Label-free molecular imaging of bacterial communities of the opportunistic pathogen Pseudomonas aeruginosa

Abstract

Biofilms, such as those formed by the opportunistic human pathogen Pseudomonas aeruginosa are complex, matrix enclosed, and surface-associated communities of cells. Bacteria that are part of a biofilm community are much more resistant to antibiotics and the host immune response than their free-floating counterparts. P. aeruginosa biofilms are associated with persistent and chronic infections in diseases such as cystic fibrosis and HIV-AIDS. P. aeruginosa synthesizes and secretes signaling molecules such as the Pseudomonas quinolone signal (PQS) which are implicated in quorum sensing (QS), where bacteria regulate gene expression based on population density. Processes such as biofilms formation and virulence are regulated by QS. This manuscript describes the powerful molecular imaging capabilities of confocal Raman microscopy (CRM) and surface enhanced Raman spectroscopy (SERS) in conjunction with multivariate statistical tools such as principal component analysis (PCA) for studying the spatiotemporal distribution of signaling molecules, secondary metabolites and virulence factors in biofilm communities of P. aeruginosa. Our observations reveal that the laboratory strain PAO1C synthesizes and secretes 2-alkyl-4-hydroxyquinoline N-oxides and 2-alkyl-4-hydroxyquinolones in high abundance, while the isogenic acyl homoserine lactone QS-deficient mutant (ΔlasIΔrhlI) strain produces predominantly 2-alkyl-quinolones during biofilm formation. This study underscores the use of CRM, along with traditional biological tools such as genetics, for studying the behavior of microbial communities at the molecular level.

Spatiotemporal Distribution of Pseudomonas aeruginosa Alkyl Quinolones under Metabolic and Competitive Stress

“…Our group and others have previously established that surface growth greatly stimulates both HQNO and PQS production ( 13 , 23 ). However, under surface motile conditions, the appearance of HQNO predictably precedes the appearance of PQS, but HQNO is not modulated equivalently to PQS ( 12 , 32 ). Here, we present evidence that at least two separate factors promote PQS production during surface growth, and we present a working model to describe the onset of PQS activation ( Fig.…”

“…3 ) and 2,800 to 3,000 cm −1 (C-H stretching region; Fig. S6 ) based on our prior work ( 12 , 25 ). The only features detected were those of P. aeruginosa cells at 746, 1,127, 1,313, and 1,583 cm −1 , independently of time and incubation pairing.…”

“…The PQS system uses 2-alkyl-4(1 H )-quinolones (AQs) ( 10 ) to mediate bacterial behaviors, including iron chelation, cytotoxicity, and other functions associated with virulence ( 11 – 13 ). The regulation of AQ production, not just PQS production, is complex.…”

Speciality Grand Challenge for “Biofilms”

“…Multiplex FISH technology has been applied to characterize the biogeography of the oral microbiota ( Valm et al., 2012 ; Valm, 2019 ) while recent microscopy imaging and analyzing technologies allowing for dynamically tracking single biofilm bacteria ( Hartmann et al., 2019 ), for example upon antibiotic stress in V. cholerae ( Díaz-Pascual et al., 2019 ). Additionally, some very promising tools have been recently adapted to metabolite identification at the single cell level in biofilm, for instance using Raman spectroscopy, Mass spectrometry or electro-chemical chip/fluorophores ( Baig et al., 2016 ; Bellin et al., 2016 ; Bodelón et al., 2016 ; Schiessl et al., 2019 ; Geier et al., 2020 ; Yang et al., 2020 ). The upcoming challenge will be, in addition to further development of these technologies, to adapt them to direct observation or characterization of in situ biofilms and to models, whether in vitro , ex vivo , or in vivo , that better feature real in situ biofilms.…”

Biological and clinical significance of quorum sensing alkylquinolones: current analytical and bioanalytical methods for their quantification

“…The use of CRM provides sub-micron spatial resolution for constructing 3D chemical maps of the biological sample under study by relying on the characteristic vibration mode of the target molecules, and SERS presents higher sensibility compared to conventional Raman spectroscopy. In several studies developed by Baig and co-workers, CRM and SERS in conjunction with principal component analysis (PCA) were applied for analysing the distribution of quinolone signalling molecules and secondary metabolites in biofilms of P. aeruginosa strains [126]. The authors succeeded in observing a distinct release profile for the different strains used in the study, highlighting the importance of these types of techniques for the study of microbial communities at molecular level for a better comprehension of bacterial pathogenicity.…”