替加環(huán)素對(duì)多重耐藥鮑曼不動(dòng)桿菌的體外抗菌活性和防耐藥突變研究
發(fā)布時(shí)間：2018-07-14 10:11
【摘要】：目的：測(cè)定替加環(huán)素對(duì)多重耐藥鮑曼不動(dòng)桿菌（MDR-AB）的最低抑菌濃度（MIC），并在此基礎(chǔ)上應(yīng)用Monte-Carlo模擬進(jìn)行PK/PD研究，評(píng)價(jià)不同劑量替加環(huán)素治療MDR-AB肺炎效果。評(píng)價(jià)替加環(huán)素聯(lián)合多粘菌素E或舒巴坦對(duì)MDR-AB的抗菌效應(yīng)；研究質(zhì)子泵抑制劑（PPIs）對(duì)替加環(huán)素體外抗菌活性的影響�；谀退幫蛔冞x擇窗（MSW）理論，測(cè)定替加環(huán)素單用及聯(lián)合多粘菌素E或舒巴坦限制MDR-AB耐藥發(fā)生的能力，初步探究MDR-AB對(duì)替加環(huán)素的耐藥機(jī)制。 方法：（1）采用瓊脂平板二倍稀釋法測(cè)定替加環(huán)素對(duì)135株MDR-AB的MIC，并結(jié)合410例肺炎患者的替加環(huán)素藥代學(xué)數(shù)據(jù)，通過(guò)Monte-Carlo模擬計(jì)算不同劑量替加環(huán)素時(shí)AUC/MIC值分布，再根據(jù)預(yù)設(shè)折點(diǎn)計(jì)算治療MDR-AB肺炎的累積反應(yīng)分?jǐn)?shù)（CFR）。（2）參照棋盤(pán)法設(shè)計(jì)，采用微量肉湯稀釋法測(cè)定替加環(huán)素與多粘菌素E、舒巴坦對(duì)70株MDR-AB的聯(lián)合MIC，計(jì)算聯(lián)合抑菌指數(shù)（FICI）并判定聯(lián)合效應(yīng)。（3）采用瓊脂平板二倍稀釋法測(cè)定替加環(huán)素單藥及聯(lián)合PPIs對(duì)6種臨床常見(jiàn)分離菌的MIC；采用菌落計(jì)數(shù)法測(cè)定替加環(huán)素單藥及聯(lián)合PPIs的體外殺菌曲線。（4）采用瓊脂平板二倍稀釋法測(cè)定替加環(huán)素、多粘菌素E及舒巴坦對(duì)30株MDR-AB的MIC和防耐藥突變濃度（MPC），計(jì)算選擇指數(shù)（SI=MPC/MIC）；隨機(jī)選取9株細(xì)菌，測(cè)定替加環(huán)素聯(lián)合用藥的MPC，計(jì)算SI。（5）測(cè)定替加環(huán)素單藥及聯(lián)合碳酰氰間氯苯腙（CCCP）對(duì)體外篩選耐藥突變菌株的MIC；利用RT-PCR技術(shù)測(cè)定耐藥突變菌株外排泵基因AdeB和AdeJ的相對(duì)表達(dá)情況。 結(jié)果：（1）135株MDR-AB中97%的菌株對(duì)替加環(huán)素敏感，3%中介，未發(fā)現(xiàn)耐藥菌株。以CFR≥90%為預(yù)設(shè)折點(diǎn)，Monte-Carlo模擬分析表明，推薦給藥劑量（50mg/Lq12h）的CFR為61.62%，低于折點(diǎn)值；給藥劑量為100mg q12h時(shí)，CFR為89.86%，接近折點(diǎn)值。（2）聯(lián)合用藥后，替加環(huán)素對(duì)MDR-AB的MIC50顯著下降，70株MDR-AB的FICI分布為：與多粘菌素E聯(lián)用時(shí)，F(xiàn)ICI≤0.5占4.3%，0.5＜FICI＜1占20%，F(xiàn)ICI=1占11.4%，1＜FICI≤2占64.3%，F(xiàn)ICI＞2為0；與舒巴坦聯(lián)用時(shí)，F(xiàn)ICI≤0.5占10%，0.5＜FICI＜1占54.3%，F(xiàn)ICI=1占25.7%，1＜FICI≤2占10%，F(xiàn)ICI＞2為0。（3）培養(yǎng)基中加入5-10mg/L的PPIs會(huì)使細(xì)菌對(duì)替加環(huán)素MIC50增高0-2倍；加入50mg/L PPIs，細(xì)菌MIC50增加4-＞128倍。加入PPIs后的殺菌曲線均位于單藥殺菌曲線上方，表明各時(shí)間點(diǎn)菌落計(jì)數(shù)較單藥出現(xiàn)不同程度增高。（4）替加環(huán)素對(duì)MDR-AB的MPC值為4-32mg/L，SI范圍為4-64；聯(lián)合4mg/L多粘菌素E后SI下降2-4倍，聯(lián)合8mg/L后SI下降4-8倍；聯(lián)合32mg/L舒巴坦后SI下降2-8倍；聯(lián)合64mg/L舒巴坦SI后下降4-64倍。（5）聯(lián)合CCCP后，原菌株MIC并無(wú)變化，除AB10號(hào)突變菌株外，，其他4株MIC下降了16-128倍。5株耐藥突變菌株外排泵基因AdeJ的表達(dá)量較原菌株相比均未見(jiàn)明顯增高，除AB10號(hào)突變菌株外，其余突變菌外排泵基因AdeB表達(dá)量增高2倍以上。 結(jié)論：（1）雖然MDR-AB對(duì)替加環(huán)素敏感率較高，但Monte-carlo模擬分析表明推薦給藥劑量對(duì)MDR-AB肺炎的療效并不理想。（2）替加環(huán)素與多粘菌素E聯(lián)用對(duì)MDR-AB以無(wú)關(guān)作用為主，與舒巴坦聯(lián)用以部分協(xié)同為主。（3）培養(yǎng)基中加入PPIs會(huì)影響替加環(huán)素的體外抗菌活性。（4）替加環(huán)素、多粘菌素E及舒巴坦MPC較高、MSW寬，單用藥易致細(xì)菌耐藥發(fā)生；聯(lián)合用藥可有效限制耐藥發(fā)生。（5）突變菌株對(duì)替加環(huán)素的耐藥機(jī)制主要為外排泵基因AdeABC過(guò)度表達(dá)。
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R96
本文編號(hào)：2121317
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R96
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