The Urinary Microbiome: Improving Diagnostics and Management of Urinary Tract Infections in Adult Females.

This article discusses the urinary microbiome in relation to urinary tract infection (UTI) in women. It makes biologic sense that the microbiota of different niches (bladder, vagina, and gut) interact with each other in health, as well as during a UTI event; however, these relationships remain poorly understood. Future research should close knowledge gaps regarding the interactions between the urinary microbiota and the host, amongst the microbiota of adjacent niches, and between the microbes within the same microbiota. The new knowledge should result in improved UTI treatment in the age of antibiotic stewardship.

Urine and Tissue Bacterial Loads Correlate With Voiding Behaviors in a Murine Urinary Tract Infection Model.

OBJECTIVES: To describe associations between voiding behavior and bacterial loads in a murine model of urinary tract infection (UTI). METHODS: Fourteen female C57BL/6J mice were transurethrally inoculated with 108colony-forming unit uropathogenic E. coli (UPEC) UTI89 in 50 µL two times, 24 hours apart. Voiding spot assays were used to measure voiding behavior. Voiding spot assays and urine cultures were performed at various time points between 1 and 28 days postinfection (dpi). Bladder and kidney bacterial loads were measured at 28 dpi. Correlations were calculated between voiding spot assay variables and bacterial loads at different dpi. In a separate experiment, 3 female mice were infected with UPEC in the same manner for histology changes at 28-dpi in chronic UTI. RESULTS: During the 28 days, among 14 mice, 8 developed chronic cystitis and 11 developed chronic pyelonephritis based on a priori definitions. All infected mice showed increased urinary frequency, polyuria, and decreased bladder capacity. Tissue fibrosis was also observed in the infected bladder. At 1 dpi and 28 dpi, the urinary bacterial loads were positively associated with frequency and polyuria. Bladder and kidney bacterial loads at 28 dpi were positively with frequency and polyuria. CONCLUSIONS: Urine and tissue bacterial loads were associated with changes of voiding behavior at both 1 and 28 dpi.

Transurethral resection of prostatic abscess.

Prostate abscess (PA) is an uncommon prostatic infection, with risk factors including indwelling catheters, acute or chronic prostatitis, bladder outlet obstruction, voiding dysfunction, recent urologic instrumentation (especially transrectal prostate biopsy), chronic kidney disease (CKD), diabetes mellitus (DM), human immunodeficiency virus (HIV), intravenous drug use (IVDU), and hepatitis C. Treatment of PA consists of antibiotics and abscess drainage via transurethral resection (TUR) or image-guided transrectal or transperineal drainage. Numerous studies have demonstrated that TUR of PA has a higher success rate and shorter hospital length of stay when compared to image-guided drainage. Despite this, TUR of PA is a relatively uncommon surgery with few useful recommendations on how to best perform this procedure. We demonstrate the TUR surgical technique for drainage of a 6 cm loculated PA in a 44-year-old man with active IVDU and hepatitis C. The patient presented with progressive voiding symptoms, urinary retention, and leukocytosis. Given the size, loculated nature of the abscess, and its proximity to the prostatic urethra, we decided to proceed to the operating room for surgical drainage as opposed to image-guided transrectal drainage. Herein we describe the trans urethral technique. He clinically improved postoperatively and repeat imaging 4 days later showed decreased abscess size. Transurethral drainage of a PA is a safe, efficient, and effective treatment option. Treatment approach should depend on abscess size, location, and presence of loculations. Combining different endourologic techniques and instruments may be necessary.

Single-cell transcriptomes of mouse bladder urothelium uncover novel cell type markers and urothelial differentiation characteristics.

OBJECTIVES: Much of the information to date in terms of subtypes and function of bladder urothelial cells were derived from anatomical location or by the expression of a small number of marker genes. To have a comprehensive map of the cellular anatomy of bladder urothelial cells, we performed single-cell RNA sequencing to thoroughly characterize mouse bladder urothelium. MATERIALS AND METHODS: A total of 18,917 single cells from mouse bladder urothelium were analysed by unbiased single-cell RNA sequencing. The expression of the novel cell marker was confirmed by immunofluorescence using urinary tract infection models. RESULTS: Unsupervised clustering analysis identified 8 transcriptionally distinct cell subpopulations from mouse bladder urothelial cells. We discovered a novel type of bladder urothelial cells marked by Plxna4 that may be involved with host response and wound healing. We also found a group of basal-like cells labelled by ASPM that could be the progenitor cells of adult bladder urothelium. ASPM+ urothelial cells are significantly increased after injury by UPEC. In addition, specific transcription factors were found to be associated with urothelial cell differentiation. At the last, a number of interstitial cystitis/bladder pain syndrome-regulating genes were found differentially expressed among different urothelial cell subpopulations. CONCLUSIONS: Our study provides a comprehensive characterization of bladder urothelial cells, which is fundamental to understanding the biology of bladder urothelium and associated bladder disease.

