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

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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.