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Background: The complex interaction between the gut and urinary microbiota underscores the importance of understanding microbial dysbiosis in pediatric urinary tract infection (UTI). However, the literature on the gut–urinary axis in pediatric UTIs is limited. This systematic review aims to summarize the current literature on the roles of gut and urinary dysbiosis in pediatric UTIs. Methods: This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. A comprehensive literature search was performed across four databases, including PubMed, Web of Science, Scopus, and EMBASE. All studies published between January 2003 and December 2023 utilizing 16S rRNA sequencing to profile the gut or urinary microbiome in children with UTIs were included. Heat map visualization was used to compare microbial profiles between UTI and control cohorts. The methodological quality assessment was performed using the Newcastle–Ottawa scale (NOS). Results: Eight studies were included in this review. While five studies compared the microbiota signatures between patients and controls, three studies focused solely on the UTI cohort. Also, the gut and urinary microbiome profiles were investigated by four studies each. The consistent loss of microbiome alpha-diversity with an enrichment of specific putative pathobiont microbes was observed among the included studies. Escherichia coli consistently emerged as the predominant uropathogen in pediatric UTIs. In addition to this, Escherichia fergusonii, Klebsiella pneumoniae, and Shigella flexneri were isolated in the urine of children with UTIs, and enrichment of Escherichia, Enterococcus, Enterobacter, and Bacillus was demonstrated in the gut microbiota of UTI patients. On the contrary, certain genera, such as Achromobacter, Alistipes, Ezakiella, Finegoldia, Haemophilus, Lactobacillus, Massilia, Prevotella, Bacteroides, and Ureaplasma, were isolated from the controls, predominantly in the fecal samples. The methodological quality of the included studies was variable, with total scores (NOS) ranging from 5 to 8. Conclusions: The enrichment of specific pathobionts, such as Escherichia coli, in the fecal or urinary samples of the UTI cohort, along with the presence of core microbiome-associated genera in the non-UTI population, underscores the critical role of the gut–urinary axis in pediatric UTI pathogenesis. These findings highlight the potential for microbiome-based strategies in pediatric UTIs. Further studies with larger cohorts, standardized healthy controls, and longitudinal profiling are essential to validate these observations and translate them into clinical practice. [ABSTRACT FROM AUTHOR]

Background: Despite a unique microbiome in urine, the relationship between nocturnal enuresis and the urobiome remains unclear. This study aimed to compare the presence of specific bacterial species in the urine of children with and without nocturnal enuresis. Methods: We used 16S ribosomal RNA gene sequencing to analyze the urobiome in urine samples obtained from the two groups of children. The presence of Propionimicrobium lymphophilum was examined using real-time PCR in the urine of 25 children diagnosed with monosymptomatic nocturnal enuresis (MNE), and 17 children without this condition. Results: Children with MNE exhibited a significantly higher prevalence of P. lymphophilum : 16 out of 25 (64.0%) compared to 4 out of 17 (23.5%) in the control group. Among children with frequent bedwetting, there was a significantly higher prevalence of P. lymphophilum ;15 out of 16 (93.8%) compared to 2 out of 9 (22.2%) in those with infrequent bedwetting. Bacterial culture tests confirmed the anaerobic growth of P. lymphophilum isolates from urine samples of two PCR-positive patients with MNE. These isolates were found to be susceptible to ampicillin. Conclusion: These findings suggest that P. lymphophilum may be associated with chronic urinary tract infections and potentially contribute to the development of MNE in children. [ABSTRACT FROM AUTHOR]

Urinary tract infections (UTIs) are one of the most common bacterial infections, affecting more than 150 million people each year in the world. UTIs have grown exponentially in the last few years. They represent a major load for both individuals and society. The highest incidence (about 55–60%) concerns women. Many pathogens are involved in UTIs, most of which are derived from the gut. Recent studies, together with recent diagnostic techniques (such as quantitative culture of urine or next-generation sequencing), have improved the knowledge of microbial communities in the urinary tract. It turned out that gut dysbiosis is strictly involved in the pathogenesis of UTIs. In particular, the human gut is the natural habitat for Escherichia coli (E. coli), the main bacterium responsible for UTIs. The overgrowth of E. coli pathogenic strains represents a risk factor for them. Furthermore, the human gut microbiota acts as a "global reservoir" for genes conferring resistance to clinically relevant antibiotics, thus influencing the treatment of UTIs. In addition, differently from the past, the idea of a sterile urinary environment has been replaced by the characterization of a urinary microbiome. The aim of our review is to explore recent studies on the association between gut microbiota and urinary microbiome and to summarize the current knowledge about the effects of interactions between gut and urinary microbial communities in the pathogenesis of UTIs, considering UTIs more as a "gut disease" and not only a urinary disease and providing new insight into the therapeutic options such as the use of probiotics. [ABSTRACT FROM AUTHOR]

Abstract Background Urolithiasis is a prevalent urological ailment characterized by increasing prevalence and recurrence rates, resulting in substantial social and economic burden. While men exhibit an incidence rate nearly twice that of women, this gender disparity is gradually diminishing. Nevertheless, the mechanisms underlying this condition remain incompletely elucidated. The identification of the urinary microbiome (urobiome) has provided a fresh perspective on urolithiasis. This study aimed to analyze the urobiome of unilateral stone formers in the renal pelvis and evaluate the variations in microbial diversity and community composition between males and females. Methods Renal pelvis urine samples were obtained from a cohort of 21 male and 9 female patients and subsequently subjected to taxonomic and functional analysis using 2bRAD sequencing for Microbiome (2bRAD-M). The collected samples were categorized into four distinct groups, namely the stone side of males (SM), stone side of females (SF), non-stone side of males (NSM), and non-stone side of females (NSF). Results Through the application of beta diversity analysis, dissimilarity was observed between NSM and NSF. Additionally, NSF exhibited a higher abundance of microbial populations, and a total of 29 distinct species were identified as differentially present between NSM and NSF using LEfSe. Lactobacillus iners, Atopobium deltae, Lawsonella clevelandensis, and Meyerozyma guilliermondii exhibited enrichment in the SF group compared to the SM group. Furthermore, we identified distinct species that differed between the SM and NSM groups, as well as the SF and NSF groups. Besides, we conducted COG annotation and KEGG pathway predictions, revealing significant differences in urobiome function across the different groups. Conclusion Variations in microbial community composition and predicted functions were observed among the various groups. Future research could potentially leverage the urobiome to personalize urolithiasis treatment based on individual microbial characteristics, taking into account gender-specific differences.

