Volatile organic compound profiling for tracking clinical Pseudomonas aeruginosa isolates.

Pseudomonas aeruginosa is an opportunistic respiratory pathogen frequently affecting hospitalized patients and individuals with chronic lung diseases such as cystic fibrosis (CF), bronchiectasis, and chronic obstructive pulmonary disease (COPD). Current diagnostic methods for airway infection are often invasive or lack sensitivity, highlighting the need for non-invasive approaches. Volatile organic compounds (VOCs) released by microbes represent promising candidates for non-invasive detection of respiratory pathogens. In this study we characterized the volatilome of clinical P. aeruginosa isolates and identified candidate VOC markers associated with bacterial metabolism. An untargeted volatilomics workflow based on gas chromatography-mass spectrometry (GC-MS) was applied to profile the in vitro VOCs produced by 48 clinical P. aeruginosa isolates associated with acute and chronic respiratory infections. We identified three previously unrecognized VOCs consistently produced by all isolates, suggesting conserved metabolic features of P. aeruginosa. Comparative analysis revealed marked metabolic differences between infection phenotypes: chronic isolates produced higher levels of aliphatic volatiles, whereas acute isolates were enriched in aromatic compounds, particularly acetophenone. Longitudinally sampled clonally related isolates from CF patients exhibited distinct and evolving volatilomic signatures, indicating metabolic adaptation over the course of chronic infection. Because LasR dysfunction is common during chronic airway colonization, we further assessed its impact on VOC production. LasR-functional isolates produced elevated levels of sulfur-containing VOCs and specific ketones, while LasR-deficient isolates displayed increased alcohol production. Overall, our findings show that isolates from chronic infections are associated with a shift from aromatic to aliphatic VOCs compared to acute isolates, and highlight volatilomics as a powerful tool for probing metabolic adaptation in P. aeruginosa and for identifying candidate VOC markers associated with infection-related phenotypes.