Objective
The purpose of this study was to characterize the urinary microbiota in women who are planning treatment for urgency urinary incontinence and to describe clinical associations with urinary symptoms, urinary tract infection, and treatment outcomes.
Study Design
Catheterized urine samples were collected from multisite randomized trial participants who had no clinical evidence of urinary tract infection; 16S ribosomal RNA gene sequencing was used to dichotomize participants as either DNA sequence-positive or sequence-negative. Associations with demographics, urinary symptoms, urinary tract infection risk, and treatment outcomes were determined. In sequence-positive samples, microbiotas were characterized on the basis of their dominant microorganisms.
Results
More than one-half (51.1%; 93/182) of the participants’ urine samples were sequence-positive. Sequence-positive participants were younger (55.8 vs 61.3 years old; P = .0007), had a higher body mass index (33.7 vs 30.1 kg/m 2 ; P = .0009), had a higher mean baseline daily urgency urinary incontinence episodes (5.7 vs 4.2 episodes; P < .0001), responded better to treatment (decrease in urgency urinary incontinence episodes, –4.4 vs –3.3; P = .0013), and were less likely to experience urinary tract infection (9% vs 27%; P = .0011). In sequence-positive samples, 8 major bacterial clusters were identified; 7 clusters were dominated not only by a single genus, most commonly Lactobacillus (45%) or Gardnerella (17%), but also by other taxa (25%). The remaining cluster had no dominant genus (13%).
Conclusion
DNA sequencing confirmed urinary bacterial DNA in many women with urgency urinary incontinence who had no signs of infection. Sequence status was associated with baseline urgency urinary incontinence episodes, treatment response, and posttreatment urinary tract infection risk.
Urgency urinary incontinence (UUI) is a heterogeneous condition that usually is attributed to abnormalities in detrusor neuromuscular functioning and/or signaling. However, most incontinence experts suspect that the UUI cause is likely more complex. Current first-line treatment for UUI is behavioral and/or pharmacologic, but many affected patients have persistent symptoms. Thus, the group of women that is affected by the generic symptom of UUI may have multiple, heterogeneous causes.
The Anticholinergic Versus Botulinum Toxin A Comparison (ABC) Trial was a 10-center, double-blind, double-placebo–controlled randomized trial in which women received either 1 intradetrusor injection of 100 U of onabotulinumtoxinA and daily oral placebo or daily oral anticholinergic medication and 1 intradetrusor injection of saline solution. The ABC study was designed and conducted before the knowledge that the female lower urinary tract (bladder and/or urethra) contains bacterial communities (termed the female urinary microbiome ).
Growing evidence suggests that the female urinary microbiota may play a role in certain urinary disorders such as UUI. Our research team previously described the female urinary microbiota of women with UUI using catheterized urine samples that were obtained at baseline from patient-participants in a randomized clinical trial for the treatment of UUI who did not have clinical urinary tract infection (UTI). Previously, we also compared various techniques to verify that transurethral catheterization was an appropriate method to collect urine samples with minimal vulvovaginal contamination. This current analysis goes beyond the initial testing of the previous samples to characterize formally the female urinary microbiota by 16S ribosomal RNA (rRNA) gene sequence analysis. Relationships among sequence status, microbiota characteristics, and clinical variables of interest, which include treatment response and posttreatment UTI risk, were also assessed.
Materials and Methods
Subjects and specimen acquisition
The full methods of the trial and the primary outcome of the ABC trial have been published. Briefly, the trial randomly assigned women without neurologic disease with moderate-to-severe UUI, which was defined as having ≥5 episodes of UUI per 3-day period. Participants were anticholinergic drug naïve or previously had used ≤2 anticholinergic medications other than the study drugs. Exclusion criteria included a postvoid residual volume ≥150 mL or previous therapy with oral study medications or onabotulinumtoxinA.
The primary outcome was reduction from baseline in mean episodes of UUI (UUIE) per day over the 6-month period, as recorded in 3-day diaries that were submitted monthly. Secondary outcomes included resolution of UUI symptoms, quality of life, use of catheters, and adverse events that included UTI, which was defined as a positive standard urine culture with >10 5 colony-forming units of a known uropathogen or treatment with antibiotic for UTI within 6 months of randomization. After treatment, intermittent catheterization was recommended if postvoid residual was either >300 mL or >150 mL with “moderate” to “quite a bit” of bother associated with incomplete voiding.
