A prospective evaluation of the prostate microbiome in malignant and benign tissue using transperineal biopsy.

BACKGROUND: The link between the prostate microbiome and prostate cancer remains unclear. Few studies have analyzed the microbiota of prostate tissue, and these have been limited by potential contamination by transrectal biopsy. Transperineal prostate biopsy offers an alternative and avoids fecal cross-contamination. We aim to characterize the prostate microbiome using transperineal biopsy. METHODS: Patients with clinical suspicion for prostate cancer who were to undergo transperineal prostate biopsy with magnetic resonance imaging (MRI) fusion guidance were prospectively enrolled from 2022 to 2023. Patients were excluded if they had Prostate Imaging Reporting and Data System lesions with scores ≤ 3, a history of prostate biopsy within 1 year, a history of prostate cancer, or antibiotic use within 30 days of biopsy. Tissue was collected from the MRI target lesions and nonneoplastic transitional zone. Bacteria were identified using 16S ribosomal RNA gene sequencing. RESULTS: Across the 42 patients, 76% were found to have prostate cancer. Beta diversity indices differed significantly between the perineum, voided urine, and prostate tissue. There were no beta diversity differences between cancerous or benign tissue, or between pre- and postbiopsy urines. There appear to be unique genera more abundant in cancerous versus benign tissue. There were no differences in alpha diversity indices relative to clinical findings including cancer status, grade, and risk group. CONCLUSIONS: We demonstrate a rigorous method to better characterize the prostate microbiome using transperineal biopsy and to limit contamination. These findings provide a framework for future large-scale studies of the microbiome of prostate cancer.

Sex-dependent effects of carbohydrate source and quantity on caspase-1 activity in the mouse central nervous system.

BACKGROUND: Mounting evidence links glucose intolerance and diabetes as aspects of metabolic dysregulation that are associated with an increased risk of developing dementia. Inflammation and inflammasome activation have emerged as a potential link between these disparate pathologies. As diet is a key factor in both the development of metabolic disorders and inflammation, we hypothesize that long term changes in dietary factors can influence nervous system function by regulating inflammasome activity and that this phenotype would be sex-dependent, as sex hormones are known to regulate metabolism and immune processes. METHODS: 5-week-old male and female transgenic mice expressing a caspase-1 bioluminescent reporter underwent cranial window surgeries and were fed control (65% complex carbohydrates, 15% fat), high glycemic index (65% carbohydrates from sucrose, 15% fat), or ketogenic (1% complex carbohydrates, 79% fat) diet from 6 to 26 weeks of age. Glucose regulation was assessed with a glucose tolerance test following a 4-h morning fast. Bioluminescence in the brain was quantified using IVIS in vivo imaging. Blood cytokine levels were measured using cytokine bead array. 16S ribosomal RNA gene amplicon sequencing of mouse feces was performed to assess alterations in the gut microbiome. Behavior associated with these dietary changes was also evaluated. RESULTS: The ketogenic diet caused weight gain and glucose intolerance in both male and female mice. In male mice, the high glycemic diet led to increased caspase-1 biosensor activation over the course of the study, while in females the ketogenic diet drove an increase in biosensor activation compared to their respective controls. These changes correlated with an increase in inflammatory cytokines present in the serum of test mice and the emergence of anxiety-like behavior. The microbiome composition differed significantly between diets; however no significant link between diet, glucose tolerance, or caspase-1 signal was established. CONCLUSIONS: Our findings suggest that diet composition, specifically the source and quantity of carbohydrates, has sex-specific effects on inflammasome activation in the central nervous system and behavior. This phenotype manifested as increased anxiety in male mice, and future studies are needed to determine if this phenotype is linked to alterations in microbiome composition.

Microbiologist in the Clinic: Postmenopausal Woman with Chronic OAB and Positive Urine Culture.

