Project Summary Urinary tract infections are among the most common healthcare associated infection, of which up to 80% are due to a urinary catheter. Catheter associated urinary tract infections (CAUTIs) are linked to an increased risk of recurrent infection, renal damage, and secondary bacteremia. These types of infections have been found to be largely polymicrobial, with 3 or more species of bacteria present during both asymptomatic colonization and active infection. We and others have shown that the most common partners are Proteus mirabilis (Pm), Escherichia coli (Ec), and Enterococcus faecalis (Ef), all of which form robust catheter. Previous work done by our lab has shown that co-culture of Pm and Ef causes a dramatic increase in biofilm biomass that is mediated by an increase in biofilm-associated proteins content, and specific adhesins, fimbria, and metabolism-related proteins were enriched in the polymicrobial biofilm. Our lab has also shown that coinfection with Pm and Ef increases disease severity in a mouse model of CAUTI, and that biofilm-enhancing interactions contribute to this process. Considering that CAUTIs often involve various combinations of Pm, Ec, and Ef, and not all dual- species interactions increase biofilm biomass, there is a gap in knowledge regarding how interactions between each of these three species influence catheter biofilm formation and infection progression. We aim to examine the impact of polymicrobial interactions on biofilm architecture, to identify protein mediators of polymicrobial biofilm enhancement, and determine their contribution to the progression and severity of polymicrobial CAUTI. Aim 1 will examine the architecture and spatial distribution of each species in single, double, and triple species biofilms using a combination of scanning electron microscopy and high-resolution fluorescence microscopy. Aim 2 will identify and characterize protein mediators of biofilm enhancement using LC-MS/MS. The top three proteins enriched in polymicrobial biofilms with increased biomass will be selected for further study, and mutants will be made in each. Using a two-pronged approach, we will determine the contribution of enriched proteins to biofilm enhancement in our standard 24- well plate assay and in our established “glass bladder” model, in which biofilms are formed directly on Foley catheters under simulated urine flow. This system is a physiologically relevant in vitro assay that closely mimics conditions occurring in a catheterized patient. Aim 3 will determine the impact of triple species interactions on infection and severity. Previous data from our lab has demonstrated that dual species interactions increase the incidence of bacteremia and urolithiasis in the CAUTI mouse model. We will examine differences in the progression and severity of triple species infection compared to each dual species infection, and will also determine the contribution of one protein mediator of biofilm enhancement to polymicro...