An exploratory study investigating the impact of the bladder tumor microbiome on Bacillus Calmette Guerin (BCG) response in non-muscle invasive bladder cancer.

PURPOSE: Intravesical Bacillus Calmette-Guerin (BCG) is standard of care for intermediate- and high-risk non-muscle invasive bladder cancer (NMIBC). The effect of the bladder microbiome on response to BCG is unclear. We sought to characterize the microbiome of bladder tumors in BCG-responders and non-responders and identify potential mechanisms that drive treatment response. MATERIALS AND METHODS: Patients with archival pre-treatment biopsy samples (2012-2018) were identified retrospectively. Prospectively, urine and fresh tumor samples were collected from individuals with high-risk NMIBC (2020-2023). BCG response was defined as tumor-free 2 years from induction therapy. Extracted DNA was sequenced for 16S rRNA and shotgun metagenomics. Primary outcomes were species richness (α-diversity) and microbial composition (ß-diversity). Paired t-tests were performed for α-diversity (Observed species/Margalef). Statistical analysis for ß-diversity (weighted and unweighted UniFrac distances, weighted Bray-Curtis dissimilarity) were conducted through Permanova, with 999 permutations. RESULTS: Microbial species richness (P < 0.001) and composition (P = 0.001) differed between BCG responders and non-responders. Lactobacillus spp. were significantly enriched in BCG-responders. Shotgun metagenomics identified possible mechanistic pathways such as assimilatory sulfate reduction. CONCLUSION: A compositional difference exists in the tumor microbiome of BCG responders and non-responders with Lactobacillus having increased abundance in BCG responders.

Template switching between the leading and lagging strands at replication forks generates inverted copy number variants through hairpin-capped extrachromosomal DNA.

Inherited and germ-line de novo copy number variants (CNVs) are increasingly found to be correlated with human developmental and cancerous phenotypes. Several models for template switching during replication have been proposed to explain the generation of these gross chromosomal rearrangements. We proposed a model of template switching (ODIRA-origin dependent inverted repeat amplification) in which simultaneous ligation of the leading and lagging strands at diverging replication forks could generate segmental inverted triplications through an extrachromosomal inverted circular intermediate. Here, we created a genetic assay using split-ura3 cassettes to trap the proposed inverted intermediate. However, instead of recovering circular inverted intermediates, we found inverted linear chromosomal fragments ending in native telomeres-suggesting that a template switch had occurred at the centromere-proximal fork of a replication bubble. As telomeric inverted hairpin fragments can also be created through double strand breaks we tested whether replication errors or repair of double stranded DNA breaks were the most likely initiating event. The results from CRISPR/Cas9 cleavage experiments and growth in the replication inhibitor hydroxyurea indicate that it is a replication error, not a double stranded break that creates the inverted junctions. Since inverted amplicons of the SUL1 gene occur during long-term growth in sulfate-limited chemostats, we sequenced evolved populations to look for evidence of linear intermediates formed by an error in replication. All of the data are compatible with a two-step version of the ODIRA model in which sequential template switching at short inverted repeats between the leading and lagging strands at a replication fork, followed by integration via homologous recombination, generates inverted interstitial triplications.

Penile prostheses harbor biofilms driven by individual variability and manufacturer even in the absence of clinical infection.