Early Increased Urinary IL-2 and IL-10 Levels Were Associated With Development of Chronic UTI in a Murine Model.

OBJECTIVES: To analyze factors during early stage of urinary tract infection (UTI) that are associated with development of chronic UTI. METHODS: Mice were inoculated with Uropathogenic Escherichia coli (UPEC) 2 times 24 hours apart. At 1, 3, 7, 10, 14, 21 and 28 days post infection (dpi), urine bacterial loads and voiding behavior (voiding spot assay, VSA) were measured. At 1 and 28 dpi, 32 urine inflammatory cytokines/chemokines were measured using enzyme-linked immunosorbent assay (ELISA). Bladder and kidney cytokines/chemokines were measured on 28 dpi. Mice that had no more than 1 episode of urine bacterial load < 104 colony forming unit/ml during the entire 4 weeks were defined as susceptible to chronic UTI, otherwise, mice were considered resistant. RESULTS: At 28 dpi, 64.3% mice developed chronic UTI (susceptible group) and 35.7% mice did not (resistant group). Factors at 1 dpi that were predictive of chronic UTI included increased urine IL-2 (OR 11.9, 95%CI 1.1-130.8, P = .043) and increased urine IL-10 (OR 14.0, 95%CI 1.0-201.2, P = .052). At 28 dpi, there were several significant differences between the susceptible vs resistant groups including urine/tissue bacterial loads and certain urine/tissue cytokines/chemokines. CONCLUSIONS: Higher urine IL-2 and IL-10 at 1 dpi predicted chronic UTI infection in this model. There have been recent publications associating both of these cytokines to UTI susceptibility. Further explorations into IL-2 and IL-10 mediated pathways could shed light on the biology of recurrent and chronic UTI which are difficult to treat.

Evaluating the safety and potential activity of URO-902 (hMaxi-K) gene transfer by intravesical instillation or direct injection into the bladder wall in female participants with idiopathic (non-neurogenic) overactive bladder syndrome and detrusor overactivity from two double-blind, imbalanced, placebo-controlled randomized phase 1 trials.

AIMS: Two phase 1 trials were performed in healthy women with the overactive bladder (OAB) syndrome and urodynamically demonstrated detrusor overactivity (DO), with the aim to demonstrate the safety and potential efficacy of URO-902, which comprises a gene therapy plasmid vector expressing the human big potassium channel α subunit. METHODS: ION-02 (intravesical instillation) and ION-03 (direct injection) were double-blind, placebo-controlled, multicenter studies without overlap in enrollment between studies. Active doses were administered and evaluated sequentially (lowest dose first) for safety. ION-02 participants received either 5000 µg or 10 000 µg URO-902, or placebo. ION-03 participants received either 16 000 or 24 000 µg URO-902, or placebo, injected directly into the bladder wall using cystoscopy. Primary outcome variables were safety parameters occurring subsequent to URO-902 administration; secondary efficacy variables also were evaluated. RESULTS: Among the safety outcomes, there were no dose-limiting toxicities or significant adverse events (AEs) preventing dose escalation during either trial, and no participants withdrew due to AEs. For efficacy, in ION-02 (N = 21), involuntary detrusor contractions on urodynamics at 24 weeks in patients receiving URO-902 (P < .0508 vs placebo) and mean urgency incontinence episodes in the 5000 µg group (P = .0812 vs placebo) each showed a downward trend. In ION-03 (N = 13), significant reduction versus placebo in urgency episodes (16 000 µg, P = .036; 24 000 µg, P = .046) and number of voids (16 000 µg, -2.16, P = .044; 24 000 µg, -2.73, P = .047) were observed 1 week after injection. CONCLUSION: Promising safety and efficacy results in these preliminary phase 1 studies suggest gene transfer may be a promising therapy for OAB/DO, warranting further investigation.

