Effect of Micro-Patterned Mucin on Quinolone and Rhamnolipid Profiles of Mucoid Pseudomonas aeruginosa under Antibiotic Stress

Abstract

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.

Metabolic insights from mass spectrometry imaging of biofilms: A perspective from model microorganisms

“…However, they lacked information that could be obtained through chemical imaging approaches. MSI-derived signal abundance and distribution patterns of quinolones from biofilm grown on engineered surfaces coated with mucin and mercaptoundecanoic acid share similar observations [ 103 , 111 ]. Further, it also shows that the effects of spatial confinement could be modulated by the properties of EPS secreted by the bacteria.…”

“…Low quinolone signal intensities were notable in the FLR01 compared to the two laboratory strains, even in the absence of the antibiotic. Differences in quinolone abundance in CF lung isolate (FRD1) compared with the laboratory strain PAO1C when allowed to grow in the same environmental conditions are also evident in other studies [ 103 ]. Highlighting that the metabolic revamping in P. aeruginosa during chronic lung infections persists even when the cells are removed and relocated to less stressful environments, as seen in both non-mucoid (FLR01) and mucoid (FRD1) isolates [ 105 ].…”

“…A few key quinolone molecules, such as 2-heptyl-3-hydroxy-4 (1H)-quinolone (PQS), 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), 2-heptyl-3-hydroxy-4(1H)-quinolone (C9-PQS), 2-nonyl-4-hydroxyquinoline N-oxide (NQNO), and biosynthetic precursors like 2-heptyl-4-hydroxy-quinoline (HHQ), and 2-nonyl-4-hydroxy-quinoline (NHQ) have been commonly detected on all surfaces. First, a comparison of the planktonic state against biofilm has indicated a higher localized abundance of quinolones and their precursors in the biofilm state [ 99 , 103 ]. However, careful consideration of media volume and cell density is necessary to establish that cells in biofilms indeed produce higher amounts of quinolones.…”

“…relays the capacity of P. aeruginosa to synthesize them [ 118 ]. MSI studies probing biofilm surfaces have reported over 10 rhamnolipid congeners, predominantly as [M + Na] + and [M + K] + adducts, with different levels of abundance and spatial heterogeneity [ 98 , 103 ]. Even though pathways to produce mono- and di-rhamnolipids have been elucidated, precise mechanisms and reasons behind the production of the wide range of rhamnolipid structures are not yet clear [ 119 , 120 ].…”

“…Congener-specific ion images also show a broader spatial distribution of rhamnolipids beyond the presence of quinolones [ 38 ]. Additionally, rhamnolipid abundance also varies between PAO1C and clinical isolate (FRD1) [ 103 ]. Considering the industrial significance of rhamnolipids as a green alternative to synthetic detergents, further characterization of these molecules could be beneficial and feasible using MSI.…”