BACKGROUND: Culture-based studies have shown that penile prostheses harbor biofilms in the presence and absence of infection, but these findings have not been adequately validated using contemporary microbiome analytic techniques. AIM: The study sought to characterize microbial biofilms of indwelling penile prosthesis devices according to patient factors, device components, manufacturer, and infection status. METHODS: Upon penile prostheses surgical explantation, device biofilms were extracted, sonicated, and characterized using shotgun metagenomics and culture-based approaches. Device components were also analyzed using scanning electron microscopy. OUTCOMES: Outcomes included the presence or absence of biofilms, alpha and beta diversity, specific microbes identified and the presence of biofilm, and antibiotic resistance genes on each prosthesis component. RESULTS: The average age of participants from whom devices were explanted was 61 ± 11 years, and 9 (45%) of 20 had a diagnosis of diabetes mellitus. Seventeen devices were noninfected, and 3 were associated with clinical infection. Mean device indwelling time prior to explant was 5.1 ± 5.1 years. All analyzed components from 20 devices had detectable microbial biofilms, both in the presence and absence of infection. Scanning electron microscopy corroborated the presence of biofilms across device components. Significant differences between viruses, prokaryotes, and metabolic pathways were identified between individual patients, device manufacturers, and infection status. Mobiluncus curtisii was enriched in manufacturer A device biofilms relative to manufacturer B device biofilms. Bordetella bronchialis, Methylomicrobium alcaliphilum, Pseudoxanthomonas suwonensis, and Porphyrobacter sp. were enriched in manufacturer B devices relative to manufacturer A devices. The most abundant bacterial phyla were the Proteobacteria, Actinobacteria, and Firmicutes. Glycogenesis, the process of glycogen synthesis, was among the predominant metabolic pathways detected across device components. Beta diversity of bacteria, viruses, protozoa, and pathways did not differ among device components. CLINICAL IMPLICATIONS: All components of all penile prostheses removed from infected and noninfected patients have biofilms. The significance of biofilms on noninfected devices remains unknown and merits further investigation. STRENGTHS AND LIMITATIONS: Strengths include the multipronged approach to characterize biofilms and being the first study to include all components of penile prostheses in tandem. Limitations include the relatively few number of infected devices in the series, a relatively small subset of devices included in shotgun metagenomics analysis, and the lack of anaerobic and other expanded conditions for culture. CONCLUSION: Penile prosthesis biofilms are apparent in the presence and absence of infection, and the composition of biofilms was driven primarily by device manufacturer, individual variability, and infection, while being less impacted by device component.

Microbe-metabolite interaction networks, antibiotic resistance, and in vitro reconstitution of the penile prosthesis biofilm support a paradigm shift from infection to colonization.

To understand differences between asymptomatic colonized and infected states of indwelling medical devices, we sought to determine penile prosthesis biofilm composition, microbe-metabolite interaction networks, and association with clinical factors. Patients scheduled for penile prosthesis removal/revision were included. Samples from swabbed devices and controls underwent next-generation sequencing, metabolomics, and culture-based assessments. Biofilm formation from device isolates was reconstituted in a continuous-flow stir tank bioreactor. 93% of 27 analyzed devices harbored demonstrable biofilm. Seven genera including Faecalibaculum and Jeotgalicoccus were more abundant in infected than uninfected device biofilms (p < 0.001). Smokers and those with diabetes mellitus or cardiac disease had lower total normalized microbial counts than those without the conditions (p < 0.001). We identified microbe-metabolite interaction networks enriched in devices explanted for infection and pain. Biofilm formation was recapitulated on medical device materials including silicone, PTFE, polyurethane, and titanium in vitro to facilitate further mechanistic studies. Nearly all penile prosthesis devices harbor biofilms. Staphylococcus and Escherichia, the most common causative organisms of prosthesis infection, had similar abundance irrespective of infection status. A series of other uncommon genera and metabolites were differentially abundant, suggesting a complex microbe-metabolite pattern-rather than a single organism-is responsible for the transition from asymptomatic to infected or painful states.

Ureteral Stents Harbor Complex Biofilms With Rich Microbiome-Metabolite Interactions.

PURPOSE: We sought to determine microbe-metabolite composition and interactions within indwelling ureteral stent biofilms, determine their association with patient factors including infection, and reconstitute biofilm formation on relevant surface materials in vitro. MATERIALS AND METHODS: Upon ureteral stent removal from patients, proximal and distal ends were swabbed. Samples were analyzed by 16S next-generation sequencing and metabolomics. A continuous-flow stir-tank bioreactor was used to reconstitute and quantify in vitro biofilm formation from stent-isolated bacteria on stent-related materials including silicone, polytetrafluoroethylene, polyurethane, polycarbonate, and titanium. Diversity, relative abundance, and association with clinical factors were analyzed with ANOVA and Bonferroni t-tests or PERMANOVA. Biofilm deposition by microbial strain and device material type were analyzed using plate counts and scanning electron microscopy following bioreactor incubation. RESULTS: All 73 samples from 37 ureteral stents harbored microbiota. Specific genera were more abundant in samples from stents wherein there was antibiotic exposure during indwelling time (Escherichia/Shigella, Pseudomonas, Staphylococcus, Ureaplasma) and in those associated with infection (Escherichia/Shigella, Ureaplasma). The enriched interaction subnetwork in stent-associated infection included Ureaplasma and metabolite 9-methyl-7-bromoeudistomin. Strains identified as clinically relevant and central to interaction networks all reconstituted biofilm in vitro, with differential formation by strain (Enterococcus faecalis most) and material type (titanium least). CONCLUSIONS: Ureteral stent biofilms exhibit patterns unique to stent-associated infection and antibiotic exposure during indwelling time. Microbes isolated from stents reconstituted biofilm formation in vitro. This work provides a platform to test novel materials, evaluate new coatings for anti-biofilm properties, and explore commensal strain use for bacterial interference against pathogens.