All subjects were free of clinical UTI before study injection, as determined by a negative nitrite and leukocyte esterase result on a urine dipstick evaluation of a catheterized specimen. Urine culture was not required. After baseline assessment and before injection, subjects provided a baseline catheterized urine sample, which was placed at –80 ° C within 1 hour; the frozen samples were batch-shipped on dry ice to Loyola University Chicago for 16S rRNA gene sequence analysis. Staff and investigators were blinded to clinical information during laboratory research analyses.
16S rRNA sequencing
DNA was isolated in a laminar flow hood to avoid contamination. Genomic DNA was extracted from 1 mL of urine with the use of previously validated protocols. Variable region 4 of the bacterial 16S rRNA gene was amplified via a 2-step nested polymerase chain reaction (PCR) protocol, with the use of modified universal primers 515F and 806R, as previously described. Two quality controls assessed the contribution of extraneous DNA from laboratory reagents: a DNA extraction–negative control with no urine added and a PCR-negative control with no template DNA added. The final PCR product was purified from unincorporated nucleotides and primers with the Qiaquick PCR purification kit (Qiagen, Valencia, CA) and Agencourt AMPure XP-PCR magnetic beads (Beckman Coulter Inc, Fullerton, CA). Samples were normalized to equal DNA concentration, as determined by Nanodrop spectroscopy (Thermo Scientific, Waltham, MA). The sample library and the PhiX sequencing control library (Illumina Inc., San Diego, CA) were denatured and added to the 2 × 250 base pair sequencing reagent cartridge, according to manufacturer’s instructions (Illumina Inc.).
DNA sequence analysis
MiSeq postsequencing software (Illumina Inc.) preprocessed sequences and removed primers and sample indices. The open-source program mothur (version 1.31.2; University of Michigan, Ann Arbor, MI; http://www.mothur.org/ ) combined paired end reads and removed contigs (overlapping sequence data) of incorrect length (<285 base pair, >300 base pair) and/or contigs that contain ambiguous bases. The remaining modified sequences were aligned to the SILVA reference database (The SILVA ribosomal RNA database project; Max Planck Institute for Marine Microbiology, Bremen, Germany). Chimeric sequences were removed with the use of the UCHIME algorithm. The remaining sequences were classified taxonomically with the use of a naïve Bayesian classifier and the RDP training set (version 9; University of Michigan, East Lansing, MI), and clustered into operational taxonomic units. METAGENassist was used to link operational taxonomic units nomenclature to taxonomic assignments.
The ABC trial was conducted with full institutional review board approval at all participating sites. All samples were processed in duplicate and characterized as sequence-positive, sequence-negative, or inconclusive. A sequence-negative sample was one in which DNA was not amplified in either technical replica. To avoid accidentally sequencing rare contaminants, we conservatively considered samples to be negative if DNA amplification was not visible on an agarose gel. A sequence-positive sample was one in which DNA was amplified from both replicas, the replicas had a Euclidian distance score <0.3, and the dominant genus (>45% sequences per sample), if present, was the same in both replicas. Samples that did not meet these criteria were deemed inconclusive and disregarded from further analysis (n = 12). For each sequence-positive sample, percent reads were calculated, and replicates were then averaged for downstream analysis.
To display the average sequence abundances, a histogram was produced, color-coded by bacterial taxa. Euclidean distance was calculated between samples, and the complete method was used for hierarchic clustering in the statistical package R (version 2.15.1; R Core Team, Vienna, Austria). The resulting dendrogram was divided into 8 urotypes. The rare urotypes were grouped into an “other” category, which condensed the original 8 urotypes to a total of 5 urotypes for use in the analysis ( Lactobacillus , Gardnerella , Gardnerella/Prevotella , Diverse, and Other) plus the negative category. These urotypes, along with the sequence-negative group, were then compared with participant demographics, symptoms at baseline, and clinical outcomes.
