A microphysiological human mini-bladder reveals urine-urothelium interplay in tissue resilience and UPEC recurrence in urinary tract infections

Domain: neuroscience
Published: 2026-02-03
Editorial outcome: major_revision

Abstract
Urine is a dynamic and highly variable biofluid. Urine-urothelium interactions are a critical yet underexplored factor in bladder homoeostasis and urinary tract infections (UTIs). Here, we report on a human ‘mini-bladder’ model that exposes a stratified urothelium to urine of defined composition, and incorporates micturition. Prolonged exposure to high-solute concentration urine weakens tight junctions, dysregulates immune responses, and reduces bladder tissue resilience. This increases susceptibility to colonisation of the bladder by uropathogenic Escherichia coli (UPEC) which reduces efficacy of antibiotic therapy. In high-solute concentration urine, Fosfomycin monotherapy – prescribed for uncomplicated UTIs, induces the formation of cell wall-deficient (CWD) UPEC in the urine (as observed in patients with recurrent UTIs) but also within deeper urothelial layers. Tissue-associated CWD UPEC directly contributes to recurrence. Our findings expand the conceptual role for CWD UPEC in UTIs, and demonstrate the power of the mini-bladder platform to capture urine-urothelial microenvironment dynamics that actively shape UTI pathogenesis and antibiotic tolerance.

Introduction
The bladder is a highly dynamic organ that undergoes substantial mechano-physiological fluctuations1–3 while retaining urine of varying composition and toxicity4,5. These fluctuations are accommodated by the bladder epithelium that functions as a critical mucosal barrier. The bladderepithelium actively modulates urine composition6–8, absorbs more salts and urea in dehydrated conditions9–12. In experiments in animal models, including rodents, urine with high concentrations of solutes in turn damages the integrity of the urothelium, causing tight junction blebbing, bladder interstitial congestion, increased apoptosis signalling and general irritation13–16. An effective and long-term colonisation of the bladder by pathogens depends on a dynamic interaction between the urothelium and urine. It is increasingly evident that this urothelial niche promotes persistence and antibiotic tolerance in urinary tract infections (UTIs)17–20, which characteristically have a high recurrence rate in susceptible individuals21.

The impact of urothelial niches and bladder dynamics is evident in the life cycle of uropathogenic Escherichia coli (UPEC), the most common causative pathogen of UTIs22. UPEC has developed multifaceted survival and propagation strategies within the complex and variable environment of urine and the urothelium. UPEC proliferates in urine, colonises the urine microbiome, and adheres strongly to the urothelium23. A subset of this bacterial population enters superficial umbrella cells, where they can grow to form larger reservoirs as intracellular bacterial communities (IBCs) that are shielded from the immune system24–26. Consequently, the upper cell layers of the urothelium actively exfoliate to reduce the bacterial load on the tissue, releasing part of the invaded UPEC back into the urine27. Deeper in the tissue, UPEC can survive in smaller intracellular clusters termed quiescent intracellular reservoir (QIRs) long after superficial inflammation has ceased18,28,29. More recent work has shown that the bladder epithelium can be rapidly populated by solitary bacteria that are intracellular or even pericellular30. Finally, UPEC in urine samples from patients treated with cell wall targeting antibiotics such as Fosfomycin have been isolated in cell wall deficient (CWD) L-forms31–33. These are particularly prominent in the urine of recurrent UTI (rUTI) patients32, suggesting a link between CWD forms and re-infection of tissue when external stressors are released. This large range of bacterial forms within a single tissue highlight the need to understand pathogenesis and therapy in a dynamic fashion across two very different microenvironments in direct interaction with each other—first, a stratified urothelium and second, urine with variable composition and solute concentration. Examination of bacteria in the tissue is only possible as an endpoint measurement in animal models, which limits dynamical information, whereas human urine has a wide range of solute concentrations (osmolarity) across healthy34 and diseased35 individuals, which is difficult to replicate in mice under laboratory conditions.

In recent years, in-vitro microphysiological models that replicate aspects of the bladder microenvironment have been used to study UTIs36–39. For example, the bladder organoid model, albeit a closed system, offers a stratified tissue with medium-throughput and possibilities to incorporate immune cells30. This model is complemented by the bladder-on-chip that lacks stratification but incorporates micturition cycles, exposure to diluted pooled urine and vascular flow40. Building on this, the 3D urine-tolerant human urothelial (3D-UHU) model has a robust stratified tissue that remains viable when exposed to undiluted pooled urine, although it doesn’t recapitulate micturition effects41. These models have complementary strengths and weaknesses, but so far, no single model incorporates a stratified, well-differentiated urothelium, an accessible lumen for urine perfusion and micturition, and the ability to stretch the tissue42—the three features that in combination are needed to capture the interaction of urine, urothelium and the respective niches formed by UPEC.

Here, we present a human ‘mini-bladder’ organoid model that meets these requirements. The tissue has multi-layer stratification and polarised differentiation, with an apical layer of umbrella cells and a tight tissue barrier exposed to urine. Micturition effects are established through induced urine flow or pressure-controlled tissue stretching. Consistent with data from animal models14,16,43, long-term exposure of the mini-bladder urothelium to urine of high solute concentration supresses epithelial immune responses, reduces barrier function and increases the incidence of cell death. Through high-resolution imaging of untreated infections over several micturition cycles, we capture the dynamics of interactions between UPEC in urine (whose solute concentration can be controlled) and the bladder tissue. Interestingly, we show that long-term exposure to urine with high solute concentration reduces tissue resilience to infection, leads to an increased accumulation of tissue-associated UPEC and reduced efficacy of antibiotic clearance. Next, we explore the differential behaviours of these bacterial sub-populations to antibiotic therapy. Cell wall targeting antibiotics like Fosfomycin generate cell-wall deficient (CWD) UPEC survivors in high concentration urine in the mini-bladder, replicating the observation from rUTI patients’ urine in a microtissue environment. We demonstrate a direct role for CWD UPEC in the urine for reseeding infections, and unexpectedly, we find that CWD UPEC that survive antibiotic can also be found in the deeper layers of the urothelium, revealing a new mechanism in the tissue that may contribute to the recurrence of infection. Collectively, this study shows that the mini-bladder serves as a powerful in vitro model for capturing the complete interplay between tissue and urine in UTIs.

Results
Mini-bladder recreates human urothelial architecture with tissue stretch and urine flow
We developed a human bladder microtissue model built on the hydrogel-based perfusable microfluidic architecture derived from Nikolaev et al.44 (Fig. 1A, Supplementary Fig 1A–D). Primary human bladder epithelial cells were seeded into the central channel of the device, and once a stratified tissue was obtained at day 15, this was exposed to urine on the apical side and nutritive media on the basal side for a further 5–7 days (Supplementary Fig 1E). The resultant tissue at day 20 (Fig. 1B, Supplementary Movie 1, Supplementary Movie 2) recreates the organisation of the human bladder epithelium. Accurate recreation of umbrella cells is critical to ensure stability of bladder tissue when exposed to urine45. In the mini-bladder, cells on the apical layer proximal to the lumen were CK20+ (Fig. 1 C, Supplementary Fig 2A, C), UP3A+ (Supplementary Fig 2D), UP1A+ (Supplementary Fig 2E), and in several instances bi-nucleated with honeycomb like structure (Supplementary Fig 2B, Fig. 1D), consistent with the known characterisation of the superficial umbrella cell layer45. In contrast, cells nearer the hydrogel scaffold expressed the basal/intermediate cell marker CK8+46 (Fig. 1E, Supplementary Fig 2F). CK13+ cells47, marking the differentiation of basal-like squamous to a transitional differentiated cells in vivo, were also observed within the stratified tissue (Fig. 1E, Supplementary Fig 2A, G). The increased expression of UP3A48, KRT20, KRT8 and KRT13 in the mini-bladder relative to the cells seeded into the device was confirmed by qRT-PCR analyses as well (Fig. 1F). An upregulation of KRT1449, a marker for a subset of basal cells capable of self-renewal and TP6350, a common progenitor cell marker, was also observed (Fig. 1F). The mini-bladder model, therefore, has appropriate stem-cell niches required for urothelial renewal, relevant for modelling the tissue response to infection or sterile injury. The stratified tissue maintains a robust barrier; strong ZO151 staining is observed on the entire apical surface lining the lumen (Fig. 1G, Supplementary Fig 2H). Barrier integrity was also confirmed by a dextran dyediffusion test (Fig. 1H). The diffusion of dextran out of the lumen into the hydrogel was 92.8 % lower in a fully differentiated mini-bladder compared to an empty device, and the diffusion decreased over time, consistent with the differentiation of a stratified tissue from a monolayer (Fig. 1I).

