Molecular Imaging of Bacterial Communities of the Opportunistic Human Pathogen Pseudomonas aeruginosa

Raman spectroscopy has carved a niche in the biological and biomedical sciences as a powerful analytical platform in disease diagnostics and in the study of biochemical phenomena. The ability to rapidly and non-invasively generate detailed molecular maps of the sample under analysis makes Raman imaging efficacious for studying communities of the bacteria Pseudomonas aeruginosa. Biofilm communities of P. aeruginosa are associated with severe and persistent infections in patients afflicted with maladies such as cystic fibrosis (CF) and severe burn wounds. Furthermore, P. aeruginosa exhibits a high tolerance to antibiotics partly due to its ability to form stable and refractory biofilms.

The research presented here describes the development of a multimodal imaging approach combining confocal Raman microscopy (CRM) and mass spectrometry imaging (MSI) and its application to the study of mechanisms involved in the formation and growth of biofilms of P. aeruginosa, principally through the characterization of alkyl quinoline (AQ) signaling molecules and secondary metabolities. Results indicate that there is a predominance of the N-oxide AQs during the early stages of biofilm growth followed by secretion of the 2-alkyl-3-hydroxy-4-quinolone family of AQs as the biofilm matures. Furthermore, Surface enhanced Raman scattering (SERS) imaging is used to explore the strain and nutrient dependent secretion of the virulence factor pyocyanin in biofilms of P. aeruginosa laboratory and CF isolate strains cultured in media supplemented with either glucose or glutamate as the source of carbon.

The multimodal imaging approach is also employed to explore bacterial swarming and twitching surface motility and the effects of antibiotics on swarm colonies of P. aeruginosa by characterizing the spatiotemporal distributions of phenazines, rhamnolipids and AQs. Dramatic changes are observed in the distributions of AQs in swarm colonies on exposure to tobramycin and carbenicillin antibiotics, which are specific to the quantity and the identity of the antibiotic.

Finally, this work discusses the possibility of employing CRM/MSI multimodal imaging approach to explore the bacterial secretome in engineered systems like fluidic devices and in multi-strain and multi-species bacterial communities in order to further the understanding of bacterial virulence, antibiotic resistance and host-pathogen interactions.