Statistical analysis
Differences were examined descriptively at baseline, as were changes in clinical outcome measures for individuals that were defined as sequence-positive and sequence-negative and for individuals classified by urotype, which is further described later. The differences in binary baseline and outcome measures across these 2 categoric measures of sequence were examined via contingency tables, with probability values that were generated from χ 2 tests; differences in mean values for continuous measures were evaluated with the use of general linear models. Because all analyses were considered descriptive, no adjustments were made for multiple comparisons, and probability values should be interpreted accordingly. To compare the mean abundance of the top 10 most abundant taxa, the Wilcoxon rank sum test was performed. Statistical analyses were conducted with SAS statistical software (version 9.3; SAS Institute Inc, Cary, NC).
Results
Approximately one-half of the urine samples (51.1%, 93/182) were sequence-positive. Table 1 displays demographics and baseline characteristics of participants relative to sequence status; the mean age was 58.5 years, and most participants were white (77%). Sequence-positive subjects were younger (55.8 ± 12.2 vs 61.3 ± 9.0 years; P = .0007), had a higher body mass index (33.7 ± 7.3 vs 30.1 ± 6.6 kg/m 2 ; P = .0009), and had a higher mean number of baseline UUIE (5.7 ± 2.5 vs 4.2 ± 2.1 per day; P < .0001). Race, ethnicity, previous anticholinergic use, or study treatment assignment did not differ between sequence-positive and sequence-negative cohorts.
| Characteristic | Sequence | P value a | |
|---|---|---|---|
| Positive (n = 93) | Negative (n = 89) | ||
| Age, y | .0007 | ||
| Mean ± SD | 55.8 ± 12.2 | 61.3 ± 9.0 | |
| Median (minimum/maximum) | 55.8 (31.1/85.4) | 61.2 (43.1/83.1) | |
| Ethnicity, n (%) | .054 | ||
| Hispanic | 23 (25) | 12 (13) | |
| Non-Hispanic | 70 (75) | 77 (87) | |
| Race, n (%) | .15 | ||
| White | 68 (73) | 73 (82) | |
| Non-white | 25 (27) | 16 (18) | |
| Body mass index, kg/m 2 | .0009 | ||
| Mean ± SD | 33.7 ± 7.3 | 30.1 ± 6.6 | |
| Median (minimum/maximum) | 32.8 (20.5/52.4) | 28.8 (18.7/48.5) | |
| Menopausal status, n (%) | .059 | ||
| Premenopausal | 17 (20) | 8 (9) | |
| Postmenopausal | 70 (80) | 77 (91) | |
| Previous anticholinergic use, n (%) | .97 | ||
| Yes | 52 (56) | 50 (26) | |
| No | 41 (44) | 39 (44) | |
| Baseline urgency urinary incontinence episodes stratum, n (%) | .0036 | ||
| 5-8 | 11 (12) | 26 (29) | |
| ≥9 | 82 (88) | 63 (71) | |
| Baseline urgency urinary incontinence episodes, per day | < .0001 | ||
| Mean ± SD | 5.66 ± 2.5 | 4.20 ± 2.1 | |
| Median (minimum/maximum) | 5.00 (1.7/12.0) | 3.67 (1.7/9.7) | |
| Overactive bladder quantitative symptom severity | .78 | ||
| Mean ± SD | 68.1 ± 18.8 | 68.8 ± 18.5 | |
| Median (minimum/maximum) | 70 (16.7/100) | 70 (26.7/100) | |
| Overactive bladder quantitative symptom severity health-related quality of life | .80 | ||
| Mean ± SD | 45.6 ± 20.7 | 46.3 ± 24.0 | |
| Median (minimum/maximum) | 47.7 (0/90.8 ) | 46.2 (6.2/98.5) | |
| Treatment, n (%) | .47 | ||
| Active onabotulinumtoxinA | 42 (45) | 45 (51) | |
| Active anticholinergic medication | 51 (55) | 44 (49) | |
Sequence-positive subjects responded better to treatment with a larger decrease in baseline UUIE (–4.4 ± 2.7 vs –3.3 ± 1.9; P = .0013) with no evidence of interaction with treatment group ( P = .92). In both groups, sequence-positive subjects were less likely to experience UTI after the initiation of the study antiincontinence treatment (9% vs 27% posttreatment UTIs; P = .0011; Table 2 ).