Pyroptosis engagement and bladder urothelial cell-derived exosomes recruit mast cells and induce barrier dysfunction of bladder urothelium after uropathogenic E. coli infection.

The specific regulatory mechanism of bladder urothelial barrier dysfunction after infection with uropathogenic Escherichia coli (UPEC) is still unclear. The cross talk between bladder urothelial cells and mast cells may play an important role during UPEC infection. In this study, the pyroptosis of urothelial cells was investigated after UPEC infection both in vivo and in vitro. The levels of IL-1ß and IL-18 in exosomes derived from bladder urothelial cells after UPEC infection were detected. The role of these processes in the recruitment and activation of mast cells was measured. The mechanism of mast cell-induced disruption of bladder epithelial barrier function was also assessed. We found that UPEC infection induced pyroptosis of bladder urothelial cells and led to the release of IL-1ß and IL-18 in the form of exosomes, which promoted the migration of mast cells. Tryptase secreted by mast cells aggravated the damage to the barrier function of the bladder urothelium by acting on protease-activated receptor 2 (PAR2). Inhibition of pyroptosis or the tryptase-PAR2 axis reduced the disruption of bladder urothelial barrier function and decreased the bacterial burden. The present study supports a novel mechanism by which pyroptosis-dependent release of exosomes from bladder urothelial cells activates mast cells and regulates bladder urothelial barrier function during UPEC infection.

Recurrent Uncomplicated Urinary Tract Infections in Women: AUA/CUA/SUFU Guideline.

PURPOSE: This document seeks to establish guidance for the evaluation and management of women with recurrent urinary tract infections (rUTI) to prevent inappropriate use of antibiotics, decrease the risk of antibiotic resistance, reduce adverse effects of antibiotic use, provide guidance on antibiotic and non-antibiotic strategies for prevention, and improve clinical outcomes and quality of life by reducing recurrence of urinary tract infection (UTI) events. MATERIALS AND METHODS: The systematic review utilized to inform this guideline was conducted by a methodology team at the Pacific Northwest Evidence-based Practice Center. A research librarian conducted searches in Ovid MEDLINE (1946 to January Week 1 2018), Cochrane Central Register of Controlled Trials (through December 2017) and Embase (through January 16, 2018). An update literature search was conducted on September 20, 2018. RESULTS: When sufficient evidence existed, the body of evidence was assigned a strength rating of A (high), B (moderate), or C (low). Such evidence-based statements are provided as Strong, Moderate, or Conditional Recommendations. In instances of insufficient evidence, additional guidance is provided as Clinical Principles and Expert Opinions. CONCLUSIONS: Our ability to diagnose, treat, and manage rUTI long-term has evolved due to additional insights into the pathophysiology of rUTI, a new appreciation for the adverse effects of repetitive antimicrobial therapy, rising rates of bacterial antimicrobial resistance (AMR), and better reporting of the natural history and clinical outcomes of acute cystitis and rUTI. As new data continue to emerge in this space, this document will undergo review to ensure continued accuracy.

A non-enzymatic method for dissection of mouse bladder urothelial tissue.

Urothelial cells contribute to bladder functions, including urine storage, urine emptying, and innate immune response. Functional studies of urothelial cells usually use either freshly isolated cells or cultured cells. Most methods of isolating urothelial cells require enzymes; however, these techniques remove proteins that connect the cells and disrupt the orientation of the cells within the multilayered urothelium. In addition, PCR or immunoblot results obtained from homogenates of bladder mucosa or whole bladder do not represent pure urothelial cells. We describe a dissection process that does not require enzymes and is able to obtain pure urothelial tissues from mice and humans. This method can isolate single urothelial cells for electrophysiology in situ and can also isolate pure urothelial tissue for PCR, microarray, and immunoblot procedures. The time required to obtain urothelial tissue from one mouse bladder is 15-20 min. This method is simple and time efficient as compared with alternative methods and therefore facilitates our understanding of urothelial biology.

Bladder urothelial BK channel activity is a critical mediator for innate immune response in urinary tract infection pathogenesis.

