Spatial distribution and structural discrimination of quorum sensing metabolites in Pseudomonas aeruginosa by MALDI-MSI.

Bacterial biofilms are complex bacterial communities embedded within an extracellular polymeric substance (EPS) that protects the cells and increases survival. Once established on an orthopaedic implant, a biofilm can cause difficult-to-treat and severe infections. Pseudomonas aeruginosa is of particular concern due to its strong biofilm-forming capability. Within biofilm communities, quorum sensing (QS) networks regulate biofilm formation, virulence, and intercellular communication. This is achieved through the production of signalling molecules, QS metabolites. In P. aeruginosa, quinolones such as the Pseudomonas quinolone signal (PQS) and its precursor 2-heptyl-4-quinolone (HHQ) are among the key regulators. However, although these signalling molecules regulate essential functions such as biofilm development, virulence regulation, and population-level communication, our understanding of their spatial distribution within P. aeruginosa biofilms is still limited. QS metabolites, also referred to as autoinducers, help in the regulation of biofilm formation and dispersion. These small molecules, including quinolones, often have an isomeric counter partner which is not involved as autoinducer. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) offers a label-free approach for the spatial detection of these metabolites directly within biofilms. To differentiate isomeric quinolones, such as PQS and 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), tandem MS (MS/MS) and trapped ion mobility spectrometry (TIMS) coupled to time-of-flight (TOF) mass spectrometry are employed in this study, with the addition of salt, sodium chloride, to enhance sodium adduct formation. This integrated approach provides spatially resolved insights into QS metabolite organisation, demonstrating an improved strategy for distinguishing isomeric species within bacterial biofilms and advancing our understanding of bacterial communication and potential anti-biofilm targets.