Urinary tract infections represent one of the most prevalent bacterial diseases, yet current diagnostic and research methodologies are hampered by inadequate culture media that fail to replicate the bladder biochemical environment. Conventional artificial urine formulations contain undefined components, lack essential nutrients, or inadequately support urinary microbiome (urobiome) growth. To address these limitations, we developed SimUrine, a fully defined synthetic urine medium that aims to replicate human bladder chemistry while supporting diverse microbial growth requirements. SimUrine was systematically developed through iterative optimization of multi-purpose artificial urine, incorporating defined concentrations of carbon sources, vitamins, trace elements, and amino acids within physiologically relevant ranges. The modular design enables component substitution without complete reformulation, facilitating customization for culturomics, antimicrobial susceptibility testing, and microbial ecology studies, while reducing batch-to-batch variability associated with authentic urine. Performance evaluation demonstrated SimUrine's capability to support the growth of fastidious urobiome members, including Lactobacillus species, Aerococcus urinae , and Corynebacterium riegelii , which fail to proliferate in conventional minimal media. Physicochemical characterization confirmed that SimUrine formulation exhibits properties within normal human urine ranges for density, conductivity, osmolarity, and viscosity, ensuring physiological relevance. Clinical applications revealed reduced antibiotic susceptibility compared to standard media, suggesting a more accurate representation of in vivo conditions. Co-culture experiments using Escherichia coli and Enterococcus faecalis demonstrated previously unobserved microbial interactions, highlighting SimUrine's utility for investigating urobiome dynamics. SimUrine represents a significant advancement in urobiome research methodology, providing a standardized, reproducible platform for investigating the urobiome under physiologically relevant conditions, potentially improving fundamental understanding and clinical diagnostic approaches. IMPORTANCE: Urinary tract infections (UTIs) affect millions globally, yet current research and diagnostic methods rely on inadequate culture media that fail to replicate the bladder's unique biochemical environment. This fundamental limitation has hindered accurate UTI research and potentially compromised clinical treatment decisions. SimUrine addresses this critical gap as the first fully defined synthetic urine medium that mimics human bladder chemistry while supporting the growth of diverse urinary microbes. The breakthrough enables the cultivation of urobiome organisms in a minimal medium that resembles natural conditions, revealing novel microbial interactions that influence urinary health. Crucially, SimUrine demonstrates different antimicrobial susceptibility patterns compared to standard clinical media, suggesting current testing protocols may inaccurately predict treatment outcomes. This standardized, reproducible platform eliminates the variability of authentic urine samples while maintaining physiological relevance, potentially transforming urobiome research methodology and providing a new tool for the study of UTIs worldwide.

Background/Objectives: Despite significant advancements in cancer treatment outcomes, side effects, such as gastrointestinal mucositis (GIM), continue to impair patients’ quality of life. The recent literature suggests L-citrulline as a novel treatment for GIM. However, no in vitro model to study the potential for L-citrulline as a treatment for GIM is currently available, and the effect of L-citrulline on the gut microbiota remains unclear. This study aims to propose a suitable in vitro model to study the effect of L-citrulline on the gut microbiota and to determine whether it can mitigate GIM. Methods: The CaCo-2 and T84 cell lines were cultured using cell impedance assays and treated with different doses of methotrexate and melphalan to select an appropriate model for L-citrulline research. The selected model was further used to investigate the impact of L-citrulline on gut microbiota cultured using microbial culture assays containing YCFAG. Results: Neither CaCo-2 nor T84 cells treated with methotrexate were suitable models for our study due to varying responses to treatment. T84 cells treated with 100 μg/mL melphalan demonstrated a consistent response, making them a suitable model for in vitro research on treatments for GIM. The use of L-citrulline demonstrated potential protective effects, as melphalan-treated enterocytes showed less cellular damage in its presence and slightly reduced enteroaggregative E. coli growth. Conclusions: L-Citrulline supplementation reduced epithelial cell injury due to melphalan, suggesting therapeutic potential. Further testing is required to determine its efficacy in vivo and clarify the mechanisms underlying this potential benefit.