In this second episode of the Microbiologist in the Clinic series, clinicians and laboratory scientists share their perspectives about a 75-year-old woman who was diagnosed with asymptomatic bacteriuria based on positive urine cultures. The patient and her GP are concerned about this laboratory finding as the patient will become immunosuppressed with planned chemotherapy. The patient has had an overactive bladder (OAB) for approximately 20 years, with good control of her urinary urgency and frequency (no incontinence) with a stable dose of OAB medication. The challenges of this clinical presentation are discussed, with evidence for evaluation and treatment.

Comparative genomic analysis of clinical Enterococcus faecalis distinguishes strains isolated from the bladder.

BACKGROUND: Enterococcus faecalis is the most commonly isolated enterococcal species in clinical infection. This bacterium is notorious for its ability to share genetic content within and outside of its species. With this increased proficiency for horizontal gene transfer, tremendous genomic diversity within this species has been identified. Many researchers have hypothesized E. faecalis exhibits niche adaptation to establish infections or colonize various parts of the human body. Here, we hypothesize that E. faecalis strains isolated from the human bladder will carry unique genomic content compared to clinical strains isolated from other sources. RESULTS: This analysis includes comparison of 111 E. faecalis genomes isolated from bladder, urogenital, blood, and fecal samples. Phylogenomic comparison shows no association between isolation source and lineage; however, accessory genome comparison differentiates blood and bladder genomes. Further gene enrichment analysis identifies gene functions, virulence factors, antibiotic resistance genes, and plasmid-associated genes that are enriched or rare in bladder genomes compared to urogenital, blood, and fecal genomes. Using these findings as training data and 682 publicly available genomes as test data, machine learning classifiers successfully distinguished between bladder and non-bladder strains with high accuracy. Genes identified as important for this differentiation were often related to transposable elements and phage, including 3 prophage species found almost exclusively in bladder and urogenital genomes. CONCLUSIONS: E. faecalis strains isolated from the bladder contain unique genomic content when compared to strains isolated from other body sites. This genomic diversity is most likely due to horizontal gene transfer, as evidenced by lack of phylogenomic clustering and enrichment of transposable elements and prophages. Investigation into how these enriched genes influence host-microbe interactions may elucidate gene functions required for successful bladder colonization and disease establishment.

Detection of Bacteria in Bladder Mucosa of Adult Females.

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.

Taxonomic considerations on Aerococcus urinae with proposal of subdivision into Aerococcus urinae, Aerococcus tenax sp. nov., Aerococcus mictus sp. nov., and Aerococcus loyolae sp. nov.

Average nucleotide identity analysis, based on whole genome sequences of 115 strains previously identified as Aerococcus urinae, an emerging uropathogen, discriminates at least six unique genomic taxa. The whole genome analysis affords clearer species boundaries over 16S rRNA gene sequencing and traditional phenotypic approaches for the identification and phylogenetic organization of Aerococcus species. The newly described species can be differentiated by matrix-assisted laser desorption ionization time-of-flight analysis of protein signatures. We propose the emendation of the description of A. urinae (type strain ATCC 51268T = CCUG 34223T=NCFB 2893) and the names of Aerococcus tenax sp. nov. (ATCC TSD-302T = DSM 115700T = CCUG 76531T=NR-58630T), Aerococcus mictus sp. nov. (ATCC TSD-301T = DSM 115699T = CCUG 76532T=NR-58629T), and Aerococcus loyolae sp. nov. (ATCC TSD-300T = DSM 115698T = CCUG 76533T=NR-58628T) for three of the newly identified genomic taxa.

Urobiome changes differ based on OAB treatment in adult females.