The robust urothelium in the mini-bladder model could be subjected to changes in bladder volume mimicking micturition by controlled hydraulic pressure differences between the inlet and outlet (S. Mov-3). When stretched over 30s, the urothelial physiology was rapidly altered from larger cuboidal cells to a tightly-packed structure with thinner, flattened apical cells (Fig. 1J, Supplementary Fig 3A, B), in good agreement with images from stretched and relaxed bladder explants52–54. Furthermore, tight junction contacts are evident in the lumenal cells (Supplementary Fig 3C), and this is enhanced overall in the tissue upon stretch53 (Fig. 1J). One mechanism by which the bladder adapts to rapid volume changes is through the presence of cytoplasmic vesicles in the umbrella cells that have been shown to transport to and fuse with the apical membrane to increase its surface area upon bladder filling52. Consistently, we observed the presence of several cytoplasmic vesicles in the unstretched tissue (Supplementary Fig 3C, D) and an overall reduction in the number of vesicles in the cytoplasm post-stretching (Fig. 1K).

Together, these results confirm that the mini-bladder microtissue model contains a stratified, differentiated urothelium exposed to urine whose morphology can react to mechanical stretching in a manner consistent with in vivo characterisations of bladder tissue.

Urine with high solute concentration alters tissue integrity and epithelial immune response
The urothelium in the mini-bladder can be stably maintained for several days in co-culture with urine. This allowed us to study how exposure to urine of variable composition over several days impacts the state of the bladder epithelium. Although not typically considered a factor in pathogenesis, human urine has a range of osmolarities, pH and compositions that vary widely between individuals over time and is very sensitive to overall health4,5,55. Over a period of 9 days, mini-bladders seeded with the same initial cells and differentiated under the same conditions showed different responses when exposed to urine pooled from different individuals (Supplementary Fig 4A). Exposure to pooled human urine (PHU) with high solute concentrations (Supplementary Table 1) typically led to accelerated tissue damage, as evidenced by an increase in the proportion of DRAQ7+ dead cells and a reduction in the overall cell numbers (Supplementary Fig 4B, C).

Ipe et al.56 developed a protocol for synthetic human urine (SHU) that closely mimics human urine in nutritional content but with well-defined ingredients. This formulation has been shown to support the growth of several uropathogens and has been used as a surrogate to pooled human urine to model antibiotic responses in UTI56,57. We hypothesised that the major driver of the variability in tissue responses to different PHU samples was the differing solute concentration, quantified through osmolarity. Accordingly, we developed two synthetic human urine formulations with osmolarities adjusted to match the upper and lower range in the pooled human urine samples (Supplementary Table 1). Both formulations contain the same amount of casamino acids, which reflects in the similar doubling time of UPEC in both formulations and are at an acidic pH=5.6 for long-term stability. Prior to use in the mini-bladder model, the pH is adjusted (with 20 mM HEPES, 1 mM CaCl2) to above 6 to match the range of PHU. The two formulations are termed high solute and low solute SHU, respectively and are used for experiments in the rest of the manuscript unless otherwise described58. Consistent with results in PHU, mini-bladders exposed to high solute SHU showed a gradual increase in the proportion of DRAQ7+ dead cells within the tissue (Fig. 2A, B, C). In contrast, mini-bladders exposed to low solute SHU showed an increase in the total number of cells and the proportion of EdU+ proliferating cells. (Fig. 2D, Supplementary Fig 4D, E).

Next, we explored the long-term consequences of urine solute concentration on tissue homoeostasis through transcriptional profiling of differentiated mini-bladder tissues exposed to high and low solute SHU for 8 days, using bulk RNAseq and subsequent gene set enrichment analyses (Fig. 2A, Supplementary Fig 5A). Long-term exposure to high solute SHU led to lower expression of cell-cell adhesion and proliferation gene sets (Fig. 2E), whereas gene sets related to developmental processes, particularly focusing on growth and differentiation, were enriched in samples exposed to low solute SHU (Supplementary Fig 5B), consistent with the morphological features noted above. In particular, genes related to urothelial differentiation, such as KRT8, KRT5, KRT20, KRT13 and UPK2 had lower expression in conditions with long-term exposure to high solute SHU (Fig. 2F), also reflected in qRT-PCR data (Supplementary Fig 5C). Genes related to intercellular junctions, such as TJP2, TJP3, CLDN1, CLDN2 and CDH2 were also downregulated by long-term exposure to high solute SHU (Fig. 2F). This transcriptional signature is corroborated by reduced ZO-1 immunostaining and higher permeability in the dextran diffusion assay (Fig. 2G, H). Interestingly, under high solute conditions, there was also a broad suppression of innate immune responses that was evident in the negative enrichment score of several gene sets for immune signalling and responses to bacterial infections (Fig. 2E, Supplementary Fig 5D). Notable amongst these are genes for toll-like receptor TLR4, CASP1, NF-κB1, cytokines IL-6, and chemokines CXCL1 and CXCL2 (Fig. 2I).

Taken together, these data show that long-term exposure to high solute urine impacts the resilience of the bladder tissue by weakening tight junctions, reducing stratified urothelial differentiation, and suppressing baseline immune responses.

Urine with high solute concentration increases UPEC invasion and lowers antibiotic clearance in the tissue
Based on the above findings, we postulated that concentrated urine could predispose the bladder towards an increased susceptibility to infection. The mini-bladder is an ideal system to test this, as UPEC pathogenesis in urine and tissue can be studied simultaneously in a way that was not previously possible. First, we established a baseline protocol for infection in PHU with a mid-range of solute concentration (~427 mOsm/kg) (Supplementary Table 1) and characterised the infection outcomes with CFT073 and UTI89, two widely used UPEC strains. Mini-bladders were inoculated with ca. 5×107 (CFT073) or 1×107 (UTI89) bacteria constitutively expressing GFP in PHU in the lumen (Supplementary Fig 6A, B). This corresponded to an approximate multiplicity of infection (MOI)=50 for CFT073 (MOI=10 for UTI89). The urinal population was sampled before and after a 10-h infection period, and the total bacterial load (within the urine and tissue) were quantified by fluorescence imaging (Supplementary Fig 6C, D). Both urinary and tissue bacterial burdens showed a steady increase over time, and characteristic features of UPEC infections such as IBCs and filamentous bacteria were visible at 10 h post-infection (hpi) (Supplementary Fig 6E, F). Similar 6-h infection periods in high and low solute SHU showed filamentous UPEC in the effluent—confirming the suitability of SHU formulation as a urine mimic (Supplementary Fig 6G).

Untreated chronic infections in humans involve a rapid turnover of bacteria with each micturition cycle, which previously could not be modelled in vitro. We can simulate this pathology in the mini-bladder by washing the lumen of the mini-bladder periodically to mimic micturition (Fig. 3A, B). This also provides a measurement of urinary bacterial load by collecting and plating for CFU. The urinary bacterial load reduced at least 10-fold after each wash (Fig. 3C), but washing did not completely clear the lumen. This is not unexpected as bacteria near the epithelial layer boundary experience lower urine flow rates59, and may also adhere better to the umbrella cells because of the catch-bond mechanism of type I pili adhesion. In each wash, we detected exfoliated epithelial cells harbouring bacterial clusters in the collected urine (Supplementary Fig 6H). The urinary population recovered to a consistent peak of ca. 5×108 CFU/mL in the intervening growth phases, a pattern consistent with a chronic, untreated infection21. By optical microscopy, we identified a population of bacteria that co-localise with the tissue after removing the urinary bacterial population by a wash cycle (hereafter referred to as tissue-associated bacteria). This population of adhered and invaded bacteria increased steadily over several micturition cycles (Fig. 3D). UPEC adheres to umbrella cells via molecular interactions between the type I pili and the mannose units of the surface protein UP1A. This interaction can be competitively inhibited by the polysaccharide D-(+)-mannose60–62. The addition of D-mannose to the initial inoculation and the sterile PHU used for subsequent washes significantly reduced the tissue-associated bacterial load, verifying that this is a population that is either attached to or invaded into the tissue (Fig. 3B, D). Interestingly, the inhibition of UPEC adhesion (a pre-requisite to bacterial invasion) also reduced the urinary bacterial load after multiple micturition cycles (Fig. 3C), demonstrating that the tissue-associated bacteria contribute to reseeding the urinary bacterial population after micturition. When the tissue is infected under similar micturition cycles with CFT073 ΔfimH mutant, the total population of tissue-associated bacteria and the urinal bacterial load at 9.5 hpi are comparable to infection with D-mannose (Fig. 3E). D-mannose administration also reduced loss of tissue permeability post infection and tissue death (Fig. 3F-I), confirming a role for tissue-associated bacteria in driving these phenotypes. The human mini-bladder model, therefore, retains viability over several micturition cycles and uniquely recapitulates the dynamics of crosstalk between urine and tissue-associated UPEC in infections.

