Mechanisms of phenotypic antibiotic resistance in Gram-negative bacteria

项目摘要/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，并威胁依赖抗生素的现代医学的重大进步 (移植、化疗等)。美国每年至少有200万例耐药感染病例， 导致超过23,000人死亡[1]。据估计，如果不采取重大行动，全球每年的死亡率将 到2050年，这些感染人数将达到1000万，超过预测的癌症死亡率。 了解耐药机制对于设计对抗耐药细菌的新疗法至关重要。 异耐药是一种神秘的抗生素耐药性形式，其中细菌分离物具有耐药性。 在抗生素存在的情况下可以快速复制的亚群，尽管在基因上相同 易感亚群被杀死[3，4]。不仅许多种类的细菌都表现出这种表型 耐药性，但据报道它对不同类别的抗生素。不幸的是，我们对 异质性耐药是极其有限的，其在感染过程中的相关性尚不清楚。使用两个临床 革兰氏阴性医院内致病菌阴沟肠杆菌的分离，我们最近发现 在体内模型中，对最后一种抗生素粘菌素的异质性耐药可能导致治疗失败。 此外，其中一个分离株具有极低频率的抗性亚群(每10,000个细胞中有1个)。 这是临床诊断试验没有发现的，导致它被错误地归类为粘菌素易感[4]。 我们未公布的国家监测数据显示，10%的人存在粘菌素异耐药 耐碳青霉烯类肠杆菌科(CRE)，包括18%的耐碳青霉烯类肠杆菌， 尽管这些分离物中的大多数被错误地归类为粘菌素敏感。这样的错误分类 可能会导致临床医生不适当地开粘菌素处方，导致治疗失败。这些加在一起， 数据突显了一种在很大程度上没有被评估的流行病，在这种流行病中，粘菌素异耐药很普遍， 绝大多数未被发现，并可能导致临床上不明原因的抗生素治疗失败。 我们将结合遗传学、生物化学、单细胞显微镜、流式细胞术和细胞分选来 对异质电阻有基础性的见解。具体地说，我们将阐明抗药性的动态 粘杆菌素耐药肠杆菌亚群及其调控的分子机制 抵抗。从这项工作中获得的知识将形成一个研究异质电阻的范例 对其他细菌和多种抗生素有抵抗力。此外，这项研究的影响可能会延伸到 在真菌[5，6]和人类癌症[7]中观察到了真核生物的异质性耐药。总的来说，这项工作将 极大地拓宽了我们对细胞亚群(表型)所表现出的特征的理解 异质性)可以控制生理。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.