INTRODUCTION AND HYPOTHESIS: Representatives of two classes of oral medication are often used to treat urgency urinary incontinence (UUI): solifenacin, an M3-receptor-selective antimuscarinic, and mirabegron, a beta-3 agonist. Two previous asynchronous drug-specific studies suggested different interactions between these medications and the urobiome despite identical methodologies, including recruitment, sample procurement, medication dose escalation strategy, determination of 12-week responders versus nonresponders, and data collection. This analysis compares data from these two studies using a uniform analytic approach. METHODS: Urine was collected aseptically via transurethral catheter from consenting participants for subsequent processing by the Expanded Quantitative Urine Culture (EQUC) protocol in two cohorts (n=50 and n=47) that were demographically similar. Species accumulation curves were generated to compare the total number of unique species detected. Indices that measure richness, evenness, and/or abundance were used to compare alpha (within sample) diversity. The Bray-Curtis Dissimilarity Index was used to determine between sample (beta) diversity. RESULTS: The majority of the 40 species detected in the pre-treatment urobiomes were detected in both cohorts. Both pre-treatment urobiomes were substantially similar in species richness, evenness, and diversity. Differences in pre-treatment urobiomes were associated with treatment response for solifenacin-treated participants only. In contrast, the pre-treatment urobiomes of mirabegron-treated participants were not associated with treatment response. Changes in the post-treatment urobiomes were detected in both cohorts with an increase in richness for both solifenacin (5-mg dose only) and mirabegron. CONCLUSIONS: Pre-treatment urobiome characteristics were associated with treatment response in participants treated with solifenacin, but not mirabegron. Differences exist in urobiome response after treatment with two medications that have known differences in mechanism of action.

Microbiologist in the clinic: coitally related symptoms with negative urine cultures.

In this first episode of the Microbiologist in the Clinic series, clinicians and laboratory scientists share their perspectives about a 30 y/o woman, who is seeking specialty consultation for frequent episodes of urinary urgency, frequency, and dysuria, which respond to short courses of antibiotics. Although her home dipsticks suggest that she has a UTI, and her urinalysis typically has a moderate number of white blood cells, her urine cultures are always negative. The challenges of this clinical presentation are discussed with evidence for evaluation and treatment.

Symptom improvement with mirabegron treatment is associated with urobiome changes in adult women.

INTRODUCTION AND HYPOTHESIS: Mirabegron, a beta-3 agonist, is prescribed for urgency urinary incontinence (UUI). We assessed the correlation of symptom improvement with urobiome characteristics in adult women participants prescribed mirabegron for UUI treatment. METHODS: We enrolled participants seeking UUI treatment who selected mirabegron and agreed to participate in this 12-week, open label study conducted at the Female Pelvic Medicine and Reconstructive Surgery Center at Loyola University Medical Center. Following eligibility screening and research consent, participants completed the overactive bladder questionnaire (OAB-Q) and provided a catheterized urine sample at baseline, 4, 8, and 12 weeks. The primary outcome, symptom improvement at 12 weeks, was based on the validated Patient Global Symptom Control questionnaire score to dichotomize symptom response (responder vs nonresponder [PGSC score ≤3]). Urine samples were processed by the Expanded Quantitative Urine Culture (EQUC) protocol. RESULTS: Eighty-three participants (mean age 68 years) completed baseline assessment. Of the 47 participants with primary outcome data and samples analysis, there were 16 responders and 31 nonresponders; responder groups were similar demographically. Living microbes were detected in most participants. There were no significant differences in alpha diversity (within sample) at baseline between groups. However, at the 12-week follow-up, the responder urobiome became significantly richer, with a larger number of genera (p = 0.027) and was significantly more diverse than the nonresponders. CONCLUSIONS: Longitudinal urobiome changes are associated with symptom improvement in adult women being treated with mirabegron for UUI. The mechanism for symptoms improvement may relate to the detected changes in the urobiome and warrants further study.

Urinary microbiota of women with recurrent urinary tract infection: collection and culture methods.