Background: Urolithiasis is a prevalent urological ailment characterized by increasing prevalence and recurrence rates, resulting in substantial social and economic burden. While men exhibit an incidence rate nearly twice that of women, this gender disparity is gradually diminishing. Nevertheless, the mechanisms underlying this condition remain incompletely elucidated. The identification of the urinary microbiome (urobiome) has provided a fresh perspective on urolithiasis. This study aimed to analyze the urobiome of unilateral stone formers in the renal pelvis and evaluate the variations in microbial diversity and community composition between males and females. Methods: Renal pelvis urine samples were obtained from a cohort of 21 male and 9 female patients and subsequently subjected to taxonomic and functional analysis using 2bRAD sequencing for Microbiome (2bRAD-M). The collected samples were categorized into four distinct groups, namely the stone side of males (SM), stone side of females (SF), non-stone side of males (NSM), and non-stone side of females (NSF). Results: Through the application of beta diversity analysis, dissimilarity was observed between NSM and NSF. Additionally, NSF exhibited a higher abundance of microbial populations, and a total of 29 distinct species were identified as differentially present between NSM and NSF using LEfSe. Lactobacillus iners, Atopobium deltae, Lawsonella clevelandensis, and Meyerozyma guilliermondii exhibited enrichment in the SF group compared to the SM group. Furthermore, we identified distinct species that differed between the SM and NSM groups, as well as the SF and NSF groups. Besides, we conducted COG annotation and KEGG pathway predictions, revealing significant differences in urobiome function across the different groups. Conclusion: Variations in microbial community composition and predicted functions were observed among the various groups. Future research could potentially leverage the urobiome to personalize urolithiasis treatment based on individual microbial characteristics, taking into account gender-specific differences. [ABSTRACT FROM AUTHOR]

Escherichia coli can colonise the urogenital tract of individuals without causing symptoms of infection, in a condition referred to as asymptomatic bacteriuria (ABU). ABU isolates can protect the host against symptomatic urinary tract infections (UTIs) by bacterial interference against uropathogenic E. coli (UPEC). The aim of this study was to investigate the genotypic and phenotypic characteristics of five ABU isolates from midstream urine samples of adults. Comparative genomic and phenotypic analysis was conducted including an antibiotic resistance profile, pangenome analysis, and a putative virulence profile. Based on the genome analysis, the isolates consisted of one from phylogroup A, three from phylogroup B2, and one from phylogroup D. Two of the isolates, PUTS 58 and SK-106-1, were noted for their lack of antibiotic resistance and virulence genes compared to the prototypic ABU strain E. coli 83,972. This study provides insights into the genotypic and phenotypic profiles of uncharacterised ABU isolates, and how relevant fitness and virulence traits can impact their potential suitability for therapeutic bacterial interference. [ABSTRACT FROM AUTHOR]

To date, the increased awareness of the impact of microbes on human health has promoted scientific interest in microbiome studies for diagnostic and therapeutic purposes, revealing correlations between specific taxa and cancer. In particular, numerous species of Porphyromonas have been associated with several types of tumors. Previously, we studied the urobiome using Next-Generation Sequencing (NGS), and found an increase in Porphyromonas somerae in first morning urine of subjects affected by bladder cancer (BCa). Here, we aimed to confirm the presence of P. somerae in BCa patients by using droplet digital Polymerase Chain Reaction (ddPCR), testing a cohort of 102 male subjects over 50 years. Our findings showed a significant increase in P. somerae in the urine of the BCa group within both ddPCR and NGS, and a correlation between the two methods was observed at a statistical level. Moreover, P. somerae's identification with ddPCR confirmed a significant association between this bacterium and the presence of BCa, highlighting its potential role as a biomarker. This allows us to propose the ddPCR as a suitable method for first-stage BCa screening and follow-up. [ABSTRACT FROM AUTHOR]

Urinary tract infection (UTI) is a term that describes an infection anywhere within the urinary system where the commonest cause is Escherichia coli, which accounts for 80% of infections and is usually found in the urinary tract (Vasudevan, 2014). Women are more susceptible to UTIs than men as their urethra is shorter, which means that infection can easily ascend the tract (Vasudevan, 2014; John et al, 2016). However, as men age, the risk becomes equal to women (Harrington and Hooton, 2000; Rowe et al, 2014) and accurate diagnosis and management of UTIs in men using guidelines is poor (Isberg et al, 2024). This article considers the risk factors for UTI in men, how they present, and how UTI can be managed in primary care. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Purpose: The recent discovery of the urinary microbiome has led to an emerging field of investigation about the potential role of microorganisms in the pathogenesis of urinary bladder cancer. Few preliminary data have been reported so far implicating urobiome as causative and prognostic factor of bladder tumorigenesis. In the present study, a review of the current evidence is presented about microbiome composition among patients with bladder cancer and healthy individuals as well as possible implications of microbiome on urothelial carcinoma of the bladder. Methods: A literature review was conducted using PubMed/MEDLINE, Scopus, and the Cochrane library until December 2023. Search algorithm was constructed using the following terms and their associated Mesh terms and Boolean operators: "urinary microbiome" and "urinary microbiota". Studies written in English language, identifying, and comparing urinary microbiome among bladder cancer patients and healthy control group were included in the review. Results: A total of 2,356 reports were identified. From this total 16 articles complied with the inclusion criteria were selected for analysis. These articles represent a total of about 486 bladder cancer patients. Conclusion: Recent studies revealed the colonization of the urinary tract and the bladder by micro-organisms using both enhanced culture- and molecular-based techniques for microbial characterization. However, several limitations exist in the literature decreasing the reliability of the current reports. As a result, urinary microbiome consist an ambitious era in bladder cancer research with an increasing number of evidence about its potential pathogenetic, prognostic and therapeutic role. [ABSTRACT FROM AUTHOR]

