Impact of Fluoroquinolone resistance on Pseudomonas virulence and patient outcome

描述（由申请人提供）：铜绿假单胞菌（Pseudomonas aeruginosa， PA）是一种对公众健康构成直接威胁的细菌，因为它在引起医院感染、出现多药耐药、毒力潜力方面起主导作用，并且在开发管道中缺乏针对该病原体的新药。在急性感染中，PA利用III型分泌系统（TTSS）将细胞毒素（即ExoU、ExoS）在接触宿主细胞后传递到宿主细胞中，最终使PA跨越上皮屏障，全身扩散或导致细胞死亡。大多数PA分离物含有exoS或exoU基因；它的存在因临床部位或疾病背景而异。exo -分泌PA和fq -耐药PA引起的急性肺炎的发病率和死亡率都很高，这提高了抗生素耐药性可能与毒性增强有关的可能性。初步数据表明，耐fq的PA群体以exoU+菌株为主，对培养的上皮细胞的细胞毒性随着耐药程度的提高而增加。我们假设exoU+ PA菌株更容易适应FQ暴露，原因是其靶酶（如DNA回转酶）发生突变，同时赋予对FQ的抗性，并由于DNA超卷曲的变化而上调TTSS基因转录。我们首先提出了一种系统的方法来确定fq耐药的PA菌株是否更具毒性，作为开发新的管理策略的必要的第一步，这些策略迫切需要用于治疗多重耐药PA引起的感染。我们将检查从PA肺炎患者获得的大量呼吸道分离样本（n=216）和已经收集的可用结局数据，以便：1)确定与exoS+菌株相比，exoU+菌株是否更频繁地观察到FQ耐药；2)评估临床分离株的耐药和毒力的共同表达在体外细胞模型中转化为生物效应的程度，以及与患者预后的相关性；3)研究反复暴露于FQ后PA菌株耐药突变积累时毒力表达的动态。如果我们能证明PA在FQ暴露后进化成一种“超级细菌”，变得更耐药，毒性更强，我们将有一个强有力的基础：1)开出更积极的治疗方案，早期和更强化的治疗；2)严格限制不加区分的FQ处方；同时，加强当前的感染控制努力，以最大限度地减少FQ耐药和毒性菌株的传播。我们将有一个强有力的基础来筛选现有的化合物，可以阻断TTSS毒力，以及基于更好地理解调节耐药性和毒力表达的分子途径来确定新的药物靶点。我们的假设如果得到证实，将有助于推进针对III型效应物（pcrV疫苗，抗pcrV抗体）的免疫治疗的发展，以及快速诊断，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铜绿假单胞菌（院内感染的主要原因）是否会随着对氟喹诺酮类抗生素产生耐药性而变得更具毒性，并证明这种共表达发生的分子模型。由于缺乏针对这种病原体的新药，因此迫切需要将这项研究作为必要的第一步，以便更好地了解耐药菌株的有效毒力策略，从而开发出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.