INTRODUCTION AND HYPOTHESIS: Many clinicians utilize standard culture of voided urine to guide treatment for women with recurrent urinary tract infections (RUTI). However, despite antibiotic treatment, symptoms may persist and events frequently recur. The cyclic nature and ineffective treatment of RUTI suggest that underlying uropathogens pass undetected because of the preferential growth of Escherichia coli. Expanded quantitative urine culture (EQUC) detects more clinically relevant microbes. The objective of this study was to assess how urine collection and culture methods influence microbial detection in RUTI patients. METHODS: This cross-sectional study enrolled symptomatic adult women with an established RUTI diagnosis. Participants contributed both midstream voided and catheterized urine specimens for culture via both standard urine culture (SUC) and EQUC. Presence and abundance of microbiota were compared between culture and collection methods. RESULTS: Forty-three symptomatic women participants (mean age 67 years) contributed specimens. Compared to SUC, EQUC detected more unique bacterial species and consistently detected more uropathogens from catheterized and voided urine specimens. For both collection methods, the most commonly detected uropathogens by EQUC were E. coli (catheterized: n = 8, voided: n = 12) and E. faecalis (catheterized: n = 7, voided: n = 17). Compared to catheterized urine samples assessed by EQUC, SUC often missed uropathogens, and culture of voided urines by either method yielded high false-positive rates. CONCLUSIONS: In women with symptomatic RUTI, SUC and assessment of voided urines have clinically relevant limitations in uropathogen detection. These results suggest that, in this population, catheterized specimens analyzed via EQUC provide clinically relevant information for appropriate diagnosis.

Phenyl-Lactic Acid Is an Active Ingredient in Bactericidal Supernatants of Lactobacillus crispatus.

Lactobacillus crispatus is a well-established probiotic with antimicrobial activity against pathogens across several niches of the human body generally attributed to the production of bacteriostatic molecules, including hydrogen peroxide and lactic acid. Here, we show that the cell-free supernatants of clinical isolates of L. crispatus harbor robust bactericidal activity. We further identify phenyl-lactic acid as a bactericidal compound with properties and a susceptibility range nearly identical to that of the cell-free supernatant. As such, we hypothesize that phenyl-lactic acid is a key active ingredient in L. crispatus supernatant. IMPORTANCE Although Lactobacillus crispatus is an established commensal microbe frequently used in probiotics, its protective role in the bladder microbiome has not been clarified. We report here that some urinary isolates of L. crispatus exhibit bactericidal activity, primarily due to its ability to excrete phenyl-lactic acid into its environment. Both cell-free supernatants of L. crispatus isolates and phenyl-lactic acid exhibit bactericidal activity against a wide range of pathogens, including several that are resistant to multiple antibiotics.

A Randomized Clinical Trial of Standard versus Expanded Cultures to Diagnose Urinary Tract Infections in Women.

PURPOSE: We compared urinary tract infection (UTI) symptom resolution rates at 7-10 days in symptomatic women randomized to treatment based on standard urine culture (SUC) versus expanded quantitative urine culture (EQUC) results. MATERIALS AND METHODS: Women ≥18 years old who responded "yes" to "do you feel you have a UTI?" agreed to urethral catheterization and followup. Symptoms were assessed using the validated UTI Symptom Assessment (UTISA) questionnaire. Culture method was randomized 2:1 (SUC:EQUC); antibiotics were prescribed to women with positive cultures. The primary outcome, UTI symptom resolution, was determined 7-10 days following enrollment on all participants regardless of treatment. RESULTS: Demographic data were similar between groups. Of the SUC and EQUC groups 63% and 74% had positive cultures (p=0.10), respectively. Of participants with positive cultures 97% received antibiotics. Primary outcome data were provided by 215 of 225 participants (SUC 143 [95%], EQUC 72 [97%]). At the primary outcome assessment, 64% and 69% in the SUC and EQUC groups, respectively, reported UTI symptom resolution (p=0.46); UTISA scores improved from baseline in the EQUC arm compared to the SUC arm (p=0.04). In the subset of women predominated by non-Escherichia coli (76), there was a trend toward more symptom resolution in the EQUC arm (21%, p=0.08). CONCLUSIONS: Symptom resolution was similar for the overall population (E. coli and non-E. coli) of women treated for UTI symptoms based on SUC or EQUC. Although the sample size limits conclusions regarding the utility of EQUC in women with non-E. coli uropathogens, the detected trend indicates that this understudied clinical subset warrants further study.

