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.

Volatile organic compounds (VOCs) have been proposed in the last two decades as biomarkers for disease detection and therapeutic monitoring. Model in vitro experiments with established cell lines are fundamental to clarify whether given VOCs originate from normal human cells or pathogens, including transformed cancer cells. Due to the trace concentrations of target metabolites, adsorptive enrichment is needed before gas chromatography-mass spectrometry (GC-MS) analysis, with solid-phase microextraction (SPME) being perfectly suited for this purpose. Here, a modification of SPME, the thin-film microextraction (TFME) technique, is proposed for analysis of cellular VOCs, which utilizes a planar mesh coated with stationary phase to increase the extraction phase volume and active surface area. In this study, four different adsorbents were compared: carboxen, divinylbenzene, hydrophobic−lipophilic balanced and polydimethylsiloxane. Amongst them, HLB sheets using poly(divinylbenzene-co-N-vinyl-pyrrolidone) skeleton structure proved to be the most versatile, enabling the most sensitive analysis of VOCs with a broad polarity and volatility. For HLB, sampling type (internal static headspace, external bi-directional headspace), extraction temperature and extraction time were also examined. An established method was successfully applied to analyze metabolites produced by A549 cells revealing five volatiles at significantly higher (additionally benzaldehyde at lower) levels in cell culture medium compared to the cell-free reference medium headspace.

β-Lactam antibiotics are most commonly used in the critically ill, but their effective dosing is challenging and may result in sub-therapeutic concentrations that can lead to therapy failure and even promote antimicrobial resistance. In this study, we present the analytical tool enabling specific and sensitive determination of the sole biologically active fraction of piperacillin and imipenem in biological material from the critically ill. Thin-film microextraction sampling technique, followed by rapid liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis, was optimized and validated for the quantitative determination of antibiotics in blood and bronchoalveolar lavage (BAL) specimens collected from intensive care unit (ICU) patients suffering from ventilation-associated pneumonia (n = 18 and n = 9, respectively). The method was optimized and proved to meet the criteria of US Food and Drug Administration (FDA) guidelines for bioanalytical method validation. Highly selective, sensitive, accurate and precise analysis by means of thin-film microextraction–LC-MS/MS, which is not affected by matrix-related factors, was successfully applied in clinical settings, revealing poor penetration of piperacillin and imipenem from blood into BAL fluid (reflecting the site of bacterial infection), nonlinearity in antibiotic binding to plasma-proteins and drug-specific dependence on creatinine clearance. This work demonstrates that only a small fraction of biologically active antibiotics reach the site of infection, providing clinicians with a high-throughput analytical tool for future studies on personalized therapeutic drug monitoring when tailoring the dosing strategy to an individual patient.