The Urinary Microbiome in Postmenopausal Women with Recurrent Urinary Tract Infections

Abstract

Purpose: The etiology of postmenopausal recurrent urinary tract infection (UTI) is not completely known, but the urinary microbiome is thought to be implicated. We compared the urinary microbiome in menopausal women with recurrent UTIs to age-matched controls, both in the absence of acute infection.

Materials and methods: This is a cross-sectional analysis of baseline data from 64 women enrolled in a longitudinal cohort study. All women were using topically applied vaginal estrogen. Women >55 years of age from the following groups were enrolled: 1) recurrent UTIs on daily antibiotic prophylaxis, 2) recurrent UTIs not on antibiotic prophylaxis and 3) age-matched controls without recurrent UTIs. Catheterized urine samples were collected at least 4 weeks after last treatment for UTI and at least 6 weeks after initiation of vaginal estrogen. Samples were evaluated using expanded quantitative urine culture (EQUC) and 16S rRNA gene sequencing.

Results: With EQUC, there were no significant differences in median numbers of microbial species isolated among groups (p=0.96), even when considering Lactobacilli (p=0.72). However, there were trends toward different Lactobacillus species between groups. With 16S rRNA sequencing, the majority of urine samples contained Lactobacillaceae, with nonsignificant trends in relative abundance among groups. Using a Bayesian analysis, we identified significant differences in anaerobic taxa associated with phenotypic groups. Most of these differences centered on Bacteroidales and the family Prevotellaceae, although differences were also noted in Actinobacteria and certain genera of Clostridiales.

Conclusions: Associations between anaerobes within the urinary microbiome and postmenopausal recurrent UTI warrants further investigation.

Keywords: microbiota; postmenopause; urinary tract infections.

Figure 1:. Urine Sample Processing

Catheterized urine was obtained using a standard research protocol. Urine was initially tested in the clinic with dipstick urinalysis (UA). If UA was suspicious for bacteruria, it was sent for standard urine culture only. If the participant had a negative (no growth) standard urine culture after suspicious UA, they were invited to provide a repeat sample. If the participant had a positive standard urine culture, the research documentation was reviewed for presence or absence of any symptoms. At this point, the participant was either: 1) excluded if there was asymptomatic bacteruria (ASB); or 2) treated for symptomatic urinary tract infection (UTI) and invited to provide a repeat sample in 4 weeks. For samples where office UA was negative, two 5mL samples were prepared and transferred to the Duke Clinical Microbiology Laboratory for standard and expanded quantitative urine culture. The remaining urine was poured into 50mL conical tubes prepared with Assay Assure, refrigerated, and transferred to the research laboratory for further processing and 16S rRNA gene sequencing. Created with BioRender.com.

Figure 2:. Comparison of the most common organisms recovered in EQUC

a) Lactobacilli: Of 64 total samples, 38 (59%) demonstrated growth of one or more organisms in expanded quantitative urine culture (EQUC). The majority of these organisms were Lactobacilli. The three most commonly isolated Lactobacillus species were L. gasseri/L. acidophilus (isolated in n=15 samples), L. casei/L. paracasei (n=6 samples), and L. crispatus (n=3 samples). Proportions of urine samples from each phenotypic group with the three most common Lactobacillus species in EQUC are depicted in the figure. For example, in women with recurrent UTI taking antibiotics, 6% contained L. gasseri/L. acidophilus in their EQUC samples compared to 29–30% in the other two phenotypic groups. These differences in proportions were not statistically significant (p=0.14, p=0.39, p=0.43, respectively). b) The most common non-Lactobacillus organisms from EQUC are identified. Proportions of urine samples from each phenotypic group where the microbe was are depicted; there were no statistically significant differences.

Figure 3:. Stacked bar plot depicting relative abundance of all microbiota per sample

Stacked bar plot where each vertical bar depicts the relative abundance of adjusted sequence variants (ASVs) and associated taxa that were recovered per sample. For each color group, the darkest shade represents the most common family identified with a phylum, with lighter shades representing other families within the same phylum. For example, the darkest shade of purple represents Lactobacillaceae while lighter purple shades represent other Firmicutes. Results obtained from V4 amplicon sequencing of the 16S rRNA gene, processed with DADA2 and mapped to the SILVA reference database.

Figure 4:. Recurrent UTI with daily antibiotics compared to controls

Bayesian GCR analysis comparing microbiota in women with recurrent UTIs taking daily antibiotics and age-matched controls (both using vaginal estrogen) while incorporating multiple clinical and technical covariates. The posterior marginal alternative probability (PMAP) was calculated at each node and shaded based on result. The highest probability of a difference between groups is at node #1 (PMAP = 99.96%), which denotes differences in Prevotella species recovered among those with rUTI+abx compared to controls. This node is a downstream branch in the taxonomic tree from two other nodes that also appear to have a high probability of differences between groups. These are identified as #4 (PMAP = 82.5%), which denotes differences in Prevotellaceae and #5 (PMAP = 80.5%), denoting differences in the order Bacteroidales. Node #2 (PMAP = 98.27%) denotes differences in Actinobacteria; this section of the tree is further exploded in Supplemental Figure 2. Node #3 (PMAP = 84.81%) denotes differences in Clostridiales, family Ruminococcaceae. Fig 4A shows the entire taxonomic tree and BCGR results; Figure 4B is an exploded section of this tree showing genus and species information.

Figure 5:. Recurrent UTIs with compared to without daily antibiotics

Bayesian GCR analysis comparing microbiota in women with recurrent UTIs taking daily antibiotics and women with recurrent UTIs not on antibiotics (both using vaginal estrogen) (a) Posterior marginal alternative probability (PMAP) was calculated at each node with one area resulting in PMAP > 60%. This corresponds to the node shaded in yellow-green where PMAP = 66.73%, indicating the probability of a true difference in taxa between groups at the location specified. (b) Exploded view of the taxa where the difference was identified, specifically showing that there are differences between the genus Ezakiella, and the other genera identified in the figure.