Next, we evaluated the role of urine solute concentration as a variable in driving favourable or deleterious outcomes in infection and therapy. We infected otherwise identical mini-bladders after 5 days of exposure to high and low solute SHU, treated with Ciprofloxacin at 100 X MIC (5 µg/mL), and observed the regrowth post-treatment (Supplementary Fig 7A, B). Prior to treatment at 3 hpi, there was no difference in the urinary bacterial burden (Supplementary Fig 7C), consistent with the nearly identical doubling time of UPEC cultured axenically in these two urine compositions (Supplementary Table 1). However, at 3 hpi, the tissue-associated bacterial burden was significantly higher under high solute concentration conditions, which can be explained by improved access to deeper layers of the tissue caused by the loss of tight junctions and differentiated cells from long-term exposure to high solute SHU (Supplementary Fig 7D). Subsequent treatment with Ciprofloxacin, an antibiotic effective even against slow-growing bacteria, showed a stark difference in treatment efficacy. Clearance of urinal bacteria was independent of SHU solute concentration, whereas tissue-associated bacteria exhibit increased survival in mini-bladders with long-term exposure to high-solute urine (at 9.5 hpi) (Supplementary Fig 7C, D). To disentangle if this effect is due to the lack of tissue resilience or a differential bacterial response to antibiotics based on spatial niche of UPEC survivors, or both, we also performed similar experiments without the 5-day high/low solute SHU exposure, with longer infection and treatment periods (Fig. 4A). At D=0, the tissue associated bacteria at 6 hpi is lower under low solute concentration conditions in comparison to high solute concentration and the difference is negated when the tissue has been pre-treated with EDTA to disrupt tight junctions (Supplementary Fig 7E). Interestingly, the protective trend against Ciprofloxacin for the tissue-associated bacteria but not the urinal bacteria was also evident even during this longer infection and treatment cycle (Fig. 4B, C, D). When normalised for bacterial burden at the start of antibiotic treatment, there is no difference in the clearance rate or recovery of tissue-associated UPEC after antibiotic treatment Supplementary Fig 7F, reinforcing the higher tissue-associated bacterial load accumulated prior to ciprofloxacin treatment as a driver for the differences in Fig. 4B–D. Unlike ciprofloxacin, experiments with Fosfomycin (FOS) administration (40X MIC, 300 µg/mL) showed not only tissue-associated bacteria but also urinary bacteria were protected from clearance in high solute concentrations (Fig. 4E, F, G). Taken together, these experiments with two antibiotics with different modes of action demonstrate that urine solute concentration can impact therapeutic outcomes through two mechanisms. First, higher solute concentration reduces tissue resilience and enables higher colonisation of the tissue by bacteria. In turn, bacteria within the tissue are further protected from clearance by antibiotics. In the case of FOS, this protection also extends to the urinal bacteria.

Tissue-invaded UPEC under urine with high solute concentration can survive in cell wall deficient form during Fosfomycin treatment
Next, we examined the effects of solute concentration on Fosfomycin (FOS) therapy in greater detail as FOS, a cell-wall targeting antibiotic, is routinely prescribed for uncomplicated UTIs and administered as a single dose that accumulates in the urine63–65 (Fig. 5A). The inefficacy of FOS in reducing urinal UPEC load in high solute SHU was clearly evident at 22 hpi (Fig. 4F) and resulted in faster regrowth and higher bacterial burdens at 29 hpi (Fig. 4G). An examination of the sampled urine at 22 hpi by confocal microscopy showed the presence of large, GFP+ forms, with a high degree of sphericity (Fig. 5B), very different from the relatively fewer (Fig. 4F) and predominantly rod-shaped bacilli obtained in low solute SHU (Supplementary Fig 8A, B, C). These large spherical morphologies are consistent with the reported structure of CWD or L-form UPEC31,32,66. CWD UPEC have been identified in the urine of human patients with chronic infections and as a response to FOS treatment in infections in zebrafish larvae32. We performed several tests to verify whether this was the case in our human model. Mickiewicz et al.32 showed that CWD forms are sensitive to hypoosmotic stress and show a defect in recovery on standard LB agar plates, which is abrogated by plating on osmoprotectant LM agar plates (containing 1x brain heart infusion and 0.58M + D-sucrose). Exponential phase UPEC cultured axenically in LB shows no difference in recoverable CFU on both formulations (Supplementary Fig 8D). The culturable urinal bacterial load at 22 hpi plated on LM plates was consistently higher than recovery on LB plates for high solute SHU in comparison to low solute SHU, consistent with our assignment of the spherical GFP+ forms as CWD UPEC (Supplementary Fig 8E, F). This difference is negated post regrowth at 29 hpi, suggesting that the CWD UPEC converted back to rod-shape once the antibiotic pressure is removed (Supplementary Fig 8G, H). As further proof, we generated CWD UPEC in axenic culture in high-solute SHU using the same treatment regimen, and imaged these structures using transmission electron microscopy (TEM) (Supplementary Fig 8I). After 16 h of FOS treatment, although a vast majority of bacteria are CWD, we also observed some bacteria in transition from rod-shaped to CWD (Supplementary Fig 8J). The size and morphology of the CWD UPEC in the TEM images are comparable to those obtained by confocal imaging (Supplementary Fig 8K). CWD UPEC were also observed axenically in high solute PHU upon similar treatment with Fosfomycin (Supplementary Fig 8L).

Intriguingly, high-resolution confocal microscopy imaging of fixed mini-bladder tissue revealed a heterogenous population of rod-shaped and spherical bacteria within the tissue (Fig. 5C). The latter population had cell sizes consistent with the morphology of CWD UPEC in the urine, and not the densely-packed smaller coccoid bacteria observed in IBCs30,40. The spatial localisation of the GFP signal overlapped significantly with that of actin, showing that these bacteria were within the tissue in either an intracellular or pericellular association (Fig. 5C, D). The exact localisation could not be resolved with the optical resolution of confocal microscopy. To examine this interaction with improved resolution, we performed correlated optical microscopy and volumetric EM on the FOS-treated tissue at 22 hpi. Potential CWD forms were identified as GFP+ via confocal imaging (Fig. 5E). Excitingly, when comparing the overall spatial orientation between the confocal and electron microscopy images, many of these GFP+ forms mapped to similar spherical structures in the 3D reconstruction of volumetric EM images (Fig. 5E, F, G). These spherical structures also had morphologies and contrast profiles consistent with axenically generated CWD UPEC imaged with the TEM (Fig. 5H, Supplementary Fig 8I). These ‘tissue-associated CWD UPECs’ were observed attached to epithelial cells in a pericellular manner at multiple independent spatial locations throughout the tissue; representative cross-sections are shown (Fig. 5I). Detailed 3D reconstruction showed a CWD UPEC residing within a crevice beneath a double-nucleated umbrella cell and positioned between two other adjacent cells (Fig. 5J). Additionally, CWD UPEC was also identified between cells deeper within the tissue away from the umbrella layer, coincident with the intermediate or basal cell population described in Fig. 1E. Our results show that CWD are not solely a urinal phenomenon, and that bacteria within the tissue can also adopt CWD forms.