Biofilms on Indwelling Artificial Urinary Sphincter Devices Harbor Complex Microbe-Metabolite Interaction Networks and Reconstitute Differentially In Vitro by Material Type.

The artificial urinary sphincter (AUS) is an effective treatment option for incontinence due to intrinsic sphincteric deficiency in the context of neurogenic lower urinary tract dysfunction, or stress urinary incontinence following radical prostatectomy. A subset of AUS devices develops infection and requires explant. We sought to characterize biofilm composition of the AUS device to inform prevention and treatment strategies. Indwelling AUS devices were swabbed for biofilm at surgical removal or revision. Samples and controls were subjected to next-generation sequencing and metabolomics. Biofilm formation of microbial strains isolated from AUS devices was reconstituted in a bioreactor mimicking subcutaneous tissue with a medical device present. Mean patient age was 73 (SD 10.2). All eighteen artificial urinary sphincter devices harbored microbial biofilms. Central genera in the overall microbe−metabolite interaction network were Staphylococcus (2620 metabolites), Escherichia/Shigella (2101), and Methylobacterium-Methylorubrum (674). An rpoB mutation associated with rifampin resistance was detected in 8 of 15 (53%) biofilms. Staphylococcus warneri formed greater biofilm on polyurethane than on any other material type (p < 0.01). The results of this investigation, wherein we comprehensively characterized the composition of AUS device biofilms, provide the framework for future identification and rational development of inhibitors and preventive strategies against device-associated infection.

Probiotic Oxalate-Degrading Bacteria: New Insight of Environmental Variables and Expression of the oxc and frc Genes on Oxalate Degradation Activity.

Oxalate, a compound produced by many edible plants and as a terminal metabolite in the liver of mammals, is a toxin that has a detrimental role to human health. Humans and other mammals do possess enzymatic systems to degrade oxalate. Moreover, numerous oxalate-degrading bacteria reside in the mammalian gut and, thus, provide an important function for hosts. The current review focuses on the environmental factors that influence the efficacy of probiotic oxalate-degrading bacteria, relative to oxalate metabolism. We describe the mechanism of oxalate catabolism and its consumption by obligate and facultative anaerobic oxalate-degrading bacteria, in both in vitro and in vivo environments. We also explore the environmental variables that impact oxalate degradation. Studies on single species degrade oxalate have not shown a strong impact on oxalate metabolism, especially in high oxalate conditions such as consumption of foods high in oxalate (such as coffee and chocolate for humans or halogeton in animal feed). Considering effective variables which enhance oxalate degradation could be used in application of effective probiotic as a therapeutic tool in individuals with hyperoxaluria. This study indicates probiotics can be considered a good source of naturally occurring oxalate degrading agent in human colon.

Mechanisms of the intestinal and urinary microbiome in kidney stone disease.

Kidney stone disease affects ~10% of the global population and the incidence continues to rise owing to the associated global increase in the incidence of medical conditions associated with kidney stone disease including, for example, those comprising the metabolic syndrome. Considering that the intestinal microbiome has a substantial influence on host metabolism, that evidence has suggested that the intestinal microbiome might have a role in maintaining oxalate homeostasis and kidney stone disease is unsurprising. In addition, the discovery that urine is not sterile but, like other sites of the human body, harbours commensal bacterial species that collectively form a urinary microbiome, is an additional factor that might influence the induction of crystal formation and stone growth directly in the kidney. Collectively, the microbiomes of the host could influence kidney stone disease at multiple levels, including intestinal oxalate absorption and direct crystal formation in the kidneys.

The Bladder Microbiome, Metabolome, Cytokines, and Phenotypes in Patients with Systemic Lupus Erythematosus.

