DESCRIPTION Catheter-associated urinary tract infection (CAUTI) is a costly clinical problem that affects millions of patients worldwide. CAUTI are characterized by infection of the bladder and pathogen colonization of the catheter surface, making them especially difficult to treat. Catheter modifications have been employed to reduce pathogen colonization, including infusion of antibiotics. However, with a rise in antibiotic resistance among uropathogen strains, there is a great need to develop alternative strategies for effective CAUTI treatment and prevention. Lactobacillus probiotics offer promise for a “bacterial interference” approach to prevent CAUTI because they not only could compete for adhesion to the catheter surface but they also produce and secrete antimicrobial compounds that are effective against uropathogens. Three-dimensional (3D)-bioprinting has enabled fabrication of well-defined, cell-laden architectures with tailored release of active agents. We hypothesized that 3D- bioprinting could offer a novel means for sustained probiotic delivery. We have capitalized on this technology to design and fabricate a prototype 3D-bioprinted catheter tubing containing the widely used probiotic strain Lactobacillus rhamnosus GG. Our preliminary data already demonstrate that our initial bioprint prototype 1) maintains probiotic viability upon extended storage, 2) displays sustained release of live L. rhamnosus, lactic acid and hydrogen peroxide in vitro, 3) develops surface-associated L. rhamnosus biofilms, 4) inhibits uropathogenic E. coli in vitro and 4) maintains L. rhamnosus viability and release in vivo in a mouse model. Here, we will test the overarching hypothesis that the combination of probiotic bacterial interference and 3D- bioprinting technologies provides an optimal framework for effective CAUTI prevention The objective of this proposal is to optimize the design and fabrication of Lactobacillus 3D bioprints and to confirm their safety and efficacy in vitro and in a preclinical mouse model, while advancing our understanding of probiotic interactions with the host and uropathogenic bacteria. Successful completion of the aims will deliver validated 3D-bioprinted prototypes that accomplish long-acting delivery of probiotic species that ultimately improve outcomes in preclinical models of CAUTI. These studies will provide the foundation for translation to reduce risks associated with urinary catheterization, while providing new insights into the effects of lactic acid-based and probiotic therapeutics on host inflammatory response and CAUTI disease markers and progression. Moreover, outcomes of this research will have a significant impact on the development of future probiotic approaches in the context of other medical device-associated infections. The successful completion of this project will deliver validated 3D-printed prototypes that enable long-acting delivery of probiotic bacteria for the prevention and treatment of CAUTI.