Next, we wondered where there is also an interplay between tissue-associated and urinal bacteria in the case of FOS treatment and CWD UPEC formation in the bladder. To this end, we examined if the presence of urinary CWD UPEC alone was sufficient to account for the overall higher burdens in mini-bladders 7 h after removal of antibiotic therapy. To achieve this, we treated UPEC in axenic cultures of high and low solute SHU with FOS and reseeded the survivors into an otherwise uninfected mini-bladder lumen (Supplementary Fig. 9A). The survivors in high solute SHU were CWD forms of similar size and morphologies as previously observed (Supplementary Fig. 9B, C, Fig. 5B). Although the FOS treatment was far more effective in low solute conditions, and therefore far fewer bacteria were seeded into the mini-bladders, this small surviving population rapidly proliferated in the lumen to reach a burden of 106–107 CFU/mL after 7 h (Supplementary Fig. 9C, D). In contrast, although the proportion of survivors was much higher in high solute SHU, these were primarily CWDs (Supplementary Fig. 9B, C), and proliferated much slower over the course of 7 h (Supplementary Fig. 9D). This is likely due to the difference in division rate of CWD UPEC compared to rod-shaped bacteria, or a significant lag period during which CWD reconverted to rod-shaped bacteria before proliferation31,67. Although more survivors were introduced in the mini-bladder in high solute SHU at 22 hpi, the final tissue-associated bacterial load post 7 h of regrowth was not significantly different in both cases (Supplementary Fig. 9E). However, this is not the case when CWD forms are generated in the mini-bladder (Fig. 4G). This demonstrates that without tissue-associated niches formed by CWD UPEC, there is no significant difference in regrowth and recurrence between high and low solute SHU after FOS treatment, highlighting the crucial contribution of intercellular CWD UPEC survivors in regrowth kinetics.

These results collectively expand the survival niches of CWD UPEC (previously considered solely a urinary population solely) to the tissue and demonstrate the role of urine solute concentration in driving the population of this niche, underscoring its critical role in recurrence. Furthermore, this highlights the significance of the mini-bladder model in capturing the dynamic interactions of different bacterial subpopulations within urine and urothelium, enhancing our understanding of recurrence after antibiotic treatment.

Methods
This study complies with the ethical regulations of Commission cantonale d'Éthique de la Recherche sur l'être humain Vaud (CER-VD)).

Microdevice design and fabrication
The microfluidic device was fabricated as previously described in Nikolaev. M et al.44 using conventional soft lithography and polydimethylsiloxane (PDMS) moulding. Briefly, the device has three main compartments: a hydrogel compartment for tissue culture in the centre, two larger open (basal) media reservoirs flanking the hydrogel compartment, and a pair of smaller inlet/outlet reservoirs (Supplementary Fig. 1A). The liquid hydrogel precursor was loaded through an additional inlet (hydrogel port) and then polymerised (details below). Medium from the two basal side reservoirs can passively diffuse into the hydrogel. The chips were UV sterilised and kept sterile until further use.

Hydrogel loading and microchannel fabrication
Extracellular matrix (ECM) solution containing neutralised 6 mg/mL bovine TeloCol®-6 type I collagen solution (Advanced Biomatrix) was injected into the hydrogel compartment of the microdevice through the hydrogel loading port and incubated at 37°C for 10 min. Thereafter, the cell loading ports and media reservoirs were filled with PBS. Laser ablation of the hydrogel was performed using a customised two-photon laser microscope co-developed with UpNano GmBH and equipped with a 10×/0.25NA objective (Zeiss), 800 nm pulsed 1.5 W laser. To generate the mini-bladder form, a 3D model of the bladder was created in Autodesk Fusion 2025, and the mesh was generated using Blender 4.5. The resulting model was imported into the microscope control software and positioned along the hydrogel compartment of the microdevice, covering its entire length, at Z=100 µm from the bottom glass coverslip. After laser ablation, the channel was perfused with PBS and then stored in the fridge (4°C) until use for up to several weeks. Overnight before use, PBS was exchanged to H6621 complete epithelial cell medium (Cell Biologics) and then maintained in the incubator (37°C, 5% CO2).

Cell culture of human bladder epithelial cells
H-6215 primary human bladder epithelial cell line (Cell Biologics) obtained from female donors was cultured in H6621 complete epithelial cell medium (with supplied kit) supplemented with 10% Heat inactivated Foetal Bovine Serum (HI- FBS) (henceforth called H6621 complete media) as recommended by the supplier. Epithelial cells were passaged by detachment with TryPLE Express Enzyme (1×) at 37°C for 3–5 min followed by neutralisation of TryPLE with H6621 complete medium. The cells used in all the experiments were at ten passages or fewer. The cells tested negative routinely for mycoplasma contamination during passaging.

Pooled and synthetic human urine
Urine was collected from 10 consenting individuals of various sex at various hour of the day, and osmolarity was measured using a micro-osmometer (Osmo1® Single-Sample Micro-Osmometer; Advanced Instruments) (BASEC ID—2025-01193 under Commission cantonale d'Éthique de la Recherche sur l'être humain Vaud (CER-VD)). The 4 samples with the highest osmolarity were pooled to obtain the PHU with high solute concentration. Similarly, the 4 samples with the lowest osmolarity were pooled to obtain PHU with low solute concentration. PHU with mid-range solute concentration was procured from BioIVT, UK (filter-sterilised human urine, mixed sex). All PHU samples were filtered using Stericup Vacuum Filters (Merck, pore size 0.22 µm) and stored at –20°C in aliquots of 5 mL.

Synthetic human urine was fabricated using the protocol from Ipe et al.56. This corresponds to high solute SHU. The detailed composition is listed in Supplementary Table 2. All components were stirred until complete dissolution, and the pH was adjusted to 5.6 with 1 M NaOH or 37% HCl for long-term stability, as neutral pH often resulted in precipitation of components when stored at 4°C. The solution was filtered using Stericup Vacuum Filters (Merck, pore size 0.22 µm). Low solute SHU was made by diluting high solute SHU 5× in ddH2O. The Iron(II) sulphate heptahydrate (FESO4.7H2O) and casamino acid concentrations were adjusted back in low solute SHU to the amount equal to high solute SHU. The pH was again adjusted to 5.6 with 1 M NaOH or 37% HCl. The SHU solutions were stored at 4°C for a maximum of 1–2 months. Osmolarity was regularly measured using a micro-osmometer (Osmo1® Single-Sample Micro-Osmometer; Advanced Instruments). Temporary buffered aliquots with additional 20 mM HEPES and 1 mM CaCl2 were made for both high and low solute SHU, this was used for all experiments related to differentiation and infection.

Mini bladder tissue culture
To seed cells into the central channel of the etched mini-bladder, H-6215 cells (between passage numbers 4 and 10) were dissociated from T75 flasks with TryPLE Express solution for 5–10 min at 37°C. The cells were washed with H6621 complete media and centrifuged at 300g for 5 min. This process was repeated twice to remove residual TryPLE. After centrifugation, the pellet was resuspended in H6621 complete medium at a density of about 108 cells/mL. Media was removed from the microchannel inlet, outlet and basal side medium reservoirs, and 10 µL of cell suspension was allowed to fill the laser-ablated microchannel by gravity-driven flow. The cells were allowed to adhere for about 1–1.5 h, followed by gentle washes through the inlet and outlet to remove all non-adherent cells. The urinal and basal channels were filled with H6621 complete media, and the device was incubated at 37°C in 5% CO2 humidified air. The cells are grown with H6621 complete media in the urinal and basal chambers for 15 days to allow for tissue stratification. From day 16, the urinal channel was replaced with high solute SHU while the basal chamber was kept under H6621 complete media. The tissue was grown with urinal high solute SHU exposure for a further 5–7 days to complete the process of stratification and differentiation. During this period, the urinal and basal medium were changed every day. Tissue defects and failure of proper mini-bladder development, detected in around 10% of cases, may be caused by technical problems related to device preparation (e.g., defective microfluidic chips, hydrogel preparation) or issues related to cell culture. These defective tissues were not used for any experiments or analysis.

Mini bladder tissue stretching
The stretching experiments were performed using the CETONI Nemesys S syringe pump system with two modules. Two 1 mL syringes (Hamilton) were connected to both inlets of the chip. The first syringe was set to deliver a constant positive (dispensing) flow rate of 10 µL/min, while the second syringe was configured for variable negative (aspiration) flow rates. When both syringes were synchronised (inlet syringe: +10 µL/min, outlet syringe: −10 µL/min), a continuous steady-state perfusion was achieved. The inflation of bladder tissue was simulated by reducing or stopping the outlet syringe while maintaining a positive flow rate in the first syringe. A cyclic rectangular-wave perfusion profile was programmed for the outlet syringe, varying the flow rate from 0 µL/min (urine accumulation, inflation phase) to −10 µL/min (bladder emptying, deflation phase). This setup enabled multiple cycles of automated perfusion with bladder stretching.