Recurrent urinary tract infection: Association of clinical profiles with urobiome composition in women.

AIMS: Clinical profiles of women with recurrent urinary tract infection (RUTI) are correlated with their urinary microbes. METHODS: This IRB-approved, cross-sectional study enrolled adult women with RUTI. Urine samples (catheterized and voided) underwent culture by expanded quantitative urine culture (EQUC) and standard urine culture (SUC) methods. A validated symptom questionnaire, relevant clinical variables, and EQUC were used to identify symptom clusters and detect associations with specific urinary microbes. RESULTS: Most (36/43) participants were postmenopausal; the average age was 67 years. 51% reported vaginal estrogen use; 51% reported sexual activity. Although single symptoms were not associated with specific urinary microbes, EQUC results were correlated with five distinct clinical profile clusters: Group A: odor, cloudiness, and current vaginal estrogen use (no culture result association). Group B: frequency, low back pain, incomplete emptying, and vaginal estrogen (significantly increased proportion of Lactobacillus-positive cultures). Group C: pain/burning, odor, cloudiness, and urgency (high proportions of UTI-associated microbe-positive cultures). Group D: frequency, urgency, pain/burning, and current vaginal estrogen use (increased number of no growth cultures). Group E: frequency, urgency, pain/burning, odor, overactive bladder, and sexually active (significantly increased proportion of Klebsiella-positive cultures). CONCLUSIONS: Distinct clinical profiles are associated with specific urinary microbes in women with RUTI. Refined assessments of clinical profiles may provide useful insights that could inform diagnostic and therapeutic considerations.

Aerococcus urinae Isolated from Women with Lower Urinary Tract Symptoms: In Vitro Aggregation and Genome Analysis.

Aerococcus urinae is increasingly recognized as a potentially significant urinary tract bacterium. A. urinae has been isolated from urine collected from both males and females with a wide range of clinical conditions, including urinary tract infection (UTI), urgency urinary incontinence (UUI), and overactive bladder (OAB). A. urinae is of particular clinical concern because it is highly resistant to many antibiotics and, when undiagnosed, can cause invasive and life-threatening bacteremia, sepsis, or soft tissue infections. Previous genomic characterization studies have examined A. urinae strains isolated from patients experiencing UTI episodes. Here, we analyzed the genomes of A. urinae strains isolated as part of the urinary microbiome from patients with UUI or OAB. Furthermore, we report that certain A. urinae strains exhibit aggregative in vitro phenotypes, including flocking, which can be modified by various growth medium conditions. Finally, we performed in-depth genomic comparisons to identify pathways that distinguish flocking and nonflocking strains.IMPORTANCEAerococcus urinae is a urinary bacterium of emerging clinical interest. Here, we explored the ability of 24 strains of A. urinae isolated from women with lower urinary tract symptoms to display aggregation phenotypes in vitro We sequenced and analyzed the genomes of these A. urinae strains. We performed functional genomic analyses to determine whether the in vitro hyperflocking aggregation phenotype displayed by certain A. urinae strains was related to the presence or absence of certain pathways. Our findings demonstrate that A. urinae strains have different propensities to display aggregative properties in vitro and suggest a potential association between phylogeny and flocking.

Characterization and spontaneous induction of urinary tract Streptococcus anginosus prophages.

Streptococcus anginosus is an often overlooked and understudied emerging pathogen inhabiting many areas of the human body. Through our sequencing of S. anginosus strains isolated from the female bladder microbiota, we detected numerous prophage sequences. Bioinformatic analysis of these sequences identified 17 distinct groups of S. anginosus prophages. The majority of these phages exhibit no sequence homology to previously characterized temperate or virulent phage sequences, indicating an unexplored diversity of Streptococcus phages. By culturing these bacterial isolates, we confirmed that the prophages of five of these groups are capable of induction. One of these putative phages was imaged, the first such evidence of an S. anginosus virus-like particle; it exhibits morphological characteristics of siphoviruses.

