Insights on the spatial distribution of the <i>Pseudomonas aeruginosa</i> secondary metabolites under swarming motility-inducing conditions using mass spectrometry imaging

<i>Pseudomonas aeruginosa</i> exhibits a variety of motility types, including twitching, swimming, and swarming motility. The latter is typically associated with growth on semi-solid surfaces (e.g., 0.4% to 0.5% agar), the formation of extending tendrils, and the secretion of rhamnolipids, a biosurfactant that aids cells in spreading along a surface. Apart from rhamnolipids, <i>P. aeruginosa</i> also secretes a variety of secondary metabolites involved in pathogenesis and defense. We developed a mass spectrometry imaging (MSI) workflow to characterize metabolites associated with swarming motility in <i>P. aeruginosa</i> and evaluated how externally applied rhamnolipids influence swarming behavior and secondary metabolite production. MSI allowed us to measure spatial profiles of prominent rhamnolipid congeners and secondary metabolites. The addition of rhamnolipids over a broad concentration range (0.0001 to 1 mg/mL) into swarming media prior to inoculation allowed for the study of concentration-dependent effects of rhamnolipids, as well as changes in the secretion profile. Unlike self-produced rhamnolipids that facilitate swarming, externally applied rhamnolipids significantly reduced swarming behavior even at low concentrations. This suggests a potential reason why tendrils from swarming cultures avoid overlap. Insights on the differential distribution of secondary metabolites within these bacterial communities on agar improve our understanding of biofilm development. The use of MSI on <i>P. aeruginosa</i> swarming cultures provides detailed spatial chemical analyses of the secreted molecules under varying conditions.IMPORTANCEThe semi-solid surface that encourages <i>Pseudomonas aeruginosa</i> to exhibit swarming motility has a similar consistency to a stationary, thickened gel commonly associated with impaired lung surfaces during infection. The effects rhamnolipids have on the spreading properties of <i>P. aeruginosa</i> under these conditions have yet to be elucidated. Here, we used mass spectrometry imaging to probe the spatial-chemical profiles of the surface to provide a more complete and unbiased understanding of the distribution of important classes of molecules secreted by <i>P. aeruginosa,</i> including rhamnolipids and a range of secondary metabolites on surfaces. The application of rhamnolipids prior to inoculation allows us to understand how these molecules affect the motility seen under moist, semi-solid conditions.