Supplementary Material List:
E-Methods 1: Protocol for the Expanded Quantitative Urine Culture (EQUC)
E-Table 1:Comparison of Demographics and Symptoms of Completed and Withdrawal Populations at Baseline
E-Table 2: Culture and Sequencing urotype listing of all samples in the study
E-Figure 1: Rarefaction Baseline and 12-Week Diversity by Response Group
E-Figure 2: Frequency of Detection of Bacterial Genera at Baseline and 12w of genera in the responders (5mg and 10mg).
E-Methods 1: Protocol for the Expanded Quantitative Urine Culture (EQUC):For standard urine culture, 0.001 mL of urine was spread quantitatively onto 5% sheep blood (BAP) and MacConkey agars (BD BBL™ Prepared Plated Media), then incubated aerobically (35°C for 24 hours). Any amount of each separate morphologic colony type was identified and counted using a detection level of 1,000 colony forming units (CFU)/mL (represented by 1 colony of growth on either plate). “No growth” was reported when no growth was observed, indicating no growth of bacteria at lowest dilution, i.e., 1:1000.
The expanded quantitative urine culture (EQUC) technique uses 0.1 mL of urine spread quantitatively onto BAP, Chocolate and Colistin, Naladixic Acid (CNA) agars (BD BBL™ Prepared Plated Media), then incubated in 5% CO2 (35°C for 48 hours). A second set of BAPs were inoculated with 0.1 mL of urine and incubated in room atmosphere at 35°C and 30°C for 48 hours, respectively. In addition, 0.1 mL of urine was inoculated onto each of two CDC Anaerobe 5% sheep blood agar (ABAP) plates (BD BBL™ Prepared Plated Media), and incubated in either a Campy gas mixture (5% O2, 10% CO2, 85% N) or in anaerobic conditions at 35°C for 48 hours. The detection level was 10 CFU/mL, represented by 1 colony of growth on any of the plates. Finally, to detect any bacterial species that may be present at quantities lower than 10 CFU/mL, 1.0 mL of urine was placed in Thioglycollate Medium (BD BBL™ Prepared Tubed Media) and incubated aerobically at 35°C for 5 days. If growth was visually detected, the Thioglycollate Medium was mixed and a few drops were plated on BAP and ABAP agars for isolation, and incubated aerobically and anaerobically (35°C for 48 hours). Each morphologically distinct colony type was isolated on a different plate of the same media to prepare a pure culture that was used for organism identification. Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) with the MALDI Biotyper 3.0 software (Bruker Daltonics, Billerica, MA) was used to identify the bacterial isolates8. To determine the false-negative rate, the following equation was used: the number of EQUC positive but standard negative samples divided by the sum of the EQUC positive and standard positive samples and the EQUC positive but standard negative samples.
E-Table1:Comparison of Demographics and Symptoms of Completed and Withdrawal Populations at Baseline
Completed Study(N=50) / Withdrawal (N=24) / p-value
Demographics
Age (years)
[Mean (Standard Deviation)] / 62.5
(SD:12.2) / 59.5
(SD:10) / 0.30a
Body Mass Index (kg/m2)
[Mean (Standard Deviation)] / 31.2
(SD:7.7) / 35.8
(SD:9.2) / 0.03a
Race/Ethnicity* / 0.40
Non Hispanic Caucasian / 38 (76%) / 17 (71%)
Hispanic Caucasian / 5 (10%) / 1 (4%)
African-American / 7 (14%) / 6 (25%)
Asian / 0 (%) / 0 (%)
Marital Status / 0.31
Married / 28 (56%) / 10 (42%)
Separated / 1 (2%) / 2 (8%)
Divorced / 7 (14%) / 2 (8%)
Widowed / 7 (14%) / 7 (29%)
Single / 7 (14%) / 3 (13%)
Estrogen Status** / 0.47
Estrogen Positive / 6 (12%) / 1 (4%)
Estrogen Negative / 43 (86%) / 22 (92%)
Unsure / 1 (2%) / 1 (4%)
Smoking / 2 (4%) / 4 (17%) / 0.08
Diabetes / 4 (8%) / 3 (13%) / 0.68
Hypertension / 21 (42%) / 11 (46%) / 0.75
Coronary Artery Disease / 5 (10%) / 4 (17%) / 0.46
Prior Treatment for OAB / 30 (60%) / 7 (29%) / 0.01
Vaginal Parity[Median (IQR)] / 2 (1-3) / 2 (0.5-3) / 0.56b
Symptoms[Median (IQR)]
Symptom Score c / 70 (53-85) / 80 (56-86) / 0.31b
Health Related Quality of Life (HRQL) c / 61 (42-82) / 37 (15-52) / 0.001b
Urinary Distress Inventory (UDI) d / 95 (56-131) / 131 (108-161) / 0.02b
Pelvic Organ Prolapse Distress Inventory (POPDI) d / 59 (15-95) / 83 (42-132) / 0.13b
Colorectal-Anal Distress Inventory (CRADI) d / 60 (20-112) / 71 (43-127) / 0.38b
Fisher's Exact Test due to small expected cell counts unless otherwise indicated.
aStudent’s T-Test
bWilcoxon rank sum test
cBased on OAB questionnaire
dBased on Pelvic Floor Disease Inventory
* Self-reported by participant
** Estrogen status is defined as negative if the patient is post-menopausal and not on hormone replacement therapy. Estrogen positive is anyone pre-menopausal and/or on hormone replacement therapy.
