Heteroresistance Interdisciplinary Research Unit (Project 2)

摘要 抗生素耐药性是我们这个时代最严重的医学挑战之一。这场危机使病人处于危险之中 无法治愈的细菌感染，并威胁到依赖抗生素的现代医学的重大进步 （移植、化疗等）。每年至少有280万例抗生素耐药性感染， 美国，导致超过35，000人死亡[1]。如果不采取重大行动， 预计到2050年感染人数将达到1000万，超过癌症的预测[2]。理解 耐药机制对于设计对抗耐药细菌的新方法和疗法至关重要。 异源耐药性（HR）是一种神秘的抗生素耐药性形式，其中细菌分离株携带一种 在抗生素存在下可以快速复制的耐药亚群，而敏感亚群可以在抗生素存在下快速复制。 亚群被杀[3，4]。我们已经观察到HR对抗生素磷霉素的作用，磷霉素是其自身的一员， 药物类别，主要在美国以口服形式用于治疗尿路感染（UTI）[5]。的 磷霉素的使用最近增加，因为细菌对其他种类的药物产生耐药性[6]， 其强大的安全性。由于其需求的增加和预期的IV使用的扩大批准， 磷霉素有望成为美国抗生素库中更重要的一部分。 因此，我们必须阐明磷霉素耐药的生物学，以指导临床使用。 值得注意的是，我们的监测数据显示，碳青霉烯类耐药患者中磷霉素HR的比例 肠杆菌科（CRE; 72%）和鲍曼不动杆菌（CRAB; 89 抗生素测试，很大一部分没有被临床诊断发现[7]。我们最近 研究表明，包括磷霉素在内的多种抗生素的HR可导致体内治疗失败[4]。 有趣的是，迄今为止在HR的研究实例中是独特的，我们发现磷霉素HR是由以下原因引起的： 两个不同的，共存的耐药亚群，两者都在药物存在下复制，而不是 持留菌，但形成抗性小（R-SM）或大（R-LG）菌落。转座子筛选的结果， 代谢组学实验揭示了R-SM和R-LG细胞代谢的潜在基础 异质性，而不是不稳定的遗传变化，如基因扩增。我们将剖析新陈代谢 信号传导驱动耐药R-SM亚群的扩张以及谷氨酸和谷胱甘肽的作用 在这个过程中。然后，我们将研究不同磷霉素耐药亚群的患病率， 不同的临床分离株。这项工作将对我们对非物质文化遗产的理解产生持续而有力的影响。 HR和代谢及表型异质性的遗传机制。这将补充项目1， 重点是不稳定的遗传机制的人力资源。新的和根本的见解获得将奠定 基金会的发现新的治疗和干预针对亚群， 减少人类疾病。

ABSTRACT Antibiotic resistance is one of the most serious medical challenges of our time. This crisis puts patients at risk of untreatable bacterial infections and threatens major advances of modern medicine that rely on antibiotics (transplants, chemotherapy, etc). There are at least 2.8 million antibiotic resistant infections each year in the US, leading to over 35,000 deaths [1]. Without significant action, worldwide annual mortality due to these infections is predicted to reach 10 million by 2050, surpassing that predicted for cancer [2]. Understanding resistance mechanisms is critical to designing novel approaches and therapeutics to combat resistant bacteria. Heteroresistance (HR) is an enigmatic form of antibiotic resistance in which a bacterial isolate harbors a resistant subpopulation that can rapidly replicate in the presence of an antibiotic, while a susceptible subpopulation is killed [3, 4]. We have observed HR to the antibiotic, fosfomycin, which is a member of its own drug class and has primarily been used in the US in an oral form to treat urinary tract infections (UTIs) [5]. The use of fosfomycin has recently increased as bacteria become resistant to other classes of drugs [6] and due to its strong safety profile. Due to its increased need and expected expanded approval for IV use, fosfomycin is expected to become a much more prominent part of the antibiotic arsenal in the US. Therefore, it is essential that we elucidate the biology of fosfomycin resistance to guide clinical use. Strikingly, our surveillance data revealed that the rate of fosfomycin HR among carbapenem-resistant Enterobacteriaceae (CRE; 72%) and Acinetobacter baumannii (CRAB; 89%) was higher than that of any other antibiotic tested, and that a large proportion was not detected by clinical diagnostics [7]. We recently demonstrated that HR to diverse antibiotics, including fosfomycin, can cause treatment failure in vivo [4]. Interestingly, and thus far unique among studied examples of HR, we found that fosfomycin HR is caused by two distinct, co-existing resistant subpopulations, both of which replicate in the presence of drug and are not persisters, but form resistant small (R-SM) or large (R-LG) colonies. Results from a transposon screen and metabolomic experiments revealed the underlying basis for the R-SM and R-LG cells to be metabolic heterogeneity, rather than unstable genetic changes such as gene amplification. We will dissect how metabolic signaling drives the expansion of the resistant R-SM subpopulation and the roles of glutamate and glutathione in this process. We will then study the prevalence of distinct fosfomycin resistant subpopulations among diverse clinical isolates. This work will have a sustained and powerful impact on our understanding of non- genetic mechanisms of HR and metabolic and phenotypic heterogeneity. This will complement Project 1 which focuses on unstable genetic mechanisms of HR. The new and fundamental insights gained will lay the foundation for the discovery of novel therapeutics and interventions targeting subpopulations to reduce human disease.