Surprising Efficacy of Discounted Antibiotics vs. MDR Gram-Negative Pathogens Occurring Through Innate Immune Sensitization

迅速增长的抗生素耐药性危机对现代医学和全球公共卫生构成了迫在眉睫的威胁。尤其是由耐碳青霉烯肺炎克雷伯菌(Kp)、大肠埃希菌(Eco)、阴沟肠杆菌(ECL)、奇异变形杆菌(Pm)、鲍曼不动杆菌(AB)和铜绿假单胞菌(PA)等多重耐药革兰氏阴性杆菌(GNR)引起的医院感染正通过水平转移和克隆性扩张传播，治疗选择有限，发病率和死亡率高。我们正在挑战目前在细菌学介质中进行的标准MIC/MBC检测，这与宿主免疫系统完全不相关。甚至在第一次与医生进行诊断性会诊之前，患者的感染就已经被许多内源性免疫效应物所对抗，包括抗菌肽、血清补体和吞噬细胞。虽然人们已经付出了巨大的努力来识别和利用不同类别的药用抗生素之间的协同作用，但对药用抗生素与内源性抗菌防御系统相互作用的研究工作很少。我们已经记录了美国最常用的处方抗生素阿奇霉素(Azm)的意想不到的活性，使高度耐多药的KP、Ab和PA菌株对先天性免疫杀伤敏感-尽管当遇到这些菌株时，这种药物甚至不包括在AST小组中。其次，我们已经记录了β-内酰胺酶抑制剂(BLI)，如他唑巴坦(TAZ)和阿维巴坦(AVI)的意想不到的活性，使高度耐药的KP和Ab对先天性免疫杀伤敏感-尽管这些药物从未被认为具有除了阻断β-内酰胺酶之外的直接作用。在目前的提案中，我们将确定AZM或BLIS使MDR GNRs对宿主AMP的杀伤、血清补体或吞噬细胞通过棋盘和动态杀伤试验增敏的协同作用，包括细菌细胞学分析(BCP)的细菌生理学分析，以及对补体、血小板和吞噬细胞增敏的研究。然后，我们将使用抗体和KP作为MDR GNRs的模型，将Azm和BLIS的先天免疫增敏转化为肺炎、败血症和尿路感染小鼠模型的有效治疗选择，利用药物或遗传靶向的小鼠在先天免疫缺陷(阳春素、补体、中性粒细胞)中识别关键的协同宿主因素。人们注意提供替代的方法来确定作用机制(如代谢前体掺入、透射电子显微镜、诱导抗性变异体的全基因组测序)，并评估Azm和BLIS对细胞因子信号转导的其他潜在的免疫调节作用。由于我们早期对耐多药革兰氏阳性病原体(耐甲氧西林金黄色葡萄球菌，VRE)和β-内酰胺类药物对天然免疫的增敏作用已在临床系列中得到证实，并影响了治疗指南，我们预计目前对耐多药GNR病原体的研究将立即产生翻译影响。

The burgeoning antibiotic resistance crisis is an imminent threat to the practice of modern medicine and global public health. In particular, nosocomial infections caused by multi-drug resistant (MDR) Gram- negative rods (GNRs) such as carbapenem-resistant strains of Klebsiella pneumoniae (Kp), Escherichia coli (Eco), Enterobacter cloacae (Ecl), Proteus mirabilis (Pm), Acinetobacter baumannii (Ab) and Pseudomonas aeruginosa (Pa) are spreading through horizontal transfer and clonal expansion with limited therapeutic options and high attendant morbidity and mortality. We are challenging the current standard MIC/MBC testing in bacteriologic media, which is totally agnostic to the host immune system. Even before the first diagnostic encounter with a physician, a patient’s infection is already being combatted by numerous endogenous immune effectors, including antimicrobial peptides, serum complement and phagocytic cells. While tremendous effort has been extended to identify and exploit synergy between different classes of pharmaceutical antibiotics, very little work has been conducted to study the way pharmaceutical antibiotics interact with endogenous antimicrobial defenses. We have documented unexpected activity of azithromycin (AZM), the most commonly prescribed antibiotic in the US, to sensitize highly MDR strains of Kp, Ab and Pa to innate immune killing – even though this drug is not even included in the AST panel when such isolates are encountered. Second, we have documented unexpected activity of β-lactamase inhibitors (BLIs) such as tazobactam (TAZ) and avibactam (AVI) to sensitize highly MDR strains of Kp and Ab to innate immune killing – even though these agents are never contemplated to have direct action beyond blocking β- lactamase enzymes. In the present proposal, we will identify synergistic interactions wherein AZM or BLIs sensitize MDR GNRs to killing by host AMPs, serum complement or phagocytic cells using checkerboard and kinetic killing assays, assays of bacterial physiology including bacterial cytological profiling (BCP), and studies of sensitization to complement, platelets, and phagocytes. Then using Ab and Kp as model MDR GNRs, we will translate innate immune sensitization by AZM and BLIs to effective therapeutic options in murine models of pneumonia, sepsis and UTI, using pharmacologically or genetically targeted mice to have defects in innate immunity (cathelicidin, complement, neutrophils) to identify key synergistic host factors. Careful attention is paid to provide alternative methods for mechanism of action determination (e.g. metabolic precursor incorporation, transmission electron microscopy, whole genome sequencing of induced resistant variants) and to assess other potential immunomodulatory effects of AZM and BLIs on cytokine signaling. As our earlier work with MDR Gram+ pathogens (MRSA, VRE) and β-lactam sensitization to innate immunity was corroborated in clinical series and influenced treatment guidelines, we foresee immediate translational impact of the current investigations on MDR GNR pathogens.