Breath Test for Pseudomonas aeruginosa in CF

描述(申请人提供)：囊性纤维化(CF)的进行性肺损伤是由于初始感染后以铜绿假单胞菌为中心的特征性细菌定植引起的，然后用野生型菌株定植，在CF宿主中发生铜绿假单胞菌向粘液表型的转化，增加细菌对抗生素的耐药性和炎症性肺损伤，导致肺功能和预后下降。对肺部铜绿假单胞菌的存在、负荷和粘液状态的快速、非侵入性诊断分析仍然没有得到满足。这样的诊断将使感染的实时监测成为可能，并显著改善目前依赖于间接测量的CF管理，例如肺功能和痰培养(即可变的，样本只是肺的一部分，不适用于年轻患者)或支气管肺泡灌洗(这是侵入性的，不适合重复使用)。我们发现，几种同位素标记的底物可以唯一地被铜绿假单胞菌转化为挥发性可呼出的标记气体，其方式取决于细菌的表型。因此，我们假设我们可以使用稳定的同位素标记化合物对呼出气体的铜绿假单胞菌特异性代谢来确定肺部感染、细菌负荷、生物膜生长和粘液状态。然而，在我们进入翻译临床试验之前，我们需要验证我们的假设，描述野生型、生物膜和粘液菌株中标记挥发物的典型比例，并在铜绿假单胞菌肺部感染的动物模型中证明有效性。因此，我们将实现这些目标：特定目标1演示和开发使用同位素比率质谱仪来测量体外反硝化作用、氰化物合成酶和尿素酶活性的同位素标记气体。具体目的2描述非粘液型和粘液型铜绿假单胞菌在体外浮游生长和生物膜中尿素酶与氰化物合成酶/反硝化产物的比率范围。具体目标3证明这种诊断方法在粘液性和非粘液性铜绿假单胞菌肺部感染小鼠中的有效性。在成功完成这一临床前阶段之后，将进行吸入剂剂型的配方工作和第一阶段临床试验。公共卫生相关性：囊性纤维化是一种高发、缩短寿命的遗传性疾病，大部分肺损伤来自铜绿假单胞菌感染。然而，监测铜绿假单胞菌的存在、负担和表型，以便能够主动和主动地管理感染的临床工具并不合适或可用。我们建议测试一种新的呼气测试的潜力，该测试依赖于一组独特的铜绿假单胞菌代谢途径，从特定的底物产生稳定的同位素标记气体。如果成功，这项工作可以转化为提供一种敏感和快速的新诊断方法，从而显著改善对这种疾病的管理。

DESCRIPTION (provided by applicant): The progressive lung damage in cystic fibrosis (CF) arises from characteristic bacterial colonization with Pseudomonas aeruginosa being central After initial infection then colonization with wild-type strains, conversion of P. aeruginosa to the mucoid phenotype in the CF host occurs, increasing bacterial resistance to antibiotics and inflammatory lung damage, and causing declining pulmonary function and prognosis. There remains an unmet need for rapid, non-invasive diagnostic analysis of lung P. aeruginosa presence, burden and mucoid status. Such a diagnostic would enable real-time monitoring of infection, and also significantly improve CF management, that currently relies upon indirect measures such as lung function and culture of expectorated sputum (that is variable, samples only a part of the lung, not applicable to younger patients) or broncoalveolar lavage (that is invasive and unsuitable for repetitive use). We show that several isotopically labeled substrates can be uniquely converted by P. aeruginosa to volatile exhalable labeled gasses, in ways that depend upon bacterial phenotype. We therefore hypothesize that We can use P. aeruginosa-specific metabolism of stable isotopically-labeled compounds to exhaled gasses to determine lung infection, bacterial load, biofilm growth and mucoid status However, before we move to a translational clinical trial, we need to test our hypothesis, delineate the typical ratios of labeled volatiles in wild-type, biofilm and mucoid strains, and also demonstrate effectiveness in an animal model of CF lung infection with P. aeruginosa. We will therefore perform these aims: Specific Aim 1 Demonstrate and develop use of isotope ratio mass spectrometry to measure isotopically labeled gasses from denitrification, cyanide synthase and urease activities in P. aeruginosa in vitro. Specific Aim 2 Delineate the range of ratios of urease to cyanide synthase/denitrification products for non- mucoid and mucoid P. aeruginosa strains in vitro growing planktonically and in biofilms. Specific Aim 3 Demonstrate effectiveness of this diagnostic approach in mice with mucoid and non-mucoid P. aeruginosa lung infection. Successful completion of this preclinical stage would be followed by formulation work on an inhaled dosage form, and Phase 1 clinical trial. PUBLIC HEALTH RELEVANCE: Cystic fibrosis is a high-prevalence, life shortening genetic disease, with much of the lung damage coming from infection with P. aeruginosa. Yet, the clinical tools to monitor for P. aeruginosa presence, burden & phenotype, so that the infection can be actively and pro-actively managed are not suitable or available. We propose to test the potential of a novel breath test, that relies upon a unique panel of P. aeruginosa metabolic pathways to produce stable isotope labeled gasses from specific substrates. If successful, this work can be translated to provide a sensitive and rapid new diagnostic that could significantly improve management of this disease.