The open probability of calcium-activated voltage-gated potassium channel (BK channel) on bladder umbrella urothelial cells is increased by lipopolysaccharide (LPS). It is hypothesized that this channel's activity is important in the urothelial innate immune response during urinary tract infection (UTI). We performed in vivo studies using female C57BL/6 mice whose bladders were inoculated with LPS (150 µl of 1 mg/ml) or uropathogenic Escherichia coli (UPEC, UTI89), without and with intravesical BK inhibitor iberiotoxin (IBTX, 1 µM). Inflammatory biomarkers (chemokines and cytokines) were measured in urine specimens collected 2 h after inoculation using a 32-multiplex ELISA. Of these 32 biomarkers, 19 and 15 were significantly elevated 2 h after LPS and UPEC exposure, respectively. IBTX significantly abrogated the elevations of 15 out of 19 biomarkers after LPS inoculation and 12 out of 15 biomarkers after UPEC inoculation. In a separate experiment, qPCR for IL-6, interferon-γ-induced protein 10 (CXCL10), and macrophage inflammatory protein 2 (CXCL2) in urothelium paralleled the changes measured in urine of these same biomarkers, supporting that urinary changes in biomarker levels reflected urothelial expression changes. These in vivo data demonstrated that BK channel activity is crucial in the urothelial host innate immune response, as measured by changes in urinary biomarkers, in UTI pathogenesis.

The Bladder is Not Sterile: an Update on the Urinary Microbiome.

PURPOSE OF REVIEW: The article discusses (1) techniques used to study bacterial urinary microbiota; (2) existence of non-bacterial urinary microbiota; (3) associations between changes in urinary microbiota and various benign lower urinary tract disorders. RECENT FINDINGS: Urine harbors a diverse microbial community that resides within it. A multitude of studies have identified differences in these communities associated with urologic conditions, suggesting that microbial communities may maintain normal bladder homeostasis. Technological advances in analytic approaches have improved our understanding of the urinary microbiome. The choice of urine sampling method (voided, catheterized, or aspirated) will significantly influence microbiome findings. Sex and age highly influence urinary microbiota; in addition to rigorous inclusion criteria, microbial studies must be sufficiently powered to overcome the substantial interindividual variability of urinary microbiota. Regardless of these complicating factors, studies have identified microbial patterns correlating with both urologic diagnoses and treatment responses. SUMMARY: Without a clear understanding of the variability of and exogenous influences on the urinary microbiota in the absence of disease, it has been challenging to reveal the microbial patterns responsible for disease pathophysiology. Host mechanisms in response to the urinary microbiome are also poorly understood. Additional research can address whether the manipulation of urinary microbiota will benefit lower urinary tract health.

Mouse urothelial genes associated with voiding behavior changes after ovariectomy and bladder lipopolysaccharide exposure.

AIMS: Symptoms from overactive bladder (OAB) and cystitis secondary to urinary tract infection (UTI) can be similar in post-menopausal women. Effects of ovariectomy (OVX) on voiding behavior after lipopolysaccharide (LPS) intravesical exposure (surrogate for cystitis) in mice were measured. Urothelial genes associated with micturition changes were identified. METHODS: Female C57BL6/J mice underwent OVX or sham surgeries (n = 10 for each). Voiding spot assays (VSA) were performed prior to surgery, 4 weeks post-surgery, and each time after 3 consecutive days of transurethral instillation of LPS. In another experiment, mice underwent either sham (n = 9) or OVX (n = 9) surgeries. Urothelial RNAs were collected 4 weeks post-surgery, day 1 and day 3 after LPS instillation. Mouse Gene 2.0 ST Arrays (entire 34 K transcripts) were used for microarray hybridization. A set of criteria was utilized to identify gene expression changes that mimicked voiding behavior changes. RESULTS: Three days after LPS exposure, OVX mice persisted with overactive whereas sham mice normalized voiding behavior. Nine urothelial paralleling voiding behavior changes were identified: IL6 (interleukin 6), IL6rα (Interleukin 6 receptor α), Ptgs2 (Prostaglandin-endoperoxide synthase 2 or COX-2), Ereg (epiregulin), Dusp6 (dual specificity phosphatase 6), Zfp948 (zinc finger protein 948), Zfp52 (Zinc finger protein 52), Gch1 (GTP cyclohydrolase 1), and Amd (S-adenosylmethionine decarboxylase). Three other genes, coding unknown proteins, were also identified: GM12840, GM23134, and GM26809. CONCLUSIONS: OVX mice persisted with increased voiding frequency after LPS. Urothelial genes that could mediate this voiding behavior include IL6, COX-2, and S-adenosylmethionine decarboxylase.