Microbial communities are dynamic, adaptive ecosystems whose collective behavior emerges from metabolic interactions such as cross-feeding, competition, and cooperation, rather than taxonomic diversity or individual metabolic potential alone. This distinction is clinically significant in the postmenopausal urinary tract, where recurrent urinary tract infections (rUTIs) are associated with complex, persistent infection dynamics including multiple contributing bacterial species. The ability of resident microbial communities to prevent pathogen establishment, known as colonization resistance, is increasingly attributed to the metabolic interactions within the urobiome itself rather than any single resident species. However, current approaches, such as taxonomic profiling and classical differential abundance analysis, can only partially describe the presence or maintenance of such interactions. Consequently, the community-level metabolic architecture determining pathogen resistance remains incompletely understood. To address this gap, we developed PhenoRewire, a network-based framework that quantifies how metabolite co-variation is rewired between biological states using untargeted metabolomics data. We applied this framework to an induced pluripotent stem cell (iPSC) urothelial organoid-derived barrier co-cultured with synthetic urobiome communities as a model of urobiome-pathogen dynamics relevant to rUTIs in two approaches. In an infection model, clinically isolated uropathogens Escherichia coli and Enterococcus faecalis, were co-cultured with a three-member urobiome community consisting of Lactobacillus gasseri , Lactobacillus crispatus , and Gardnerella vaginalis . Here we show how E. coli drove the metabolic reorganization, while E. faecalis amplified it disproportionately. PhenoRewire disentangled the 6-fold metabolic network amplification mediated by E. faecalis as a metabolic facilitator, revealing an emergent urobiome-pathogen co-variation architecture (1,781 vs 227 edges) not recapitulated by either community alone. Moreover, in a six-member urobiome single-strain dropout experiment, we revealed that removal of the sole Actinomycete Winkia anitrata caused significant network collapse (Louvain modularity falls from 0.707 to 0.038), identifying it as the single non-redundant keystone of the community. More broadly, these results demonstrate how untargeted metabolomics co-variation network analysis can be applied to defined synthetic urobiomes in combination with a urothelial host model to elucidate community dynamics. This framework provides a template that can be extended beyond the urobiome to investigate any complex microbial community where ecological behavior remains an open question.

Urinary tract infections represent one of the most prevalent bacterial diseases, yet current diagnostic and research methodologies are hampered by inadequate culture media that fail to replicate the bladder biochemical environment. Conventional artificial urine formulations contain undefined components, lack essential nutrients, or inadequately support urinary microbiome (urobiome) growth. To address these limitations, we developed SimUrine, a fully defined synthetic urine medium that aims to replicate human bladder chemistry while supporting diverse microbial growth requirements. SimUrine was systematically developed through iterative optimization of multi-purpose artificial urine, incorporating defined concentrations of carbon sources, vitamins, trace elements, and amino acids within physiologically relevant ranges. The modular design enables component substitution without complete reformulation, facilitating customization for culturomics, antimicrobial susceptibility testing, and microbial ecology studies, while reducing batch-to-batch variability associated with authentic urine. Performance evaluation demonstrated SimUrine’s capability to support growth of fastidious urobiome members, including Lactobacillus species, Aerococcus urinae , and Corynebacterium riegelii , which fail to proliferate in conventional minimal media. Physicochemical characterization confirmed that SimUrine formulation exhibits properties within normal human urine ranges for density, conductivity, osmolarity, and viscosity, ensuring physiological relevance. Clinical applications revealed reduced antibiotic susceptibility compared to standard media, suggesting more accurate representation of in vivo conditions. Co-culture experiments using Escherichia coli and Enterococcus faecalis demonstrated previously unobserved microbial interactions, highlighting SimUrine’s utility for investigating urobiome dynamics. SimUrine represents a significant advancement in urobiome research methodology, providing a standardized, reproducible platform for investigating urobiome under physiologically relevant conditions, potentially improving fundamental understanding and clinical diagnostic approaches. IMPORTANCE: Urinary tract infections affect millions globally, yet current research and diagnostic methods rely on inadequate culture media that fail to replicate the bladder’s unique biochemical environment. This fundamental limitation has hindered accurate UTI research and potentially compromised clinical treatment decisions. SimUrine addresses this critical gap as the first fully defined synthetic urine medium that mimics human bladder chemistry while supporting growth of diverse urinary microbes. The breakthrough enables cultivation of urobiome organisms in a minimal medium that resembles natural conditions, revealing novel microbial interactions that influence urinary health. Crucially, SimUrine demonstrates different antimicrobial susceptibility patterns compared to standard clinical media, suggesting current testing protocols may inaccurately predict treatment outcomes. This standardized, reproducible platform eliminates the variability of authentic urine samples while maintaining physiological relevance, potentially transforming urobiome research methodology and improving clinical diagnostic accuracy for urinary tract infections worldwide.