Pseudomonas aeruginosa (P. aeruginosa) is commonly implicated in hospital-acquired infections where its capacity to form biofilms on a variety of surfaces and the resulting enhanced antibiotic resistance seriously limit treatment choices. Because surface attachment sensitizes P. aeruginosa to quorum sensing (QS) and induces virulence through both chemical and mechanical cues, we investigate the effect of surface properties through spatially patterned mucin, combined with sub-inhibitory concentrations of tobramycin on QS and virulence factors in both mucoid and non-mucoid P. aeruginosa strains using multi-modal chemical imaging combining confocal Raman microscopy and matrix-assisted laser desorption/ionization-mass spectrometry. Samples comprise surface-adherent static biofilms at a solid-water interface, supernatant liquid, and pellicle biofilms at an air-water interface at various time points. Although the presence of a sub-inhibitory concentration of tobramycin in the supernatant retards growth and development of static biofilms independent of strain and surface mucin patterning, we observe clear differences in the behavior of mucoid and non-mucoid strains. Quinolone signals in a non-mucoid strain are induced earlier and are influenced by mucin surface patterning to a degree not exhibited in the mucoid strain. Additionally, phenazine virulence factors, such as pyocyanin, are observed in the pellicle biofilms of both mucoid and non-mucoid strains but are not detected in the static biofilms from either strain, highlighting the differences in stress response between pellicle and static biofilms. Differences between mucoid and non-mucoid strains are consistent with their strain-specific phenology, in which the mucoid strain develops highly protected biofilms.
Keywords: Pseudomonas aeruginosa; Raman imaging; antibiotic resistance; biofilm; mass spectrometry imaging; rhamnolipids.