Research Project
10346595
SPECIFIC AIMS Urinary tract infection (UTI) is among the most common bacterial infections and often recurs. Postmenopausal (PM) women, an understudied and growing demographic, are severely affected by recurrent UTI (rUTI). In this population, rUTI can persist for years, greatly reducing quality of life and imposing a healthcare burden. Antibiotic treatment of rUTI is hindered by increasing rates of antibiotic resistance and allergy. A potential source of rUTI therapies is the urinary microbiome, termed here the urobiome. 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 niche-specific urobiome functions or metabolic requirements. We have begun to address these gaps in knowledge, with striking results. As preliminary data we have completed shotgun metagenomic sequencing (MGS) of urine from a cross- sectional cohort (N=76) of three groups of PM women: No UTI history, rUTI remission, and active rUTI relapse. We identified compositional urobiome differences between groups and discovered that urinary lactobacilli (Lb) abundance in PM women was associated with specific modalities of estrogen hormone therapy (EHT), suggesting that EHT type or dosage may be important. The most enriched species among EHT users was L. crispatus, a protective member of the vaginal microbiota. Our data support the hypothesis that EHT enriches urinary Lb in PM women. To begin to test this hypothesis, we developed a sensitive LC-MS/MS method to measure EHT metabolites in urine. Application of this method to No UTI history and rUTI remission urine revealed group-specific correlations between urinary taxa and estrogen metabolites. Specifically, we observed direct correlations between L. crispatus, L. gasseri, and L. iners abundance and urinary estrone and 17?-estradiol metabolite concentration in the No UTI history group. Strikingly, this correlation was lost in the rUTI Remission group. One hypothesis to explain this observation is that rUTI and its treatment permanently alter urobiome composition and its response to EHT. However, to robustly test this hypothesis, longitudinal studies are needed. Functional analysis of cross-sectional cohort MGS data revealed differential enrichment of key metabolic pathways between the metagenomes of the No UTI history, rUTI relapse and rUTI remission groups. These data suggest that the urobiomes of PM women with both active rUTI and rUTI history are functionally different than those of PM women without UTI history. Because MGS only reveals genetic potential, the relevance of differentially enriched pathways to the urinary environment must be assessed. We will build on these exciting preliminary data to interrogate urobiome functions and dynamics associated with bladder health, a state defined here as No UTI history, and rUTI in PM women through the following aims: Aim 1. Define metabolic differences between the urobiomes of women with different rUTI histories relevant to the urinary environment. Functional (pathway enrichment) analysis of MGS data indicates distinct differences in metabolic potential between the No UTI history, rUTI remission and rUTI relapse groups. We hypothesize that the urinary metabolic environment is also distinct between these groups. This aim will use metabolomic analysis of biobanked cross-sectional cohort urine to identify enriched metabolic pathways that are relevant to the urinary environment and to measure associations between urinary metabolites and microbial taxa within the urobiome. (1A) We will first identify pathway inputs (e.g. carbohydrates, amino acids) that are relevant to the urinary environment in each group via targeted MS. (1B) We will then perform unbiased quantitation of urinary metabolites in each group by untargeted MS. (1C) Finally, we will use these data to identify interactions between urinary metabolites and taxa in No UTI history, rUTI remission and rUTI relapse groups. Completing this aim will define key, relevant metabolic differences between women with different rUTI histories and identify metabolite-taxa associations related to rUTI. Aim 2. Establish the spatiotemporal dynamics of urobiome composition and function in healthy PM women and during rUTI. Urobiome dynamics are poorly understood and longitudinal studies have not been conducted in PM women. Based on our preliminary data, we hypothesize that the urobiome of PM women using EHT is more stable than that of non-EHT users. (2A) We will assess changes in urobiome composition, encoded function, and urinary metabolites over time in PM women with no UTI history using or not using EHT. (2B) We will test the recent model of interconnected vaginal and urinary microbiomes by analyzing compositional co- variation between the urobiome and the vaginal and peri-anal microbiomes over time. We also hypothesize that urobiome dynamics of PM women using EHT will be distinct from non-users during rUTI. (2C) We will analyze urobiome composition, function, and urinary metabolites during rUTI relapse, treatment, and recovery in PM women with rUTI using or not using EHT and quantify the effect of EHT on rUTI outcome. Finally, how the microbiomes of proximal anatomical sites vary during the rUTI cycle is unknown. (2D) We will measure compositional changes within vaginal and peri-anal microbiomes during rUTI relapse and recovery. Completing this aim will define the effect of EHT on urobiome dynamics in healthy PM women and during rUTI. This proposal will generate foundational knowledge of the function and spatiotemporal dynamics of the urobiome of PM women necessary for the rational design of urobiome-targeted therapies for rUTI. Project Summary 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. 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 niche-specific 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 discovered that urinary lactobacilli (Lb) abundance was associated with specific modalities of estrogen hormone therapy (EHT) in PM women without active rUTI. We then defined the correlation between urinary Lb abundance and estrogen metabolite concentration measured by LC-MS/MS and found that estrone and 17?-estradiol concentration directly correlated with Lb abundance in women without UTI history but not in women with rUTI history but no current UTI (rUTI remission). Further, functional MGS analysis revealed differentially enriched metabolic pathways between no UTI history versus rUTI remission and rUTI relapse. Together, these data suggest that urobiomes of healthy women are functionally different than those with rUTI history. To robustly test this hypothesis and address critical gaps in knowledge in the field, our multidisciplinary, collaborative team proposes to 1) Define metabolic differences between the urobiomes of healthy women and those with rUTI relevant to the urinary environment, and 2) Establish the spatiotemporal dynamics of urobiome composition and function in healthy PM women and during rUTI. In completing these aims, we will define key, relevant metabolic differences between urobiomes and identify metabolite-taxa associations related to rUTI. Importantly, 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 foundational knowledge of the function and spatiotemporal dynamics of the urobiome of PM women necessary for the rational design of urobiome-targeted therapies for rUTI.
