Volatile organic compounds in headspace characterize isolated bacterial strains independent of growth medium or antibiotic sensitivity

PLoS One. 2024 Jan 26;19(1):e0297086. doi: 10.1371/journal.pone.0297086. eCollection 2024.


Introduction: Early and reliable determination of bacterial strain specificity and antibiotic resistance is critical to improve sepsis treatment. Previous research demonstrated the potential of headspace analysis of volatile organic compounds (VOCs) to differentiate between various microorganisms associated with pulmonary infections in vitro. This study evaluates whether VOC analysis can also discriminate antibiotic sensitive from resistant bacterial strains when cultured on varying growth media.

Methods: Both antibiotic-sensitive and -resistant strains of Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumonia were cultured on 4 different growth media, i.e. Brain Heart Infusion, Marine Broth, Müller-Hinton and Trypticase Soy Agar. After overnight incubation at 37°C, the headspace air of the cultures was collected on stainless steel desorption tubes and analyzed by gas chromatography time-of-flight mass spectrometry (GC-tof-MS). Statistical analysis was performed using regularized multivariate analysis of variance and cross validation.

Results: The three bacterial species could be correctly recognized based on the differential presence of 14 VOCs (p<0.001). This discrimination was not influenced by the different growth media. Interestingly, a clear discrimination could be made between the antibiotic-resistant and -sensitive variant of Pseudomonas aeruginosa (p<0.001) based on their species-specific VOC signature.

Conclusion: This study demonstrates that isolated microorganisms, including antibiotic-sensitive and -resistant strains of Pseudomonas aeruginosa, could be identified based on their excreted VOCs independent of the applied growth media. These findings suggest that the discriminating volatiles are associated with the microorganisms themselves rather than with their growth medium. This study exemplifies the potential of VOC analysis as diagnostic tool in medical microbiology. However, validation of our results in appropriate in vivo models is critical to improve translation of breath analysis to clinical applications.

MeSH terms

  • Anti-Bacterial Agents / pharmacology
  • Bacteria
  • Culture Media
  • Humans
  • Pseudomonas Infections*
  • Pseudomonas aeruginosa
  • Staphylococcus aureus
  • Volatile Organic Compounds* / analysis
  • Volatile Organic Compounds* / pharmacology


  • Volatile Organic Compounds
  • Anti-Bacterial Agents
  • Culture Media

Grants and funding

T. Lubbers (MLDS) Maag Lever Darm Stichting career development grant CDG16-12 supported by Dutch Digestive Foundation (https://www.mlds.nl/) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.