Metabolic Pathways of Pathogenic Planktonic & Biofilm Bacterial Communities: A Bottom-up Proteomic Study

The cause for persistence of chronic wounds is identified as antibiotic resistance which is caused by the mixed species of microbes that live together in synergy. The basic design of these consortia biofilms is poorly understood, and in-depth characterization of these complex systems is necessary. This dissertation presents the application of tandem mass spectrometric methodology to determine proteins that predict the phenotypic changes in pathogenic bacterial monocultures, cocultures and biofilm aggregates. These complex systems undergo adaptation to perturbations in their environment and they are observed to be resistive in the presence of other species that aid survival of these multiple species. A detailed description of the current understanding of their survival and threat is highlighted along with improvements in sample preparation, availability of high-resolution commercial instruments and their capability to help determine hundreds of protein identities. Data manipulation and interpretation methods with statistical software availability are elaborated in detail. The next part of this work presents proteomic data to validate the existence of pathogenic bacteria in oxygen deprived or excessive lactate supplemented diverse environments. These are simulations of natural environments in which these organisms thrive including the lungs of cystic fibrosis patients or the wound surfaces of diabetes mellitus patients. The highlight of this work is presented in the latter half of the thesis, where an application and development of a method of sampling was tested to remove biofilm material from surfaces using laser irradiation. This method enabled determination of the variations in cells that grow as layers within a biofilm to distinguish differences as they experience an oxygen and nutrient gradient. Finally, it was deemed necessary to determine methods to elucidate the presence or absence of contaminants (especially viral) in bacterial cultures to maintain reproducibility of results interpreted through the protein data obtained. Current practices were tested on already available data as a proof of concept of the work presented. Overall, these experiments were conducted to examine the growth phenotype of planktonic cultures and biofilms of disease-causing pathogenic bacteria Pseudomonas aeruginosa and Staphylococcus aureus. The data obtained in the thesis is to cater a larger goal of supporting computational models that is preliminarily dedicated to a three species model of a chronic wound biofilm which includes Pseudomonas aeruginosa (an aerobe), Staphylococcus aureus (a facultative anaerobe) and Clostridium perfringens (an anaerobe) pathogenic bacterial species. The refinement of these models and the applicability is enhanced by the data provided based on spatially resolved analytical methods developed and implemented.