Mechanisms of phenotypic antibiotic resistance in Gram-negative bacteria

项目总结/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）。在美国，每年至少有200万例抗生素耐药性感染， 导致23,000多人死亡[1]。据估计，如果不采取重大行动， 到2050年，这些感染将达到1000万，超过癌症的预测死亡率[2]。 了解耐药机制对于设计对抗耐药细菌的新疗法至关重要。 异源耐药性是抗生素耐药性的一种神秘形式，其中细菌分离物具有耐药性。 在抗生素存在下可以快速复制的亚群，而基因相同但 易感亚群被杀死[3，4]。不仅许多细菌物种表现出这种形式的表型 耐药性，但据报道，它对不同类别的抗生素。不幸的是，我们对 异源耐药性极其有限，其在感染期间的相关性尚不清楚。使用两个临床 革兰氏阴性医院病原体阴沟肠杆菌的分离株，我们最近表明， 在体内模型中，对最后一线抗生素粘菌素的异源抗性可导致治疗失败[4]。 此外，其中一个分离物具有非常低频率的耐药亚群（<1/10，000个细胞） 临床诊断试验未检测到，导致其被错误分类为粘菌素敏感[4]。 我们未发表的国家监测数据显示，粘菌素异质耐药性存在于10%的 碳青霉烯类耐药肠杆菌科（CRE），包括18%的碳青霉烯类耐药肠杆菌， 尽管这些分离株中的大多数被错误地分类为粘菌素敏感的。这种错误分类 可能导致临床医生不适当地开具粘菌素，导致治疗失败。综上所述各项 数据突出了一种很大程度上未被重视的流行病，其中粘菌素异源抗性是普遍的， 绝大多数未被发现，并可能导致临床上无法解释的抗生素治疗失败。 我们将使用遗传学、生物化学、单细胞显微镜、流式细胞术和细胞分选的组合， 对异源抗性有基础性的认识。具体来说，我们将阐明耐药的动力学 粘杆菌素异质耐药肠杆菌内的亚群，以及控制的分子机制 阻力从这项工作中获得的知识将形成一个研究异源抗性的范例， 其他细菌和各种抗生素。此外，这项研究的影响可能会扩大到 在真菌[5，6]和人类癌症[7]中观察到真核生物作为异源抗性。总体而言，这项工作将 显著拓宽了我们对细胞亚群（表型 异质性）可以控制生理学。这将是我们对抗抗生素耐药性的关键一步 并将为发现减轻人类痛苦的新疗法奠定基础。

PROJECT SUMMARY/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 million antibiotic resistant infections each year in the US, leading to over 23,000 deaths [1]. It is estimated that without significant action, worldwide annual mortality due to these infections will reach 10 million by 2050, surpassing the predicted mortality from cancer [2]. Understanding resistance mechanisms is critical to designing novel therapeutics to combat resistant bacteria. Heteroresistance 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 genetically identical yet susceptible subpopulation is killed [3, 4]. Not only do many species of bacteria exhibit this form of phenotypic resistance, but it has been reported against different classes of antibiotics. Unfortunately, our understanding of heteroresistance is extremely limited and its relevance during infection has been unclear. Using two clinical isolates of the Gram-negative nosocomial pathogen Enterobacter cloacae, we recently showed that heteroresistance to the last-line antibiotic colistin can cause treatment failure in an in vivo model [4]. Furthermore, one of the isolates harbored a very low frequency resistant subpopulation (<1 in 10,000 cells) that was undetected by clinical diagnostic tests, leading to its incorrect classification as colistin susceptible [4]. Our unpublished national surveillance data reveal that colistin heteroresistance is present in 10% of carbapenem-resistant Enterobacteriaceae (CRE), including 18% of carbapenem-resistant Enterobacter, although the majority of these isolates are incorrectly classified as colistin susceptible. Such misclassification could lead clinicians to prescribe colistin inappropriately, leading to treatment failures. Taken together, these data highlight a largely unappreciated epidemic in which colistin heteroresistance is prevalent, overwhelmingly undetected, and may cause unexplained antibiotic treatment failure in the clinic. We will use a combination of genetics, biochemistry, single cell microscopy, flow cytometry and cell sorting to make foundational insights into heteroresistance. Specifically, we will elucidate the dynamics of the resistant subpopulation within colistin heteroresistant Enterobacter, as well as the molecular mechanism controlling resistance. The knowledge gained from this work will form a paradigm with which to study heteroresistance in other bacteria and against diverse antibiotics. Further, the impact of this research will likely extend to eukaryotes as heteroresistance has been observed in fungi [5, 6] and human cancers [7]. Overall, this work will significantly broaden our understanding of how traits exhibited by a subpopulation of cells (phenotypic heterogeneity) can control physiology. This will represent a critical step in our fight against antibiotic resistant bacteria and will lay the foundation for the discovery of novel therapeutics that alleviate human suffering.