E-Table 2: Culture and Sequencing urotype listing of all samples in the study
- Culture and Sequencing Urotypes for UUI at baseline and 4 weeks.
UUI: Baseline Samples / UUI: 4 week Samples
Study ID / Sequencing Urotype / Culture Urotype / Study ID / Sequencing Urotype / Culture Urotype
OAB001_B / Sneathia / Not Cultured / OAB003_4 / Clostridium / Not Cultured
OAB002_B / Lactobacillus / Not Cultured / OAB004_4 / Undetermined / Not Cultured
OAB003_B / Diverse / Not Cultured / OAB005_4 / Diverse / Not Cultured
OAB004_B / Lactobacillus / Not Cultured / OAB006_4 / Lactobacillus / Not Cultured
OAB005_B / Diverse / Not Cultured / OAB007_4 / Enterobacteriaceae / Not Cultured
OAB006_B / Lactobacillus / Not Cultured / OAB008_4 / Gardnerella / Not Cultured
OAB007_B / Undetermined / Not Cultured / OAB010_4 / Gardnerella / Not Cultured
OAB008_B / Gardnerella / Not Cultured / OAB012_4 / Undetermined / Not Cultured
OAB010_B / Gardnerella / Not Cultured / OAB013_4 / Undetermined / Lactobacillus
OAB011_B / Sneathia / Not Cultured / OAB014_4 / Lactobacillus / Not Cultured
OAB012_B / Undetermined / Not Cultured / OAB015_4 / Aerococcus / Aerococcus
OAB013_B / Undetermined / Not Cultured / OAB016_4 / Undetermined / Bacillus
OAB014_B / Lactobacillus / Not Cultured / OAB017_4 / Gardnerella / Diverse
OAB015_B / Undetermined / Not Cultured / OAB018_4 / Lactobacillus / Lactobacillus
OAB016_B / Undetermined / Not Cultured / OAB020_4 / Diverse / Actinobaculum
OAB017_B / Lactobacillus / Lactobacillus / OAB021_4 / Lactobacillus / Staphylococcus
OAB018_B / Lactobacillus / Diverse / OAB022_4 / Undetermined / Streptococcus
OAB019_B / Diverse / Alloscardovia / OAB023_4 / Gardnerella / Gardnerella
OAB020_B / Staphylococcus / Staphylococcus / OAB024_4 / Lactobacillus / Aerococcus
OAB021_B / Lactobacillus / Lactobacillus / OAB025_4 / Diverse / Lactobacillus
OAB022_B / Undetermined / Streptococcus / OAB026_4 / Gardnerella / Gardnerella
OAB023_B / Gardnerella / Diverse / OAB027_4 / Undetermined / Bifidobacterium
OAB024_B / Undetermined / Lactobacillus / OAB028_4 / Undetermined / Bacillus
OAB025_B / Diverse / Actinobaculum / OAB029_4 / Undetermined / Undetermined
OAB026_B / Gardnerella / Gardnerella / OAB030_4 / Lactobacillus / Lactobacillus
OAB027_B / Undetermined / Bifidobacterium / OAB031_4 / Undetermined / Lactobacillus
OAB028_B / Undetermined / Enterobacteriaceae / OAB032_4 / Lactobacillus / Lactobacillus
OAB029_B / Undetermined / Streptococcus / OAB033_4 / Undetermined / Undetermined
OAB030_B / Lactobacillus / Lactobacillus / OAB034_4 / Undetermined / Undetermined
OAB031_B / Undetermined / Diverse / OAB035_4 / Undetermined / Diverse
OAB032_B / Gardnerella / Gardnerella / OAB037_4 / Undetermined / Streptococcus
OAB033_B / Undetermined / Enterobacteriaceae / OAB039_4 / Undetermined / Lactobacillus
OAB034_B / Undetermined / Undetermined / OAB040_4 / Undetermined / Streptococcus/ Actinomyces
OAB035_B / Undetermined / Diverse / OAB041_4 / Gardnerella / Diverse
OAB036_B / Undetermined / Streptococcus / OAB042_4 / Enterobacteriaceae / Enterobacteriaceae
OAB037_B / Undetermined / Undetermined / OAB043_4 / Diverse / Diverse
OAB038_B / Undetermined / Undetermined / OAB044_4 / Undetermined / Diverse
OAB039_B / Lactobacillus / Lactobacillus / OAB045_4 / Gardnerella / Gardnerella
OAB040_B / Undetermined / Diverse / OAB047_4 / Undetermined / Alloscardovia/ Rothia
OAB041_B / Undetermined / Diverse / OAB048_4 / Diverse / Diverse