Simple Summary: The microbiome refers to the vast community of microorganisms such as bacteria that inhabit a particular host or ecosystem. Recently, it was discovered that the urine from healthy dogs hosts its own unique microbiome, known as the urobiome. These microbial organisms play important roles in maintaining urinary system health and preventing disease. Although the gut microbiome is heavily influenced by diet, it remains unknown how nutritional features impact the urobiome. Therefore, the purpose of this work was to determine how dietary features alter the urobiome's composition in clinically healthy dogs. Specifically, we examined how nutrient intake (protein, fat, and crude fiber), commercial diet brands, and dietary diversity (i.e., the number of unique food sources consumed each day) altered the abundance and diversity of bacteria present in the canine urobiome. We discovered that both commercial diet brands and dietary diversity were associated with distinct shifts in the composition of the urobiome. This discovery highlights the complex relationships between diet and urinary microbes, and these findings could ultimately lead to novel dietary strategies to promote urobiome health. Nutrition plays an important role in shaping the gut microbiome composition, although the impact of diet on the urinary microbiome (i.e., urobiome) remains unknown. The aim of this pilot study was to discover how nutritional features affect the diversity and composition of the urobiome in dogs. Dietary histories were obtained for 15 clinically healthy adult dogs, including limited nutrient (protein, fat, crude fiber), commercial diet brand, and dietary diversity profiles. The urine samples were collected via cystocentesis, followed by sequencing of the bacterial 16S rRNA gene. The data were analyzed to determine associations between major nutrients and dietary sources with the urobiome's composition. The protein, fat, and crude fiber contents had no statistically significant effect on the alpha or beta diversity. However, the beta diversity values differed (PERMANOVA; p = 0.017, R2 = 0.10) between dogs fed one commercial diet brand compared to dogs consuming any other brand. The beta diversity values also differed (p = 0.019, R2 = 0.10) between dogs consuming more diverse daily diets compared to those consuming less diverse diets (≥3 or <3 unique food sources, respectively). Overall, the results of this pilot study suggest that diet might impact the urobiome and support further exploration of the relationship between diet and the urobiome's composition in dogs. [ABSTRACT FROM AUTHOR]

BackgroundDespite a unique microbiome in urine, the relationship between nocturnal enuresis and the urobiome remains unclear. This study aimed to compare the presence of specific bacterial species in the urine of children with and without nocturnal enuresis.MethodsWe used 16S ribosomal RNA gene sequencing to analyze the urobiome in urine samples obtained from the two groups of children. The presence of Propionimicrobium lymphophilum was examined using real-time PCR in the urine of 25 children diagnosed with monosymptomatic nocturnal enuresis (MNE), and 17 children without this condition.ResultsChildren with MNE exhibited a significantly higher prevalence of P. lymphophilum: 16 out of 25 (64.0%) compared to 4 out of 17 (23.5%) in the control group. Among children with frequent bedwetting, there was a significantly higher prevalence of P. lymphophilum;15 out of 16 (93.8%) compared to 2 out of 9 (22.2%) in those with infrequent bedwetting. Bacterial culture tests confirmed the anaerobic growth of P. lymphophilum isolates from urine samples of two PCR-positive patients with MNE. These isolates were found to be susceptible to ampicillin.ConclusionThese findings suggest that P. lymphophilum may be associated with chronic urinary tract infections and potentially contribute to the development of MNE in children.

ABSTRACT The impact of chronic indwelling urinary catheters (IUCs) on the composition and stability of the urinary microbiota remains unknown. The primary aim of this study was to describe the urinary microbiomes of geriatric males with chronic IUCs. A secondary aim was to explore clinical catheter-associated urinary tract infection (CAUTI) courses of the participants. Geriatric male patients with chronic IUCs were followed longitudinally. Catheterized urine, catheter tips, and both urethral and periurethral swabs were collected from participants at monthly intervals. Microbes were isolated and identified from each specimen using an enhanced culture method called expanded quantitative urine culture (EQUC) and targeted 16S rRNA gene DNA sequencing. Microbial outcomes were examined both in the absence of urinary symptoms and in the context of clinical diagnosis of CAUTI. Ten male participants (mean age 86 years) were enrolled. Urinary microbiomes differed for each participant. However, within each individual, microbiomes were similar over time and across niches (bladder, catheter, urethra, and periurethra). Within-niche microbiomes differed across individuals, and this was observed over time. The most abundant bacteria isolated from all niches were known uropathogens. Six of 10 individuals met diagnostic criteria for CAUTI at least once during the 12-month observation period, but no evidence of this or antibiotic treatment/response was discernable in our monthly samples. The microbiomes of each participant were unique and remained similar over time and across niches. Longitudinal EQUC or 16S rRNA gene sequencing data could be useful to clinicians when diagnosing or treating possible CAUTI.IMPORTANCECatheter-associated urinary tract infections (CAUTIs) are serious but preventable nosocomial infections. The most common risk factor for developing CAUTI is prolonged use of indwelling urinary catheters (IUCs). This study provides the first longitudinal description of the urinary microbiomes of geriatric males with chronic IUCs, in the absence of urinary signs and symptoms, as a first step toward enhancing our knowledge of the impact of chronic IUCs on the composition and stability of the urinary microbiota. This is an understudied area, particularly for males.

Urinary tract infections (UTIs) are among the most common bacterial infections in humans, particularly affecting women, with significant clinical and socioeconomic impacts. Despite advances in medical research, the diagnostic criteria for UTI have remained practically unchanged since Kass’s seminal work, emphasizing the need for a reevaluation in light of new scientific insights. Recent studies have highlighted the importance of the urobiome, a previously underappreciated community of microorganisms within the urinary tract (UT), and its role in maintaining urogenital health. The gut-bladder axis has emerged as a critical pathway in understanding UTI as a dysbiosis, where imbalances in the microbial community and its relation with the host contribute to infection susceptibility. This review explores the evolving definitions and diagnostic challenges of UTI, particularly in women, and examines the implications of recent discoveries on the urobiome and the gut-bladder axis. Additionally, we discuss the potential of novel therapeutic strategies to restore microbial balance, offering a promising avenue for the therapeutic management of UTIs.

Replicability is a well-established challenge in microbiome research with a variety of contributing factors at all stages, from sample collection to code execution. Here, we focus on voided urine sample storage conditions for urogenital microbiome analysis. Using urine samples collected from 10 adult females, we investigated the microbiome preservation efficacy of AssayAssure Genelock (Genelock), compared with no preservative, under different temperature conditions. We varied temperature over 48 h in order to examine the impact of conditions samples may experience with home voided urine collection and shipping to a central biorepository. The following common lab and shipping conditions were investigated: -20°C, ambient temperature, 4°C, freeze-thaw cycle, and heat cycle. At 48 h, all samples were stored at -80°C until processing. After generating 16S rRNA gene amplicon sequencing data using the highly sensitive KatharoSeq protocol, we observed individual variation in both alpha and beta diversity metrics below interhuman differences, corroborating reports of individual microbiome variability in other specimen types. While there was no significant difference in beta diversity when comparing Genelock versus no preservative, we did observe a higher concordance with Genelock samples shipped at colder temperatures (-20°C and 4°C) when compared with the samples shipped at -20°C without preservative. Our results indicate that Genelock does not introduce a significant amount of microbial bias when used on a range of temperatures and is most effective at colder temperatures. IMPORTANCE The urogenital microbiome is an understudied yet important human microbiome niche. Research has been stimulated by the relatively recent discovery that urine is not sterile; urinary tract microbes have been linked to health problems, including urinary infections, incontinence, and cancer. The quality of life and economic impact of UTIs and urgency incontinence alone are enormous, with $3.5 billion and $82.6 billion, respectively, spent in the United States. annually. Given the low biomass of urine, novelty of the field, and limited reproducibility evidence, it is critical to study urine sample storage conditions to optimize scientific rigor. Efficient and reliable preservation methods inform methods for home self-sample collection and shipping, increasing the potential use in larger-scale studies. Here, we examined both buffer and temperature variation effects on 16S rRNA gene amplicon sequencing results from urogenital samples, providing data on the consequences of common storage methods on urogenital microbiome results.

