Project Summary The chemical interplay between cells within pathogenic microbial communities has profound effects on phenotypic states; numerous chemical signals, virulence factors, and redox-active species influence both the virulence potential of bacterial constituents and the response of immune cells. Detailed understanding how ?micro-geography? impacts cellular behavior via transmission of social cues, chemical warfare, and other system attributes such as oxygen and resource limitation would therefore offer potentially valuable new long-term strategies key for combatting infection. A capability to precisely position small, bacterial aggregates having defined physical and phenotypic attributes within microbial communities of interest would enable the study time-variant spatial interactions between cells, such as those that may occur in the spread and progression of infections, as well as dynamic reorganization within established microbiomes. Here, we propose development of a technology platform for investigating sociomicrobiology and other chemically based attributes in pathogenic microbial communities in which bacterial colonies and, in some instances, cells from mammalian lines of defined population sizes/densities, shapes, arrangements, and sociomicrobiological status will be organized in porous protein-based microcontainers 3D printed on maneuverable optical fiber tips. This optrode platform will provide capabilities both for reporting on and controllably perturbing, microbial environments of interest by facile chemical exchange through microcontainer walls and will acquire detailed molecular information on microscopic ecosystems via detection of fluorescent transcriptional reporters and other cellular probes, as well as engineered chemical reporters for nitric oxide and reactive oxygen species incorporated in microcontainer walls. As validation of the platform, we will evaluate spatiotemporal attributes for transmission of quorum sensing and population-dependent antibiotic resistance bi-directionally between fiber-mounted 3D-printed Pa aggregates and model microbiome communities maintained as a physically stationary ensemble of aggregates in visually accessible cultures maintained on an inverted microscope system. As proof-of-concept for extending methodologies to delivery of bacterial communities to in vivo environments, we will 3D fabricate defined populations of Pa on large-format imaging fiber-optic bundles. Phenotypic interplay will be evaluated between fiber-mounted populations and stationary cultures of Pa/Sa. If successful, capabilities that derive from these high-risk, high-benefit studies will have medium- and long-term applications to various in vitro models and in vivo microbomes, including chronic infections in wounds, gastrointestinal tract, and respiratory system.