Impact of Fluoroquinolone resistance on Pseudomonas virulence and patient outcome

描述(申请人提供)：铜绿假单胞菌(PA)是一种对公众健康构成直接威胁的细菌，因为它在导致医院感染、出现多药耐药、毒力潜力以及开发过程中缺乏针对这种病原体的新药方面发挥着主导作用。在急性感染期间，PA利用III型分泌系统(TTSS)与宿主细胞接触时将细胞毒素(即ExoU、EXOS)传递到宿主细胞，最终允许PA跨越上皮屏障进行全身传播或导致细胞死亡。大多数PA分离株要么含有exos基因，要么含有exoU基因；其存在因临床部位或疾病背景而异。已证实由ExoU分泌型PA和FQ耐药PA引起的急性肺炎的发病率和死亡率过高，这增加了抗生素耐药性可能与毒力增强有关的可能性。初步数据显示，耐FQ的PA群体以exoU+菌株为主，耐药程度较高的菌株对培养的上皮细胞具有更强的细胞毒性。我们假设，exoU+PA菌株通过在目标酶(例如DNA旋转酶)上发生突变来更容易地适应FQ暴露，这些突变同时赋予对FQ的抗性，并由于DNA超螺旋的变化而上调TTSS基因的转录。我们首先提出了一种系统的方法来确定耐FQ的PA菌株是否更具毒力，作为开发由多重耐药PA引起的感染迫切需要的新管理策略的必要的第一步。我们将检测从PA肺炎患者那里获得的大样本(n=216)以及已经收集的现有结果数据，以：1)确定与EXOS+菌株相比，在exoU+菌株中是否更常观察到FQ耐药性，2)评估临床菌株中耐药性和毒力的共同表达在多大程度上转化为体外细胞模型中的生物效应并与患者的预后相关，以及3)研究随着反复暴露于FQ后PA菌株中耐药性突变的积累，毒力表达的动态。如果我们证明PA通过变得更具抗药性和更强的毒力而演变为应对FQ暴露的“超级细菌”，我们将有强有力的基础来1)通过早期和更密集的治疗来开出更具侵略性的治疗，2)严格限制FQS的不分青红皂白地开药，同时加强当前的感染控制努力，以最大限度地减少FQ耐药和毒力菌株的传播。我们将有坚实的基础来筛选现有的能够阻断TTSS毒力的化合物，并基于对调节耐药性和毒力表达的分子途径的更好理解来识别新的药物靶点。我们的假设如果得到证实，将有助于推进针对III型效应器(pcrV疫苗、抗PcrV抗体)的免疫治疗的发展，并有助于快速诊断以确定毒力潜力作为患者护理的重要组成部分。公共卫生相关性：该项目的目标是确认院内感染的主要原因铜绿假单胞菌是否在对氟喹诺酮类抗生素产生抗药性时变得更加毒力，并证明这种共同表达的分子模型。缺乏针对这种病原体的新药正在开发中，这突显了迫切需要进行这项研究，作为必要的初始步骤，以便更好地了解耐药株的有效毒力策略，以便开发旨在解除毒力的新疗法。

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa (PA) is a bacterium that poses an immediate threat to public health because of its leading role in causing nosocomial infections, emergence of multidrug-resistance, virulence potential, and the lack of new drugs targeting this pathogen in the development pipeline. During acute infections, PA utilizes the type III secretion system (TTSS) to deliver cytotoxins (i.e. ExoU, ExoS) into host cells upon contact, ultimately allowing PA to cross epithelial barrier and spread systemically or cause cell death. Most PA isolates contain either exoS or exoU gene; its presence varies by clinical sites or disease backgrounds. Excess morbidity and mortality have been demonstrated for acute pneumonia caused by ExoU-secreting PA and FQ-resistant PA, raising the possibility that antibiotic resistance may be linked with enhanced virulence. Preliminary data indicates that FQ-resistant PA population is predominated by exoU+ strains, with increased cytotoxicity towards cultured epithelial cells for those showing higher degree of resistance. We hypothesize that exoU+ PA strains adapt more readily to FQ exposure by developing mutations at target enzymes (e.g. DNA gyrase) which simultaneously confer resistance to FQ and upregulate TTSS gene transcription due to changes in DNA supercoiling. We are first to propose a systematic approach to determine if FQ-resistant PA strains are more virulent as a necessary initial step towards the development of novel management strategies urgently needed for infections caused by multi-drug resistant PA. We will examine a large sample of respiratory isolates (n=216) obtained from patients with PA pneumonia and available outcome data already collected to: 1) determine if FQ resistance is more frequently observed in exoU+ compared to exoS+ strains, 2) assess the extent to which co-expression of resistance and virulence in clinical isolates translates into a biologic effect in in vitro cellular model and correlates with outcomes of patients, and 3) study the dynamics of virulence expression as resistance mutations accumulate in PA strains following repeated exposure to FQ. If we prove that PA evolves into a "superbug" in response to FQ exposure by becoming more resistant and more virulent, we will have a strong basis to 1) prescribe more aggressive treatment with early and more intensive therapy, 2) severely limit indiscriminant prescribing of the FQs and at the same time, intensify current infection control efforts to minimize spread of FQ-resistant and virulent strains. We will have a strong basis to screen existing compounds that can block TTSS virulence as well as identify novel drug targets based on a better understanding of the molecular pathways that regulate resistance and virulence expression. Our hypothesis if proven will help advance the development of immunotherapy against the type III effectors (pcrV vaccine, anti-PcrV antibody) and also rapid diagnostics to determine virulence potential as an essential part of patient care. PUBLIC HEALTH RELEVANCE: The goal of this project is to confirm if Pseudomonas aeruginosa, a leading cause of nosocomial infections, becomes more virulent as it becomes resistant to the fluoroquinolone antibiotics and prove the molecular model by which this co-expression occurs. The lack of new drugs targeting this pathogen in the development pipeline underscores the urgent need to perform this study as a necessary initial step in order to gain a better understanding of the operative virulence strategies in the resistant strains so that novel therapeutics aim at disarming virulence can be developed.