Abstract Background Although the human bladder is reported to harbor unique microbiota, our understanding of how these microbial communities interact with their human hosts is limited, mostly owing to the lack of isolates to test mechanistic hypotheses. Niche-specific bacterial collections and associated reference genome databases have been instrumental in expanding knowledge of the microbiota of other anatomical sites, such as the gut and oral cavity. Results To facilitate genomic, functional, and experimental analyses of the human bladder microbiota, we present a bladder-specific bacterial isolate reference collection comprising 1134 genomes, primarily from adult females. These genomes were culled from bacterial isolates obtained by a metaculturomic method from bladder urine collected by transurethral catheterization. This bladder-specific bacterial isolate reference collection includes 196 different species, including representatives of major aerobes and facultative anaerobes, as well as some anaerobes. It captures 72.2% of the genera found when re-examining previously published 16S rRNA gene sequencing of 392 adult female bladder urine samples. Comparative genomic analysis finds that the taxonomies and functions of the bladder microbiota share more similarities with the vaginal microbiota than the gut microbiota. Whole-genome phylogenetic and functional analyses of 186 bladder Escherichia coli isolates and 387 gut Escherichia coli isolates support the hypothesis that phylogroup distribution and functions of Escherichia coli strains differ dramatically between these two very different niches. Conclusions This bladder-specific bacterial isolate reference collection is a unique resource that will enable bladder microbiota research and comparison to isolates from other anatomical sites.

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The human urinary bladder hosts a complex microbial community of low biomass referred to as the urobiome. While the composition of the urobiome has been investigated in adults for over a decade now, only a few studies have considered the presence and composition of the urobiome in children. It is critical to explore how the urobiome develops throughout the life span and how it changes in the presence of various health conditions. Therefore, we set to review the available data on pediatric urobiome composition and its development with age and disease. In addition, we focused on identifying and reporting specific gaps in our knowledge of the pediatric urobiome that we hope will be addressed by future studies in this swiftly developing field with fast-improving methods and consensus.

Urinary tract infections (UTIs) are one of the most common bacterial infections, affecting more than 150 million people each year in the world. UTIs have grown exponentially in the last few years. They represent a major load for both individuals and society. The highest incidence (about 55–60%) concerns women. Many pathogens are involved in UTIs, most of which are derived from the gut. Recent studies, together with recent diagnostic techniques (such as quantitative culture of urine or next-generation sequencing), have improved the knowledge of microbial communities in the urinary tract. It turned out that gut dysbiosis is strictly involved in the pathogenesis of UTIs. In particular, the human gut is the natural habitat for Escherichia coli (E. coli), the main bacterium responsible for UTIs. The overgrowth of E. coli pathogenic strains represents a risk factor for them. Furthermore, the human gut microbiota acts as a “global reservoir” for genes conferring resistance to clinically relevant antibiotics, thus influencing the treatment of UTIs. In addition, differently from the past, the idea of a sterile urinary environment has been replaced by the characterization of a urinary microbiome. The aim of our review is to explore recent studies on the association between gut microbiota and urinary microbiome and to summarize the current knowledge about the effects of interactions between gut and urinary microbial communities in the pathogenesis of UTIs, considering UTIs more as a “gut disease” and not only a urinary disease and providing new insight into the therapeutic options such as the use of probiotics.

To date, the increased awareness of the impact of microbes on human health has promoted scientific interest in microbiome studies for diagnostic and therapeutic purposes, revealing correlations between specific taxa and cancer. In particular, numerous species of Porphyromonas have been associated with several types of tumors. Previously, we studied the urobiome using Next-Generation Sequencing (NGS), and found an increase in Porphyromonas somerae in first morning urine of subjects affected by bladder cancer (BCa). Here, we aimed to confirm the presence of P. somerae in BCa patients by using droplet digital Polymerase Chain Reaction (ddPCR), testing a cohort of 102 male subjects over 50 years. Our findings showed a significant increase in P. somerae in the urine of the BCa group within both ddPCR and NGS, and a correlation between the two methods was observed at a statistical level. Moreover, P. somerae’s identification with ddPCR confirmed a significant association between this bacterium and the presence of BCa, highlighting its potential role as a biomarker. This allows us to propose the ddPCR as a suitable method for first-stage BCa screening and follow-up.

Urinary tract infections (UTIs) are a common health issue affecting individuals worldwide. Recurrent urinary tract infections (rUTI) pose a significant clinical challenge, with limited understanding of the underlying mechanisms. Recent research suggests that the urobiome, the microbial community residing in the urinary tract, may play a crucial role in the development and recurrence of urinary tract infections. However, the specific virulence factor genes (VFGs) driven by urobiome contributing to infection recurrence remain poorly understood. Our study aimed to investigate the relationship between urobiome driven VFGs and recurrent urinary tract infections. By analyzing the VFGs composition of the urinary microbiome in patients with rUTI compared to a control group, we found higher alpha diversity in rUTI patients compared with healthy control. And then, we sought to identify specific VFGs features associated with infection recurrence. Specifically, we observed an increased abundance of certain VGFs in the recurrent infection group. We also associated VFGs and clinical data. We then developed a diagnostic model based on the levels of these VFGs using random forest and support vector machine analysis to distinguish healthy control and rUIT, rUTI relapse and rUTI remission. The diagnostic accuracy of the model was assessed using receiver operating characteristic curve analysis, and the area under the ROC curve were 0.83 and 0.75. These findings provide valuable insights into the complex interplay between the VFGs of urobiome and recurrent urinary tract infections, highlighting potential targets for therapeutic interventions to prevent infection recurrence. [ABSTRACT FROM AUTHOR]

Introduction and hypothesis: Our objective was to evaluate if botox alters the urinary microbiome of patients with overactive bladder and whether this alteration is predictive of treatment response. Methods: This multicenter prospective cohort study included 18–89-year-old patients undergoing treatment for overactive bladder with 100 units of botox. Urine samples were collected by straight catheterization on the day of the procedure (S1) and again 4 weeks later (S2). Participants completed the Patient Global Impression of Improvement form at their second visit for dichotomization into responders and nonresponders. The microbiome was sequenced using 16s rRNA sequencing. Wilcoxon signed rank and Wilcoxon rank sum were used to compare the microbiome, whereas chi-square, Wilcoxon rank sum, and the independent t-test were utilized for clinical data. Results: Sixty-eight participants were included in the analysis. The mean relative abundance and prevalence of Beauveria bassiana, Xerocomus chrysenteron, Crinipellis zonata, and Micrococcus luteus were all found to increase between S1 and S2 in responders; whereas in nonresponders the mean relative abundance and prevalence of Pseudomonas fragi were found to decrease. The MRA and prevalence of Weissella cibaria, Acinetobacter johnsonii, and Acinetobacter schindleri were found to be greater in responders than nonresponders at the time of S1. Significant UM differences in the S1 of patients who did (n = 5) and did not go on to develop a post-treatment UTI were noted. Conclusions: Longitudinal urobiome differences may exist between patients who do and do not respond to botox. [ABSTRACT FROM AUTHOR]

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.

