Bladder bacterial diversity differs in continent and incontinent women: a cross-sectional study

doi:10.1016/j.ajog.2020.04.033

American Journal of Obstetrics and Gynecology

Volume 223, Issue 5, November 2020, Pages 729.e1-729.e10

Some of the data were previously presented as “The female urinary microbiota differ by primary lower urinary tract disorder” at the International Continence Society, Florence, Italy, Sept. 12–15, 2017, and at 39th Annual American Urogynecologic Society Meeting, Providence, RI, Oct. 3–7, 2017.

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Background

Since the discovery of the bladder microbiome (urobiome), interest has grown in learning whether urobiome characteristics have a role in clinical phenotyping and provide opportunities for novel therapeutic approaches for women with common forms of urinary incontinence.

Objective

This study aimed to test the hypothesis that the bladder urobiome differs among women in the control cohort and women affected by urinary incontinence by assessing associations between urinary incontinence status and the cultured urobiome.

Study Design

With institutional review board oversight, urine specimens from 309 adult women were collected through transurethral catheterization. These women were categorized into 3 cohorts (continent control, stress urinary incontinence [SUI], and urgency urinary incontinence [UUI]) based on their responses to the validated Pelvic Floor Distress Inventory (PFDI) questionnaire. Among 309 women, 150 were in the continent control cohort, 50 were in the SUI cohort, and 109 were in the UUI cohort. Symptom severity was assessed by subscale scoring with the Urinary Distress Inventory (UDI), subscale of the Pelvic Floor Distress Inventory. Microbes were assessed by expanded quantitative urine culture protocol, which detects the most common bladder microbes (bacteria and yeast). Microbes were identified to the species level by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. Alpha diversity indices were calculated for culture-positive samples and compared across the 3 cohorts. The correlations of UDI scores, alpha diversity indices, and species abundance were estimated.

Results

Participants had a mean age of 53 years (range 22–90); most were whites (65%). Women with urinary incontinence were slightly older (control, 47; SUI, 54; UUI, 61). By design, UDI symptom scores differed (control, 8.43 [10.1]; SUI, 97.95 [55.36]; UUI, 93.71 [49.12]; P<.001). Among 309 participants, 216 (70%) had expanded quantitative urine culture–detected bacteria; furthermore, the urinary incontinence cohorts had a higher detection frequency than the control cohort (control, 57%; SUI, 86%; UUI, 81%; P<.001). In addition, the most frequently detected species among the cohorts were as follows: continent control, Lactobacillus iners (12.7%), Streptococcus anginosus (12.7%), L crispatus (10.7%), and L gasseri (10%); SUI, S anginosus (26%), L iners (18%), Staphylococcus epidermidis (18%), and L jensenii (16%); and UUI, S anginosus (30.3%), L gasseri (22%), Aerococcus urinae (18.3%), and Gardnerella vaginalis (17.4%). However, only Actinotignum schaalii (formerly Actinobaculum schaalii), A urinae, A sanguinicola, and Corynebacterium lipophile group were found at significantly higher mean abundances in 1 of the urinary incontinence cohorts when compared with the control cohort (Wilcoxon rank sum test; P<.02), and no individual genus differed significantly between the 2 urinary incontinence cohorts. Both urinary incontinence cohorts had increased alpha diversity similar to continent control cohort with indices of species richness, but not evenness, strongly associated with urinary incontinence.

Conclusion

In adult women, the composition of the culturable bladder urobiome is associated with urinary incontinence, regardless of common incontinence subtype. Detection of more unique living microbes was associated with worsening incontinence symptom severity. Culturable species richness was significantly greater in the urinary incontinence cohorts than in the continent control cohort.

Introduction

Symptoms, urodynamic findings, and presumed etiologic mechanisms are used to subgroup women with urinary incontinence (UI) into categories, including the 2 most common forms of UI, stress urinary incontinence (SUI) and urgency urinary incontinence (UUI). These subgroups are used to initiate clinical treatment. To further refine clinical algorithm and treatment efficacy, multiple experts have expressed the need for additional phenotyping of affected patients. Since the discovery and confirmation of the human bladder urobiome (an umbrella term used to describe the microbiota and their genomes),1, 2, 3, 4 interest has grown in learning whether bladder urobiome characteristics play a role in clinical phenotyping and provide opportunities for novel therapeutic approaches.5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 However, investigations of a single UI subgroup have focused predominantly on UUI, and thus, comparisons with SUI have been lacking. Two complementary techniques have been used to characterize the human urobiome, expanded culture and DNA sequencing.⁷ In this study, we tested the hypothesis that the urobiome differs among women in the continent control cohort and women affected by UI. To assess the associations between UI status and urobiome composition, we used an enhanced culture method called expanded quantitative urine culture (EQUC) coupled with matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonics, Billerica, MA). The former detects the most common microbes (bacteria and yeast), whereas the latter identifies those microbes to the species level.³^,¹⁷

AJOG at a Glance

This study aimed to determine whether bladder urobiome characteristics have a role in clinical phenotyping and provide opportunities for novel therapeutic approaches for common forms of urinary incontinence.