Immunofluorescence staining
Mini-bladders were rinsed with PBS (Gibco) and fixed in 4% paraformaldehyde (PFA; Thermofisher) in PBS overnight at 4°C. After rinsing with PBS (Gibco), the hydrogel compartment was cut around its perimeter using a razor blade to extract the block of hydrogel containing mini-bladder tissue. The samples were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS (Gibco) (30 min, RT) and blocked in 1% Bovine Serum Albumin in PBS (Gibco) (blocking buffer) at 4°C for at least 8 h. Samples were subsequently incubated overnight at 4°C with primary antibodies diluted in blocking buffer. The following primary antibodies were used: rabbit anti-human Cytokeratin 20 (1:50, Biorbyt, orb256650), rabbit anti-human Uroplakin 3A (1:50, Thermofisher, PA5-87581), rabbit anti-human Alexa Fluor 488-Cytokeratin 13 (1:50, Abcam, ab92551), rabbit anti-human Alexa Fluor 647-Cytokeratin 8 (1:50, Abcam, ab53280), goat anti-human ZO-1 (1:50, Abcam, ab190085), rabbit anti-human UP1A (1:50, Thermofisher, 11045-RBM9-P1ABX) diluted in blocking buffer. The samples were then incubated in blocking buffer for at least 5 h to wash unbound primary antibodies, then incubated overnight at 4°C with secondary antibodies. The following secondary antibodies were used: Alexa Fluor 488 donkey anti-goat (1:1000, Thermofisher, A-11055), Alexa Fluor 700 goat anti-rabbit (1:1000, Thermofisher, A-21038) and Alexa Fluor 647 donkey anti-rabbit (1:1000, Thermofisher, A-31573) diluted in blocking buffer. The samples were then stained for Hoechst solution 33342 (1: 300, Thermofisher, 62249), DAPI (1:300, Thermofisher, 62248) or Alexa Fluor 555-Phalloidin (1:400, Thermofisher, A34055) diluted in blocking buffer for 1 h at RT (based on the experiment).

RNA extraction and quantitative real-time PCR (qRT-PCR)
The hydrogel block containing mini bladder tissue was extracted by cutting the hydrogel compartment using a razor blade. The hydrogel block was then incubated in Type I Collagenase (150 U/mL, Thermofisher, 17018029) diluted in PBS (Gibco) for 30 min at 37°C. Additionally, the hydrogel was periodically sheared using a 1 mL pipette to accelerate disaggregation of the matrix. The sample was then centrifuged at 600g for 10 min and the pellet was incubated in 700 µL of RNA lysis buffer with 0.14 M β-mercaptoethanol (β-ME) (RNAeasy Plus Micro Kit, QIAGEN). RNA was isolated following the manufacturer’s instructions and resuspended in 14 µL of DEPC-treated water. 11 µL of the RNA-containing solution was used to generate cDNA using the SuperScript® IV First-Strand Synthesis System with random hexamers (Invitrogen), which was stored at –20°C. qRT-PCR primer sequences are listed in Supplementary Table 3. qRT-PCR reactions were prepared with SYBR Green PCR Master Mix (Applied Biosystems) with 500 nM primers, and 1 µL cDNA. Reactions were run for quantification on QuantStudio 6 or 7 Flex Real-Time PCR System (Thermofisher), and amplicon specificity was confirmed by melting-curve analysis.

RNA-sequencing and data analysis
Libraries were prepared from ribodepleted RNA isolated from human bladder microtissue and sequenced using the AVITI sequencer (PE75). A mean of 53.3 million 75-base-pair paired-end reads was obtained among the 11 samples (43.23–75.74 million reads). The quality of sequenced Fastq files was analysed using FastQC (version 0.11.10), and reads were mapped on the human reference genome assembly hg38 using STAR (v2.7.10b)69. The uniquely mapped reads were between 82.6 and 94.3%. Gene counts were generated using FeatureCounts70, and differential expression analysis was performed with the DESeq2 (v.1.42.1)71 package from Bioconductor (v3.18)72. Genes were considered differentially expressed based on an adjusted p-value cutoff of <0.05. Libraries normalisation was performed using the variance stabilising transformation method of DESeq2. This normalisation produces transformed data on a log (2) scale. The point of these transformations is to remove the dependence of the variance on the mean, particularly the high variance of the logarithm of count data when the mean is low. VST is very similar to the regularised logarithm or rlog. Gene Set Enrichment analysis was carried out using the GSEA72 function in ClusterProfiler73 (v4.10.1), and the following annotated gene sets from MSigDB v6.274: the Hallmark gene set75.

UPEC infection of mini-bladder
The UPEC CFT073 strain was originally isolated from a patient with bacteraemia of urinary tract origin76 and procured from ATCC. A derivative strain expressing green fluorescent protein (GFP) was generated by introducing the sfGFP sequence from plasmid pSLC293 under the control of a strong Pσ70 promoter at the chromosomal attHK022 site (between nucleotides 1,090,927 and 1,090,928) using a recently described method77 (adapted from Simonet et al.78). The fimH locus (between nucleotides 5,143,529 and 5,144,440) was deleted from CFT073 sfGFP using the λRed recombineering system (adapted from Simonet et al.78). The UPEC UTI89 strain, recovered from patients with acute cystitis, was gifted from the Guet lab at IST Austria. Similar to CFT073, a derivative strain expressing green fluorescent protein (GFP) was generated by introducing the sfGFP sequence from plasmid pSLC293 under the control of a strong Pσ70 promoter at the chromosomal attHK022 site (between nucleotides 1044461 and 1044640). To induce expression of type 1 pili, UPEC was grown in LB media under non-shaking conditions at 37°C overnight prior to the experiment, to achieve a stationary phase culture (for CFT073–OD600 = 1.5 and corresponding to a concentration of ~1.5×109 bacteria/mL). The bacteria were diluted 3-fold (final concentration of ~5×108 cells/mL for CFT073) by centrifuging (2500g, 5 min) and resuspended in either pooled or high/low solute synthetic human urine (temporary aliquots made with additional 20 mM HEPES, 1 mM CaCl2), based on the experiment. For all infection experiments, 100 µL of this suspension was introduced into the lumen through one reservoir. This was flowed through the channel, collected from the other reservoir and reintroduced into the original reservoir. After two such rounds, the mini-bladder was considered infected. For CFT073, this corresponded to an inoculum of ~5×107 bacteria and an MOI of 1:50 (assuming that the superficial cell layer is populated by the same number of epithelial cells that initially form a monolayer in the mini-bladder, ca. 106 cells). Similarly, for experiments with UTI89 (Supplementary Fig. 6B–D), this corresponded to an inoculum of ~107 bacteria and an MOI of 1:10. UPEC was then allowed to grow undisturbed for 3–10 h, depending on experimental conditions. For Supplementary Fig. 7E, the mini-bladder was lumenally pre-treated with 0.5 mM EDTA diluted in low solute SHU for 30 min before infection.

Micturition in the mini-bladder and CFU measurements
To simulate micturition, bacteria that had grown in the lumen were first removed by pipetting out the contents from the outlet port and collecting the washout. Thereafter, 100 µL of sterile urine each was perfused through the lumen twice and the washout collected. The total washout was then plated for CFU. This is referred to as the CFU count pre-micturition. Thereafter, a further two washes with 100 µL of sterile urine each were performed, and the washout collected to enumerate any remaining UPEC in the lumen. This is referred to as the CFU count post-micturition. CFU was counted by plating on LB or LM32 agar plates and incubating overnight at 37°C. LM agar was made by diluting 120 g D (+)-sucrose (0.58 M), 22.2 g Bacto Brain heart infusion (Thermofisher; 237300), 6 g Bacto Agar (Becton Dickinson; BD0214010), 1.2 g MgSO4 in 600 mL of ddH2O. For experiments to test the impact of the D-mannose on UPEC adhesion, D-mannose was added in PHU at a final concentration of 11.1 mM, and this urine formulation was used for infection and washes.

