Microfabricated environments for the study of bacterial group behavior

This thesis describes the application of micro three dimensional printing (µ3DP) techniques to create protein microstructures for the study of bacterial group behavior in small populations. Studies involving aggregates of ~10^1 to ~10^5 cells have shown extensive and complex communication and spatial organization. Multiphoton lithography (MPL) provides a means to quickly design and execute the fabrication of microscale structures with submicron resolution from a variety of biocompatible polymers. Using this technique, intricate spatial arrangements of bacteria can be achieved while maintaining small population sizes at high cell density (≥10^8 cells/mL), providing in vitro culture conditions which better simulate in vivo settings. As a result, valuable information can be obtained about bacterial social interactions through the coupling of additional analytical techniques to detect the presence or absence of extracellular signaling molecules. While quorum sensing (QS) remains the most extensively studied means of bacterial communication, it is becoming increasingly apparent that additional factors are necessary to effect certain changes in population-wide genetic expression which can lead to increased virulence, pathogenicity, and the development of antibiotic resistance. The work presented in this thesis addresses the influences of cell density, chemical heterogeneity of the environment within cell aggregates, and level of cell surface attachment as mechanisms to induce or influence the development of antibiotic resistance. Building upon previous work presented by members of the Shear lab, BSA-gelatin protein microstructures were used to study the behavior and response of the opportunistic pathogen Pseudomonas aeruginosa under these conditions. Antibiotic resistance was observed in low cell number/high density populations in agreement with previous work presented by the Shear lab. In addition, it was found that localized regions of oxygen depletion do not correlate directly with antibiotic resistance development, as the population size required for depletion far exceeded that for development of resistance. Finally, a new technique directed at simultaneous biofilm inhibition and cell removal from solution was explored.