Nutrition plays an important role in shaping the gut microbiome composition, although the impact of diet on the urinary microbiome (i.e., urobiome) remains unknown. The aim of this pilot study was to discover how nutritional features affect the diversity and composition of the urobiome in dogs. Dietary histories were obtained for 15 clinically healthy adult dogs, including limited nutrient (protein, fat, crude fiber), commercial diet brand, and dietary diversity profiles. The urine samples were collected via cystocentesis, followed by sequencing of the bacterial 16S rRNA gene. The data were analyzed to determine associations between major nutrients and dietary sources with the urobiome’s composition. The protein, fat, and crude fiber contents had no statistically significant effect on the alpha or beta diversity. However, the beta diversity values differed (PERMANOVA; p = 0.017, R2 = 0.10) between dogs fed one commercial diet brand compared to dogs consuming any other brand. The beta diversity values also differed (p = 0.019, R2 = 0.10) between dogs consuming more diverse daily diets compared to those consuming less diverse diets (≥3 or

Continued efforts to isolate and sequence bacteria of the urinary tract has increased representation of these species in publicly available databases. This in turn has improved taxonomic classifications of the urinary microbiome (urobiome). Short-read sequencing targeting a variable region(s) of the 16S rRNA gene sequence has been fundamental in characterizing the urobiomes of males and females with and without lower urinary tract symptoms, as well as cancers of the urinary tract. Here, we have compiled a data set of full-length or near-full-length 16S rRNA gene sequences for the urobiome. To generate this data set, we first plated 203 isolates from the bladder on differential media and sequenced their full-length 16S rRNA gene sequence. We combined this data set with publicly available genomes from primarily the female urinary tract. The final data set includes 399 sequences representative of 160 different species from 73 genera. We assessed the ability of publicly available databases to correctly predict these sequences based on the V1-V3, V4, and V4-V6 variable regions. As expected, species designations based upon these variable regions is often not possible or incorrect. We also detected incorrect genus-level classifications. This data set can be used to supplement existing databases, by increasing urobiome species variation, and thus improve future studies characterizing urobiomes.

Background: Microbiome dysbiosis plays a role in the pathogenesis of many urological diseases, including bladder cancer (BC). The aim of the study was to compare the urinary and gut microbiota of patients with BC with a healthy control (HC) group. Methods: The study group included patients hospitalized in 2020 to 2021 with diagnosed BC and HC. Prior to the transurethral resection of bladder tumor, patients collected their urine and stool which was then subjected to 16S rRNA gene sequencing. Results: Overall, 25 patients were enrolled in the study: 18 in the BC group and 7 in the HC group. Analysis of the urine and stool microbiome showed no statistically significant differences between patients with BC and HC in alpha diversity, beta diversity, and difference in taxa relative abundance. Detailed analysis of urine and stool microbiome depending on patient- and tumor-related characteristics also showed no statistically significant differences in alpha diversity and beta diversity. Differences in abundance (ANCOM) were noted in both types of samples in patients with BC. In the urine test, genus Lactobacillus was more common in patients with a positive history of Bacillus Calmette–Guérin (BCG) therapy, while genus Howardella and the strain Streptococcus anginosus were more common in women. In stool samples, abundance of phylum Desulfobacterota was most abundant in Grade G1 and least in G2. Class Alphaproteobacteria, order Rhodospirillales, order Flavobacteriales, and family Flavobacteriaceae were more common in women. Conclusions: The microbiome of urine and stool of patients with BC does not differ significantly from that of HC; however, its composition in patients with BC varies according to the patient's sex. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Antibiotic therapy alone is unable to control recurrent urinary tract infection (UTI); uropathogens have become multiresistant, and alternative strategies are needed. Far from sterile, the urinary tract contains various low-biomass microbiota, some of whose members appear to protect against clinical UTI. This narrative review summarizes (a) the current knowledge of male and female urobiomes in healthy and diseased states, as well as their interplay among sexual partners and (b) clinical trials to date assessing probiotic and other nonantibiotic measures to reduce UTI. We used the PubMed interface to search Ovid Medline for articles describing urogenital flora, UTI, UTI dysbiosis, the effects of sexual intercourse on urogenital flora, and clinical trials of probiotics as UTI prophylaxis. The healthy urobiome of women contains several Lactobacillus species, some of which may impede Escherichia coli growth in the urinary tract. Although Lactobacilli have been found in male urethral microbiota, their presence in male bladder microbiota is less certain. Distal male urethral and vaginal microbiomes of male and sexual female partners influence one another, but more research is needed on the direct interplay of their full urobiomes. Clinical trials assessing the therapeutic potential of Lactobacilli have been largely underpowered and highly varied in tested formulations and routes and frequencies of administration; as such, they have failed to show a clear benefit. Faecal microbiota transplantation for recurrent Clostridium difficile infection was shown, in a retrospective study of seven patients, to reduce recurrent UTI as a side effect. The urobiome in men and women is complex, variable, and still understudied. Although there is hope that Lactobacilli and faecal microbial transplantation could be future nonantibiotic options for recurrent UTI, both require more pharmacologic and clinical research to identify optimal preparations and routes of administration. [ABSTRACT FROM AUTHOR]

The microbiome is the totality of microorganisms found in a specific biological niche. It has been proven that in the human body, the microbiome is responsible for its proper functioning. Dysbiosis, i.e., a disturbance in the composition of the microbiome, may be associated with the pathogenesis of many human diseases. Until recently, studies did not focus on the microbiome of the urinary tract, because, since the 19th century, there had been a dogma that urine in healthy people is sterile. Yet, advances in molecular biology techniques have allowed this dogma to be overthrown. The use of DNA sequencing has shown that the urinary tract has its own endogenous microbiome. This discovery enabled further research on the characteristics of the urine microbiomes of healthy people, as well as on the role of the urine microbiome in the pathogenesis of many urological diseases, including bladder diseases. The aim of this review is to summarize the current knowledge on the urinary microbiome in bladder diseases and to identify potential directions for further research. [ABSTRACT FROM AUTHOR]

