Attacking the Global Problem of Antimicrobial Resistance

项目摘要 除了对医疗保健的影响外，抗菌素耐药性对经济的影响也很大。多过 每年有200万例感染是由至少对一线抗生素产生抗药性的细菌引起的，造成 美国医疗保健系统每年耗资200亿美元。铜绿假单胞菌(PA)导致超过51,000人健康 美国每年与护理相关的感染，其中13%是多药耐药(MDR)。非抗生素 预防和治疗细菌感染的方法提供了抗生素的有吸引力的替代品/附件 对耐多药PA分离株不再有效。其中之一是甘草酸苷(GLY)，它是一种从甘草根中提取的物质 (光果甘草)，对脓毒症、结肠炎、肺和脑损伤的动物模型有效； 用于慢性肝炎的临床治疗。感染后，GLY减少细胞外释放的高水平 运动蛋白组盒1(HMGB1)，一种激活细胞表面天然免疫受体的原型警报蛋白 影响宿主炎症反应。甘氨酸对PA实验性角膜炎的直接抑菌作用 由非耐多药临床(角膜炎)分离株(如KEI 1025)和非眼部分离株MDR9诱导。 初步/最近公布的数据支持，在MDR9诱导的PA角膜炎中，Gly：a)渗透细菌 膜，b)降低外排泵的活性，增加杀菌作用，d)与环丙沙星在 活体，最佳减少中性粒细胞渗入和板数。因此，我们的首要假设是 甘氨酸对角膜和眼附属器无毒，PA感染后与HMGB1结合可防止 激活先天免疫受体。为了检验这一假设，本文提出了两个具体目标。 具体目标1：测试Gly耐受性良好且不会改变细胞结构或 正常角膜、结膜和眼附属物的生理参数。在这个目标中，我们将测试 GLY局部给药对未感染的正常角膜的影响，如果眼压(IOP)，泪量， 角膜神经模式、角膜敏感度、常驻免疫细胞和/或结膜改变。 特定目标2：验证GLY对PA角膜炎的保护作用是通过抑制介导的假设 髓系细胞中TLR4/RAGE信号通路的HMGB1扩增这一目标将测试GLY是否与 HMGB1，抑制HMGB1-TLR4(-RAGE)相互作用和信号通路，其下游作用于 未成熟髓系树突状细胞(DC)成熟，巨噬细胞产生促炎细胞因子和 趋化因子和中性粒细胞的渗透和功能。Aim 2a将在GLY处理的小鼠身上测试这一假说 利用TLR4和RAGE KO小鼠和髓系特异性感染非耐药PA角膜炎分离株KEI1025 HMGB1KO小鼠。Aim 2b是翻译的，并将在GLY治疗的小鼠身上验证这一假设 与莫西沙星联合应用，在感染MDR PA后18h开始。

Project Summary Besides the impact on health care, the economic impact of antimicrobial resistance is significant. More than two million infections a year are caused by bacteria that are resistant to at least first-line antibiotics, costing the US health care system 20 billion dollars each year. Pseudomonas aeruginosa (PA) causes over 51,000 health care associated infections per year in the US, of which 13% are multi-drug resistant (MDR). Non-antibiotic approaches to prevent and treat bacterial infections provide attractive alternatives/adjuncts to antibiotics no longer effective against MDR PA isolates. One of these, glycyrrhizin (GLY), an extract from the licorice root (Glycyrrhiza glabra), is effective in treatment of animal models of sepsis, colitis, lung and brain injury; and is used in the clinical management of chronic hepatitis. After infection, GLY reduces extracellularly released high mobility group box 1 (HMGB1), a prototypic alarmin that activates cell surface innate immune receptors affecting host inflammatory responses. GLY has direct antimicrobial effects in PA experimental keratitis induced by non-MDR clinical (keratitis) isolates (e.g., KEI 1025) and by MDR9, a non-ocular isolate. Preliminary/recently published data support that in MDR9 induced PA keratitis, GLY: a) permeabilizes bacterial membranes, b) reduces efflux pump activity, increases bacterial killing and d) combined with Ciprofloxacin in vivo, reduces the neutrophil infiltrate and plate count optimally. Therefore, our overarching hypothesis is that GLY is non-toxic to the cornea and ocular adnexa and after PA infection, binds to HMGB1 preventing activation of innate immune receptors. To test this hypothesis two Specific aims are proposed. Specific Aim 1: Tests the hypothesis that GLY is well-tolerated and does not alter the cytoarchitectue nor the physiological parameters of the normal cornea, conjunctiva and ocular adnexa. In this aim, we will test the effects of GLY given topically on the uninfected, normal cornea, and if intraocular pressure (IOP), tear volume, corneal nerve pattern, corneal sensitivity, resident immune cells and/or conjunctiva are altered. Specific Aim 2: Tests the hypothesis that GLY's protective effect on PA keratitis is mediated by inhibiting HMGB1 amplification of TLR4/RAGE signaling pathways in myeloid cells. This aim will test if GLY binding to HMGB1, inhibits HMGB1-TLR4 (-RAGE) interactions and signaling pathways, with downstream effects on immature myeloid dendritic cell (DC) maturation, macrophage production of proinflammatory cytokines and chemokines and neutrophil infiltration and function. Aim 2a will test this hypothesis in GLY treated mice after infection with KEI1025, a non-MDR PA keratitis isolate using TLR4 and RAGE KO mice and myeloid specific HMGB1 KO mice. Aim 2b is translational and will test this hypothesis in mice in which GLY treatment is combined with Moxifloxacin and initiated 18h after infection with MDR PA.