The female urinary microbiome: a comparison of women with and without urgency urinary incontinence

Abstract

Bacterial DNA and live bacteria have been detected in human urine in the absence of clinical infection, challenging the prevailing dogma that urine is normally sterile. Urgency urinary incontinence (UUI) is a poorly understood urinary condition characterized by symptoms that overlap urinary infection, including urinary urgency and increased frequency with urinary incontinence. The recent discovery of the urinary microbiome warrants investigation into whether bacteria contribute to UUI. In this study, we used 16S rRNA gene sequencing to classify bacterial DNA and expanded quantitative urine culture (EQUC) techniques to isolate live bacteria in urine collected by using a transurethral catheter from women with UUI and, in comparison, a cohort without UUI. For these cohorts, we demonstrated that the UUI and non-UUI urinary microbiomes differ by group based on both sequence and culture evidences. Compared to the non-UUI microbiome, sequencing experiments revealed that the UUI microbiome was composed of increased Gardnerella and decreased Lactobacillus. Nine genera (Actinobaculum, Actinomyces, Aerococcus, Arthrobacter, Corynebacterium, Gardnerella, Oligella, Staphylococcus, and Streptococcus) were more frequently cultured from the UUI cohort. Although Lactobacillus was isolated from both cohorts, distinctions existed at the species level, with Lactobacillus gasseri detected more frequently in the UUI cohort and Lactobacillus crispatus most frequently detected in controls. Combined, these data suggest that potentially important differences exist in the urinary microbiomes of women with and without UUI, which have strong implications in prevention, diagnosis, or treatment of UUI. Importance: New evidence indicates that the human urinary tract contains microbial communities; however, the role of these communities in urinary health remains to be elucidated. Urgency urinary incontinence (UUI) is a highly prevalent yet poorly understood urinary condition characterized by urgency, frequency, and urinary incontinence. Given the significant overlap of UUI symptoms with those of urinary tract infections, it is possible that UUI may have a microbial component. We compared the urinary microbiomes of women affected by UUI to those of a comparison group without UUI, using both high-throughput sequencing and extended culture techniques. We identified statistically significant differences in the frequency and abundance of bacteria present. These differences suggest a potential role for the urinary microbiome in female urinary health.

Trial registration: ClinicalTrials.gov NCT01642277.

FIG 1

Urinary microbiome profile by cohort based on 16S rRNA gene V4 sequencing. Stacked bar plots depict the sequence abundances of the 15 most abundant genus- or family-level taxa in the UUI and non-UUI cohorts. Taxa were ranked according to mean abundance across all samples. The y axis represents the percentage of sequences for a particular bacterial taxa; the x axis represents the study participants separated by cohort. The family Enterobacteriaceae could not be classified to the genus level. The remainder of sequences were combined in the category labeled Other.

FIG 2

Clustering of the urinary microbiome into urotypes. The dendrogram was based on hierarchical clustering of the Euclidean distance between samples in the combined UUI and non-UUI cohorts. The dashed line depicts where the clades were divided into 6 urotypes: Gardnerella, Sneathia, Diverse, Staphylococcus, Enterobacteriaceae, and Lactobacillus. The stacked bar plot below the dendrogram depicts the sequence abundances of the overall most abundant taxa.

FIG 3

Comparison of sequence-based urinary microbiome by cohort. The frequency (A) and median sequence abundance (B) of the overall most abundant taxa detected by sequencing were calculated. The families Enterobacteriaceae, Lachnospiraceae, and Ruminococcaceae could not be classified to the genus level. In panel A, a combination of Pearson chi-square and Fisher’s exact tests was used to compare the frequency of genera detected by sequencing between the cohorts. “*” represents a P value of <0.05. In panel B, a Wilcoxon rank sum test was used to compare the median sequence abundances between the cohorts. IQR, interquartile range. “*” represents P values of <0.05.

FIG 4

Rarefaction curves of the cultured bacterial species by cohort. The plot depicts the number of species cultured via EQUC by the number of urine samples assayed.

FIG 5

Genus-level comparison of cultured urinary microbiota by cohort. The Pearson chi-square and Fisher’s exact tests were used to compare the frequencies of the genera isolated from urine via EQUC. *, P <0.05; **, P < 0.001.

FIG 6

Species-level comparison of cultured urinary microbiota by cohort. The Pearson chi-square and Fisher’s exact tests were used to compare the frequencies of the species isolated from urine via EQUC. *, P < 0.05; **, P  < 0.01.

FIG 7

Comparison of taxa detected by 16S rRNA gene sequencing and EQUC. (A) Comparison of sequence status and EQUC status for the 52 urine samples that were assayed by both methods. (B) Comparison of the taxa detected by sequencing and culture of the sequence-positive, EQUC-positive urine samples (n = 30). Each square was color coded based on whether the taxa were detected by sequence only (green), EQUC only (red), sequence and EQUC (yellow), or neither sequence nor EQUC (gray).