| Characteristic | Sequence | P value | |
|---|---|---|---|
| Positive (n = 90) | Negative (89) | ||
| Urinary tract infection, n (%) | .0011 | ||
| Yes | 8 (9) | 24 (27) | |
| No | 85 (91) | 65 (73) | |
| Change in urgency urinary incontinence, episodes per day | .0013 | ||
| Mean ± SD | –4.36 ± 2.7 | –3.32 ± 1.9 | |
| Median (minimum/maximum) | –4.03 (–11.7/0.7) | –3.17 (–9.3/2.5) | |
| Overactive bladder quantitative symptom severity change | .37 | ||
| Mean ± SD | –46.8 ± 23.8 | –43.7 ± 22.7 | |
| Median (minimum/maximum) | –48.1 (–86.7/11.1) | –44.4 (–93.3/9.2) | |
| Overactive bladder quantitative health-related quality of life change | .60 | ||
| Mean ± SD | 39.2 ± 21.9 | 37.3 ± 25.2 | |
| Median (minimum/maximum) | 37.5 (–9.0/96.7) | 36.9 (–28.8/93.8) | |
In sequence-positive urine samples, hierarchic clustering revealed 8 major urotypes ( Figure 1 , top). In 7 of these urotypes, a single bacterial genus or family dominated (ie, represented >45% total sequences in a sample; Figure 1 , bottom). For example, in the first urotype, Enterobacteriaceae accounted for >90% of the total sequences in all of the 8 samples. Nearly one-half of the sequence-positive urine samples were dominated by the genus Lactobacillus (45%), followed by Gardnerella (17%), Gardnerella/Prevotella (9%), Enterobacteriaceae (9%), Staphylococcus (3%), Aerococcus (2%) and Bifidobacterium (2%). The remaining cluster was labeled “Diverse” to signify those (13%) without a dominant genus. Although these more diverse samples were often composed of different genera, they grouped together ( Table 1 , bottom). Each urotype was observed in samples from ≥2 performance sites, and several urotypes were observed at multiple clinical sites ( Table 3 ).
| Urotype | n | Site identification |
|---|---|---|
| Aerococcus | 2 | 08,22 |
| Bifidobacterium | 2 | 02,16 |
| Diverse | 12 | 02,07,15,17,18,21 |
| Enterobacteriaceae | 8 | 02,06,07,17,18 |
| Gardnerella | 16 | 02,06,07,15,16,17,18,22 |
| Gardnerella/Prevotella | 8 | 02,07,08,18,22 |
| Lactobacillus | 42 | 02,06,07,14,15,16,17,18,21,22 |
| Staphylococcus | 3 | 02,17 |
Most sequence-positive samples had a dominant genus ( Figure 1 ). To assess the prevalence of these genera in samples where they did not dominate, we calculated the proportion of sequences that belonged to the dominant taxon of each urotype across all sequence-positive samples. Figure 2 demonstrates that nearly all the sequence-positive samples contained Lactobacillus (with a median 20% Lactobacillus sequences per urine sample). With the exception of Gardnerella , the other urotype-defining taxa were detected less commonly in samples outside those they dominated.
Cluster analysis revealed 8 major urotypes ( Figure 1 ): Lactobacillus, Gardnerella, Gardnerella/Prevotella, Enterobacteriaceae, Staphylococcus , Aerococcus , Bifidobacterium , and Diverse. Although some major clusters (especially Lactobacillus , Gardnerella, and Diverse) branched into subclusters, for further analyses, we treated these subclusters as 1 urotype. We also combined the less common urotypes ( Gardnerella/Prevotella, Enterobacteriaceae, Staphylococcus , Aerococcus, and Bifidobacterium ) together (Other). We compared these 5 urotypes and the sequence-negative group with baseline demographic and clinical variables. Whereas race, ethnicity, and menopausal status were similar across urotypes, age ( P = .005) and body mass index ( P = .014) differed ( Table 4 ). Additional analysis detected an upward trend for age with the Enterobacteriaceae-dominant and Staphylococcus -dominant urotypes and a downward trend with the Gardnerella/Prevotella- dominant urotype ( Figure 3 , A). Body mass index tended to be lower in the Enterobacteriaceae-dominant , Staphylococcus -dominant, and sequence-negative groups ( Figure 3 , B). Whereas symptom severity was similar across urotypes, baseline UUIE differed by incontinence severity ( P = .046) and frequency ( P < .0001; Table 4 ). Although Staphylococcus -dominant and sequence-negative groups showed a trend towards lower baseline UUIE ( Figure 3 , C), the number of urine samples that represented these urotypes was small, and the analysis lacked statistical power. Treatment response (change in UUIE; P = .0017) and development of posttreatment UTI ( P = .0058) differed among the 5 urotypes ( Table 5 ).