In this study, bladder urobiome composition was associated with urinary incontinence regardless of urinary incontinence subtype. Women with urinary incontinence were more likely to have detectable microbes than women in the continent control cohort. Detection of more unique living microbes was associated with worsening incontinence symptom severity.

Women with urinary incontinence have urobiomes that differ from unaffected women in the continent control cohort. Once replicated with appropriate controls for clinical variables of interest, these findings should prompt investigators to address whether the detected changes are etiologic factors for or consequences of urinary incontinence.

Section snippets

Study design and patient population

After institutional review board approval, we enrolled 309 women seeking care at Loyola University Medical Center between December 2013 and December 2015 using identical study protocols, procedures, and equipment. On the day of enrollment, each participant was without clinical evidence of urinary tract infection (UTI) (ie, negative result for standard urine culture test and absence of clinical diagnosis or treatment of UTI).

Using the responses to the Pelvic Floor Distress Inventory (PFDI)

Demographics

Table 1 indicates the demographic characteristics of the 3 cohorts (continent control, SUI, and UUI). The mean ages (years) differed (control, 47; SUI, 54; UUI, 61; P<.001), and a larger proportion of the continent control cohort reported being sexually active (P<.001). Minor demographic differences were noted in race or ethnicity, UUI medication, hormonal treatment, and some comorbidities. In the Appendix, other than age (P=.001), the UUI and SUI cohorts were similar (Supplemental Table). By

Principal findings

There are differences in the cultured bladder urobiome among adult women with the major subtypes of UI and women in the continent control cohort. These differences, which relate to detection and richness, may be useful for UI phenotyping. Women with UI were more likely to have detectable microbes than women in the control cohort, and detection of more unique living microbes was associated with worsening incontinence symptom severity (ie, richness indices were positively correlated with UDI

Acknowledgments

We thank Mary Tulke, RN, for her assistance with participant recruitment and sample collection and Cynthia Brincat, MD, who greatly assisted with recruitment efforts of the study population.

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Cited by (29)

