Community-acquired urinary tract infection (UTI) is among the most common bacterial infections worldwide, affecting at least 150 million people annually. When a patient suffers 3 UTIs within a 12-month period, the infection is termed recurrent UTI (rUTI). rUTI severely diminishes quality of life and has become one of the most difficult urologic diseases to manage in women. Rates of range from 19-36% in premenopausal women and increasing to 50% in postmenopausal (PM) women. The higher incidence of rUTI in PM women compared to premenopausal women suggests that the host environment plays a role in rUTI susceptibility. rUTI management relies on antibiotic therapy but many front-line antibiotics have become ineffective due to widespread antimicrobial resistance. To combat the increasing rates of antibiotic-refractory rUTI, new therapies must be developed. A promising source of new rUTI therapies is the urinary microbiome, termed here the urobiome, which has been recently identified as an important component of the urinary environment. A critical step in the development of probiotic therapies is defining niche colonization and maintenance requirements. However, to date, urobiome studies have been largely focused on compositional classification and have not identified nichespecific urobiome functions or metabolic requirements. Furthermore, the effect of rUTI and the hormonal changes on urobiome composition and function has not been assessed in PM women. We have completed shotgun metagenomic sequencing (MGS) of urine from a cross-sectional cohort of PM women. In this work we have discovered that urinary lactobacilli (Lb) abundance in women without rUTI was associated with specific modalities of estrogen hormone therapy (EHT). Our functional analysis of the MGS data revealed differential enrichment in central carbon metabolism pathways in the metagenomes of women with no history of UTI versus women with active rUTI suggesting that urobiomes of healthy women are functionally different than those with rUTI. We have assembled a multidisciplinary, collaborative team to conclusively fill fundamental gaps in knowledge through the following specific aims: 1) Define the correlation between urinary Lb abundance and estrogen concentration in PM women using EHT, 2) Define metabolic differences between the urobiomes of healthy women and those with rUTI relevant to the urinary environment, and 3) Establish the spatiotemporal dynamics of urobiome composition and function in healthy PM women and during rUTI. In completing these aims, we will quantify excreted EHT metabolites and define their correlation with urinary Lb abundance. We will define key, relevant metabolic differences between urobiomes and identify metabolite-taxa associations related to rUTI. Finally, our longitudinal analysis of urinary metagenomes and metabolites in matched patient samples will define the effect of EHT on urobiome dynamics in healthy PM women and during rUTI. These findings will generate...