Staining exfoliated epithelial cells
Exfoliated epithelial cells were collected by centrifuging (600g, 10 min) the pre and post micturition washouts. The pellet was resuspended in 100 µL of PBS. This suspension was fixed with 4% paraformaldehyde (PFA; Thermofisher) in PBS overnight at 4°C. The sample was washed (by centrifugation, 600g, 10 min) with PBS, and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS (Gibco) (30 min, RT). This was followed by blocking in 1% Bovine Serum Albumin in PBS (Gibco) (blocking buffer, 1 h, RT). The samples were stained for DAPI (1: 300, Thermofisher, 62248) and Alexa Fluor 555–Phalloidin (1:400, Thermofisher, A34055) diluted in blocking buffer for 1 h at RT. The exfoliated epithelial cells were resuspended in 50 µL of PBS and placed on a slide for imaging.

Staining proliferating and dead cells in the mini-bladder
For live-imaging dead cells (e.g., Fig. 2B), the tissues were exposed to DRAQ7 (1:1000, Thermofisher, D15106) diluted in urine and the basal media for 30 min at 37°C pre-imaging. Overall nuclei were stained for live-imaging using Hoechst solution 33342 (1: 1000, Thermofisher, 62249) diluted in urine and the basal media for 1–2 h at 37°C. For staining proliferating cells (e.g., Supplementary Fig. 4D), the tissues were exposed to 10 µM of EdU labelling solution (Click-iT Plus EdU Imaging Kit, Thermofisher) diluted in urine and the basal media for 3 h at 37°C. The tissues were then fixed with 4% paraformaldehyde (PFA; Thermofisher) in PBS overnight at 4°C. The tissue was permeabilised with 0.1% Triton X-100 (Sigma-Aldrich) in PBS (Gibco) (30 min, RT). This was followed by blocking in 1% Bovine Serum Albumin in PBS (Gibco) (blocking buffer, 1 h, RT). To detect EdU, the Click-iT® Plus reaction cocktail was freshly prepared (as per the recommended protocol by the manufacturer; 0.5 mL cocktail was made with 440 µL 1X Click-iT® reaction buffer, 10 µL copper protectant, 1.2 µL Alexa Fluor® picolyl azide, 50 µL 1X Click-iT® EdU buffer additive). The tissue was exposed to 0.5 mL of Click-iT® Plus reaction cocktail for 30 min at RT.

Antibiotic treatment in mini-bladder
For antibiotic treatment, Fosfomycin (300 µg/mL, 40X MIC in high solute SHU; 300 µg/mL, 20X MIC in PHU high) or Ciprofloxacin (5 µg/mL, 100X MIC in high solute SHU) diluted in sterile urine and basal media was introduced into the devices. The total UPEC remaining post treatment was quantified by collecting urinal bacteria with 2 washes of 100 µL sterile urine with antibiotics. The lumen was then replaced with sterile urine and the basal chamber with basal media, both without antibiotics to allow bacterial regrowth. For the experiment with Fosfomycin treatment in Fig. 4F, the lumen was washed twice with 100 µL of sterile urine without antibiotics at different timepoints during the regrowth phase, and the washout was used for counting CFU.

Microscopy and image processing
Immunostained mini bladder tissues. Immunostained tissue was treated with FocusClear (CelExplorer, FC-101) for 30 min at RT to enhance the transparency of the hydrogel. The sample was then mounted on a ibidi dish with 90% Glycerol in PBS (Gibco).

For whole tissue imaging, related to Fig. 1B, C, E, G; Supplementary Fig. 2A, the sample was imaged using an inverted Leica SP8 confocal microscope with a white light laser and 25x water immersion objective (NA=0.95, Leica). The voxel size was 0.91 µm×0.91 µm×1 µm in X, Y, and Z. The laser lines 405 nm, 488 nm, 555 nm, and 647 nm were used with intensity between 5 and 20%, adjusting for image saturation. The whole tissue has a thickness of ~300 µm in Z. Owing to scattering within the tissue, it was not possible to image the whole tissue with the same illumination settings. Instead, the tissue was imaged in 3 sections, each of 100 µm thickness in the Z plane. To account for the increased scattering within the deeper sections of the tissue, the overall laser intensities were adjusted for each section to maintain similar image saturation. The 3 sections were then stitched together along the Z axis using ImageJ to obtain the overall shape of the tissue in 3D. Background subtraction of 50 pixels and a median filter of 0.5 pixels was applied to the stack in ImageJ. Imaris 9.9 (Bitplane) was used for rendering 3D images and orthogonal views.

For magnified sections, related to Supplementary Fig. 2B–H, Fig. 2G and Fig. 1D, the sample was imaged using an inverted Leica SP8 confocal microscope. Supplementary Fig. 2B was imaged white light laser and a 40x glycerol immersion objective (NA=1.25, Leica). The pixel size was 0.14 µm×0.14 µm in X-Y. The laser lines 405 nm and 555 nm were used with intensity between 5 and 20%, adjusting for image saturation. Supplementary Fig. 2C, H and Fig. 2G were imaged with a white light laser and 40x glycerol immersion objective (NA=1.25, Leica). The voxel size was 0.28 µmx0.28 µmx0.36 µm in X, Y, and Z. The laser lines 405 nm, 488 nm, and 647 nm were used with intensity between 5 and 20%, adjusting for image saturation. Supplementary Fig. 2D, E, F, G were imaged with a white light laser and 25x water immersion objective (NA=0.95, Leica). The voxel size was 0.23 µmx0.23 µmx0.57 µm in X, Y, and Z. The laser lines 405 nm, 488 nm, 647 nm and 696nm were used with intensity between 5 and 20%, adjusting for image saturation. Figure 1D was imaged white light laser and a 63x oil immersion objective (NA=1.40, Leica). The pixel size was 0.3 µm×0.3 µm in X-Y. The 405 nm and 555 nm laser lines were used with intensity between 5 and 20%, adjusting for image saturation. Imaris 9.9 (Bitplane) was used for rendering 3D images.

For infected tissue, related to Fig. 5C, D, E and Supplementary Fig. 6E, F, the sample was imaged using an inverted Leica SP8 confocal microscope. Supplementary Fig. 6E, F were imaged with a white light laser and 63× water immersion objective (NA=1.20, Leica). The voxel size was 0.18 µm×0.18 µm×0.36 µm in X, Y, and Z. Figure 5C, D, E were imaged with a white light laser and 63x water immersion objective (NA=1.20, Leica). The voxel size was 0.36 µm×0.36 µm×0.36 µm in X, Y, and Z plane. The 405 nm, 488 nm, and 555 nm laser lines were used with intensity between 5 and 20%, adjusting for image saturation. Imaris 9.9 (Bitplane) was used for rendering 3D images.

For EdU measurements, related to Supplementary Fig. 4D, the sample was imaged using an inverted Leica SP8 confocal microscope with a white light laser and 25× water immersion objective (NA=0.95, Leica). The pixel size was 0.45 µm×0.45 µm in X-Y with 1 µm in Z plane. The laser lines 405 nm and 647 nm were used with 10% intensity. Imaris 9.9 (Bitplane) was used for rendering 3D images (surface grain size 1 µm; auto thresholding) and analysing the volume of EdU+ and Hoechst+ sections.

Live imaging DRAQ7+ cells in mini bladder tissues. For live-imaging DRAQ7+ cells in the mini bladder, related to Fig. 3H and Supplementary Fig. 4B, the sample was imaged using a Leica Thunder widefield imaging system with a 25× water immersion objective (NA=0.95, Leica). The voxel size was 0.52 µm×0.52 µm×1 µm in X, Y, and Z. The LED source was used with 5–10% and 16% intensity, respectively, with bandpass dichroic filters at 405 nm and 647 nm. The samples are imaged to capture a Z-stack of 100 µm from the bottom. The image was deconvoluted using Leica Thunder software with Large Volume Computational Clearing (LVCC). Imaris 9.9 (Bitplane) was used for rendering 3D images (surface grain size 1 µm; auto thresholding) and analysing the volume of DRAQ7+ and Hoechst+ sections.

