Temporal dynamics of certain human microbiotas have been described in longitudinal studies; variability often relates to modifiable factors or behaviors. Early studies of the urinary microbiota preferentially used samples obtained by transurethral catheterization to minimize vulvovaginal microbial contributions. Whereas voided specimens are preferred for longitudinal studies, the few studies that reported longitudinal data were limited to women with lower urinary tract (LUT) symptoms, due to ease of accessing a clinical population for sampling and the impracticality and risk of collecting repeated catheterized urine specimens in a nonclinical population. Here, we studied the microbiota of the LUT of nonsymptomatic, premenopausal women using midstream voided urine (MSU) specimens to investigate relationships between microbial dynamics and personal factors. Using 16S rRNA gene sequencing and a metaculturomics method called expanded quantitative urine culture (EQUC), we characterized the microbiotas of MSU and periurethral swab specimens collected daily for approximately 3 months from a small cohort of adult women. Participants were screened for eligibility, including the ability to self-collect paired urogenital specimens prior to enrollment. In this population, we found that measures of microbial dynamics related to specific participant-reported factors, particularly menstruation and vaginal intercourse. Further investigation of the trends revealed differences in the composition and diversity of LUT microbiotas within and across participants. These data, in combination with previous studies showing relationships between the LUT microbiota and LUT symptoms, suggest that personal factors relating to the genitourinary system may be an important consideration in the etiology, prevention, and/or treatment of LUT disorders. IMPORTANCE Following the discovery of the collective human urinary microbiota, important knowledge gaps remain, including the stability and variability of this microbial niche over time. Initial urinary studies preferentially utilized samples obtained by transurethral catheterization to minimize contributions from vulvovaginal microbes. However, catheterization has the potential to alter the urinary microbiota; therefore, voided specimens are preferred for longitudinal studies. In this report, we describe microbial findings obtained by daily assessment over 3 months in a small cohort of adult women. We found that, similarly to vaginal microbiotas, lower urinary tract (LUT) microbiotas are dynamic, with changes relating to several factors, particularly menstruation and vaginal intercourse. Our study results show that LUT microbiotas are both dynamic and resilient. They also offer novel opportunities to target LUT microbiotas by preventative or therapeutic means, through risk and/or protective factor modification.

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245/250) 24MANUSCRIPT: (5000/5000) 25 ACKNOWLEDGMENTS: We thank Mary Tulke RN for her assistance with participant 35 recruitment. We also acknowledge funding from NIH (R01 DK104718 awarded to AJW 36and LB). The funders did not play a part in the design or conduct of the study. 37 TKP, BW, LB, ERM, and AJW designed the study. TKP and BW recruited participants. 38TKP processed the specimens. TH prepared them for DNA sequencing. RL sequenced 39 the specimens. TKP, BW, QD and AJW analyzed the data. TKP and AJW wrote the 40 manuscript. TKP, BW, TH, RL, LB, QD, ERM, and AJW reviewed and edited the 41 manuscript. 42 43 ABSTRACT 59Temporal dynamics of certain human microbiotas have been described in 60 longitudinal studies; variability often relates to modifiable factors or behaviors. Early 61 studies of the urinary microbiota preferentially used samples obtained by transurethral 62 catheterization to minimize vulvo-vaginal microbial contributions. Whereas voided 63 specimens are preferred for longitudinal studies, the few studies that reported 64 longitudinal data were limited to women with lower urinary tract (LUT) symptoms, due to 65 ease of accessing a clinical population for sampling and the impracticality and risk of 66 collecting repeated catheterized urine specimens in a non-clinical population. Here, we 67 studied the microbiota of the LUT of non-symptomatic, pre-menopausal women using 68 mid-stream voided urine (MSU) specimens to investigate relationships between 69 microbial dynamics and personal factors. Using 16S rRNA gene sequencing and a 70 metaculturomics method called Expanded Quantitative Urine Culture (EQUC), we 71 characterized the microbiotas of MSU and peri-urethral swab specimens collected daily 72 for approximately three months from a small cohort of adult women. Participants were 73 screened for eligibility, including ability to self-collect paired urogenital specimens prior 74 to enrollment. In this population, we found that measures of microbial dynamics related 75 to specific participant-reported factors, particularly menstruation and vaginal 76intercourse. Further investigation of the trends revealed differences in composition and 77 diversity of LUT microbiotas within and across participants. These data, in combination 78 with previous studies showing relationships between the LUT microbiota and LUT 79 symptoms, suggest that personal factors relating to the genitourinary system may be an 80 important consideration in the etiology, prevention, and/or treatment of LUT disorders.

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Introduction-Previous work has shown that the vaginal microbiome decreases in Lactobacilluspredominance and becomes more diverse following menopause. It has also been shown that estrogen therapy restores Lactobacillus-dominance in the vagina, and that topical estrogen is associated with OAB symptom improvement. We now know that the bladder contains a unique microbiome, and increased bladder microbiome diversity is associated with OAB. However, there is no understanding of how quickly each pelvic floor microbiome responds to estrogen or if those changes are associated with symptom improvement.Study Design-Analysis of data from post-menopausal participants in two trials (NCT02524769 and NCT02835846) who chose vaginal estrogen as their primary OAB treatment and used 0.5 grams of conjugated estrogen (Premarin Cream, (Pfizer, New York City, NY)) twice weekly for 12 weeks. Baseline and 12-week follow-up data included the OAB-q questionnaire and participants provided catheterized urine, vaginal swabs, perineal swabs, and voided urine. Microbes were detected by an enhanced culture protocol. Linear mixed models were used to estimate microbiome changes over time. Urinary AMP activity was assessed by a bacterial growth inhibition assay and correlated with relative abundance of members of the urobiome.Results-Twelve weeks of estrogen treatment resulted in decreased microbial diversity within the vagina (Shannon, p=0.047; Richness, p=0.043), but not in the other niches. A significant increase in Lactobacillus was detected in the bladder (p=0.037), but not the vagina (p=0.33), perineum (p=0.56), or voided urine (p=0.28). The change in Lactobacillus levels in the bladder was associated with modest changes in urgency incontinence symptoms (p=0.02). The relative abundance of the genus Corynebacterium correlated positively with urinary AMP activity after estrogen treatment. Conclusion-Estrogentherapy may change the microbiome of different pelvic floor niches. The vagina begins to decrease in diversity and the bladder experiences a significant increase in Lactobacillus levels; the latter is correlated with a modest improvement in the symptom severity sub-scale of the OAB-q. CondensationEstrogen therapy for overactive bladder resulted in decreased bladder bacterial diversity and increased bladder Lactobacillus, which were associated with modest changes in urgency incontinence symptoms.

