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Background In women, compelling evidence associates lower urinary tract microbiota (LUTM) with lower urinary tract symptoms (LUTS); a similar association in men with benign prostate enlargement (BPE) is not established. Objective To determine whether associations exist between LUTM and LUTS. Design, setting, and participants Forty-nine male volunteers, aged 40–85 yr, were recruited from one academic tertiary care center. Twenty-eight patients undergoing BPE/LUTS surgery and 21 undergoing non-BPE/LUTS surgery were stratified by International Prostate Symptom Score (IPSS), and paired voided/catheterized urine specimens were collected for expanded quantitative urine culture (EQUC) and 16S ribosomal RNA gene sequencing. Outcome measurements and statistical analysis Primary and secondary outcomes were presence of detectable LUTM and specific bacterial members of the LUTM, respectively. Baseline data were compared. Univariable logistic regression models were used to calculate odds ratios (ORs) for IPSS category associated with the presence of bladder microbiota. Relative LUTM proportions were compared with IPSS using chi-square tests. Results and limitations Thirty-nine percent of catheterized and 98% of voided specimens contained LUTM. Catheterized and voided LUTM differed significantly. LUTM was detected in catheterized urine of 22.2% of men with mild LUTS, 30.0% with moderate LUTS, and 57.1% with severe LUTS (p = 0.024). Increased IPSS category was associated with significantly higher odds of detectable bacteria (OR: 2.21, 95% confidence interval: 1.09–4.49). Small sample size limited this study, making it unable to identify significant differences in specific bacterial taxa based on IPSS. Conclusions Voided urine does not adequately characterize the male bladder microbiome. In males with and without BPE, IPSS severity was associated with detectable bacteria in catheterized urine, which samples the bladder. Additional studies are needed to identify specific bladder bacteria associated with LUTS. Patient summary To study bladder bacteria, urine should be collected with a catheter. Men with severe urinary symptoms are more likely to have detectable bladder bacteria than those with less severe symptoms.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA). JSRV infection is usually detected post-mortem by macroscopic and histological examination of lungs for lesions of OPA. Subsequently, the presence of JSRV may be confirmed using polymerase chain reaction (PCR) on tumour tissue. Our goal was to determine the most effective way of combining macroscopic and histological examination with reverse transcriptase PCR (RT-PCR) to detect JSRV infection post-mortem. Lungs of slaughtered sheep (n = 369) with macroscopic lesions were examined macroscopically and histologically to identify lesions consistent with OPA, and subsequently subjected to RT-PCR for JSRV. Positive (Ppos) and negative (Pneg) agreement and Cohen’s Kappa were calculated between RT-PCR and: 1) macroscopic examination; 2) histological examination; 3) macroscopic and histological examinations combined in series, and 4) in parallel. The highest Ppos was between macroscopic and histological examination in parallel and RT-PCR (0.38). Conversely, Pneg for all combinations of RT-PCR and macroscopic and histological examinations was high (0.95-0.96). All Kappa values were low (0.1-0.33). This indicates that macroscopic and histological examination combined in parallel is the most effective way to identify animals that should be tested using RT-PCR for JSRV. If a positive result is obtained on macroscopic examination and/or histological examination, RT-PCR for JSRV should be carried out. The high Pneg indicates that if a negative result is obtained on macroscopic and histological examination, RT-PCR testing is not merited, as the result is likely to be negative. This provides an evidence-base for the diagnosis of JSRV infection.

In this issue of Cell Host & Microbe, Adebayo et al. (2020) examine the urobiome of older community-dwelling women within the TwinUK Cohort. They define a core genitourinary microbiome for older women with many heritable microbial taxa. Some taxa appear to co-occur, suggesting the existence of specific microbial consortia.

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.

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.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.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.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.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.

The ability of bacteria to adapt to environmental changes has allowed these organisms to thrive in all parts of the globe. By monitoring their extracellular and intracellular environments, bacteria assure their most appropriate response for each environment. Post-translational modification of proteins is one mechanism by which cells respond to their changing environments. Here, we report that two post-translational modifications regulate transcription of the extracytoplasmic stress-responsive promoter cpxP: (i) acetyl phosphate-dependent phosphorylation of the response regulator CpxR and (ii) acetyl coenzyme A-dependent acetylation of the α subunit of RNA polymerase. Together, these two post-translational modifications fine-tune cpxP transcription in response to changes in the intracellular environment.

Most bacterial promoters are regulated by several signals. This is reflected in the complexity of their organization, with multiple binding sites for different transcription factors. Studies of a small number of complex promoters have revealed different distinct mechanisms that integrate the effects of multiple transcription factors.

In their letter to the editor, Sianou et al questioned our conclusion [1] that the female bladder is not sterile because, they argue, the use of a transurethral catheter to collect urine could introduce bacterial contaminants into the bladder, contaminants that could be detected by sensitive techniques such as 16S ribosomal RNA (rRNA) gene sequencing and expanded quantitative urine culture (EQUC). Given the possibility of contamination, they contend that suprapubic aspiration is the only validmethod of collecting urine formicrobiological studies of the bladder. Given the rapidly evolving scientific ability to detect and describe bacterial communities, these are valid questions that we have carefully considered. Clinical diagnosis and treatment should be improved to detect and treat meaningful uropathogens, with an understanding of the normal urinary resident bacterial community. We have also grown increasingly concerned about the validity of the clinical practices used for diagnosis and treatment of urinary tract disorders based, most commonly, on voided urine samples. Contamination is a risk inherent in any samplingmethod; however, suprapubic aspiration is clearly not feasible for routine clinical care. Nonetheless, we began our studies by analyzing paired urine collected by both suprapubic and transurethral methods. As suggested by Sianou et al, we also collected and analyzed controls—a swab of the skin was performed where the needle puncture would occur prior to the application of antiseptic and a needle subsequently inserted at that site that did not enter the bladder. The samples obtained by suprapubic aspirate (SPA) and transurethral catheter (TUC) resembled each other, whereas the control samples did not. We concluded that the bacteria detected in the SPA and TUC samples had indeed been present in the bladder [2]. To determine whether the DNAdetected came from live bacteria,we developed EQUC, which identified live bacteria in urine obtained by the TUC method [3]. Subsequently, we performed EQUC and 16S

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.We sought to study the cross-sectional relationships between urinary microbiota features and demographic and clinical characteristics of women undergoing stress urinary incontinence surgery.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.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.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.

Adult human urine is not sterile. The resident bacterial community may contribute to urinary health and disease in undiscovered ways. Bacterial genomic sequencing and expanded urine culture techniques are major complementary tools for scientific exploration in urologic research.