The Urethral Microbiota: A Missing Link in the Female Urinary Microbiota.

PURPOSE: We examined the urethral microbiota, determined if it differs from the bladder urinary microbiota, and assessed if its composition differs based on patient demographic factors and presence of lower urinary tract symptoms. MATERIALS AND METHODS: Patients presenting to our urogynecology clinic were enrolled in the study. Demographic information and responses to the Pelvic Floor Distress Inventory questionnaire were collected. All participants provided midstream voided urine, periurethral swab, transurethral swab and catheterized urine samples, which were analyzed by Expanded Quantitative Urine Culture and MALDI-TOF mass spectrometry. Bray-Curtis dissimilarity analysis assessed diversity between sample types for each participant. Kruskal-Wallis, chi-square, McNemar, Wilcoxon signed rank and Fisher's exact tests tested for significance. RESULTS: A total of 49 patients participated in the study. Bladder microbiota were dissimilar to urethral, periurethral and voided urine microbiota (p <0.0001). Urethral and periurethral microbiota were similar (p >0.05), but the urethral microbiota were dissimilar to voided urine microbiota (p=0.001) while the periurethral microbiota were not (p >0.05). Women less than 55 years old were more likely to be sexually active, premenopausal and Hispanic compared to women 55 years old or older. Women in the younger cohort had Lactobacillus and Gardnerella cultured from urethral samples more frequently and more abundantly than women in the older cohort. There was no significant association between lower urinary tract symptoms and the frequency or abundance of urethral bacteria species. CONCLUSIONS: Niches of microbiota along the female lower urinary tract may be influenced by age, menopausal status and sexual activity. More research is needed to determine the function and clinical significance of the urethral microbiome.

Vaginal estrogen therapy is associated with increased Lactobacillus in the urine of postmenopausal women with overactive bladder symptoms.

BACKGROUND: Previous work has shown that the vaginal microbiome decreases in Lactobacillus predominance and becomes more diverse after menopause. It has also been shown that estrogen therapy restores Lactobacillus dominance in the vagina and that topical estrogen is associated with overactive bladder symptom improvement. We now know that the bladder contains a unique microbiome and that increased bladder microbiome diversity is associated with overactive bladder. However, there is no understanding of how quickly each pelvic floor microbiome responds to estrogen or if those changes are associated with symptom improvement. OBJECTIVE: This study aimed to determine if estrogen treatment of postmenopausal women with overactive bladder decreases urobiome diversity. STUDY DESIGN: We analyzed data from postmenopausal participants in 2 trials (NCT02524769 and NCT02835846) who chose vaginal estrogen as the primary overactive bladder treatment and used 0.5 g of conjugated estrogen (Premarin cream; Pfizer, New York City, NY) twice weekly for 12 weeks. Baseline and 12-week follow-up data included the Overactive Bladder questionnaire, and participants provided urine samples via catheter, vaginal swabs, perineal swabs, and voided urine samples. Microbes were detected by an enhanced culture protocol. Linear mixed models were used to estimate microbiome changes over time. Urinary antimicrobial peptide activity was assessed by a bacterial growth inhibition assay and correlated with relative abundance of members of the urobiome. RESULTS: In this study, 12 weeks of estrogen treatment resulted in decreased microbial diversity within the vagina (Shannon, P=.047; Richness, P=.043) but not in the other niches. A significant increase in Lactobacillus was detected in the bladder (P=.037) but not in the vagina (P=.33), perineum (P=.56), or voided urine (P=.28). The change in Lactobacillus levels in the bladder was associated with modest changes in urgency incontinence symptoms (P=.02). The relative abundance of the genus Corynebacterium correlated positively with urinary antimicrobial peptide activity after estrogen treatment. CONCLUSION: Estrogen therapy 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 subscale of the Overactive Bladder questionnaire.