Urinary microbiome community types associated with urinary incontinence severity in women
2024, American Journal of Obstetrics and Gynecology
Urinary microbiome (urobiome) studies have previously reported on specific taxa and community differences in women with mixed urinary incontinence compared with controls. Therefore, a hypothesis was made that higher urinary and vaginal microbiome diversity would be associated with increased urinary incontinence severity.
This study aimed to test whether specific urinary or vaginal microbiome community types are associated with urinary incontinence severity in a population of women with mixed urinary incontinence.
This planned secondary, cross-sectional analysis evaluated associations between the urinary and vaginal microbiomes and urinary incontinence severity in a subset of Effects of Surgical Treatment Enhanced With Exercise for Mixed Urinary Incontinence trial participants with urinary incontinence. Incontinence severity was measured using bladder diaries and Urinary Distress Inventory questionnaires collected at baseline. Catheterized urine samples and vaginal swabs were concurrently collected before treatment at baseline to assess the urinary and vaginal microbiomes. Of note, 16S rRNA V4 to V6 variable regions were sequenced, characterizing bacterial taxa to the genus level using the DADA2 pipeline and SILVA database. Using Dirichlet multinomial mixtures methods, samples were clustered into community types based on core taxa. Associations between community types and severity measures (Urinary Distress Inventory total scores, Urinary Distress Inventory subscale scores, and the number of urinary incontinence episodes [total, urgency, and stress] from the bladder diary) were evaluated using linear regression models adjusted for age and body mass index. In addition, alpha diversity measures for richness (total taxa numbers) and evenness (proportional distribution of taxa abundance) were analyzed for associations with urinary incontinence episodes and community type.
Overall, 6 urinary microbiome community types were identified, characterized by varying levels of common genera (Lactobacillus, Gardnerella, Prevotella, Tepidimonas, Acidovorax, Escherichia, and others). The analysis of urinary incontinence severity in 126 participants with mixed urinary incontinence identified a Lactobacillus-dominated reference group with the highest abundance of Lactobacillus (mean relative abundance of 76%). A community characterized by fewer Lactobacilli (mean relative abundance of 19%) and greater alpha diversity was associated with higher total urinary incontinence episodes (2.67 daily leaks; 95% confidence interval, 0.76–4.59; P=.007) and urgency urinary incontinence episodes (1.75 daily leaks; 95% confidence interval, 0.24–3.27; P=.02) than the reference group. No significant association was observed between community type and stress urinary incontinence episodes or Urogenital Distress Inventory total or subscores. The composition of vaginal community types and urinary community types were similar but composed of slightly different bacterial taxa. Vaginal community types were not associated with urinary incontinence severity, as measured by bladder diary or Urogenital Distress Inventory total and subscale scores. Alpha diversity indicated that greater sample richness was associated with more incontinence episodes (observed genera P=.01) in urine. Measures of evenness (Shannon and Pielou) were not associated with incontinence severity in the urinary or vaginal microbiomes.
In the urobiome of women with mixed urinary incontinence, a community type with fewer Lactobacilli and more diverse bacteria was associated with more severe urinary incontinence episodes (total and urgency) compared with a community type with high predominance of a single genus, Lactobacillus. Whether mixed urinary incontinence severity is due to lesser predominance of Lactobacillus, greater presence of other non–Lactobacillus genera, or the complement of bacteria consisting of urobiome community types remains to be determined.
Comparative Genomic Study of Streptococcus anginosus Reveals Distinct Group of Urinary Strains
2023, mSphere
Whole-genome analysis of S. anginosus strains provides greater insight into the diversity of this species than from marker genes alone. Our investigation of 166 publicly available S. anginosus genomes via average nucleotide identity and core genome analysis revealed two phylogenomically distinct groups of this species, with one group almost exclusively consisting of isolates from the urinary tract.
Streptococcus anginosus is a prevalent member of the human flora. While it has been found in the microbiota of “healthy” asymptomatic individuals, it has also been associated with genitourinary tract infections and bacteremia. Based upon multilocus sequence analysis, two subspecies and two genomosubspecies have been characterized for the species. We previously conducted whole-genome sequencing of 85 S. anginosus isolates from the urinary tract. Here, we present genomic analysis of this species, including isolates from the urinary tract as well as gut and fecal, vaginal, oral, respiratory, and blood and heart samples. Average nucleotide identity and core genome analysis revealed that these strains form two distinct groups. Group 1 is comprised of the S. anginosus type strain and other previously identified S. anginosus subspecies and genomosubspecies, including isolates from throughout the human body. In contrast, group 2 consists of predominantly urinary streptococci (n = 77; 85.6%). Both of these S. anginosus groups are distinct from other members of the Streptococcus anginosus group (SAG) species S. intermedius and S. constellatus. Genes conserved among all strains of one group but not in any strains in the other group were next identified. Group 1 strains included genes found in S. intermedius and S. constellatus, suggesting that they were lost within the ancestor of the group 2 strains. In contrast, genes unique to the group 2 strains were homologous to more distant streptococci, indicative of acquisition via horizontal gene transfer. These genes are ideal candidates for use as marker genes to distinguish between the two groups in the human microbiota.
IMPORTANCE Whole-genome analysis of S. anginosus strains provides greater insight into the diversity of this species than from marker genes alone. Our investigation of 166 publicly available S. anginosus genomes via average nucleotide identity and core genome analysis revealed two phylogenomically distinct groups of this species, with one group almost exclusively consisting of isolates from the urinary tract. In contrast, only 8 urinary strains were identified within the other group, which contained the S. anginosus type strain, as well as all identified subspecies and genomosubspecies. While genomic analysis suggested that this urinary group of S. anginosus is genomically different from the previously characterized S. anginosus subspecies, phenotypic characterization is still needed. Given prior reports of the prevalence of S. anginosus in the urinary tract of both continent and incontinent females, future studies are needed to investigate if the symptom state of the urinary tract is associated with these two different groups.
A Child's urine is not sterile: A pilot study evaluating the Pediatric Urinary Microbiome
2022, Journal of Pediatric Urology
Citation Excerpt :
We observed a similar trend, especially in post-pubertal girls, with Lactobacillus, Prevotella and/or Bifidobacterium predominating urethral and vaginal microbiomes (Fig. 1H, Supplemental Figures 3, 4), and Lactobacillus, Bifidobacterium, or a Lactobacillus/Gardnerella combination predominating the urobiome (Fig. 1C and D, Supplemental Figures 1, 2). These microbial profiles resemble adult female urobiomes [3,10,11,28], supporting the hypothesis that adult female urobiomes establish around puberty and persist into adulthood. What drives these changes remains unclear.
A bladder microbiome (urobiome) exists in adults. Data supports the effects of the adult urobiome on urinary tract health with associations between dysbiotic urobiomes and lower urinary tract disorders. Understanding urobiome origin is important since other microbiomes establish around birth and microbiome alterations are linked to disease development. However, the pediatric urobiome has not been well studied.
We sought to determine the age when the urobiome develops, compare the pediatric urobiome to microbiomes of adjacent urogenital niches, and compare the urobiomes between boys and girls and across age groups.
Seventy-four children less than 18 years of age without recent antibiotic exposure were recruited, including 48 males and 26 females, aged 2 weeks to 209 months of age. Transurethral catheterized urine samples and samples from the perineum, urethra, vagina, and foreskin were collected. Specimens were assessed using the expanded quantitative urine culture protocol and by 16S rRNA gene sequencing. Dada2 was used to profile microbial compositions, and BLCA was used to identify microbial taxa.
Bacteria were detected in 90.5% of urine samples and identified in children as young as 2 weeks of age. Microbial communities and compositions of the female bladder and other urogenital niches (urethra, perineum, and vagina) differed significantly by age. Lactobacillus predominated the bladder, urethral, and vaginal microbiomes in post-pubertal girls. Compared to female urinary microbiomes, those of males differed less substantially. Only perineal microbiomes differed significantly by age, whereas male urethral and foreskin microbiomes did not differ significantly.
We identified that a urinary microbiome is established as early as infancy. In addition, the female urobiome changes throughout childhood, until the post-pubertal bacterial taxa becomes consistent with that seen in adult females. Whereas in boys, the urinary microbiome changed very little over time. In addition, the surrounding urogenital microbiomes differed less in boys as compared to females. Microbiomes established at a young age may have long-term influences on immune, metabolic, and neurobehavioral traits. The same may be true for the urobiome. Our study provides a foundation for future research to determine the influence of the pediatric urobiome on the development of urinary and even non-urinary disorders.
A pediatric urobiome exists, with differences between males and females and can be detected at a young age with changes occurring throughout childhood. Similarities and differences are also seen between the pediatric urobiome and adjacent niches.
Draft genomes of Lactobacillus jensenii UMB0908, UMB1545, and UMB5777 from the urinary tract
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THE URINARY MICROBIOTA IN HEALTHY PRE-AND POSTMENOPAUSAL WOMEN AND ITS CHANGES IN OVERACTIVE BLADDER SYNDROME
2024, Akusherstvo i Ginekologiya (Russian Federation)
Survey of the infant male urobiome and genomic analysis of Actinotignum spp.
2023, npj Biofilms and Microbiomes

