Identifying breath biomarkers for S. aureus methicillin resistance and small colony variants

项目摘要：大多数囊性纤维化（CF）患者的主要死因是呼吸道疾病 由慢性肺部感染和炎症引起的衰竭。金黄色葡萄球菌是最 是美国CF人群肺部感染的常见原因，70%的患者S.金黄色 2018年S有两种亚型。金黄色葡萄球菌，特别令人担忧，因为它们与较低的 肺功能和更高的死亡风险：耐甲氧西林S。金黄色葡萄球菌（或MRSA）和小菌落变体（或 SCV）。大约50%的CF患者MRSA检测阳性，28%的SCV检测阳性。测试 S.金黄色葡萄球菌，MRSA和SCV严重依赖于痰液的收集，但随着CF护理的改善和使用 CFTR调节剂的增加，痰液的产生正在下降，尽管肺部感染的风险 持续存在。因此，迫切需要用于诊断无痰肺部感染的新方法。 我们的长期目标是开发一种检测S的呼吸测试。慢性CF中的金黄色葡萄球菌MRSA和SCV 肺部感染该提案的总体目标是确定MRSA的推定呼吸生物标志物， SCV并确定其诊断准确性。我们的核心假设是呼吸中的挥发性生物标志物 可用于快速和灵敏地检测MRSA和SCV，不依赖于与其他病原体的合并感染。 种或其他S.金黄色葡萄球菌分离株。我们将通过充分利用- 特征为S.金黄色葡萄球菌分离物来自小菌落变异株S.金黄色葡萄球菌（SCVSA）研究（目的1），以及配对 使用呼吸法（IMPACT-Breath）进行痰液和呼吸标本的改良呼吸支原体检测 研究（目标2）。该项目的预期成果是S.金黄色葡萄球菌MRSA和SCV， 并估计它们在CF患者呼吸中的敏感性和特异性。 目的1：鉴定体外培养的MRSA、SCV和MRSA-SCV的推定挥发性生物标志物。我们将 表征甲氧西林敏感正常菌落变体（MSSA-NCV）、MRSA- 体外培养的NCV、MSSA-SCV和MRSA-SCV分离株，使用100株测序和表型分析分离株 SCVSA的研究。我们假设MRSA与MSSA有不同的挥发性生物标志物， 对于SCV与NCV分离株，并且由MRSA-SCV分离株产生一组新的生物标志物。 目的2：完善MRSA和SCV的推定挥发性生物标志物，并评估其在呼吸中的准确性 有CF的人。我们将分析来自100 S的配对痰和呼吸样本。金葡菌阳性CF肺 IMPACT-Breath研究中的感染。我们将使用文化依赖和文化无关的方法 鉴定和量化痰中的MRSA和SCV，并将这些数据与每个受试者的呼吸相关联 挥发物我们假设MRSA和SCV的挥发性生物标志物将在人的呼吸中检测到 对这些沙门氏菌培养呈阳性金黄色葡萄球菌亚型，即使在其他微生物物种的存在下， MSSA和NCV的存在。

PROJECT ABSTRACT: A primary cause of death for most persons with cystic fibrosis (CF) is respiratory failure caused by damaging chronic lung infections and inflammation. Staphylococcus aureus is the most common cause of lung infections in the US CF population, with 70% of patients testing positive for S. aureus in 2018. There are two subtypes of S. aureus that are particularly worrying because they are linked to lower lung function and higher risk of death: methicillin-resistant S. aureus (or MRSA) and small colony variants (or SCVs). Approximately 50% of CF patients test positive for MRSA and 28% test positive for SCVs. Testing for S. aureus, MRSA, and SCVs heavily relies upon the collection of sputum, but as CF care improves and use of CFTR modulators increases, the production of sputum is declining, though the risk of lung infections persists. Therefore, novel methods for diagnosing lung infections without sputum are urgently needed. Our long-term goal is to develop a breath test for the detection of S. aureus MRSA and SCV in chronic CF lung infections. The overall objective of this proposal is to identify putative breath biomarkers of MRSA and SCVs and to determine their diagnostic accuracy. Our central hypothesis is that volatile biomarkers in breath can be used to rapidly and sensitively detect MRSA and SCVs, independent of co-infections with other species, or the presence of other S. aureus isolates. We will test our hypothesis by leveraging well- characterized S. aureus isolates from the Small Colony Variant S. aureus (SCVSA) study (Aim 1), and paired sputum and breath specimens from the IMproving P. Aeruginosa deteCTion using Breath (IMPACT-Breath) study (Aim 2). The expected outcomes of this project are breath biomarkers of S. aureus MRSA and SCVs, and estimates of their sensitivity and specificity in the breath of persons with CF. Aim 1: Identify putative volatile biomarkers of MRSA, SCVs, and MRSA-SCVs grown in vitro. We will characterize the volatile metabolomes of methicillin sensitive-normal colony variant (MSSA-NCV), MRSA- NCV, MSSA-SCV, and MRSA-SCV isolates cultured in vitro, utilizing 100 sequenced and phenotyped isolates from the SCVSA study. We hypothesize that there are distinct volatile biomarkers for MRSA vs. MSSA and for SCV vs. NCV isolates, and that a novel set of biomarkers are produced by MRSA-SCV isolates. Aim 2: Refine putative volatile biomarkers of MRSA and SCV and estimate their accuracy in the breath of persons with CF. We will analyze paired sputum and breath samples from 100 S. aureus positive CF lung infections from the IMPACT-Breath study. We will use culture-dependent and culture-independent methods to identify and quantify MRSA and SCVs in the sputum and correlate these data to each subject’s breath volatiles. We hypothesize that volatile biomarkers for MRSA and SCV will be detected in the breath of persons that are culture-positive for these S. aureus subtypes, even in the presence of other microbial species, and in the presence of MSSA and NCVs.