Urolithiasis is a common urological disease with increasing incidence and a high recurrence rate, whose etiology is not fully understood. The application of sequencing and culturomics has revealed that urolithiasis is closely related to the urinary microbiome (urobiome), shedding new light on the pathogenesis of stone formation. In this study, we recruited 30 patients with unilateral stones and collected their renal pelvis urine from both sides. Then, we performed 2bRAD-M, a novel sequencing technique that provides precise microbial identification at the species level, to characterize the renal pelvis urobiome of unilateral stone formers in the both sides. We first found that the urobiome in the stone side could be divided into two clusters (Stone1 and Stone2) based on distance algorithms. Stone2 harbored higher microbial richness and diversity compared to Stone1. The genera Cupriavidus and Sphingomonas were overrepresented in Stone1, whereas Acinetobacter and Pseudomonas were overrepresented in Stone2. Meanwhile, differential species were identified between Stone1 and Stone2. We further constructed a random forest model to discriminate two clusters which achieved a powerful diagnostic potential. Moreover, the urobiome of the non-stone side (Control1/2) was compared with that of the stone side (Stone1/2). Stone1 and Control1 showed different microbial community distributions, while Stone2 was similar to Control2 based on diversity analysis. We also identified differentially abundant species among all groups. We assumed that there might be different mechanisms of how microbiota contribute to stone formation in two clusters. Our findings might assist in the selection of suitable medical treatments for urolithiasis. [ABSTRACT FROM AUTHOR]

"Expanded quantitative urine culture (EQUC)" is an enhanced culture protocol for the detection of viable microbes in urine specimens. Using a large volume of urine and different sets of cultural conditions, EQUC is able to uncover a wide range of bacteria and fungi (yeasts) that were otherwise undetected by the standard urinary culture. In addition to common urinary pathogens, EQUC has been shown to detect emerging and new pathogens, and commensal microbiota. Although the usefulness of EQUC protocol in clinical set up has not yet been fully established, recent studies have demonstrated that EQUC can provide valuable information regarding symptom resolution, treatment responses and diagnosis of major urinary disorders including urinary tract infections, urinary incontinence and other lower urinary tract symptoms. EQUC may also help in evaluating the utility of beneficial microbiota as biotherapeutics. This narrative minireview describes the current research findings regarding the clinical utility of EQUC in characterizing the role of urinary microbiome and uropathogens in health and disease. The literature which are written in English, available on "PubMed" and contain any of the terms- "expanded quantitative urine culture", "enhanced quantitative urine culture" and "EQUC" in the abstracts were used as the source articles to prepare this minireview. [ABSTRACT FROM AUTHOR]

While interest in developing the human microbiome as a biomarker for attention-deficit hyperactivity disorder (ADHD) is increasing, there has been limited exploration in utilizing urine samples. In this study, we analysed urine microbiome profiles by extracting 16S ribosomal DNA from purified bacteria-derived extracellular membrane vesicles obtained from urine samples. Sequencing libraries were constructed by amplifying V3–V4 hypervariable regions sequenced using Illumina MiSeq. Profiles of male Korean children and adolescents with ADHD (n = 33) were compared with healthy sex-matched controls (n = 39). Statistically controlling for age, we found decreased alpha diversity in the urine bacteria of the ADHD group, as evidenced by reduced Shannon and Simpson indices (p < 0.05), and significant differences in beta diversity between the two groups (p < 0.001). The phyla Firmicutes and Actinobacteriota, as well as the genera Ralstonia and Afipia, were relatively more abundant in the ADHD group. The phylum Proteobacteria and the genera Corynebacterium and Peptoniphilus were more abundant in the control group. Notably, the genus Afipia exhibited significant correlations with the Child Behavior Checklist Attention Problems score and DSM-oriented ADHD subscale. This study is the first to propose the urine microbiome as a potential biomarker for pediatric ADHD. [ABSTRACT FROM AUTHOR]

Introduction: The development of molecular biology methods and their application in microbial research allowed the detection of many new pathogens that cause urinary tract infections (UTIs). Despite the advances of using new research techniques, the etiopathogenesis of UTIs, especially in patients undergoing dialysis and patients after kidney transplantation, is still not fully understood. Methods: This study aimed to characterize and compare the composition of the bacterial element of the urinary tract microbiome between the groups of patients undergoing dialysis (n = 50) and patients after kidney transplantation (n = 50), with positive or negative urine culture, compared to healthy individuals (n = 50). Results: Asymptomatic bacteriuria was observed in 30% of the urine cultures of patients undergoing dialysis and patients after kidney transplantation, with Escherichia coli as the most dominant microorganism (73%) detected with the use of classical microbiology techniques. However, differences in the bacterial composition of the urine samples between the evaluated patient groups were demonstrated using the amplicon sequencing. Finegoldia, Leptotrichia, and Corynebacterium were found to be discriminative bacteria genera in patients after dialysis and kidney transplantation compared to the control group. In addition, in all of urine samples, including those without bacteriuria in classical urine culture, many types of bacteria have been identified using 16S rRNA sequencing. Discussion: The revealed microbial characteristics may form the basis in searching for new diagnostic markers in treatment of patients undergoing dialysis and patients after kidney transplantation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Urinary tract infection is one of the most common bacterial infections and can cause major burdens, not only to individuals but also to an entire society. Current knowledge of the microbial communities in the urinary tract has increased exponentially due to next-generation sequencing and expanded quantitative urine culture. We now acknowledge a dynamic urinary tract microbiome that we once thought was sterile. Taxonomic studies have identified the normal core microbiota of the urinary tract, and studies on the changes in microbiome due to sexuality and age have set the foundation for microbiome studies in pathologic states. Urinary tract infection is not only caused by invading uropathogenic bacteria but also by changes to the uromicrobiome milieu, and interactions with other microbial communities can also contribute. Recent studies have provided insights into the pathogenesis of recurrent urinary tract infections and antimicrobial resistance. New therapeutic options for urinary tract infections also show promise; however, further research is needed to fully understand the implications of the urinary microbiome in urinary tract infections. [ABSTRACT FROM AUTHOR]

