替加环素对多重耐药鲍曼不动杆菌的体外抗菌活性和防耐药突变研究

发布时间：2018-07-14 10:11

【摘要】：目的：测定替加环素对多重耐药鲍曼不动杆菌（MDR-AB）的最低抑菌浓度（MIC），并在此基础上应用Monte-Carlo模拟进行PK/PD研究，评价不同剂量替加环素治疗MDR-AB肺炎效果。评价替加环素联合多粘菌素E或舒巴坦对MDR-AB的抗菌效应；研究质子泵抑制剂（PPIs）对替加环素体外抗菌活性的影响。基于耐药突变选择窗（MSW）理论，测定替加环素单用及联合多粘菌素E或舒巴坦限制MDR-AB耐药发生的能力，初步探究MDR-AB对替加环素的耐药机制。方法：（1）采用琼脂平板二倍稀释法测定替加环素对135株MDR-AB的MIC，并结合410例肺炎患者的替加环素药代学数据，通过Monte-Carlo模拟计算不同剂量替加环素时AUC/MIC值分布，再根据预设折点计算治疗MDR-AB肺炎的累积反应分数（CFR）。（2）参照棋盘法设计，采用微量肉汤稀释法测定替加环素与多粘菌素E、舒巴坦对70株MDR-AB的联合MIC，计算联合抑菌指数（FICI）并判定联合效应。（3）采用琼脂平板二倍稀释法测定替加环素单药及联合PPIs对6种临床常见分离菌的MIC；采用菌落计数法测定替加环素单药及联合PPIs的体外杀菌曲线。（4）采用琼脂平板二倍稀释法测定替加环素、多粘菌素E及舒巴坦对30株MDR-AB的MIC和防耐药突变浓度（MPC），计算选择指数（SI=MPC/MIC）；随机选取9株细菌，测定替加环素联合用药的MPC，计算SI。（5）测定替加环素单药及联合碳酰氰间氯苯腙（CCCP）对体外筛选耐药突变菌株的MIC；利用RT-PCR技术测定耐药突变菌株外排泵基因AdeB和AdeJ的相对表达情况。结果：（1）135株MDR-AB中97%的菌株对替加环素敏感，3%中介，未发现耐药菌株。以CFR≥90%为预设折点，Monte-Carlo模拟分析表明，推荐给药剂量（50mg/Lq12h）的CFR为61.62%，低于折点值；给药剂量为100mg q12h时，CFR为89.86%，接近折点值。（2）联合用药后，替加环素对MDR-AB的MIC50显著下降，70株MDR-AB的FICI分布为：与多粘菌素E联用时，FICI≤0.5占4.3%，0.5＜FICI＜1占20%，FICI=1占11.4%，1＜FICI≤2占64.3%，FICI＞2为0；与舒巴坦联用时，FICI≤0.5占10%，0.5＜FICI＜1占54.3%，FICI=1占25.7%，1＜FICI≤2占10%，FICI＞2为0。（3）培养基中加入5-10mg/L的PPIs会使细菌对替加环素MIC50增高0-2倍；加入50mg/L PPIs，细菌MIC50增加4-＞128倍。加入PPIs后的杀菌曲线均位于单药杀菌曲线上方，表明各时间点菌落计数较单药出现不同程度增高。（4）替加环素对MDR-AB的MPC值为4-32mg/L，SI范围为4-64；联合4mg/L多粘菌素E后SI下降2-4倍，联合8mg/L后SI下降4-8倍；联合32mg/L舒巴坦后SI下降2-8倍；联合64mg/L舒巴坦SI后下降4-64倍。（5）联合CCCP后，原菌株MIC并无变化，除AB10号突变菌株外，，其他4株MIC下降了16-128倍。5株耐药突变菌株外排泵基因AdeJ的表达量较原菌株相比均未见明显增高，除AB10号突变菌株外，其余突变菌外排泵基因AdeB表达量增高2倍以上。结论：（1）虽然MDR-AB对替加环素敏感率较高，但Monte-carlo模拟分析表明推荐给药剂量对MDR-AB肺炎的疗效并不理想。（2）替加环素与多粘菌素E联用对MDR-AB以无关作用为主，与舒巴坦联用以部分协同为主。（3）培养基中加入PPIs会影响替加环素的体外抗菌活性。（4）替加环素、多粘菌素E及舒巴坦MPC较高、MSW宽，单用药易致细菌耐药发生；联合用药可有效限制耐药发生。（5）突变菌株对替加环素的耐药机制主要为外排泵基因AdeABC过度表达。
[Abstract]:Objective: to determine the minimum inhibitory concentration (MIC) of tegacycline on multidrug resistant Acinetobacter Bauman (MDR-AB), and to evaluate the effect of different doses of tegicycline in the treatment of MDR-AB pneumonia by Monte-Carlo simulation on the basis of PK/PD, and evaluate the antibacterial effect of tegacycline combined with polymyxin E or sulbactam on MDR-AB. The effect of pump inhibitor (PPIs) on the antiseptic activity of tegagin in vitro. Based on the resistance mutation selection window (MSW) theory, the ability of tegagin single use and combined polymyxin E or sulbactam to restrict the occurrence of MDR-AB resistance was determined, and the mechanism of MDR-AB resistance to tegagin was preliminarily explored.
