SUMMARY/ABSTRACT The rising rate of antibiotic resistance is increasingly complicating treatment for infections that, not long ago, were easily treated. Prominent among these are catheter-associated urinary tract infections (CAUTIs), which are the most common hospital-associated infection (HAI), and infections of ureteral stents. Over 15 million UTIs occur in the USA each year with accompanying deterioration in the quality of life and increased health care costs. As a result, 15% of all antibiotics prescribed are for the treatment of UTIs, making these infections a leading cause of antibiotic use. Complicating treatment decisions for CAUTI/stent-related infections is that most of these infections are polymicrobial, with multiple bacterial species simultaneously colonizing the implant. Current treatment guidelines typically focus on a single infecting organism and do not consider the underlying polymicrobial foundation of the infection, as reflected by the fact that treatment is often ineffective, requires the removal and replacement with a new catheter/stent, which typically then becomes infected by the same consortia of bacterial species. There is a significant gap in knowledge of the specific composition of these consortia, including fastidious species not amenable to detection by traditional approaches. Other knowledge gaps include: i) the mechanisms that promote and sustain these polymicrobial infections; ii) which communities/members are responsible for symptoms; iii) why certain consortia show resilience and reappear even in the presence of antibiotic treatment; and iv) development of antibiotic and antibiotic-sparing strategies that efficiently disrupt the cycle of polymicrobial infection on urinary catheters and stents. To address this problem, experimental murine CAUTI models have been developed for several prominent Gram-negative and Gram-positive uropathogens, including important multi-drug resistant (MDR) genera such as Enterococcus, Staphylococcus, Klebsiella and Acinetobacter and cutting-edge small molecule therapeutics and immunotherapies have been developed that are highly effective in murine monomicrobial CAUTI models. This proposal will build upon this foundation to: i) characterize human samples from patients with long-term indwelling catheters or stents using a third-generation metagenomic sequencing approach to determine (in <7 hours from sample collection) all members of the infecting consortia and their antibiotic resistance profiles; as proof of concept same day diagnosis and antibiotic prescription in the clinical setting; ii) correlate measures of UTI symptomatology and disease outcomes with specific community members; iii) use mouse and in vitro biofilm models to investigate mechanisms of bacterial-bacterial interactions critical in catheter-associated polymicrobial communities; and iv) investigate the ability of therapeutics, known to be efficacious against one species, to treat polymicrobial CAUTIs. These studies will...