肺炎克雷伯菌耐药突变选择窗的体内外研究

发布时间：2018-01-09 19:20

本文关键词：肺炎克雷伯菌耐药突变选择窗的体内外研究　出处：《中国医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的体外测定氟喹诺酮类药物左氧氟沙星对临床分离肺炎克雷伯菌的最低抑菌浓度(Minimum Inhibitory Concentration, MIC)和防突变浓度(Mutant Prevention Concentration, MPO)。建立小鼠肺部肺炎克雷伯菌感染模型,测定相关药代／药效动力学(PK/PD)参数,观察药物浓度对耐药突变菌株富集程度的影响,结合体外实验测定结果,在动物体内初步验证突变选择窗(MSW)理论,并通过MSW理论与已有的药代动力学参数,对临床左氧氟沙星治疗肺炎克雷伯菌肺炎的给药策略进行评价。方法采用CLSI规定的标准琼脂平皿二倍稀释法,测定氟喹诺酮药物左氧氟沙星对临床分离肺炎克雷伯菌的MIC和MPC,计算选择指数(SI),选择铜绿假单胞菌ATCC27853作为质控菌株；鼻腔滴注法建立小鼠肺部肺炎克雷伯菌感染模型,比较不同接种剂量组死亡情况,确定最佳造模剂量；根据最终确定造模剂量建立小鼠肺部肺炎克雷伯菌感染模型,通过灌胃方法分别给予15mg/kg、30 mg/kg、60 mg/kg和90mg/kg左氧氟沙星治疗,考察不同剂量组耐药突变株的出现频率及治疗后MIC变化情况,评价不同剂量组与细菌耐药突变株富集扩增的关系；采用高效液相色谱法(HPLC)测定模型组小鼠治疗一天、三天、五天、七天后的左氧氟沙星血药浓度,参考相关PK/PD常数,结合体外测定MPC和MSW值评价临床应用左氧氟沙星治疗细菌性感染的剂量与疗效关系。结果体外测定临床分离肺炎克雷伯菌的MIC和MPC分别为0.0625μg／ml和1μg／ml,SI=16,根据CLSI药敏标准,此菌株为对左氧氟沙星敏感菌株；三个接种剂量组死亡率无明显差异,综合考虑接种时间、接种剂量等方面因素,确定最终接种浓度为108CFU/ml,接种剂量为30μl；给予不同剂量左氧氟沙星治疗过程中,15mg/kg剂量组和90mg/kg剂量组,突变率较恒定,无明显升高,MIC值多无变化,而30mg/kg和60mg/kg剂量组突变率明显升高,MIC值升高的动物数明显增多,30mg/kg主要选择低水平突变,MIC值较接种时升高2倍,而60mg/kg剂量组则主要选择高水平突变,MIC值较接种时升高8倍；HPLC测定结果表明,在治疗期间内,血药浓度位于MSW内的时间越长,细菌的突变率越高。结论不同剂量左氧氟沙星治疗肺炎克雷伯菌感染的细菌突变率不同,不适当剂量的药物虽可以达到治疗目的,却会引起细菌突变株扩增,在一定范围内水平传播,加速细菌耐药。根据本文的研究结果,推荐临床应用左氧氟沙星治疗细菌性感染疾病时,在兼顾安全的前提下采用大剂量冲击疗法。
[Abstract]:objective
In vitro determination of fluoroquinolone levofloxacin and minimum inhibitory concentration of clinical isolates of Klebsiella pneumoniae (Minimum Inhibitory, Concentration, MIC) and mutant prevention concentration (Mutant Prevention Concentration, MPO). The establishment of the lungs of mice infected with Klebsiella pneumoniae model, determination of pharmacokinetic / pharmacodynamic (PK/PD) parameters, to observe the drug the concentration of drug resistance mutations affect the enrichment degree of strain, combined with the determination results of experiments in vitro and in vivo animal preliminary validation of mutant selection window (MSW) theory, and the pharmacokinetic parameters of MSW theory and the evaluation of clinical levofloxacin in the treatment of pneumonia caused by Klebsiella pneumoniae dosing strategy.
Method
Using standard agar CLSI prescribed two times dilution method, determination of fluoroquinolone levofloxacin in clinical isolates of Klebsiella pneumoniae MIC and MPC, calculate the selection index (SI), selection of Pseudomonas aeruginosa ATCC27853 as control bacteria; nasal instillation method to establish mouse lung of Klebsiella pneumoniae infection model. The comparison of the death of different inoculation doses to determine the optimum dose; according to the final dose of mice lung Klebsiella pneumoniae infection model, 15mg/kg, were given by gavage for 30 mg/kg, 60 mg/kg and 90mg/kg of levofloxacin in the treatment of resistant strains of different dosage groups, investigated the frequency and changes of MIC after treatment the mutation, evaluation of different dose groups and bacteria resistant mutants were amplified by relationship enrichment; high performance liquid chromatography (HPLC) determination of model mice for a day, three days, five days, seven days After the levofloxacin blood concentration, refer to the relevant PK/PD constant, combined with MPC and MSW values in vitro to evaluate the relationship between the dose and efficacy of levofloxacin in the treatment of bacterial infections.
Result
Determination of clinical isolates of Klebsiella pneumoniae MIC and MPC were 0.0625 g / ml and 1 g / ml, SI=16 in vitro, drug sensitivity according to the CLSI standard, this strain is susceptible to levofloxacin strains; no significant differences between the three dose group mortality, considering the time of inoculation, inoculation amount etc. factors that determine the final inoculation concentration is 108CFU/ml, with the dose of 30 L; given different doses of levofloxacin in the treatment process, 15mg/kg and 90mg/kg groups, the mutation rate is relatively constant, significantly increased, MIC value had no change, while 30mg/kg and 60mg/kg dose group mutation rate was significantly increased, the MIC value of the number of animal elevated 30mg/kg increased significantly, the main choice of low level mutation, the MIC value is 2 times higher when inoculated, while the 60mg/kg group mainly choose high levels of mutation, the MIC value is vaccination increased 8 times; the results of HPLC analysis show that in the treatment period, blood concentration The longer the time in MSW, the higher the rate of mutation of bacteria.
conclusion
Different doses of levofloxacin in the treatment of Klebsiella pneumoniae infections in different mutation rate, improper drug dose can achieve the goal of treatment, but can cause bacterial mutant amplification and transmission within the certain range, accelerate the bacterial resistance. According to the results of this study, the recommended clinical application of levofloxacin in the treatment of bacterial infection disease and the impact of high-dose therapy on the premise of safety.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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