GC-MS profiling of volatile metabolites produced by Klebsiella pneumoniae

Wojciech Filipiak; Karolina Żuchowska; Marta Marszałek; Dagmara Depka; Tomasz Bogiel; Natalia Warmuzińska; Barbara Bojko

doi:10.3389/fmolb.2022.1019290

GC-MS profiling of volatile metabolites produced by Klebsiella pneumoniae

Front Mol Biosci. 2022 Oct 18:9:1019290. doi: 10.3389/fmolb.2022.1019290. eCollection 2022.

Authors

Wojciech Filipiak¹, Karolina Żuchowska¹, Marta Marszałek¹, Dagmara Depka², Tomasz Bogiel², Natalia Warmuzińska¹, Barbara Bojko¹

Affiliations

¹ Department of Pharmacodynamics and Molecular Pharmacology, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Bydgoszcz, Poland.
² Department of Microbiology, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Bydgoszcz, Poland.

Abstract

Currently used methods for diagnosing ventilator-associated pneumonia (VAP) are complex, time-consuming and require invasive procedures while empirical antibacterial therapy applies broad spectrum antibiotics that may promote antimicrobial resistance. Hence, novel and fast methods based on alternative markers are needed for VAP detection and differentiation of causative pathogens. Pathogenic bacteria produce a broad range of volatile organic compounds (VOCs), some of which may potentially serve as biomarkers for microorganism identification. Additionally, monitoring of dynamically changing VOCs concentration profiles may indicate emerging pneumonia and allow timely implementation of appropriate antimicrobial treatment. This study substantially extends the knowledge on bacterial metabolites providing the unambiguous identification of volatile metabolites produced by carbapenem-resistant and susceptible strains of Klebsiella pneumoniae (confirmed with pure standards in addition to mass spectra match) but also revealing their temporary concentration profiles (along the course of pathogen proliferation) and dependence on the addition of antibiotic (imipenem) to bacteria. Furthermore, the clinical strains of K. pneumoniae isolated from bronchoalveolar lavage specimens collected from mechanically ventilated patients were investigated to reveal, whether bacterial metabolites observed in model experiments with reference strains could be relevant for wild pathogens as well. In all experiments, the headspace samples from bacteria cultures were collected on multibed sorption tubes and analyzed by GC-MS. Sampling was done under strictly controlled conditions at seven time points (up to 24 h after bacteria inoculation) to follow the dynamic changes in VOC concentrations, revealing three profiles: release proportional to bacteria load, temporary maximum and uptake. Altogether 32 VOCs were released by susceptible and 25 VOCs by resistant strain, amongst which 2-pentanone, 2-heptanone, and 2-nonanone were significantly higher for carbapenem-resistant KPN. Considerably more metabolites (n = 64) were produced by clinical isolates and in higher diversity compared to reference KPN strains.

Keywords: Klebsiella pneumoniae; bacteria markers; breath markers; gas chromatography -mass spectrometry (GC-MS); headspace analysis; ventilator-associated pneumonia (VAP); volatile metabolites; volatile organic compounds (VOC).