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The identification of specific volatile organic compounds (VOCs) microorganisms may assist in developing a fast and accurate methodology determination of pulmonary bacterial infections in exhaled air. As a pulmonary bacteria were cultured and their headspace analyzed for the amount of excreted VOCs to select those compounds which are exclusively associated with specific microorganisms. Development of a rapid, methodology for identification of bacterial species may improve antibiotic therapy, ultimately leading to controlling the antibiotic problem. Two hundred bacterial headspace samples from four different microorganisms (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus and Klebsiella pneumoniae) were analyzed by gas chromatography-mass to detect a wide array of VOCs. Statistical analysis of these volatiles the characterization of specific VOC profiles indicative for each Differences in VOC abundance between the bacterial types were determined ANalysis of VAriance-principal component analysis (ANOVA-PCA). These were visualized with PCA. Cross validation was applied to validate the We identified a large number of different compounds in the various thus demonstrating a highly significant difference in VOC occurrence of cultures compared to the medium and between the cultures themselves. Additionally, a separation between a methicillin-resistant and a methicillin-sensitive isolate of S. aureus could be made due to differences between compounds. ANOVA-PCA analysis showed that 25 VOCs differently profiled across the various microorganisms, whereas a PCA enabled the visualization of these clear differences between the We demonstrated that identification of the studied microorganisms, antibiotic susceptible and resistant S. aureus substrain, is possible selected number of compounds measured in the headspace of these in vitro results may translate into a breath analysis approach that has potential to be used as a diagnostic tool in medical microbiology.