菌株中表达上调，可能参与结核分枝杆菌耐喹诺酮药物的发生。更多还原

【Abstract】 Objective: To study the Beijing area isolates of Mycobacterium tuberculosis katG,inhA, oxyR, ahpC, ndh, dfrA, kasA, inhA promoter region, oxyR-ahpC intergene,rpoB, rpsL, rrs, gyrA, gyrB, tlyA and thyA genes mutation characteristics, and discussthe Mycobacterium tuberculosis to isoniazid, rifampicin, streptomycin, ethambutol,quinolones, amikacin, capreomycin and sodium para-aminosalicylic resistantmolecular mechanism. Methods:303cases of isoniazid-resistant strains and61casesof isoniazid-susceptible strains,195cases of rifampicin-resistant strains and169cases of rifampicin-susceptible strains,360cases of streptomycin-resistant strains and53cases of streptomycin-susceptible strains,266cases of ethambutol-resistant strainsand32cases of ethambutol-susceptible strains,118cases of quinolone-resistantstrains and128cases of quinolone-susceptible strains,10cases of amikacin resistantstrains,25cases of capreomycin-resistant strains,15cases of amikacin andcapreomycin cross-resistant strains and30cases of double-susceptible strains,31cases of sodium para-aminosalicylic resistant strains and65cases on the sodiumpara-aminosalicylic susceptible of clinical isolates related genes were amplified byPCR and DNA sequencing. Results:(1) The303cases of isoniazid-resistantMycobacterium tuberculosis, katG gene mutations rate was84.8%and97.4%,mutation was focus of katG85-463codon district, including the S315T sites mutationrate was up to55.4%, while80.3%of the R463L mutation sites rate in the susceptiblestrains was higher than62.7%in drug-resistant strains (P <0.05);18.8%pointmutations of inhA the promoter region in the-15(C→T) was higher than4.9%of thesusceptible strains (P <0.05);9.6%of oxyR-ahpC intergenic region mutant inresitance strains was higher than1.6%of susceptible strains (P <0.05).②In the195cases of rifampicin resistant strains,168cases (86.2%) had rpoB gene mutation, themutations were mainly in the531(41%),526(22.5%),516(9.7%) sites, while25cases (92.6%) were rifampicin resistant strains in27multisite mutations strains.③The360cases of streptomycin resistant strains, the rpsL gene mutation rate was71.7%and rrs gene mutation rate was11.6%, respectively; the rpsL gene of K43R and K88R point mutation proportion was60.6%and11.1%, respectively, while therrs gene A1401G site (2.2%) in the resistant strains was less than the susceptiblestrains (9.4%), statistically significant was different between the two groups (P <0.05).④The266cases of ethambutol resistant strains were detected, mutation rate of theembB gene was45.1%,98.3%of mutations was in the codon region of319-497,306codon is most common (26.1%).⑤In the118cases of quinolone resistant strainswere detected, mutation rate of gyrA gene was74.6%, the most common was94codon (49.9%) and90codon (19.5%).⑥The tlyA gene mutation results showed thatamikacin and susceptible strains had any mutant; capreomycin had six mutations(24%) in single drug-resistant strains, amikacin and capreomycin cross-resistantstrains had one case of synonymous mutations; mutation rate of A1401G in amikacin,capreomycin, amikacin and capreomycin cross resistant strains were10%,8%,13.3%,respectively; C1402T in amikacin, amikacin and capreomycin cross-resistance drugshad one case, respectively.⑦T he31cases of para-aminosalicylic resistant strains,mutation rate of thyA gene was71%,19deletion base C and168deletion bp Cmutation were most common, the mutation rate were54.8%,25.8%in the resistantstrains,35.5%and12.3%in susceptible strains; two sites mutation differences in theresistant and susceptible strains were not statistically significant (P>0.05).Conclusions: This study further clarifies the relationship between the eight kinds ofanti-TB drugs of Mycobacterium tuberculosis with gene in Beijing, and contribute tostudy the popular molecular characteristics of the Beijing and establish rapid, specific,and accurate drug susceptibility molecular diagnostic methods. Objective: Selecting of levofloxacin (LFX) resistant and sensitive clinicalisolates，to analyse the effect of efflux pump systems in the LFX resistance and screenof the LFX drug efflux pump genes. Methods: The minimum inhibitoryconcentration (MIC) changes was analysing before and after adding the three pumpinhibitor reserpine (Res),carbonyl cyanide-chlorophenyl hydrazone (CCCP) andverapamil (Ver) by the resazurin susceptibility testing methods,13kinds of effluxpump gene expression were detected by quantitative PCR after drug stimulating.Results:59cases were the LFX resistant strains, and four cases were sensitive strains.Before using the pump inhibitors,52drug-resistant strains of the MIC were greaterthan2mg/l (containing2mg/l),7strains of the MIC were less than2mg/l; afterapplication of pump inhibitors Res,43resistant strains of MIC were greater than2mg/l,16strains of the MIC were less than2mg/l, there was significant differencebefore and after application of pump inhibitors (X2=4.374, P<0.05), MIC values waslower2-16times; after application of pump inhibitor CCCP, all the59resistantstrains of MIC values were less than2mg/l, difference was statistically significantbefore and after the application of pump inhibitor (X2=970, P <0.05), MIC valueswas lower2-64times; after application of pump inhibitors Ver,31resistant strains ofMIC were greater than2mg/l,28strains of the MIC were less than2mg/l,resistance rate has dropped significantly， the difference was statistically significant(X2=17.913, P <0.05) before and after the application of pump inhibitors, the MICvalues was lower2-64times; pump inhibitors also had effected the four cases ofsusceptible strains.The effect of CCCP and Ver was most obvious: after applicationof pump inhibitors only a MIC value was2mg/l, while the application of the pumpinhibitors Res, MIC changes was not obvious. The gyrA and gyrB genes weredetected in59LFX resistant strains and4LFX-sensitive strains,45(95.8%,45/47)mutations strains of the MIC values were greater than2mg/l, only the two D94Gmutant strains MIC values were2mg/l. Furthermore,12resistant strains had no any mutant, all63strains of gyrB gene had no mutant.12resistant mutant strains MICvalues results show that the12resistant strains of the MIC values were lower2-16times after the application of pump inhibitors CCCP; the10resistant strains of MICvalues decreased2-4times after the application of pump inhibitor Ver, only451and575strains of the MIC values did not change;729,777,896,838,529,642and325strains of the MIC values reduced by2-4times after application of pump inhibitorsRes, the other five strains of the MIC values did not change after the application ofpump inhibitors. After the LFX drugs to stimulate, the results showed that the10kinds of pump gene level were decreased after drug treatment, including Rv1348andRv2686c, level of genes were0.52and0.42times comparing pre-drug treatment. Thelevel of three pump gene were increased, Rv2846c gene level was1.21timescomparing pre-drug treatment; Rv3239c gene level was1.17times comparingpre-drug treatment; Rv2936gene level was1.06times comparing pre-drug treatment.Conclusions:Target genes of gyrA mutation was the main mechanism of quinoloneresistance and associated with high level of quinolone resistance. Mycobacterialefflux pump systems involved in the occurrence of quinolone resistance, pumpinhibitor could inhibited efflux pump system, which CCCP of effect is the mostsignificant. Potential efflux pump genes level of Rv2846c, Rv3239c and Rv2936areincreased in the resistant strains, may be involved in the occurrence ofMycobacterium tuberculosis resistant to quinolone drugs.更多还原