Dextran based diffusion assay. The mini-bladder lumen was perfused with fluorescein isothiocyanate (FITC)-tagged dextran (average mol wt. 4000, Sigma Aldrich, 46944) diluted to 1 mg/mL in PBS (related to Figs. 1H, 3F). The centre Z-slice of the tissue (approx. 150 µm from the bottom of the lumen) was imaged immediately with Leica Thunder widefield imaging system using a 25x water immersion objective (NA=0.95, Leica). The pixel size was 0.26 µm×0.26 µm in X-Y. The LED source was used at ca. 5–10% intensity adjusted for saturation with bandpass dichroic filters at 475 nm. Intensity variation was plotted at multiple cross-sectional lines perpendicular to the urinal axis. At most 3 cross-sectional lines were measured per device. The intensity is normalised between the maximum and minimum values. The normalised intensity values at Z=525 µm away from the central urinal axis on both sides are plotted for the relative dextran permeability graph (in Figs. 1I, 2H, 3G).

Time lapse of infected mini bladder tissue. Timelapse snapshot images of infected tissue samples in Figs. 3B, 4B, Supplementary Fig. 6B, Supplementary Fig. 7B were imaged with Leica Thunder widefield imaging system using a 25x water immersion objective (NA=0.95, Leica). The stage-top incubator was connected to a gas mixer (Okolab) to maintain 5% CO2 and a temperature of 37°C throughout the imaging period. For Fig. 3B, the voxel size was 0.26 µm×0.26 µm×1 µm in the X-Y and Z plane. The LED source was used with 10% intensity with bandpass dichroic filters at 475 nm. For Fig. 4B, Supplementary Fig. 6B (UTI89), Supplementary Fig. 7B, the pixel size was 0.52 µm×0.52 µm in X-Y with 1 µm in Z plane. The LED source was used with 10–15% intensity with bandpass dichroic filters at 475 nm, intensity adjusted for saturation.

Timelapse snapshot images of infected tissue samples in Fig. 4E, Supplementary Fig. 6B (CFT073) were imaged using an inverted Leica SP8 confocal microscope using a 25x water immersion objective (NA=0.95, Leica). The stage-top incubator was connected to a gas mixer (Okolab) to maintain 5% CO2 and a temperature of 37°C throughout the imaging period. For Supplementary Fig. 6B (CFT073), the voxel size was 0.91 µm×0.91 µm×1 µm in X, Y, and Z. The laser line 500 nm was used with 6% intensity. For Fig. 4E, the voxel size was 0.45 µm×0.45 µm×1 µm in X, Y and Z plane. The laser line 500 nm was used with 15% intensity.

All samples were also imaged using brightfield illumination. A total Z-stack of 120 µm from the bottom was captured. Tissue-associated bacteria were analysed using Imaris 9.9 (Bitplane) (an example of the analysis pipeline is shown in Supplementary Fig. 10). Imaris 9.9 (Bitplane) was used to create a surface using the bacterial signal in a 650 µm×650 µm×100 µm cuboid centred on the bottom layer of tissue (surface grain size 0.5 µm; auto thresholding). The total volume of the surface was exported as the tissue-associated bacterial volume.

Bacterial imaging. Bacteria related Supplementary Fig. 6G, H, Supplementary Fig. 8L and Supplementary Fig. 9B were imaged using an inverted Leica SP8 confocal microscope. Supplementary Fig. 6G, H were imaged with a white light laser and 63x oil immersion objective (NA=1.4, Leica). The pixel size was 0.1 µm×0.1 µm in X and Y. Supplementary Fig. 8L was imaged with a white light laser and 63x oil immersion objective (NA=1.4, Leica). The pixel size was 0.14 µm×0.14 µm in X and Y. Supplementary Fig. 9B was imaged with a white light laser and 63x oil immersion objective (NA=1.4, Leica). The pixel size was 0.36 µm×0.36 µm in X and Y. The 488 nm laser line was used with intensity between 5 and 20%, adjusting for image saturation. Bacteria related to Fig. 5B and Supplementary Fig. 8C were imaged using Leica Thunder widefield imaging system. Figure 5B was imaged with 40x dry objective (NA=0.95, Leica). The pixel size was 0.32 µm×0.32 µm in X and Y. Supplementary Fig. 8C was imaged with a 40x dry objective (NA=0.95, Leica). The pixel size was 0.16 µm×0.16 µm in X and Y. LED source was used at 5–20% intensity with bandpass dichroic filters at 475 nm.

Array tomography SEM: sample preparation and imaging (stretched and unstretched tissue)
For array tomography SEM of stretched and unstretched tissue (Fig. 1J, Supplementary Fig. 3), a fixative solution of 1% PFA and 2.5% glutaraldehyde in 0.1 M phosphate buffer was added to the basal side reservoirs while tissues were stretched to their maximum state or in unstretched conditions. The tissues were then left for at least 2 h in the “inflated” or normal state while connected to the syringes. Samples were then incubated for 1 h in 2% (w/v) osmium tetroxide and 1.5% (w/v) K4[Fe(CN)6] in 100 mM PB buffer. Samples were incubated for 1 h in 1% (w/v) tannic acid in 100 mM PB buffer, then 30 min in 2% (w/v) aqueous solution of osmium tetroxide, followed by 1% (w/v) uranyl acetate for 2 h at room temperature. At the end of gradual dehydration cycles, samples were flat-embedded in Epon-Araldite mix.

Polymerised flat blocks were trimmed using a 90° diamond trim tool (Diatome, Biel, Switzerland), and the arrays of 100 nm sections were obtained using a 35° ATC diamond knife (Diatome, Biel, Switzerland) mounted on a Leica UC7 microtome. Arrays were transferred on wafers as described in the following protocols79,80.

Wafers were analysed using Helios SEM microscope (Thermo Fisher Scientific) at 2 keV landing energy and 0.8 nA beam current at 2 mm distance using a Mirror Detector (MD-BSA). Images were collected semi-automatically with 5 µs dwell time using Maps 3.11 software (Thermo Fisher Scientific). Images were collected at or 5 mm/s (6084×2044) to generate 5 nm resolution images, respectively. To cover a larger area of the sample, several images were collected at a given resolution and stitched together using Maps 3.17 software (Thermo Fisher Scientific)79,80.

Analysis of vesicles. Vesicles in array tomography SEM cross sections were identified manually based on contrast differences (example—Supplementary Fig. 3C, D). The total number of vesicles within each lumen facing the cell was enumerated manually in both stretched and unstretched tissue (Fig. 1K).

TEM: sample preparation and imaging (axenic CWD UPEC)
For transmission electron microscopy of axenic UPEC samples (Supplementary Fig. 8I, J), the bacteria pellet was resuspended in a buffered mix of 1.0% glutaraldehyde and 2.0% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) for 2 h. After gentle centrifugation, the pellet was then post fixed 1.0% osmium tetroxide with 1.5% potassium ferrocyanide, and then 1.0% osmium tetroxide alone. It was finally stained for 30 min in 1% uranyl acetate in water before being dehydrated through increasing concentrations of alcohol and then embedded in Durcupan ACM (Fluka, Switzerland) resin before being placed between glass slides, and the resin was cured at 60°C for 24 h. Once hardened, the resin-embedded pellet was glued to blank resin blocks with cyanoacrylate glue, and thin (50 nm thick) sections were cut with a diamond knife. These were collected onto pioloform support films on single slot copper grids, contrasted with lead citrate and uranyl acetate. Sections were imaged in a transmission electron microscope (FEI Tecnai Spirit) operating at 80 kV using a digital camera (FEI Eagle).

Serial block face-scanning electron microscopy (SBEM): sample preparation of mini-bladder tissue
Mini-bladders at 22 hpi post Fosfomycin treatment (Fig. 5F–J) were fixed in 1% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4 for 1 h. The hydrogel block containing mini bladder tissue was extracted by cutting the PDMS using a razor blade. A diagonal cut was made across the sample to ensure complete flow of fixative to the apical side of the tissue. The sample was then blocked in 1% Bovine Serum Albumin in PBS (Gibco) for 30 min followed by staining with Hoechst solution 33342 (1: 300, Thermofisher, 62249) and Alexa Fluor 555—Phalloidin (1:400, Thermofisher, A34055) for 1 h at RT. Post wash, the sample was screened using Leica SP8 confocal microscope with a 63× water immersion objective to identify suitable sites of CWD bacteria colocalising with the urothelial cells and image series were collected through the entire structure. The sample was left overnight in the same fixative and then post-fixed in potassium ferrocyanide (1.5%) and osmium (2%), followed by thiocarbohydrazide (1%), and then osmium tetroxide (2%). It was then stained overnight in uranyl acetate (1%), washed in distilled water at 50°C, before final staining with lead aspartate at the same temperature. They were finally dehydrated in increasing concentrations of ethanol and then embedded in Durupan resin and hardened at 65°C for 24 h between glass slides.