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.

Extracellular Acidic pH Inhibits Acetate Consumption by Decreasing Gene Transcription of the Tricarboxylic Acid Cycle and the Glyoxylate Shunt.

Escherichia coli produces acetate during aerobic growth on various carbon sources. After consuming the carbon substrate, E. coli can further grow on the acetate. This phenomenon is known as the acetate switch, where cells transition from producing acetate to consuming it. In this study, we investigated how pH governs the acetate switch. When E. coli was grown on a glucose-supplemented medium initially buffered to pH 7, the cells produced and then consumed the acetate. However, when the initial pH was dropped to 6, the cells still produced acetate but were only able to consume it when little (<10 mM) acetate was produced. When significant acetate was produced in acidic medium, which occurs when the growth medium contains magnesium, amino acids, and sugar, the cells were unable to consume the acetate. To determine the mechanism, we characterized a set of metabolic mutants and found that those defective in the tricarboxylic acid (TCA) cycle or glyoxylate shunt exhibited reduced rates of acetate consumption. We further found that the expression of the genes in these pathways was reduced during growth in acidic medium. The expression of the genes involved in the AckA-Pta pathway, which provides the principal route for both acetate production and consumption, was also inhibited in acidic medium but only after glucose was depleted, which correlates with the acetate consumption phase. On the basis of these results, we conclude that growth in acidic environments inhibits the expression of the acetate catabolism genes, which in turn prevents acetate consumption.IMPORTANCE Many microorganisms produce fermentation products during aerobic growth on sugars. One of the best-known examples is the production of acetate by Escherichia coli during aerobic growth on sugars. In E. coli, acetate production is reversible: once the cells consume the available sugar, they can consume the acetate previously produced during aerobic fermentation. We found that pH affects the reversibility of acetate production. When the cells produce significant acetate during growth in acidic environments, they are unable to consume it. Unconsumed acetate may accumulate in the cell and inhibit the expression of pathways required for acetate catabolism. These findings demonstrate how acetate alters cell metabolism; they also may be useful for the design of aerobic fermentation processes.

Global Lysine Acetylation in Escherichia coli Results from Growth Conditions That Favor Acetate Fermentation.

Lysine acetylation is thought to provide a mechanism for regulating metabolism in diverse bacteria. Indeed, many studies have shown that the majority of enzymes involved in central metabolism are acetylated and that acetylation can alter enzyme activity. However, the details regarding this regulatory mechanism are still unclear, specifically with regard to the signals that induce lysine acetylation. To better understand this global regulatory mechanism, we profiled changes in lysine acetylation during growth of Escherichia coli on the hexose glucose or the pentose xylose at both high and low sugar concentrations using label-free mass spectrometry. The goal was to see whether lysine acetylation differed during growth on these two different sugars. No significant differences, however, were observed. Rather, the initial sugar concentration was the principal factor governing changes in lysine acetylation, with higher sugar concentrations causing more acetylation. These results suggest that acetylation does not target specific metabolic pathways but rather simply targets accessible lysines, which may or may not alter enzyme activity. They further suggest that lysine acetylation principally results from conditions that favor accumulation of acetyl phosphate, the principal acetate donor in E. coliIMPORTANCE Bacteria alter their metabolism in response to nutrient availability, growth conditions, and environmental stresses using a number of different mechanisms. One is lysine acetylation, a posttranslational modification known to target many metabolic enzymes. However, little is known about this regulatory mode. We investigated the factors inducing changes in lysine acetylation by comparing growth on glucose and xylose. We found that the specific sugar used for growth did not alter the pattern of acetylation; rather, the amount of sugar did, with more sugar causing more acetylation. These results imply that lysine acetylation is a global regulatory mechanism that is responsive not to the specific carbon source per se but rather to the accumulation of downstream metabolites.