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: Ms Lin’s current affiliation is Duchossosis Family Institute, University of Chicago, Chicago, IL.

: K.J.T.W. discloses membership on the advisory board of Live UTI Free. E.R.M. discloses research support from the National Institutes of Health (NIH), Astellas Pharma, and Boston Scientific. A.J.W. discloses research support from the NIH, Astellas Pharma, and Kimberly-Clark. L.B. discloses research funding from the NIH and editorial stipends from the Female Pelvic Medicine and Reconstructive Surgery, UpToDate, and the Journal of the American Medical Association. The remaining authors (T.K.P. H.L., X.G., E.E.H., and Q.D.) report no conflict of interest.

: This research was supported by research grant numbers R01 DK104718, R56 DK104718, R21 DK097435, and P20 DK108268 and by a grant from the Falk Foundation (LU#202567). The funders did not play a part in the design of the study.

: Cite this article as: Price TK, Lin H, Gao X, et al. Bladder bacterial diversity differs in continent and incontinent women: a cross-sectional study. Am J Obstet Gynecol 2020;223:729.e1-10.

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Published by Elsevier Inc.

Original ResearchGynecologyBladder bacterial diversity differs in continent and incontinent women: a cross-sectional study

Background

Objective

Study Design

Results

Conclusion

Introduction

AJOG at a Glance

Section snippets

Study design and patient population

Demographics

Principal findings

Acknowledgments

Am J Obstet Gynecol

Am J Obstet Gynecol

Am J Obstet Gynecol

Evidence of uncultivated bacteria in the adult female bladder

J Clin Microbiol

Spectrum of bacterial colonization associated with urothelial cells from patients with chronic lower urinary tract symptoms

J Clin Microbiol

Urine is not sterile: use of enhanced urine culture techniques to detect resident bacterial flora in the adult female bladder

J Clin Microbiol

Integrated next-generation sequencing of 16S rDNA and metaproteomics differentiate the healthy urine microbiome from asymptomatic bacteriuria in neuropathic bladder associated with spinal cord injury

J Transl Med

The bladder is not sterile: history and current discoveries on the urinary microbiome

Curr Bladder Dysfunct Rep

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

mBio

Does the urinary microbiome play a role in urgency urinary incontinence and its severity?

Front Cell Infect Microbiol

The urinary microbiome in women with mixed urinary incontinence compared to similarly aged controls

Int Urogynecol J

Original Research
Gynecology
Bladder bacterial diversity differs in continent and incontinent women: a cross-sectional study