| Characteristic | Lactobacillus (n = 42) | Gardnerella (n = 16) | Diverse (n = 12) | Other (n = 23) | Negative (n = 89) | P value |
|---|---|---|---|---|---|---|
| Age, y | .0005 | |||||
| Mean ± SD | 53.2 ± 11.5 | 53.7 ± 10.0 | 61.0 ± 11.2 | 59.5 ± 14.0 | 61.3 ± 9.0 | |
| Median | 53.5 | 54.2 | 60.3 | 58.4 | 61.2 | |
| Ethnicity, n (%) | .24 | |||||
| Hispanic | 10 (24) | 5 (31) | 4 (33) | 4 (17) | 12 (13) | |
| Non-Hispanic | 32 (76) | 11 (69) | 8 (67) | 19 (83) | 77 (87) | |
| Race, n (%) | .20 | |||||
| White | 28 (67) | 11 (69) | 11 (92) | 18 (78) | 73 (82) | |
| Nonwhite | 14 (33) | 5 (31) | 1 (8) | 5 (22) | 16 (18) | |
| Body mass index, kg/m 2 | .014 | |||||
| Mean ± SD | 33.8 ± 7.6 | 32.6 ± 5.2 | 35.2 ± 8.7 | 33.3 ± 7.3 | 30.1 ± 6.6 | |
| Median | 32.5 | 32.5 | 36.0 | 32.8 | 28.8 | |
| Menopausal status, n (%) | .22 | |||||
| Premenopausal | 10 (26) | 4 (22) | 2 (17) | 1 (6) | 8 (9) | |
| Postmenopausal | 29 (74) | 14 (78) | 10 (83) | 17 (94) | 77 (91) | |
| Previous anticholinergic use, n (%) | .71 | |||||
| Yes | 23 (55) | 8 (50) | 9 (75) | 12 (52) | 50 (56) | |
| No | 19 (45) | 8 (50) | 3 (25) | 11 (48) | 39 (44) | |
| Baseline urgency urinary incontinence stratum, n (%) | .046 | |||||
| 5-8 | 6 (14) | 2 (12) | 0 (0) | 3 (13) | 26(29) | |
| ≥9 | 36 (86) | 14 (88) | 12 (100) | 20 (87) | 63 (71) | |
| Baseline urgency urinary incontinence, episodes per day | < .0001 | |||||
| Mean ± SD | 5.0 ± 2.1 | 6.0 ± 2.8 | 6.2 ± 1.9 | 6.3 ± 2.9 | 4.2 ± 2.1 | |
| Median | 4.50 | 5.17 | 6.50 | 6.00 | 3.67 | |
| Overactive bladder quantitative symptom severity | .49 | |||||
| Mean ± SD | 65.2 ± 17.4 | 66.6 ± 19.1 | 69.2 ± 17.5 | 73.8 ± 21.4 | 68.8 ± 18.5 | |
| Median | 60.0 | 71.67 | 70.0 | 76.7 | 70.0 | |
| Overactive bladder quantitative health-related quality of life | .32 | |||||
| Mean ± SD | 49.0 ± 17.7 | 49.5 ± 21.2 | 42.9 ± 24.1 | 37.5 ± 22.5 | 46.3 ± 24.0 | |
| Median | 50.8 | 53.7 | 40.0 | 43.1 | 46.2 |
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