Most bacteria in natural and clinical settings grow as surface-attached biofilms, which are bacterial communities that have self-assembled into an encased matrix of extracellular polymeric substances (EPS). To form these bacterial biofilm communities and infect host cells, an intercellular signaling process described as quorum sensing (QS) is very important. For the opportunistic pathogen, Pseudomonas aeruginosa, QS regulates the expression of many genes important to biofilm initiation, EPS production, and virulence. While much has been learned about select factors that regulate biofilm formation in vitro and in animal models, the specifics by which multispecies groups of cells form biofilms are not yet clear. Correlated mass spectrometric and Raman imaging will be used to develop a multiplex analysis method for studying host-associated microbial communities. These methods allow the determination of individual bacterial species and their microbial products within a mixed bacterial community growing in situ on surfaces. A long-term goal is the design of detection and diagnostic strategies informed by an understanding of bacterial interactions and signature biomolecule production. Work toward this goal will begin by conducting experiments with the opportunistic pathogen Pseudomonas aeruginosa, specifically developing methods to distinguish P. aeruginosa from other bacterial species, distinguish separate P. aeruginosa strains from each other, and determine the spatial distribution of four types of P. aeruginosa molecules associated with virulence: acyl- homoserine lactones (AHL), quinolones, such as pseudomonas quinolone signal (PQS), rhamnolipids, and exopolymeric substances (EPS). Successfully implementing this research program will result in bacterial mapping methods with improved sensitivity and resolution, substantially enhancing our ability to identify specific species of bacteria in mixed samples, and to identify and map specific chemical products produced by these bacteria that are critical to host colonization, infection, and virulence.
Most infections are the result of surface-attached biofilm communities of bacteria that colonize host surfaces and the bacterium Pseudomonas aeruginosa is an opportunistic pathogen that readily forms biofilms on many surfaces. We propose to use P. aeruginosa as a model species for developing correlated mass spectrometric and Raman imaging to perform multiplex analysis, enabling determination of individual bacterial species and many microbial products produced by these bacteria within a mixed bacterial community growing in situ on surfaces. We will use our new methodology to distinguish separate P. aeruginosa strains from each other, and determine the spatial distribution of acyl-homoserine lactone (AHL) and quinolone signaling molecules, biosurfactant rhamnolipids, and exopolymeric substances (EPS) produced by P. aeruginosa, each of which contributes to biofilm formation affecting virulence of P. aeruginosa.
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