Emerging studies reveal unique bacterial communities in the human bladder, with alteration of composition associated to disease states. Systemic lupus erythematosus (SLE) is a complex autoimmune disease that is characterized by frequent impairment of the kidney. Here, we explored the bladder microbiome, metabolome, and cytokine profiles in SLE patients, as well as correlations between microbiome and metabolome, cytokines, and disease profiles. We recruited a group of 50 SLE patients and 50 individually matched asymptomatic controls. We used transurethral catheterization to collect urine samples, 16S rRNA gene sequencing to profile bladder microbiomes, and liquid chromatography-tandem mass spectrometry to perform untargeted metabolomic profiling. Compared to controls, SLE patients possessed unique bladder microbial communities and increased alpha diversity. These differences were accompanied by differences in urinary metabolomes, cytokines, and patients' disease profiles. The SLE-enriched genera, including Bacteroides, were positively correlated with several SLE-enriched metabolites, including olopatadine. The SLE-depleted genera, such as Pseudomonas, were negatively correlated to SLE-depleted cytokines, including interleukin-8. Alteration of the bladder microbiome was associated with disease profile. For example, the genera Megamonas and Phocaeicola were negatively correlated with serum complement component 3, and Streptococcus was positively correlated with IgG. Our present study reveals associations between the bladder microbiome and the urinary metabolome, cytokines, and disease phenotypes. Our results could help identify biomarkers for SLE. IMPORTANCE Contrary to dogma, the human urinary bladder possesses its own unique bacterial community with alteration of composition associated with disease states. Systemic lupus erythematosus (SLE) is a complex autoimmune disease often characterized by kidney impairment. Here, we explored the bladder microbiome, metabolome, and cytokine profiles in SLE patients, as well as correlations between the microbiome and metabolome, cytokines, and disease profiles. Compared to controls, SLE patients possessed a unique bladder microbial community and elevated alpha diversity. These differences were accompanied by differences in bladder metabolomes, cytokines, and patients' disease profiles. SLE-enriched genera were positively correlated with several SLE-enriched metabolites. SLE-depleted genera were negatively correlated to SLE-depleted cytokines. Alteration of the bladder microbiome was associated with disease profile. Thus, our study reveals associations between the bladder microbiome and the bladder metabolome, cytokines, and disease phenotypes. These results could help identify biomarkers for SLE.

Delineating the Role of the Urinary Metabolome in the Lithogenesis of Calcium-Based Kidney Stones.

OBJECTIVE: To delineate the role of the urinary metabolome in the genesis of urinary stone disease (USD). METHODS: Untargeted metabolomics was utilized in comparative analyses of calcium-based stones (CBS) and spot urine samples from patients with a history of USD with or without urinary stone activity based on radiologic imaging. Stone and urine metabolomes were stratified by composition and radiographic stone-activity, respectively. Additionally, we quantified highly abundant metabolites that were present in either calcium oxalate (CaOx) or calcium phosphate (CaPhos) stones and also significantly enriched in the urine of active stone formers (SF) compared to non-active SF. These data were used to delineate either a direct involvement of urinary metabolites in lithogenesis or the passive uptake of biomolecules within the stone matrix. RESULTS: Urinary metabolomes were distinct based on radiographic stone-activity and the 2 types of CBS. Stratification by radiologic stone activity was driven by the enrichment of 14 metabolites in the urine of active SF that were also highly abundant in both CaOx and CaPhos stones, indicative of a potential involvement of these metabolites in lithogenesis. Using the combination of these 14 metabolites in total, we generated a model that correctly classified patients as either active vs non-active SF in a prospectively recruited cohort with 73% success. CONCLUSION: Collectively, our data suggest specific urinary metabolites directly contribute to the formation of urinary stones and that active SF may excrete higher levels of lithogenic metabolites than non-active patients. Future studies are needed to confirm these findings and establish the causative mechanisms associated with these metabolites.

Transperineal Prostate Biopsy is Associated With Lower Tissue Core Pathogen Burden Relative to Transrectal Biopsy: Mechanistic Underpinnings for Lower Infection Risk in the Transperineal Approach.

OBJECTIVE: To understand the mechanistic basis for reduced infectious complications in transperineal (TP) prostate biopsy, we sought to determine whether TP prostate biopsy is associated with a lower degree of pathogen introduction into the prostate relative to transrectal (TR) biopsy. MATERIALS AND METHODS: In men scheduled for prostate biopsy for standard clinical indications, rectal and perineal skin swabs, and 2 extra biopsy cores, were obtained. Specimens underwent DNA extraction followed by next-generation sequencing and standard laboratory culture. Microbial quantity and composition were determined and compared between prostate core biopsy tissue from individuals who underwent TP vs TR biopsy. RESULTS: Twenty-three men were accrued to the study. Biopsy core tissue from the TP group had less microbial diversity (15.0 vs 25.8 phylogenetic clades/sample, P = .0004) and had a lower quantity of known pathogens (36.3 vs 104.2 normalized counts of pathogens/sample, P = .018) relative to the TR group. TP group tissue core flora was more attributable to the perineal than rectal source (P = .047). Viable Escherichia coli was isolated from 45% of the TR group cores, but none in the TP group (P = .014). CONCLUSION: Biopsy tissue from individuals who undergo TP biopsy harbors a lower human pathogenic bacterial load than those who undergo TR biopsy, with a minimal risk of viable E. coli. Our results elucidate a possible mechanism for reduced infectious risk associated with TP biopsy relative to TR biopsy and a rational basis for widespread implementation of TP biopsy.

