Linking volatile metabolites from bacterial pathogens to exhaled breath condensate of people with cystic fibrosis

Obtaining sputum samples from people with cystic fibrosis (pwCF) for microbiology has become challenging due to the positive clinical effects of the cystic fibrosis transmembrane conductance regulator modulator therapy, elexacaftor-tezacaftor-ivacaftor (ETI). Although ETI improves lung function and reduces sputum production, recent data shows that bacterial pathogens persist, making continued monitoring of infection important. As an alternative to sputum sampling, this study developed a non-invasive technique called 'Cough Breath' (CB) to identify volatile organic compounds (VOCs) in exhaled breath condensate (EBC) and link them to cystic fibrosis (CF) bacterial pathogens using purge and trap GC-MS. The CB culturing approach was able to isolate pathogens from expectorated particulates simultaneously with EBC collection; however, culturing positivity was low, with 6% of samples collected (<i>n</i>=47) positive for either <i>Pseudomonas aeruginosa</i> or <i>Staphylococcus aureus</i>. From EBC, we identified VOCs matching those uniquely produced by <i>P. aeruginosa</i> (7), <i>S. aureus</i> (12), <i>Achromobacter xylosoxidans</i> (8) and <i>Granulicatella adiacens</i> (2); however, the overall detection rate was also low. Expanding to VOCs produced across multiple pathogens identified 30 frequently detected in the EBC of pwCF, including 2,3-pentanedione, propyl pyruvate, oxalic acid diallyl ester, methyl isobutyl ketone, methyl nitrate, 2-propenal, acetonitrile, acetoin and 2,3-butanedione. Comparing isolate volatilomes and EBC samples from the same pwCF enhanced detection rates with key VOCs, such as 2,3-pentanedione (86%) and propyl pyruvate (83%), in <i>P. aeruginosa</i> isolates. Further investigation showed that VOC production differed across strains and at different growth phases, creating variability that may explain the overall low EBC detection rate. Although this study successfully cultured CF pathogens from cough particulates and matched their unique VOCs in EBC samples, our results indicate that microbial volatiles more generally indicative of infection, such as 2,3-pentanedione, may have the most utility in aiding diagnostics in pwCF on ETI who have reduced sputum production in the clinic.