Abstract Background The pathogenesis of kidney stone disease (KSD) is not fully understood, and potential contributing factors remain to be explored. Several studies have revealed that the urinary microbiome (urobiome) of stone formers was distinct from that of healthy individuals using 16S rRNA gene sequencing, most of which only provided microbial identification at the genus level. 2bRAD sequencing for Microbiome (2bRAD-M) is a novel sequencing technique that enables accurate characterization of the low-biomass microbiome at the species resolution. We aimed to apply 2bRAD-M to profile the renal pelvis urobiome of unilateral kidney stone patients and compared the urobiome with and without stone(s). Method A total of 30 patients with unilateral stones were recruited, and their renal pelvis urine from both sides was collected. A ureteroscope was inserted into the renal pelvis with stone(s) and a ureteral catheter was placed into the ureteroscope to collect renal pelvis urine. This procedure was repeated again with new devices to collect the urine of the other side. 2bRAD-M was performed to characterize the renal pelvis urobiome of unilateral stone formers to explore whether microbial differences existed between the stone side and the non-stone side. Results The microbial community composition of the stone side was similar to that of the non-stone side. Paired comparison showed that Corynebacterium was increased and Prevotella and Lactobacillus were decreased in the stone side. Four species (Prevotella bivia, Lactobacillus iners, Corynebacterium aurimucosum, and Pseudomonas sp_286) were overrepresented in the non-stone side. 24 differential taxa were also identified between two groups by linear discriminant analysis effect size (LEfSe). Extensive and close connections among genera and species were observed in the correlation analysis. Moreover, a random forest classifier was constructed using specific enriched species, which can distinguish the stone side from the non-stone side with an accuracy of 71.2%. Conclusion This first 2bRAD-M microbiome survey gave an important hint towards the potential role of urinary dysbiosis in KSD and provided a better understanding of mechanism of stone formation.

“Expanded quantitative urine culture (EQUC)” is an enhanced culture protocol for the detection of viable microbes in urine specimens. Using a large volume of urine and different sets of cultural conditions, EQUC is able to uncover a wide range of bacteria and fungi (yeasts) that were otherwise undetected by the standard urinary culture. In addition to common urinary pathogens, EQUC has been shown to detect emerging and new pathogens, and commensal microbiota. Although the usefulness of EQUC protocol in clinical set up has not yet been fully established, recent studies have demonstrated that EQUC can provide valuable information regarding symptom resolution, treatment responses and diagnosis of major urinary disorders including urinary tract infections, urinary incontinence and other lower urinary tract symptoms. EQUC may also help in evaluating the utility of beneficial microbiota as biotherapeutics. This narrative minireview describes the current research findings regarding the clinical utility of EQUC in characterizing the role of urinary microbiome and uropathogens in health and disease. The literature which are written in English, available on “PubMed” and contain any of the terms- “expanded quantitative urine culture”, “enhanced quantitative urine culture” and “EQUC” in the abstracts were used as the source articles to prepare this minireview.

Introduction: In complex microbial communities, the importance of microbial species at very low abundance levels and their prevalence for overall community structure and function is increasingly being recognized. Clinical microbiome studies on urinary stone disease (USD) have indicated that both the gut and urinary tract microbiota are associated with the onset of the disease and that kidney stones them-selves harbor a complex, yet consistent and viable, microbiome. However, how rare phylotypes contribute to this association remains unclear. Delineating the contribution of rare and common phylotypes to urinary stone disease is important for the development of bacteriotherapies to promote urologic health.Methods: The objectives of the current report were to conduct a metaanalysis of 16S rRNA datasets derived from the kidney stone, stool, and urine samples of participants with or without urinary stone disease. To delineate the impact of rare and common phylotypes, metaanalyses were conducted by first separating rare and common taxa determined by both the frequency and abundance of amplicon sequence variants.Results: Consistent with previous analyses, we found that gut, upper urinary, and lower urinary tract microbiomes were all unique. Rare phylotypes comprised the majority of species observed in all sample types, with kidney stones exhibiting the greatest bias toward rarity, followed by urine and stool. Both rare and common fractions contributed significantly to the differences observed between sample types and health disparity. Furthermore, the rare and common fractions were taxonomically unique across all sample types. A total of 222 and 320 unique rare phylotypes from urine and stool samples were found to be significantly associated with USD. A co-occurrence correlation analysis revealed that rare phylotypes are most important for microbiome structure in stones, followed by urine and stool.Discussion: Collectively, the results indicate that rare phylotypes may be important for the pathophysiology of USD, particularly in the kidney stone matrix, which is inherently a very low microbial biomass niche that can have implications for the diagnosis and treatment of kidney stones. Further studies are needed to investigate the functional significance of rare phylotypes in kidney stone pathogenesis.

Diagnosis and treatment of urinary tract infections (UTIs) remains stagnant. The presumption that a patient either has a UTI or does not (binary choice) is inappropriately simplistic. Laboratory diagnostic tests have not advanced for decades. The goal of UTI treatment has not been rigorously defined and may increase the prescription of potentially harmful, inappropriate antibiotics. Despite the high incidence of UTI diagnoses, the high cost of UTI treatment, and increasing concerns associated with antimicrobial resistance, the development of novel and more accurate UTI tests has not been considered a priority, in part due to the general perception that current UTI care is already sufficient. In this review, we discuss the importance of improving UTI diagnostic testing to improve treatment outcomes. We discuss the problems associated with UTI diagnosis. Urinary microbes are alive and exist in both healthy and symptomatic individuals—urine is not sterile. We specifically outline the limitations of standard urine culture methods used by clinical microbiology laboratories, explaining clearly why such methods cannot be considered to be the “gold standard,” as standard culture methods underreport most of the urinary tract microbes, including some acknowledged and many emerging uropathogens. We do not recommend abandonment of this test, as no universally accepted substitute yet exists. However, we strongly encourage the development of new and improved diagnostic tests that can both improve outcomes and preserve antibiotic stewardship.

IntroductionThe development of molecular biology methods and their application in microbial research allowed the detection of many new pathogens that cause urinary tract infections (UTIs). Despite the advances of using new research techniques, the etiopathogenesis of UTIs, especially in patients undergoing dialysis and patients after kidney transplantation, is still not fully understood.MethodsThis study aimed to characterize and compare the composition of the bacterial element of the urinary tract microbiome between the groups of patients undergoing dialysis (n = 50) and patients after kidney transplantation (n = 50), with positive or negative urine culture, compared to healthy individuals (n = 50).ResultsAsymptomatic bacteriuria was observed in 30% of the urine cultures of patients undergoing dialysis and patients after kidney transplantation, with Escherichia coli as the most dominant microorganism (73%) detected with the use of classical microbiology techniques. However, differences in the bacterial composition of the urine samples between the evaluated patient groups were demonstrated using the amplicon sequencing. Finegoldia, Leptotrichia, and Corynebacterium were found to be discriminative bacteria genera in patients after dialysis and kidney transplantation compared to the control group. In addition, in all of urine samples, including those without bacteriuria in classical urine culture, many types of bacteria have been identified using 16S rRNA sequencing.DiscussionThe revealed microbial characteristics may form the basis in searching for new diagnostic markers in treatment of patients undergoing dialysis and patients after kidney transplantation.