Comparative functional analysis of the urinary tract microbiome for individuals with or without calcium oxalate calculi.

Individuals with urinary stone disease (USD) exhibit dysbiosis in the urinary tract and the loss of Lactobacillus that promote urinary tract health. However, the microbial metabolic functions that differentiate individuals with USD from healthy individuals are unknown. The objective of the current study was to determine the microbial functions across prokaryotic, viral, fungal, and protozoan domains that are associated with calcium oxalate (CaOx) stone formers through comparative shotgun metagenomics of midstream, voided urine samples for a small number of patients (n = 5 CaOx stone formers, n = 5 healthy controls). Results revealed that CaOx stone formers had reduced levels of genes associated with oxalate metabolism, as well as transmembrane transport, proteolysis, and oxidation-reduction processes. From 17 draft genomes extracted from the data and > 42,000 full length reference genomes, genes enriched in the Control group mapped overwhelming to Lactobacillus crispatus and those associated with CaOx mapped to Pseudomonas aeruginosa and Burkholderia sp. The microbial functions that differentiated the clinical cohorts are associated with known mechanisms of stone formation. While the prokaryotes most differentiated the CaOx and Control groups, a diverse, trans-domain microbiome was apparent. While our sample numbers were small, results corroborate previous studies and suggest specific microbial metabolic pathways in the urinary tract that modulate stone formation. Future studies that target these metabolic pathways as well as the influence of viruses, fungi, and protozoa on urinary tract physiology is warranted.

Tranexamic acid in patients with complex stones undergoing percutaneous nephrolithotomy: a randomised, double-blinded, placebo-controlled trial.

OBJECTIVES: To assess the efficacy and safety of single-dose tranexamic acid on the blood transfusion rate and outcomes of patients with complex kidney stones undergoing percutaneous nephrolithotomy (PCNL). PATIENTS AND METHODS: In a randomised, double-blinded, placebo-controlled trial, 192 patients with complex kidney stone (Guy's Stone Scores III-IV) were prospectively enrolled and randomised (1:1 ratio) to receive either one dose of tranexamic acid (1 g) or a placebo at the time of anaesthetic induction for PCNL. The primary outcome measure was the occurrence rate of perioperative blood transfusion. The secondary outcome measures included blood loss, operative time, stone-free rate (SFR), and complications. ClinicalTrials.gov identifier: NCT02966236. RESULTS: The overall risk of receiving a blood transfusion was reduced in the tranexamic acid group (2.2% vs 10.4%; relative risk, 0.21, 95% confidence interval [CI] 0.03-0.76, P = 0.033; number-needed-to-treat: 12). Patients randomised to the tranexamic acid group had a higher immediate and 3-month SFR compared with those in the placebo group (29% vs 14.7%, odds ratio [OR] 2.37, 95% CI 1.15-4.87, P = 0.019, and 46.2% vs 28.1%, OR 2.20, 95% CI 1.20-4.02, P = 0.011, respectively). Faster haemoglobin recovery occurred in patients in the tranexamic acid group (mean, 21.3 days; P = 0.001). No statistical differences were found in operative time and complications between groups. CONCLUSIONS: Tranexamic acid administration is safe and reduces the need for blood transfusion by five-times in patients with complex kidney stones undergoing PCNL. Moreover, tranexamic acid may contribute to better stone clearance rate and faster haemoglobin recovery without increasing complications. A single dose of tranexamic acid at the time of anaesthetic induction could be considered standard clinical practice for patients with complex kidney stones undergoing PCNL.

Meta-analysis of Clinical Microbiome Studies in Urolithiasis Reveal Age, Stone Composition, and Study Location as the Predominant Factors in Urolithiasis-Associated Microbiome Composition.

