Forming Consensus To Advance Urobiome Research.

Urobiome research has the potential to advance the understanding of a wide range of diseases, including lower urinary tract symptoms and kidney disease. Many scientific areas have benefited from early research method consensus to facilitate the greater, common good. This consensus document, developed by a group of expert investigators currently engaged in urobiome research (UROBIOME 2020 conference participants), aims to promote standardization and advances in this field by the adoption of common core research practices. We propose a standardized nomenclature as well as considerations for specimen collection, preservation, storage, and processing. Best practices for urobiome study design include our proposal for standard metadata elements as part of core metadata collection. Although it is impractical to follow fixed analytical procedures when analyzing urobiome data, we propose guidelines to document and report data originating from urobiome studies. We offer this first consensus document with every expectation of subsequent revision as our field progresses.

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

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.

Urinary microbes and postoperative urinary tract infection risk in urogynecologic surgical patients.

INTRODUCTION AND HYPOTHESIS: Women have a 20% risk of developing a urinary tract infection (UTI) following urogynecologic surgery. This study assessed the association of postoperative UTI with bacteria in preoperative samples of catheterized urine. METHODS: Immediately before surgery, vaginal swabs, perineal swabs, and catheterized urine samples were collected, and the V4 region of the 16S ribosomal RNA (rRNA) gene was sequenced. The cohort was dichotomized in two ways: (1) standard day-of-surgery urine culture result (positive/negative), and (2) occurrence of postoperative UTI (positive/negative). Characteristics of bladder, vaginal, and perineal microbiomes were assessed to identify factors associated with postoperative UTI. RESULTS: Eighty-seven percent of the 104 surgical patients with pelvic organ prolapse/urinary incontinence (POP/UI) were white; mean age was 57 years. The most common genus was Lactobacillus, with a mean relative abundance of 39.91% in catheterized urine, 53.88% in vaginal swabs, and 30.28% in perineal swabs. Two distinct clusters, based on dispersion of catheterized urine (i.e., bladder) microbiomes, had highly significant (p < 2.2-16) differences in age, microbes, and postoperative UTI risk. Postoperative UTI was most frequently associated with the bladder microbiome; microbes in adjacent pelvic floor niches also contributed to UTI risk. UTI risk was associated with depletion of Lactobacillus iners and enrichment of a diverse mixture of uropathogens. CONCLUSIONS: Postoperative UTI risk appears to be associated with preoperative bladder microbiome composition, where an abundance of L. iners appears to protect against postoperative UTI.

Urinary symptoms are associated with certain urinary microbes in urogynecologic surgical patients.

INTRODUCTION AND HYPOTHESIS: Persistent and de novo symptoms decrease satisfaction after urogynecologic surgery. We investigated whether the preoperative bladder microbiome is associated with urinary symptoms prior to and after urogynecologic surgery. METHODS: One hundred twenty-six participants contributed responses to the validated OABq symptom questionnaire. Catheterized (bladder) urine samples and vaginal and perineal swabs were collected immediately preoperatively. Bacterial DNA in the urine samples and swabs was sequenced and classified. RESULTS: Preoperative symptom severity was significantly worse in sequence-positive patients. Higher OABq Symptom Severity (OABqSS) scores (more symptomatic) were associated with higher abundance in bladder urine of two bacterial species: Atopobium vaginae and Finegoldia magna. The presence of Atopobium vaginae in bladder urine also was correlated with its presence in either the vagina or perineum. CONCLUSIONS: Two specific bacterial species detected in bladder urine, Atopobium vaginae and Finegoldia magna, are associated with preoperative urinary symptom severity in women undergoing POP/SUI surgery. The reservoir for Atopobium vaginae may be adjacent pelvic floor niches. This observation should be validated in a larger cohort to determine whether there is a microbiologic etiology for certain preoperative urinary symptoms.

Microorganisms Identified in the Maternal Bladder: Discovery of the Maternal Bladder Microbiota.