OAB042_B / Gardnerella / Diverse / OAB049_4 / Lactobacillus / Lactobacillus/ Gardnerella
OAB043_B / Undetermined / Diverse / OAB050_4 / Enterobacteriaceae / Enterobacteriaceae
OAB044_B / Undetermined / Candida / OAB051_4 / Diverse / Streptococcus
OAB045_B / Enterobacteriaceae / Enterobacteriaceae/ Bacillus / OAB052_4 / Lactobacillus / Lactobacillus
OAB046_B / Undetermined / Undetermined / OAB054_4 / Undetermined / Undetermined
OAB047_B / Undetermined / Streptococcus / OAB055_4 / Undetermined / Actinomyces
OAB048_B / Diverse / Diverse / OAB056_4 / Lactobacillus / Lactobacillus
OAB049_B / Lactobacillus / Gardnerella / OAB057_4 / Enterobacteriaceae / Diverse
OAB050_B / Undetermined / Diverse / OAB058_4 / Undetermined / Streptococcus
OAB051_B / Diverse / Diverse / OAB060_4 / Enterobacteriaceae / Enterobacteriaceae
OAB052_B / Lactobacillus / Diverse / OAB061_4 / Undetermined / Diverse
OAB053_B / Lactobacillus / Diverse / OAB062_4 / Undetermined / Gardnerella
OAB054_B / Undetermined / Undetermined / OAB066_4 / Undetermined / Streptococcus
OAB055_B / Undetermined / Undetermined / OAB072_4 / Lactobacillus / Lactobacillus
OAB056_B / Lactobacillus / Lactobacillus / OAB073_4 / Undetermined / Bifidobacterium
OAB057_B / Diverse / Undetermined / OAB074_4 / Diverse / Enterobacteriaceae
OAB058_B / Undetermined / Streptococcus / OAB075_4 / Enterobacteriaceae / Enterobacteriaceae
OAB059_B / Diverse / Diverse
OAB060_B / Enterobacteriaceae / Enterobacteriaceae
OAB061_B / Undetermined / Streptococcus
OAB062_B / Gardnerella / Gardnerella
OAB063_B / Undetermined / Lactobacillus
OAB064_B / Lactobacillus / Streptococcus
OAB065_B / Enterococcus / Enterobacteriaceae
OAB066_B / Undetermined / Streptococcus
OAB067_B / Undetermined / Streptococcus
OAB068_B / Undetermined / Lactobacillus
OAB069_B / Undetermined / Diverse
OAB070_B / Undetermined / Undetermined
OAB071_B / Gardnerella / Lactobacillus
OAB072_B / Lactobacillus / Lactobacillus
OAB073_B / Undetermined / Undetermined
OAB074_B / Undetermined / Lactobacillus
OAB075_B / Undetermined / Diverse
- Culture and Sequencing Urotypes for UUI at 12 weeks and Controls at Baseline
UUI: 12 week Samples / Controls Samples
Study ID / Sequencing Urotype / Culture Urotype / Study ID / Sequencing Urotype / Culture Urotype
OAB003_12 / Diverse / Not Cultured / C001 / Undetermined / Not Cultured
OAB004_12 / Gardnerella / Not Cultured / C002 / Undetermined / Not Cultured
OAB006_12 / Diverse / Not Cultured / C003 / Gardnerella / Not Cultured
OAB007_12 / Undetermined / Not Cultured / C004 / Lactobacillus / Not Cultured
OAB008_12 / Not Sequenced / Not Cultured / C005 / Lactobacillus / Not Cultured
OAB010_12 / Not Sequenced / Not Cultured / C006 / Undetermined / Not Cultured
OAB012_12 / Undetermined / Undetermined / C007 / Undetermined / Not Cultured
OAB013_12 / Undetermined / Lactobacillus / C008 / Undetermined / Lactobacillus
OAB014_12 / Lactobacillus / Lactobacillus / C009 / Undetermined / Alloscardovia
OAB015_12 / Aerococcus / Aerococcus / C010 / Undetermined / Undetermined
OAB016_12 / Undetermined / Streptococcus / C011 / Undetermined / Undetermined
OAB017_12 / Undetermined / Lactobacillus / C012 / Undetermined / Lactobacillus
OAB018_12 / Lactobacillus / Diverse / C013 / Lactobacillus / Lactobacillus
OAB020_12 / Undetermined / Streptococcus/ Actinomyces / C014 / Undetermined / Staphylococcus
OAB021_12 / Lactobacillus / Lactobacillus / C015 / Lactobacillus / Lactobacillus
OAB022_12 / Undetermined / Diverse / C016 / Undetermined / Lactobacillus
