(647h) Engineering Hydrogels to Study Phage-Bacteria Interactions in Biologically-Relevant Environments

Bacteriophages (i.e., “phages”) are viruses that infect, replicate in, and ultimately kill bacteria. Phage-bacteria interactions play a key role in determining microbiome formation and functioning, with critical implications in biomedical applications. Phage-bacteria interactions are constantly occurring within our bodies, influencing the healthy and diseased states of our microbiomes. Further, phages are being explored as therapeutics to treat antibiotic-resistant infections, with great success in ongoing “phage therapy” clinical trials. Typically, studies of phage-bacteria interactions use “well-mixed” cultures in test tubes or Petri dishes, which do not mimic many of the complexities of biomedically-relevant environments. For example, in natural environments, bacteria and phages often exist in crowded, spatially heterogenous, and porous microenvironments such as mucus, biofilms, and biological tissue, where spatial heterogeneity and interactions with their surrounding environment fundamentally alter their behavior. Furthermore, phage-bacteria interactions are typically studied at a limited number of timepoints due to the experimental challenges of real-time analysis, further limiting understanding of how they influence microbiomes in the real world.

Hydrogel biomaterials have been extensively explored as 3D cell culture platforms to study mammalian cell behavior for applications across biomedical sciences, such as tissue repair and mechanobiology. Here, we extend the field of hydrogel biomaterials engineering to create microenvironments for investigating phage-bacteria interactions in biologically-mimicking conditions, where we investigate how physiologically-relevant confinement and porosity influences phage-bacteria dynamics. We create transparent porous matrices (~10^-1 - 10⁰ μm pore distributions) consisting of packed hydrogel microparticles (i.e., “granular hydrogels”). Using time-lapse confocal microscopy, we investigate the growth and migration behavior of E. coli communities interacting with purely lytic T4 phages in real-time as a function of initial spatial distribution of phages and bacteria, initial concentrations of phages and bacteria, and the degree of confinement imposed by the surrounding porous matrix (modulated by packing density of our granular hydrogel biomaterials). We use embedded bioprinting to impose spatial distribution and heterogeneities in our investigations. Further, we investigate how hydrogels can be used to deliver phages to bacterial communities, unraveling the dynamics of phage delivery in biologically-relevant 3D environments. Through these studies, we uncover fundamental differences between phage-bacteria interactions in 3D microenvironments that mimic their native habitats, compared to traditional well-mixed studies. Ultimately, this work will provide new quantitative insights to inform the design of phage-based engineering solutions across biomedical disciplines.