Objective The objective of this study was to characterize the bladder microbiota in pregnancy. Methods A prospective observational study of 51 pregnant women, admitted to a tertiary care hospital, who underwent straight catheterization urine collection or transurethral Foley catheter placement. 16S rRNA gene sequencing and enhanced quantitative urine culture assessed the maternal bladder microbiota with comparisons made to standard urine culture results. Results Enhanced quantitative urine culture and 16S rRNA gene sequencing detected bacteria in the majority of participants. Lactobacillus and Gardnerella were the most commonly detected microbes. In contrast, standard urine culture had a 100% false-negative rate and failed to detect several known or emerging urinary pathogens. Conclusion There are live bacteria in the bladders of most pregnant women. This challenges the definition of asymptomatic bacteriuria.

Urinary Symptoms and Their Associations With Urinary Tract Infections in Urogynecologic Patients.

OBJECTIVE: To assess urinary symptoms associated with urinary tract infection (UTI) in a urogynecologic population of women. METHODS: In this cohort study, we enrolled 150 urogynecologic patients who completed the validated UTI Symptom Assessment questionnaire and contributed transurethral catheterized urine samples. The primary measure (UTI diagnosis) was defined in three ways. Self-report (a nonculture-based UTI diagnosis) was defined by a yes or no response to the query "Do you think you have a UTI?" Two culture-based UTI diagnoses also were analyzed: standard urine culture (10 colony-forming units [CFU]/mL or greater) and enhanced quantitative urine culture (10 CFU/mL or greater) of any uropathogen. Statistical analyses were performed on patient demographics and urinary symptom prevalence among patient groups. RESULTS: Although the presence of the urinary symptoms of frequency and urgency (respectively) differ somewhat between UTI-positive and UTI-negative women (self-report [P=.005 and P<.001], standard urine culture [P=.038 and P=.044], or enhanced quantitative urine culture [P=.059 and P=.098]), the presence of dysuria (pain or burning) during urination was significantly more prevalent in UTI-positive women for all UTI definitions (self-report P<.001, standard urine culture P<.001, and enhanced quantitative urine culture P=.010). Furthermore, women reporting dysuria had higher severity and bother scores for all other urinary symptoms assessed by the UTI Symptom Assessment questionnaire compared with women not reporting dysuria (frequency P=.001, urgency P=.006, dysuria P<.001). CONCLUSION: Our findings show that, in women seeking urogynecologic care, the presence of frequency and urgency of urination does not confirm a culture-based UTI diagnosis. Instead, clinicians can more readily detect UTI using the presence of dysuria, which more effectively discriminates UTI-positive and UTI-negative individuals, regardless of the culture-based method used to diagnose UTI.

Evaluation of the urinary microbiota of women with uncomplicated stress urinary incontinence.

BACKGROUND: Female urinary microbiota are associated with urgency urinary incontinence and response to medication. The urinary microbiota of women with stress urinary incontinence has not been described. OBJECTIVE: We sought to study the cross-sectional relationships between urinary microbiota features and demographic and clinical characteristics of women undergoing stress urinary incontinence surgery. STUDY DESIGN: Preoperative urine specimens were collected from women without urinary tract infection and were available from 197 women (174 voided, 23 catheterized) enrolled in a multicenter prospective randomized trial, the Value of Urodynamic Evaluation study. Demographic and clinical variables were obtained including stress and urgency urinary incontinence symptoms, menopausal status, and hormone use. The bacterial composition of the urine was qualitatively assessed by sequencing the bacterial 16S ribosomal RNA gene. Phylogenetic relatedness and microbial alpha diversity were compared to demographics and symptoms using generalized estimating equation models. RESULTS: The majority of 197 urine samples (86%) had detectable bacterial DNA. Bacterial diversity was significantly associated with higher body mass index (P = .02); increased Medical, Epidemiologic, and Social Aspects of Aging urge index score (P = .04); and hormonal status (P < .001). No associations were detected with stress urinary incontinence symptoms. Increased diversity was also associated with a concomitant lower frequency of Lactobacillus in hormone-negative women. CONCLUSION: Women undergoing stress urinary incontinence surgery have detectable urinary microbiota. This cross-sectional analysis revealed that increased diversity of the microbiota was associated with urgency urinary incontinence symptoms, hormonal status, and body mass index. In contrast, the female urinary microbiota were not associated with stress urinary incontinence symptoms.

