胍基修饰的抗菌高分子与抗生素的协同效应及其对细菌耐药性的影响

Due to the recent surge of antibiotic resistance, antibiotic monotherapy is no longer sufficient to treat multidrug-resistant (MDR) bacterial infections. Combination therapy of different antibiotics or antibiotic with nonantibiotic adjuvant is a potential solution. However, the clinical efficacy of the antibiotic combinations remains unclear, and their effects on development of antibiotic resistance are still unknown. Recently, we developed antimicrobial guanidinium-functionalized polycarbonates which exhibited strong in vivo antimicrobial activity and high selectivity (therapeutic index for A. baumannii infection: ED50/LD50=1473). More importantly, the polymers induced no drug resistance even after repeated treatment, which is probably due to the unique membrane lytic and membrane translocation action mechanisms of the poly(guanidiniums). Interestingly, the RNA-seq results further revealed that poly(guanidiniums) and antibiotic exerted opposite effects on regulation of ~78% common gene shared by bacteria treated with polymer and antibiotic, respectively. Inspired by these interesting findings, we propose combining antimicrobial poly(guanidiniums) and antibiotics to increase antibiotic uptake in bacteria with purpose of enhancing antibacterial activity and mitigation of antibiotic resistance. Our preliminary results indeed confirmed that poly(guanidiniums) have synergistic antimicrobial effect with four common antibiotics against MDR A. baumannii. In particular, the minimum inhibitory concentration of antibiotic rifampicin was reduced by ~2000 fold when it was combined with poly(guanidiniums). Further systematic investigation will be carried out to study in vitro and in vivo synergistic effects as well as action mechanism of antimicrobial poly(guanidiniums) and antibiotics against MDR Gram-negative bacteria, and to explore whether this combination can mitigate antibiotic resistance. We aim to provide a novel and effective antimicrobial polymer/antibiotic combination therapy for treatment of MDR infections.

单一抗生素疗法已不足以应对大量涌现的多重耐药菌，多抗生素或抗生素/非抗生素组合疗法是极具研究前景的解决方案，但抗生素组合对耐药菌的临床功效及其对耐药性影响仍然存疑。申请人与合作团队近期研发了兼具优异杀菌性能和低毒性的胍基修饰可降解高分子(治疗指数高达1473)，且其不会导致耐药性(Nature Communication,2018)。研究还发现高分子和抗生素对两者分别处理过的耐药菌有着相反的基因表达调控。受上述研究启发，我们提出高分子/抗生素组合疗法，以提高抗生素的摄入而增强抗生素对耐药菌的抗菌能力，并同时抑制细菌耐药性的产生和进化。预实验结果表明高分子可与四种抗生素产生协同抗菌效应，其中抗生素利福平的最低抑菌浓度可降低高达~2000倍。因此我们拟系统研究胍基修饰的抗菌高分子/抗生素组合对耐药菌的协同效应及其成因，并探索其对耐药性的抑制作用，以期为耐药菌感染提供一个全新有效的治疗方案。

细菌的抗生素耐药性导致单一抗生素疗法已不能有效治疗许多由耐药菌导致的感染疾病。本项目通过将胍基功能化的碳酸酯（pEt_20）与抗生素进行联合用药，实现增强抗生素抗菌活性并逆转多重耐药性革兰氏阴性菌的耐药表型的目的。本研究团队首先通过有机催化开环聚合的方法成功合成了pEt_20。抗菌测试结果表明大分子pEt_20能够显著降低多种不同类型的抗生素（包括阿奇霉素、庆大霉素、亚胺培南、四环素、多粘菌素等）对多重耐药性革兰氏阴性菌（鲍曼不动杆菌）的最低抑菌浓度（MIC），使其从耐药范围降到敏感范围。pEt_20不仅能够提高传统抗生素对耐药菌的抗菌活性，而且还能够极大程度地提高抗肺结核药物利福平和抗风湿类药物金诺芬这两种原本并不用于治疗革兰氏阴性菌感染的药物的抗菌功能。研究表明在与pEt_20联用后，这两种药物的MIC均降低高达511倍。此外，pEt_20与利福平药物组合不仅对利福平耐药菌具有优异的抗菌作用，而且相对于利福平单独用药更不容易导致细菌的耐药性。更为重要的是，研究团队进一步利用多重耐药性鲍曼不动杆菌引起的小鼠血液感染模型证明了这种联合疗法在体内的有效性。pEt_20和利福平这一组合疗法极大程度地提高感染小鼠的生存率并降低了血液内的细菌浓度。本项目同时对高分子的协同抗耐药菌机制进行了探索，研究发现pEt_20能够在不破坏细菌细胞膜的情况下与胞内的蛋白质和核酸结合，并在与利福平药物联用后显著提升菌内的活性氧水平。这可能是pEt_20与多种抗菌药物产生协同抗耐药菌作用的重要原因。此外，体外和体内实验均表明pEt_20与利福平联用具有较好的生物安全性，其在有效杀菌浓度的剂量下并不会产生系统毒性。该工作有望为多重耐药性革兰氏阴性菌感染提供了新的治疗思路。

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