Lipopolysaccharide stimulates BK channel activity in bladder umbrella cells.

Bladder urothelium plays an active role in response to bacterial infection. There is little known about the electrophysiological activity in urothelial cells in this process. We used a nonenzymatic method to isolate bladder urothelial tissue and to patch clamp umbrella cells in situ. A 200 pS conductance potassium (K+) channel was detected from female C57BL6 mice. Of 58 total patches, 17.2% patches displayed the 200 pS K+ conductance channel. This K+ conductance channel showed Ca2+ sensitivity and voltage dependence. Specific big-conductance potassium channel (BK) inhibitors (paxilline, iberiotoxin) blocked the 200 pS K+ conductance channel activity. RT-PCR and immunoblot confirmed BK channel pore-forming α-subunit (BK-α) mRNA and protein in urothelium. Immunohistochemistry also showed the BK-α located in urothelium. The above data provided evidence that the 200 pS K+ conductance channel was a BK channel. Lipopolysaccharide (LPS), a component of uropathogenic Escherichia coli, was used to investigate the role of BK channel in the pathogenesis of urinary tract infection. BK channel activity as NPo increased threefold within 30 min of exposure to LPS. mRNAs for LPS receptors (TLR4, CD14, MD-2) were expressed in the urothelium but not in lamina propria or detrusor. Blockade of the receptors by an antagonist (polymyxin B) abrogated LPS's effect on BK channel. The involvement of protein kinase A (PKA) on BK channel activity was demonstrated by applying PKA blockers (H89 and PKI). Both PKA inhibitors abolished the BK channel activity induced by LPS. In conclusion, BK channel was identified in bladder umbrella cells, and its activity was significantly increased by LPS.

New therapeutic directions to treat underactive bladder.

Underactive bladder (UAB) is a term used to describe a constellation of symptoms that is perceived by patients suggesting bladder hypocontractility. Urodynamic measurement that suggest decreased contractility of the bladder is termed detrusor underactivity (DUA). Regulatory approved specific management options with clinically proven ability to increase bladder contractility do not currently exist. While DUA specific treatments presumably will focus on methods to increase efficiency of bladder emptying capability relying on augmenting the motor pathway in the micturition reflex, other approaches include methods to augment the sensory (afferent) contribution to the micturition reflex which could result in increased detrusor contractility. Another method to induce more efficient bladder emptying could be to induce relaxation of the bladder outlet. Using cellular regenerative techniques, the detrusor smooth muscle can be targeted so the result is to increase detrusor smooth muscle function. In this review, we will cover areas of potential new therapies for DUA including: drug therapy, stem cells and regenerative therapies, neuromodulation, and urethral flow assist device. Paralleling development of new therapies, there also needs to be clinical studies performed that address how DUA relates to UAB.

Premature contractions of the bladder are suppressed by interactions between TRPV4 and SK3 channels in murine detrusor PDGFRα+ cells.

During filling, urinary bladder volume increases dramatically with little change in pressure. This is accomplished by suppressing contractions of the detrusor muscle that lines the bladder wall. Mechanisms responsible for regulating detrusor contraction during filling are poorly understood. Here we describe a novel pathway to stabilize detrusor excitability involving platelet-derived growth factor receptor-α positive (PDGFRα+) interstitial cells. PDGFRα+ cells express small conductance Ca2+-activated K+ (SK) and TRPV4 channels. We found that Ca2+ entry through mechanosensitive TRPV4 channels during bladder filling stabilizes detrusor excitability. GSK1016790A (GSK), a TRPV4 channel agonist, activated a non-selective cation conductance that coupled to activation of SK channels. GSK induced hyperpolarization of PDGFRα+ cells and decreased detrusor contractions. Contractions were also inhibited by activation of SK channels. Blockers of TRPV4 or SK channels inhibited currents activated by GSK and increased detrusor contractions. TRPV4 and SK channel blockers also increased contractions of intact bladders during filling. Similar enhancement of contractions occurred in bladders of Trpv4 -/- mice during filling. An SK channel activator (SKA-31) decreased contractions during filling, and rescued the overactivity of Trpv4 -/- bladders. Our findings demonstrate how Ca2+ influx through TRPV4 channels can activate SK channels in PDGFRα+ cells and prevent bladder overactivity during filling.