Research into the lower urinary tract (LUT) microbiota has primarily focused on its relationship to LUT symptoms (LUTS), taking snapshots of these communities in individuals with and without LUTS. While certain bacterial taxa have been associated with LUTS, or the lack thereof, the temporal dynamics of this community were largely unknown. Recently, we conducted a longitudinal study and found that vaginal intercourse resulted in a shift in species richness and diversity within the LUT microbiota. This is particularly relevant as frequent vaginal intercourse is a major risk factor for urinary tract infection (UTI) in premenopausal women (Aydin et al. Int Urogynecol J 2015;26:795–804). To further investigate the relationship between vaginal intercourse and LUT microbiota, here we present the results of a 3 week study in which daily urogenital specimens were collected from a female participant and her male sexual partner. Consistent with our previous findings, the LUT microbiota changed after vaginal intercourse, most notably a high abundance of Streptococcus mitis was observed post-coitus. We isolated and sequenced S. mitis from both sexual partners finding that: (i) the S. mitis isolates from the female partner’s urogenital tract were genomically similar throughout the duration of the study, and (ii) they were related to one isolate from the male partner’s oral cavity collected at the end of the study, suggesting transmission between the two individuals. We hypothesize that blooms in S. mitis after vaginal intercourse may play a role in coitus-related UTI. We found that a S. mitis isolate, in contrast to a Lactobacillus jensenii isolate displaced after vaginal intercourse, cannot inhibit the growth of uropathogenic Escherichia coli . Thus, this bloom in S. mitis may provide a window of opportunity for a uropathogen to colonize the LUT.

Purpose:Interstitial cystitis/bladder pain syndrome is a chronic urological condition diagnosed in nearly 8 million females in the United States. Whether urinary microbiota play an etiological role remains controversial. Most studies assessed the microbiota of interstitial cystitis/bladder pain syndrome patients with voided or catheterized urine as a proxy for bladder urothelium; however, urine may not be a true reflection of the bladder microbiota. Bladder biopsy tissue may provide a more accurate, and thus more clinically relevant, picture of bladder microbiota.Materials and Methods:Bladder biopsy tissues were obtained from: (1) 30 females with interstitial cystitis/bladder pain syndrome (18-80 years old) via cystoscopically guided cold-cup biopsy following therapeutic bladder hydrodistention, and (2) 10 non–interstitial cystitis/bladder pain syndrome females undergoing pelvic organ prolapse repair. To detect bacteria, technical duplicates of each RNAlater-preserved biopsy were subjected to 16S rRNA gene sequencing. To visualize bacteria, paraformaldehyde-fixed, paraffin-embedded biopsies were subjected to a combined multiplexed fluorescence in situ hybridization and fluorescence immunohistochemistry assay and confocal microscopy.Results:Bacteria were detected by 16S rRNA gene sequencing in at least 1 technical duplicate of most biopsies. The most abundant genus was Staphylococcus, followed by Lactobacillus; Escherichia was common but not abundant. There was no significant difference between interstitial cystitis/bladder pain syndrome patients and controls (P > .05). Combined fluorescence in situ hybridization and immunohistochemistry reproducibly detected 16S rRNA in epithelial cells and shed cells in the urothelium and lesioned areas and capillary walls in the lamina propria of human bladder biopsy tissue.Conclusions:We conclude that urothelial and urinary microbiota are similar but not identical in adult females.

Although the human bladder is reported to harbor unique microbiota, our understanding of how these microbial communities interact with their human hosts is limited, mostly owing to the lack of isolates to test mechanistic hypotheses. Niche-specific bacterial collections and associated reference genome databases have been instrumental in expanding knowledge of the microbiota of other anatomical sites, e.g., the gut and oral cavity. To facilitate genomic, functional, and experimental analyses of the human bladder microbiota, here we present a bladder-specific bacterial reference collection comprised of 1134 genomes. These genomes were culled from bacterial isolates obtained by a metaculturomic method from bladder urine collected by transurethral catheterization. This bladder-specific bacterial reference collection includes 196 different species, including representatives of major aerobes and facultative anaerobes, as well as some anaerobes. It captures 72.2 % of the genera found when we reexamined previously published 16S rRNA gene sequencing of 392 adult female bladder urine samples. Comparative genomic analysis found that the taxonomies and functions of the bladder microbiota shared more similarities with the vaginal microbiota than the gut microbiota. Whole-genome phylogenetic and functional analyses of 186 bladderE. coliisolates and 387 gutE. coliisolates supports the hypothesis that phylogroup distribution and functions ofE. colistrains differ dramatically between these two very different niches. This bladder-specific bacterial reference collection is a unique resource that will enable hypothesis-driven bladder microbiota research and comparison to isolates from other anatomical sites.