Leucine degradation metabolite ratio as a measure of meropenem susceptibility in carbapenem-resistant Klebsiella pneumoniae

Rapid detection of carbapenem-resistant Klebsiella pneumoniae (CRKP) is essential for effective clinical management and surveillance. This study investigated volatile organic compounds (VOCs) as phenotypic biomarkers of carbapenem resistance, with the goal of developing a translationally viable diagnostic metric. Sixteen clinical K. pneumoniae isolates comprising both CRKP and carbapenem-susceptible K. pneumoniae (CSKP) strains were cultured for 6 h in headspace vials, with and without meropenem, and VOCs were extracted using polydimethylsiloxane-coated probes. Volatiles were analyzed by thermal desorption-gas chromatography-mass spectrometry. Under meropenem stress, CRKP and CSKP exhibited distinct VOC profiles, with two leucine-derived metabolites, 3-methyl-1-butanol (syn. isoamyl alcohol) and 3-methylbutanal (syn. isovaleraldehyde), emerging as key discriminatory features. The ratio of these two metabolites accurately classified resistance phenotype, achieving 100% sensitivity and 94.1% specificity. The ratio also correlated significantly with minimum inhibitory concentration and zone of inhibition values. Stable isotope tracing confirmed their origin from leucine catabolism, and metabolic supplementation experiments suggested a functional role for 3-methyl-1-butanol in promoting growth under antibiotic stress. These findings show that antimicrobial resistance is accompanied by detectable shifts in volatile metabolism and that VOCs can serve as reliable indicators of resistance phenotype. The 3-methyl-1-butanol/3-methylbutanal ratio presents a robust biomarker of meropenem resistance, detectable within 6 h of culture growth, and supports the further development of volatilome-based diagnostics for clinical microbiology. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae pose a major threat to patient outcomes and hospital infection control, yet current diagnostic methods for carbapenem resistance are time-consuming or require specific molecular testing. This study demonstrates that meropenem resistance is associated with reproducible and quantifiable changes in volatile metabolism that can be detected within 6 h of culture. We identify a simple ratio of two leucine-derived volatile organic compounds, 3-methyl-1-butanol and 3-methylbutanal, that discriminates resistant from susceptible K. pneumoniae strains and correlates with conventional phenotypic susceptibility measures. Importantly, this ratio reflects a functional metabolic response to antibiotic stress rather than the presence of specific resistance genes, supporting its broad applicability across resistance mechanisms. These findings establish volatile metabolites as rapid, phenotypic indicators of carbapenem resistance and provide a foundation for developing volatilome-based diagnostics that could accelerate antimicrobial susceptibility testing in routine clinical microbiology laboratories.