OAB023_12 / Gardnerella / Diverse / C017 / Undetermined / Lactobacillus
OAB024_12 / Lactobacillus / Lactobacillus / C018 / Undetermined / Streptococcus/ Bifidobacterium
OAB025_12 / Undetermined / Bifidobacterium / C019 / Lactobacillus / Staphylococcus
OAB026_12 / Gardnerella / Gardnerella / C020 / Lactobacillus / Gardnerella
OAB028_12 / Undetermined / Undetermined / C021 / Undetermined / Not Cultured
OAB029_12 / Undetermined / Undetermined / C022 / Undetermined / Micrococcus
OAB030_12 / Undetermined / Enterobacteriaceae / C023 / Lactobacillus / Lactobacillus
OAB031_12 / Lactobacillus / Lactobacillus / C024 / Undetermined / Undetermined
OAB032_12 / Lactobacillus / Lactobacillus / C025 / Undetermined / Unknown
OAB033_12 / Gardnerella / Enterobacteriaceae / C026 / Undetermined / Undetermined
OAB034_12 / Undetermined / Streptococcus / C027 / Atopobium / Diverse
OAB035_12 / Undetermined / Undetermined / C028 / Undetermined / Enterococcus
OAB037_12 / Undetermined / Streptococcus / C029 / Undetermined / Diverse
OAB039_12 / Lactobacillus / Lactobacillus / C030 / Lactobacillus / Lactobacillus
OAB040_12 / Undetermined / Streptococcus/ Actinomyces / C031 / Enterobacteriaceae / Enterobacteriaceae
OAB041_12 / Gardnerella / Lactobacillus / C032 / Diverse / Streptococcus/ Bifidobacterium
OAB042_12 / Undetermined / Diverse / C033 / Undetermined / Enterobacteriaceae
OAB043_12 / Undetermined / Diverse / C034 / Lactobacillus / Undetermined
OAB044_12 / Undetermined / Undetermined / C035 / Gardnerella / Gardnerella
OAB045_12 / Gardnerella / Gardnerella / C036 / Diverse / Undetermined
OAB047_12 / Undetermined / Corynebacterium / C037 / Lactobacillus / Lactobacillus
OAB049_12 / Lactobacillus / Lactobacillus/ Gardnerella / C038 / Undetermined / Streptococcus
OAB051_12 / Undetermined / Streptococcus / C039 / Lactobacillus / Lactobacillus
OAB054_12 / Undetermined / Gardnerella / C040 / Undetermined / Undetermined
OAB055_12 / Undetermined / Diverse / C041 / Lactobacillus / Enterococcus
OAB056_12 / Lactobacillus / Lactobacillus / C042 / Lactobacillus / Undetermined
OAB057_12 / Diverse / Enterobacteriaceae / C043 / Diverse / Undetermined
OAB058_12 / Undetermined / Undetermined / C044 / Undetermined / Undetermined
OAB060_12 / Enterobacteriaceae / Enterobacteriaceae / C045 / Gardnerella / Candida
OAB062_12 / Enterobacteriaceae / Enterobacteriaceae / C046 / Undetermined / Lactobacillus
OAB066_12 / Undetermined / Streptococcus / C047 / Undetermined / Streptococcus
OAB072_12 / Lactobacillus / Lactobacillus / C048 / Undetermined / Undetermined
OAB073_12 / Lactobacillus / Lactobacillus / C049 / Undetermined / Undetermined
OAB074_12 / Undetermined / Diverse / C050 / Undetermined / Undetermined
C051 / Diverse / Undetermined
C052 / Gardnerella / Undetermined
C053 / Undetermined / Lactobacillus
C054 / Undetermined / Lactobacillus
C055 / Lactobacillus / Undetermined
C056 / Lactobacillus / Lactobacillus
C057 / Undetermined / Undetermined
C058 / Lactobacillus / Lactobacillus
C059 / Undetermined / Undetermined
C060 / Undetermined / Undetermined
E-Figure 1: Rarefaction Baseline and 12-Week Diversity by Response Group
E-Figure 2: Frequency of Detection of Bacterial Genera at Baseline and 12w of genera in the responders (5mg and 10mg).
Comparison of the frequency of detection for baseline and 12 week urines in responders (including both 5mg and 10mg responders). There is no one genus that decreases significantly over that timeframe in individuals that respond to solifenicin.