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

迅速发展的抗生素耐药性危机是对现代医学和全球公共卫生实践的迫在眉睫的威胁。特别地，由多重耐药（MDR）革兰氏阴性杆菌（GNR）引起的医院感染，例如肺炎克雷伯氏菌（Kp）、大肠杆菌（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）通过水平转移和克隆扩张传播，治疗选择有限，伴随着高发病率和死亡率。我们正在挑战目前在细菌培养基中进行的标准MIC/MBC测试，该测试对宿主免疫系统完全不可知。甚至在第一次与医生诊断之前，患者的感染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。虽然已经做出了巨大的努力来识别和利用不同类别的药物抗生素之间的协同作用，但很少有工作来研究药物抗生素与内源性抗微生物防御相互作用的方式。我们已经记录了阿奇霉素（AZM）（美国最常用的处方抗生素）的意外活性，使Kp、Ab和Pa的高MDR菌株对先天性免疫杀伤敏感-即使当遇到此类分离株时，该药物甚至不包括在AST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））的意外活性，使Kp和Ab的高MDR菌株对先天性免疫杀伤敏感-即使这些药物从未被认为具有阻断β-内酰胺酶以外的直接作用。在本提案中，我们将确定协同相互作用，其中AZM或BLI敏化MDR GNRs杀死宿主AMP，血清补体或吞噬细胞使用棋盘和动力学杀伤试验，细菌生理学试验，包括细菌细胞学分析（BCP），和研究敏化补体，血小板和吞噬细胞。然后使用Ab和Kp作为模型MDR GNR，我们将AZM和BLI的先天免疫致敏转化为肺炎、脓毒症和UTI小鼠模型中的有效治疗选择，使用具有先天免疫缺陷（cathelicidin、补体、中性粒细胞）的靶向或遗传靶向小鼠来鉴定关键的协同宿主因子。仔细注意提供用于确定作用机制的替代方法（例如，代谢前体掺入、透射电子显微镜、诱导的抗性变体的全基因组测序），并评估AZM和BLI对细胞因子信号传导的其他潜在免疫调节作用。由于我们早期对MDR革兰氏阳性病原体（MRSA，VRE）和β-内酰胺致敏先天免疫的研究在临床系列中得到证实，并影响了治疗指南，因此我们预见了当前对MDR GNR病原体的研究的直接转化影响。

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.