Abstract Background Calcium oxalate (CaOx) uroliths are common in dogs. Humans with CaOx urolithiasis exhibit alterations of the urinary and urogenital microbiomes that might mediate urolith formation. Detection of urogenital microbes associated with CaOx in dogs could inform disease pathophysiology. Objective To identify compositional differences in the urogenital microbiome of Miniature Schnauzers with and without CaOx uroliths. Animals Nineteen midstream, voided urine samples from Miniature Schnauzers with (n = 9) and without (n = 10) a history of CaOx urolithiasis. Methods Analytical cross‐sectional study. Microbial DNA was extracted from previously frozen urine samples and sequenced for the bacterial 16S rRNA V3‐V4 hypervariable regions. Diversity and composition of microbial populations were compared between urolith formers and controls. Results Alpha and beta diversity measures were similar between groups. Five individual bacterial taxa differed in abundance (indicator values >0.5 and P

ABSTRACT Streptococcus anginosus is a prevalent member of the human flora. While it has been found in the microbiota of “healthy” asymptomatic individuals, it has also been associated with genitourinary tract infections and bacteremia. Based upon multilocus sequence analysis, two subspecies and two genomosubspecies have been characterized for the species. We previously conducted whole-genome sequencing of 85 S. anginosus isolates from the urinary tract. Here, we present genomic analysis of this species, including isolates from the urinary tract as well as gut and fecal, vaginal, oral, respiratory, and blood and heart samples. Average nucleotide identity and core genome analysis revealed that these strains form two distinct groups. Group 1 is comprised of the S. anginosus type strain and other previously identified S. anginosus subspecies and genomosubspecies, including isolates from throughout the human body. In contrast, group 2 consists of predominantly urinary streptococci (n = 77; 85.6%). Both of these S. anginosus groups are distinct from other members of the Streptococcus anginosus group (SAG) species S. intermedius and S. constellatus. Genes conserved among all strains of one group but not in any strains in the other group were next identified. Group 1 strains included genes found in S. intermedius and S. constellatus, suggesting that they were lost within the ancestor of the group 2 strains. In contrast, genes unique to the group 2 strains were homologous to more distant streptococci, indicative of acquisition via horizontal gene transfer. These genes are ideal candidates for use as marker genes to distinguish between the two groups in the human microbiota. IMPORTANCE Whole-genome analysis of S. anginosus strains provides greater insight into the diversity of this species than from marker genes alone. Our investigation of 166 publicly available S. anginosus genomes via average nucleotide identity and core genome analysis revealed two phylogenomically distinct groups of this species, with one group almost exclusively consisting of isolates from the urinary tract. In contrast, only 8 urinary strains were identified within the other group, which contained the S. anginosus type strain, as well as all identified subspecies and genomosubspecies. While genomic analysis suggested that this urinary group of S. anginosus is genomically different from the previously characterized S. anginosus subspecies, phenotypic characterization is still needed. Given prior reports of the prevalence of S. anginosus in the urinary tract of both continent and incontinent females, future studies are needed to investigate if the symptom state of the urinary tract is associated with these two different groups.

SUMMARYUrinary tract infection (UTI) is among the most common infections in clinical practice. In some cases, if left untreated, it can lead to pyelonephritis and urosepsis. In other cases, UTI resolves without treatment. Clinical diagnosis is typically based on patient symptoms and/or urinalysis, including urine dipsticks. The standard urine culture method is sometimes employed to identify the suspected urinary pathogen (uropathogen) and/or guide antimicrobial choice, but results are rarely available before 24 h. The standard urine culture method also misses fastidious, anaerobic, and slow-growing uropathogens and rarely reports polymicrobial infections. The unexplained combination of negative urine cultures with persistent urinary tract symptoms is distressing to both patients and clinicians. Given the broad appreciation of the advantages provided by rapid testing (e.g., for COVID-19 or influenza A), a rapid, accurate diagnostic test is needed to deliver timely treatment to patients seeking care for UTI that optimizes antibiotic stewardship. Herein, we discuss progress being made toward an accessible, timely (i.e., within hours), accurate assay with results that are clinically useful for the treating clinician within the timeframe of the infection (i.e., the growth rate of the pathogen(s)). New and emerging uropathogens often overlooked by current diagnostic techniques are also reviewed.

ABSTRACT Replicability is a well-established challenge in microbiome research with a variety of contributing factors at all stages, from sample collection to code execution. Here, we focus on voided urine sample storage conditions for urogenital microbiome analysis. Using urine samples collected from 10 adult females, we investigated the microbiome preservation efficacy of AssayAssure Genelock (Genelock), compared with no preservative, under different temperature conditions. We varied temperature over 48 h in order to examine the impact of conditions samples may experience with home voided urine collection and shipping to a central biorepository. The following common lab and shipping conditions were investigated: −20°C, ambient temperature, 4°C, freeze-thaw cycle, and heat cycle. At 48 h, all samples were stored at −80°C until processing. After generating 16S rRNA gene amplicon sequencing data using the highly sensitive KatharoSeq protocol, we observed individual variation in both alpha and beta diversity metrics below interhuman differences, corroborating reports of individual microbiome variability in other specimen types. While there was no significant difference in beta diversity when comparing Genelock versus no preservative, we did observe a higher concordance with Genelock samples shipped at colder temperatures (–20°C and 4°C) when compared with the samples shipped at −20°C without preservative. Our results indicate that Genelock does not introduce a significant amount of microbial bias when used on a range of temperatures and is most effective at colder temperatures. IMPORTANCE The urogenital microbiome is an understudied yet important human microbiome niche. Research has been stimulated by the relatively recent discovery that urine is not sterile; urinary tract microbes have been linked to health problems, including urinary infections, incontinence, and cancer. The quality of life and economic impact of UTIs and urgency incontinence alone are enormous, with $3.5 billion and $82.6 billion, respectively, spent in the United States. annually. Given the low biomass of urine, novelty of the field, and limited reproducibility evidence, it is critical to study urine sample storage conditions to optimize scientific rigor. Efficient and reliable preservation methods inform methods for home self-sample collection and shipping, increasing the potential use in larger-scale studies. Here, we examined both buffer and temperature variation effects on 16S rRNA gene amplicon sequencing results from urogenital samples, providing data on the consequences of common storage methods on urogenital microbiome results.