Fitness of gram-negative pathogens during bacteremia

我们在医院里正在失去与耐药性革兰氏阴性细菌病原体的斗争， 临床护理设施。CDC估计，每年有超过200万人感染17种 抗药性细菌病原体，每年造成23，000人死亡。超过一半的物种是革兰氏- 阴性病原体，包括碳青霉烯类耐药肠杆菌科（CRE）和非发酵 条件致病菌鲍曼不动杆菌尽管CDC描述了碳青霉烯类耐药（CR） 细菌作为"噩梦细菌"，这些病原体或其抗生素的毒力所需的因素- 在血流感染期间的易感对应物在很大程度上是未知的。因此，迫切需要 确定独特（物种特异性）和共同（六种革兰氏阴性菌中的大多数所需）适应性， 毒力因子，并绘制关键代谢途径和必需基因图谱 这些病原体在体内使用的装置。我们的长期目标是阐明 引起医院感染的革兰氏阴性菌。本申请的总体目标是 进行RNA-seq并测量E.大肠杆菌，肺炎克雷伯菌， 粘质沙雷氏菌、弗氏柠檬酸杆菌和阴沟肠杆菌以及A.鲍曼不动杆菌，在鼠中 菌血症模型，其中Tn-seq已在很大程度上完成了这些分离株。我们的核心假设是 根据肠杆菌科（Enterobacteriaceae）和A.鲍曼不动杆菌）水平， 这些病原体需要矫形核心功能和种特异性适应因子的组合， 在菌血症期间获得营养并逃避宿主反应。这些拟议研究的理由是， 在我们的健康中，引起血流感染的革兰氏阴性病原体的抗生素耐药性正在迅速上升 护理系统。我们计划通过以下方法客观地识别对血流感染至关重要的独特和常见基因： 敏感细菌和CR细菌，并测量它们的体内基因表达。独特和常见毒力 将研究决定因素以确定发病机制。我们将检验我们的中心假设 并通过完成这些具体目标来达到本申请的目的：1）定义活性代谢 途径和由此产生的生长动力学在菌血症期间跨越六个革兰氏阴性病原体。2)识别 六种革兰氏阴性病原体菌血症所需的共同和独特的途径。具体的预期 结果将包括：（a）菌血症期间每种病原体生长动力学的精确测量，（B） 在体内同等生长阶段确定优选和所需途径，以及（c） 这些途径用病原体特异性的或在多种病原体中共有的基因注释。的 这些研究的积极影响将是巨大的。我们将揭示我们的六个发病机制， 大多数涉及耐药性医院病原体，并确定开发新疗法的目标。

We are losing the battle against antibiotic-resistant Gram-negative bacterial pathogens in our hospitals and clinical care facilities. The CDC estimates that over 2 million people are infected annually with 17 species of antibiotic-resistant bacterial pathogens, killing 23,000 people per year. Over half of the species are Gram- negative pathogens including carbapenem-resistant Enterobacteriaceae (CRE) and the non-fermenting opportunistic pathogen Acinetobacter baumannii. Although the CDC described carbapenem-resistant (CR) bacteria as “nightmare bacteria”, the factors required for virulence of these pathogens or their antibiotic- susceptible counterparts during bloodstream infections are largely unknown. Thus, there is an urgent need to identify unique (species-specific) and common (required by most of the six Gram-negative species) fitness and virulence factors required by these species for bacteremia, and map key metabolic pathways and essential gene sets used by these pathogens in vivo. Our long-term goal is to delineate the mechanisms of pathogenesis in Gram-negative bacteria that cause hospital-acquired infections. The overall objective of this application is to conduct RNA-seq and measure in vivo growth rates in representative isolates of E. coli, Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, and Enterobacter cloacae as well as A. baumannii, in the murine model of bacteremia in which Tn-seq has been largely completed for these isolates. Our central hypothesis is that based on the relatedness of CR species at the family (Enterobacteriaceae) and class (A. baumannii) levels, these pathogens require a combination of orthologous core functions and species-specific fitness factors to acquire nutrients and evade host responses during bacteremia. The rationale for these proposed studies is that antibiotic resistance is rising rapidly in Gram-negative pathogens that cause bloodstream infections in our health care systems. We plan to objectively identify unique and common genes critical for bloodstream infection by antibiotic-susceptible and CR bacteria and measure their in vivo gene expression. Unique and common virulence determinants will be investigated to determine mechanisms of pathogenesis. We will test our central hypothesis and attain the objective of this application by completing these specific aims: 1) Define the active metabolic pathways and resultant growth kinetics across six Gram-negative pathogens during bacteremia. 2) Identify shared and unique pathways required for bacteremia by six Gram-negative pathogens. Specific expected outcomes will include: (a) precise measurement of growth kinetics of each pathogen during bacteremia, (b) identification of preferred and required pathways at equivalent growth phases in vivo, and (c) detailed maps of these pathways annotated with genes that are pathogen-specific or shared across multiple pathogens. The positive impact of these studies will be substantial. We will uncover mechanisms of pathogenesis for six of our most concerning antibiotic-resistant hospital pathogens and identify targets for developing new therapeutics.