Simplified molecular imaging analysis of excreted microbial metabolites using a benchtop MALDI-TOF system

Significance of the Topic

The ability to map microbial metabolites directly in biofilms provides critical insights for healthcare, environmental and industrial microbiology. Understanding spatial distributions of quorum sensing molecules and lipids informs infection diagnostics, biofilm control strategies and interspecies interaction studies.

Study Objectives and Overview

This work evaluates a cost-effective benchtop MALDI-TOF system for imaging excreted metabolites from Pseudomonas aeruginosa biofilms, both in mono- and polymicrobial cultures with Staphylococcus aureus and Candida albicans. Complementary LESA-MS profiling on Orbitrap platforms delivers high-accuracy mass measurements and structural assignments.

Used Instrumentation

• Shimadzu MALDI-8030 dual polarity benchtop MALDI-TOF (200 Hz solid-state laser, 355 nm; load-lock chamber; <55 dB operation)
• iMLayer™ automated 9-aminoacridine matrix sublimation device
• Advion LESA extraction system
• Thermo Orbitrap Exactive and Q Exactive mass spectrometers for LESA-MS and MS/MS

Methodology

Biofilms were grown on a thin agar layer deposited over MALDI targets. After 24 h incubation, colonies were removed and slides dried. Matrix deposition used automated sublimation of 9-aminoacridine. Imaging data were acquired in negative ion mode on the MALDI-8030. LESA sampling employed 1:1 methanol:water for quorum sensing metabolites and 2:1 chloroform:methanol for rhamnolipids, with MS and MS/MS analyses performed on Orbitrap instruments.

Key Results and Discussion

MALDI imaging visualized homogenous distributions of HHQ, NHQ, PQS and rhamnolipids beneath biofilm sites. Co-culture with S. aureus or C. albicans significantly decreased signal intensities, reflecting competitive interactions. LESA-MS data corroborated these reductions and provided accurate mass values (±5 ppm) and MS/MS confirmation of compound identities. Notably, HHQ levels rose in the presence of S. aureus by LESA-MS, although MALDI images showed low HHQ signal during co-culture.

Benefits and Practical Applications

• Accessible imaging of microbial metabolites using affordable benchtop hardware
• Combined spatial and compositional analysis enhances understanding of biofilm chemistry
• Potential application in infection biomarker mapping and antimicrobial development

Future Trends and Opportunities

Advances may include higher spatial resolution imaging, integration of real-time MALDI monitoring, multiplexed detection of broader metabolite classes and application to clinical diagnostics. Coupling imaging with deeper MS/MS workflows and machine learning-based data analysis will further elucidate microbial interactions.

Conclusion

This study demonstrates that entry-level MALDI-TOF imaging, combined with LESA-MS profiling, can effectively map and characterize microbial exometabolites in biofilms. The approach offers a practical platform for spatial metabolomics in microbiology research and applied biofilm analysis.

References

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