Methods: (1) the two times dilution method of agar plate was used to determine the MIC of tegagin on 135 strains of MDR-AB and the data of tegadin in 410 patients with pneumonia. The distribution of AUC/MIC value at different doses of tegagin was simulated by Monte-Carlo, and the cumulative reaction fraction (CFR) for the treatment of MDR-AB pneumonia was calculated according to the presupposed fold point. (2) reference Chessboard method, tegagarin and polymyxin E were measured with micro broth dilution method, combined MIC of 70 strains of MDR-AB by Shubatan, combined bacteriostasis index (FICI) was calculated and combined effect was determined. (3) MIC of tigocycline single drug and combined PPIs against 6 clinical common isolates were measured by agar plate two times dilution method; colony counting method was used. The bactericidal curves of tegagin single drug and combined PPIs were measured in vitro. (4) two times dilution method of agar plate was used to determine the MIC and anti drug resistance mutation concentration (MPC) of 30 strains of MDR-AB and the selection index (SI=MPC/MIC), 9 strains of bacteria were selected and MPC of tegicycline combined use was measured, and SI. (5) was calculated. The MIC of resistant mutant strains was screened by CN and CCCP, and the relative expression of AdeB and AdeJ of the efflux pump gene of the mutant strain was determined by RT-PCR technique.
Results: (1) 97% of the 135 strains of MDR-AB were sensitive to tigocyclin, 3% medium, and no drug resistant strains were found. CFR > 90% as a preset point. Monte-Carlo simulation analysis showed that the recommended dosage (50mg/Lq12h) CFR was 61.62%, lower than the fold value; when the dosage was 100mg q12h, CFR was 89.86%, close to the fold value. (2) after the combination, TIG. The MIC50 of MDR-AB decreased significantly, and the FICI distribution of 70 strains of MDR-AB was as follows: when combined with polymyxin E, FICI < 0.5 was 4.3%, 0.5 < FICI < 1, 11.4%, 1 < FICI < 2, 64.3%, FICI > 2 as 0. The addition of 5-10mg/L to PPIs in the nutrient group increased the bacteria to tegatin MIC50 0-2 times; adding 50mg/L PPIs, the bacterial MIC50 increased by 4- > 128 times. The bactericidal curves after adding PPIs were above the bactericidal curve of the single drug, indicating that the colony count at each time point was higher than that of the single drug. (4) the MPC value of tegocyclin to MDR-AB was 4-32mg/L, SI. The range was 4-64, SI decreased 2-4 times after combined 4mg/L polymyxin E, SI decreased 4-8 times after combined 8mg/L, SI decreased by 2-8 times after combined with sulbactam and 4-64 times after SI. (5) after CCCP, the original strain MIC was not changed. Except for AB10 number mutant strain, his 4 strains dropped 16-128 times the resistant mutant strain of mutant strain. Compared with the original strain, the expression of the pump gene AdeJ was not significantly higher than that of the original strain. In addition to the mutant strain AB10, the expression of the pump gene AdeB of the other mutant strains increased by more than 2 times.
Conclusions: (1) although the sensitivity of MDR-AB to tegicycline is high, Monte-carlo simulation analysis shows that the recommended dosage is not ideal for the efficacy of MDR-AB pneumonia. (2) the combination of tegocycline and polymyxin E is independent of MDR-AB, and combined with sulbactam in combination with partial synergy. (3) the addition of PPIs in the medium affects tegocycline. In vitro antibacterial activity. (4) tegatocin, polymyxin E and sulbactam MPC are higher, MSW is wide, and single drug is easy to cause bacterial resistance. (5) the resistance mechanism of the mutant strain to tegagin is mainly overexpression of the outer row pump gene AdeABC.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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