The Clinical Urine Culture: Enhanced Techniques Improve Detection of Clinically Relevant Microorganisms.

Enhanced quantitative urine culture (EQUC) detects live microorganisms in the vast majority of urine specimens reported as "no growth" by the standard urine culture protocol. Here, we evaluated an expanded set of EQUC conditions (expanded-spectrum EQUC) to identify an optimal version that provides a more complete description of uropathogens in women experiencing urinary tract infection (UTI)-like symptoms. One hundred fifty adult urogynecology patient-participants were characterized using a self-completed validated UTI symptom assessment (UTISA) questionnaire and asked "Do you feel you have a UTI?" Women responding negatively were recruited into the no-UTI cohort, while women responding affirmatively were recruited into the UTI cohort; the latter cohort was reassessed with the UTISA questionnaire 3 to 7 days later. Baseline catheterized urine samples were plated using both standard urine culture and expanded-spectrum EQUC protocols: standard urine culture inoculated at 1 µl onto 2 agars incubated aerobically; expanded-spectrum EQUC inoculated at three different volumes of urine onto 7 combinations of agars and environments. Compared to expanded-spectrum EQUC, standard urine culture missed 67% of uropathogens overall and 50% in participants with severe urinary symptoms. Thirty-six percent of participants with missed uropathogens reported no symptom resolution after treatment by standard urine culture results. Optimal detection of uropathogens could be achieved using the following: 100 µl of urine plated onto blood (blood agar plate [BAP]), colistin-nalidixic acid (CNA), and MacConkey agars in 5% CO2 for 48 h. This streamlined EQUC protocol achieved 84% uropathogen detection relative to 33% detection by standard urine culture. The streamlined EQUC protocol improves detection of uropathogens that are likely relevant for symptomatic women, giving clinicians the opportunity to receive additional information not currently reported using standard urine culture techniques.

Incontinence medication response relates to the female urinary microbiota.

INTRODUCTION AND HYPOTHESIS: Many adult women have resident urinary bacteria (urinary microbiome/microbiota). In adult women affected by urinary urgency incontinence (UUI), the etiologic and/or therapeutic role of the urinary microbiome/microbiota remains unknown. We hypothesized that microbiome/microbiota characteristics would relate to clinically relevant treatment response to UUI medication per os. METHODS: Adult women initiating medication treatment orally for UUI and a comparator group of unaffected women were recruited in a tertiary care health-care system. All participants provided baseline clinical data and urine samples. Women with UUI were given 5 mg solifenacin, with potential dose escalation to 10 mg for inadequate UUI symptom control at 4 weeks. Additional data and urine samples were collected from women with UUI at 4 and 12 weeks. The samples were assessed using 16S ribosomal RNA (rRNA) gene sequencing and enhanced quantitative urine culturing. The primary outcome was treatment response as measured by the validated Patient Global Symptom Control (PGSC) questionnaire. Clinically relevant UUI symptom control was defined as a 4 or 5 score on the PGSC. RESULTS: Diversity and composition of the urinary microbiome/microbiota of women with and without UUI differed at baseline. Women with UUI had more bacteria and a more diverse microbiome/microbiota. The clinical response to solifenacin in UUI participants was related to baseline microbiome/microbiota, with responders more likely to have fewer bacteria and a less diverse community at baseline. Nonresponders had a more diverse community that often included bacteria not typically found in responders. CONCLUSIONS: Knowledge of an individual's urinary microbiome/microbiota may help refine UUI treatment. Complementary tools, DNA sequencing, and expanded urine culture provide information about bacteria that appear to be related to UUI incontinence status and treatment response in this population of adult women.

The female urinary microbiome in urgency urinary incontinence.

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.