【作者】 李晓波；

【导师】 黄鹤；

【作者基本信息】 天津大学 ， 化学工程与技术， 2021， 硕士

【摘要】 多重耐药鲍曼不动杆菌（Multidrug-resistant Acinetobacter baumannii,MDR-Ab）可引发严重感染,且对多数临床抗生素耐药,严重威胁全球公共健康安全。盐酸小檗碱（berberine hydrochloride,BBH）是一种从小檗科等植物中提取的异喹啉生物碱,被广泛用作抗菌药物。本研究旨在通过基于BBH的联合用药对抗MDR-Ab,使其对抗生素复敏,为临床解决多重耐药/泛耐药难题提供新的干预策略。本研究首先通过药敏试验和棋盘试验探讨了BBH分别与舒巴坦、环丙沙星、替加环素和美罗培南4种抗生素联合用药对不同MDR-Ab菌株的体外协同效应。结果表明,BBH对MDR-Ab菌株的MIC≥256 mg/L,有较弱的抗菌活性,但其可以显著提高MDR-Ab对不同抗生素的敏感性,具有协同效应,甚至使抗生素复敏。此外,小鼠大腿MDR-Ab感染模型结果表明,BBH/舒巴坦联合用药可降低2.53 Log₁₀CFU/大腿的细菌负荷,显著提高了舒巴坦的体内抗菌效能。同时,为在单细胞水平验证联合用药的效果,本研究搭建了原核微生物单细胞微流控芯片系统来探究在不同联合用药作用下单个细菌的形态及其生长速率μ和滞后时间λ的变化。结果表明BBH与靶向青霉素结合蛋白2的美罗培南和美西林联用能降低菌群的异质性,清除休眠细菌并阻止细菌通过形态变化逃逸抗生素的攻击。最后通过敲除ade B基因并检测BBH的细菌体内积累量,探究了BBH对抗生素的复敏机制,结果发现BBH在△ade B菌株的积累量增加2倍以上,且BBH对其抗生素复敏活性丧失,证明了BBH通过竞争AdeABC外排泵外排抗生素的结合位点来协助抗生素发挥抑菌活性。综上,本研究设计了一种针对MDR-Ab的新型联合用药策略,并在单细菌水平对其抑菌活性进行了评估,为解决临床耐药菌感染问题提供了有益借鉴。更多还原

【Abstract】 Multidrug-resistant（MDR）Acinetobacter baumannii strains can cause severe infections,and are rapidly developing resistance to the last-resort of existing antibiotics,posing a major and global threat to health care system.Berberine hydrochloride（BBH）,a kind of isoquinoline alkaloids extracted from Berberis and other plants,has been widely used as an antibacterial medicine.This study aims to achieve the resensitization of MDR-Ab to antibiotics through the combination of BBH and antibiotics,and provide a novel approach for clinic to solve the problem caused by multi-drug/pan-drug resistance.Firstly,in vitro synergistic effects of BBH with antibiotics（sulbactam,ciprofloxacin,tigecycline,and meropenem）against MDR A.baumannii were determined by susceptibility tests and checkerboard assays.The results showed that BBH alone had weak antimicrobial activity（e.g.,MIC≥256 mg/L）against MDR A.baumannii.However,it dramatically increased the susceptibility of antibiotics against MDR strains（e.g.,synergistic effects;FICI<0.5）,even reversed their resistance to antibiotics.In vivo study suggested BBH with sulbactam had strong antimicrobial efficiency than monotherapy,reducing 2.53 Log₁₀CFU/thigh bacterial burdens.In addition,to verify the efficiency of combination therapy against MDR-Ab in a single-cell level,we utilized a single–cell microfluidic to appraise the changes of single cell’s growth rateμ,lag timeλ,and morphology caused by the BBH/antibiotic treatment.Using this system,it was demonstrated that BBH with meropenem or mecillinam targeted penicillin binding protein 2 was more effective against MDR-TJ,resulting in decrease of homogeneous population,clearage of dormant cells,and preventing bacteria from escaping the treatment of drugs through morphological changes.Further more,the antibiotic-sensitizing mechanism of action of BBH was evaluated via ade B gene knockout and BBH accumulation studies.In△ade B strains,BBH accumulated more than 2 folds and the synergistic effects eliminated,proving that BBH boosted the antibiotics activities against MDR-Ab by competing the binding sites of AdeABC to increase the high accumulation of antibiotics contributed to kill the bacteria.In sum,this study provides a novel combination therapy against MDR A.baumannii,and evaluated its antibacterial activity at a level of single bacteria,which may offer an approach to combat relevant infection caused by MDR-Ab.更多还原