Application of GC Orbitrap Mass Spectrometry for Untargeted Metabolomics of Pathogenic Microorganisms

Significance of the Topic

Mixed infections of Candida albicans and Staphylococcus aureus cause severe morbidity and mortality due to enhanced virulence, tissue invasion and antimicrobial resistance. Understanding the metabolic interactions within mono- and co-cultured biofilms can reveal novel targets for treatment, improve comprehension of pathogen survival strategies, and guide anti-biofilm therapy development.

Aims and Study Overview

This study aimed to apply an untargeted metabolomics workflow using high-resolution GC-Orbitrap mass spectrometry to characterize intracellular and extracellular metabolites from mono- and co-cultures of C. albicans and S. aureus. Key objectives included:

• Comparing metabolite profiles of single-species biofilms and growth media.
• Identifying metabolic shifts induced by interkingdom interactions.
• Demonstrating the capabilities of the Q Exactive GC Orbitrap system for untargeted discovery.

Methodology

Biofilm cultures of C. albicans, S. aureus and their co-culture were grown alongside fresh and spent growth media. Samples were dried, chemically derivatized by methoxymation and trimethylsilylation, and analyzed on a Q Exactive GC Orbitrap GC-MS/MS platform.

• Run time: 24.5 minutes per injection.
• Data acquisition: full-scan at high resolving power, sub-ppm mass accuracy.
• Data processing: peak alignment and statistics in Compound Discoverer; identification in TraceFinder.

Applied Instrumentation

• Thermo Scientific Q Exactive GC Orbitrap GC-MS/MS.
• Thermo Scientific Compound Discoverer software for untargeted workflow.
• Thermo Scientific TraceFinder for targeted compound matching.

Results and Discussion

Untargeted analysis detected 465 medium and 405 intracellular compounds, including 22 novel high-scoring metabolites. Principal component analysis separated mono- and co-culture intracellular profiles and distinguished fresh medium from culture samples. Key findings:

• Rapid consumption of glucose and fructose across all cultures; trehalose depletion specific to C. albicans-containing media.
• Enhanced pentose phosphate pathway activity in co-culture biofilms, suggesting sedoheptulose-7-phosphate as a metabolic interaction node.
• Validation targets: pyroglutamate (5-oxoproline) uptake, myristic acid depletion in co-culture, and intracellular sugar phosphate levels consistent with active biosynthesis without cell lysis.

Practical Applications and Benefits

• The high mass accuracy and chromatographic resolution enable discrimination of isomeric metabolites for broad untargeted profiling.
• Combined targeted and untargeted workflows allow database-driven quantification alongside novel compound discovery.
• Data archiving permits retrospective re-interrogation as reference libraries expand.

Future Trends and Opportunities

Advances in high-resolution GC-Orbitrap metabolomics will support in-depth studies of complex microbiomes and interkingdom interactions. Integration with multi-omics and real-time data processing can drive precision antimicrobial strategies and identify metabolic vulnerabilities in pathogen consortia.

Conclusion

This work demonstrates the power of GC-Orbitrap MS for untargeted metabolomics of pathogenic biofilms. The approach reveals metabolic crosstalk between C. albicans and S. aureus, highlighting pathways for potential therapeutic intervention and underscoring the technique’s broad applicability in microbial research.

References

1. Stipetic L.H. et al. A novel metabolomic approach used for the comparison of Staphylococcus aureus planktonic cells and biofilm samples. Metabolomics. 12(4):1–11 (2016).
2. Stipetic L.H. et al. Draft genome sequence of isolate Staphylococcus aureus LHSKBClinical, isolated from an infected hip. Genome Announcements. 3(2):e00336-15 (2015).
3. Weidt S. et al. A novel targeted/untargeted GC-Orbitrap metabolomics methodology applied to Candida albicans and Staphylococcus aureus biofilms. Metabolomics. 12:189 (2016). doi:10.1007/s11306-016-1134-2.