To determine whether functionally relevant questions associated with the urinary or gut microbiome and urinary stone disease (USD) can be answered from metagenome-wide association studies (MWAS), we performed the most comprehensive meta-analysis of published clinical MWAS in USD to date, using publicly available data published prior to April 2021. Six relevant studies met inclusion criteria. For alpha-diversity, significant differences were noted between USD status, stone composition, sample type, study location, age, diet, and sex. For beta-diversity, significant differences were noted by USD status, stone composition, sample type, study location, antibiotic use (30 days and 12 months before sampling), sex, hypertension, water intake, body habitus, and age. Prevotella and Lactobacillus in the gut and urinary tract, respectively, were associated with healthy individuals, while Enterobacteriaceae was associated with USD in the urine and stones. Paradoxically, other Prevotella strains were also strongly associated with USD in the gut microbiome. When data were analyzed together, USD status, stone composition, age group, and study location were the predominant factors associated with microbiome composition. Meta-analysis showed significant microbiome differences based on USD status, stone composition, age group or study location. However, analyses were limited by a lack of public data from published studies, metadata collected, and differing study protocols. Results highlight the need for field-specific standardization of experimental protocols in terms of sample collection procedures and the anatomical niches to assess, as well as in defining clinically relevant metadata and subphenotypes such as stone composition. IMPORTANCE Studies focused on the microbiome broadly support the hypothesis that the microbiome influences the onset of chronic diseases such as urinary stone disease. However, it is unclear what environmental factors shape the microbiome in ways that increase the risk for chronic disease. In addition, it is unclear how differences in study methodology can impact the results of clinical metagenome-wide association studies. In the current meta-analysis, we show that age, stone composition, and study location are the predominant factors that associate with the microbiome and USD status. Furthermore, we reveal differences in results based on specific analytical protocols, which impacts the interpretation of any microbiome study.

Multiplexing mutation rate assessment: determining pathogenicity of Msh2 variants in Saccharomyces cerevisiae.

Despite the fundamental importance of mutation rate as a driving force in evolution and disease risk, common methods to assay mutation rate are time-consuming and tedious. Established methods such as fluctuation tests and mutation accumulation experiments are low-throughput and often require significant optimization to ensure accuracy. We established a new method to determine the mutation rate of many strains simultaneously by tracking mutation events in a chemostat continuous culture device and applying deep sequencing to link mutations to alleles of a DNA-repair gene. We applied this method to assay the mutation rate of hundreds of Saccharomyces cerevisiae strains carrying mutations in the gene encoding Msh2, a DNA repair enzyme in the mismatch repair pathway. Loss-of-function mutations in MSH2 are associated with hereditary nonpolyposis colorectal cancer, an inherited disorder that increases risk for many different cancers. However, the vast majority of MSH2 variants found in human populations have insufficient evidence to be classified as either pathogenic or benign. We first benchmarked our method against Luria-Delbrück fluctuation tests using a collection of published MSH2 missense variants. Our pooled screen successfully identified previously characterized nonfunctional alleles as high mutators. We then created an additional 185 human missense variants in the yeast ortholog, including both characterized and uncharacterized alleles curated from ClinVar and other clinical testing data. In a set of alleles of known pathogenicity, our assay recapitulated ClinVar's classification; we then estimated pathogenicity for 157 variants classified as uncertain or conflicting reports of significance. This method is capable of studying the mutation rate of many microbial species and can be applied to problems ranging from the generation of high-fidelity polymerases to measuring the frequency of antibiotic resistance emergence.

Standardization of microbiome studies for urolithiasis: an international consensus agreement.

Numerous metagenome-wide association studies (MWAS) for urolithiasis have been published, leading to the discovery of potential interactions between the microbiome and urolithiasis. However, questions remain about the reproducibility, applicability and physiological relevance of these data owing to discrepancies in experimental technique and a lack of standardization in the field. One barrier to interpreting MWAS is that experimental biases can be introduced at every step of the experimental pipeline, including sample collection, preservation, storage, processing, sequencing, data analysis and validation. Thus, the introduction of standardized protocols that maintain the flexibility to achieve study-specific objectives is urgently required. To address this need, the first international consortium for microbiome in urinary stone disease - MICROCOSM - was created and consensus panel members were asked to participate in a consensus meeting to develop standardized protocols for microbiome studies if they had published an MWAS on urolithiasis. Study-specific protocols were revised until a consensus was reached. This consensus group generated standardized protocols, which are publicly available via a secure online server, for each step in the typical clinical microbiome-urolithiasis study pipeline. This standardization creates the benchmark for future studies to facilitate consistent interpretation of results and, collectively, to lead to effective interventions to prevent the onset of urolithiasis, and will also be useful for investigators interested in microbiome research in other urological diseases.