Serial block face electron microscopy (SBEM)
The resin-embedded device was glued with conductive cement to an aluminium stub oriented in the same way as when it was imaged in the confocal microscope. It was then trimmed to expose only the region of interest for imaging in the electron microscope. This region was approximately 300 µm×300 µm in the x and y directions. Care was taken to compare images of the block with those taken in the confocal microscopy so that the corresponding regions could be imaged. The final block was mounted inside a scanning electron microscope (Zeiss Merlin, Zeiss NTS), holding a block face cutting microtome (3View, Gatan). Layers of resin, 50 nm thick, were cut from the block surface, and sequential images collected after each layer was removed. An acceleration voltage of the 1.6 kV was used with a pixel size of 8 nm with a dwell time of 1 µs. Series of nearly aligned images was collected, with final alignment being carried out in the FIJI imaging software (www.fiji.sc). Segmentation of different cells and structures was carried out on the Webknossos platform. Final models were exported to 3D modelling software for reconstruction (Blender.org).

Quantification and statistical analysis
All statistical analysis was performed using GraphPad Prism. Details of the statistical tests and exact numbers of biological and technical replicates are provided in the figure legends, which also provide information about the depiction of the mean or median values and standard deviations or standard errors of the mean and confidence intervals where appropriate.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, Paduthol & Nikolaev et al. developed a mini-bladder model that simulates urine-urothelium interactions, incorporating a stratified urothelium and micturition. Using this system, the authors demonstrate that exposure to high-solute urine disrupts tight junctions, alters immune responses, and reduces tissue resilience, increasing susceptibility to uropathogenic Escherichia coli (UPEC) colonization. They also show that bacteria evade killing by ciprofloxacin and fosfomycin in this environment and that fosfomycin promotes the formation of cell wall-deficient UPEC, which could contribute to recurrent infections. This model is novel and represents a significant advancement in the field. The results are compelling and of interest to the field.\nMajor comments:\n-The term organoid refers to self-organized three-dimensional tissue cultures that are derived from stem cells. Normally these are grown in 3D dimensions (in ECM – e.g. Matrigel). I find it misleading since the authors use here a primary cell line.\n-Line 175 “the mini-bladders exposed to low solute SHU continued to support active proliferation”: The images show that the mini-bladders are not destroyed by the low solute SHU but they do not show active proliferation (e.g., EdU staining). In fact, if the model accurately mimics the bladder, proliferation should be very low after differentiation and in the absence of any insult.\n-Line 244: “which can be explained by improved access to deeper layers of the tissue caused by the loss of tight junctions…”. The authors do not demonstrate whether the loss of tight junctions is directly related. They could either provide evidence (e.g., loosening tight junctions with EDTA in the presence of low or mild solute) or adjust the wording to indicate that it \"could\" be explained by this mechanism.\n-In Fig. 4: the authors state that when infecting with high or low solute SHU, 6 hpi is already sufficient to observe differences in tissue-associated bacteria. Do tight junctions disrupt that quickly? Only nuclei counts are shown in Fig. 2 (for SHU) and Fig. S2 (for PHU). Given the importance the authors place on tight junction integrity throughout the manuscript, it would be helpful to include ZO-1 staining over time to better assess these changes.\n-It is unusual that the mini-bladders were grown in different media depending on their appearance. The authors mentioned that in about 10% of the chips, they had to supplement the medium with growth factors (Y-27632, A83-01, CHIR99021, B27, FGF7, 10, and 2). This could have significant effects on the tissue or its response, as for example, Y-27632 and A83-01 are inhibitors of key host pathways. It may also explain why some replicates appear very different from others in some experiments. Defective mini-bladders should be excluded from the analysis.\nMinor comments:\n-Fig.1. (C,E,G):A close-up in the figures would improve visualization.\n-Since the quantification is image-based for tissue-associated bacteria, it would be helpful to include some close-up images. Were these quantified from images similar to those in Fig. 3, 4, S5, and S6?\n-It would be helpful to see the DRAQ7 staining in the mini-bladders exposed to the PHU (Fig. 2S) to compare it with those in Fig. 2 with SHU.\n-I am sure there is a reason for using SHU or PHU in different experiments, but it is not always clearly stated in the text. Some clarification would help the reader understand.\n-Does the permeability of the model change with exposure to high-solute PHU or SHU?\n-It is interesting that while the bacterial doubling time in SHU remains the same between high and low solute conditions, in PHU, there is a significant difference between high and low solute but not as much between mild and high. Could this be due to a lack of nutrients in the low (more diluted) solute PHU?\n-Representative images corresponding to the measurements in Fig. S6A-B would be good as well.\n-Reference 46 is missing.", "reviewer_2": "The paper from Paduthol and colleagues presents an intriguing new human urothelial model, which is used to explore several interesting biological questions including the effect of urine concentration on tissue damage and antibiotic response. It’s a tremendous achievement to manage to get (mostly) differentiated, urine-tolerant urothelium, flow and stretch all into one model, which is a great advance from this lab’s previous work with Emulate. Overall, the model has potential for discovering some worthwhile new host/pathogen biology beyond what has been explored here, and is impactful for the field. There are, however, some concerns, both major and minor, which we’ve outlined below in the hopes that these comments might prove useful for improving the manuscript.\nMajor concerns\n1. The premise of the work is based on literature which the authors use to lay out as a general fact that high-solute urine damages the urothelium (“gradual tissue death”, tight junction depletion, apoptosis etc.). I was surprised to see this as I was not familiar with such facts, especially as it seems counterintuitive that any sort of urine would gradually kill the very organ that has been highly evolved and equipped to deal with it. Dehydration would have been a common occurrence in the human race (and all mammals) over millennia, and still is. I looked up each of the papers supporting the idea that urine degrades the bladder (13,14,15,16 and 43). Three of the studies were in rats, one was in rat cell culture, and the last was in toads. Cell cultures cannot tolerate urine, not having the requisite protection, so this may not be the best model system to study cytopathic effects of urine. I’m not familiar with the toad urothelium, but it’s well known that rodents have more concentrated urine than humans, and of course they have thinner urothelia, which might make them more delicate. I think it’s misleading to lay out these “facts” without acknowledging that they are done in model systems that might not be as relevant to the human bladder – the way it is phrased, the reader may assume these are human studies. I certainly did before checking the references.\n2. Line 117. “The resultant tissue at day 20 recreates the organization of the human bladder epithelium.” Authors are entirely silent, both here in the Results and later in the Discussion, on the fact that the normal urothelium does not have stretches of undifferentiated, CK20-negative tissue punctuated by blooms of differentiated (possibly hypertrophic?) tissue. The human urothelium should have a uniform layer of intermediate cells 5-6 layers deep. In the example shown in Fig 1, it looks like this ranges from 0 to 5 intermediate cells depending on location. Don’t get me wrong; this model is still an impressive achievement, but its limitations need to be acknowledged up front in the results text and not waved away under the guise of a perfect “recreation”. The fact that a significant subset of the bladder urothelia is undifferentiated also makes us take the high-solute damage with a grain of salt – no pun intended. Maybe these undifferentiated zones are more prone to infection as well.\nGiven the above, please qualify all sentences such as that in line 151-2: “Together, these results confirm that the mini-bladder microtissue model contains a stratified, differentiated urothelium [with the implication that this is 100% true across the tissue]…” See also Discussion, line 344.\n3. Line 162. Experiments are said to be done here with mini-bladders that had “differentiated uniformly”: this is interesting, as it confirms that perhaps they often don’t, as demonstrated in Figure 1 – but we should be shown an image or two of fully/uniformly differentiated and stratified bladders in Figure 1 as well, just so we can see for ourselves. By extension, are we to assume that all the other experiments were done with the incompletely developed mini-bladders shown in Figure 1?\n4. Fig 1K. Knowing whether fusiform vesicles are present in the model, meaning that the recycling that has been shown to allow bacterial entry during the stretch/relax cycle (e.g. Truschel et al.) might take place, is crucial to understand how physiological your dynamic bladder model is. We as readers cannot be satisfied with Figure 1K without seeing any images of these vesicles. There is nothing evident in Figure 1J – not surprising at that resolution. Representative images of the correct resolution that were used to quantify these vesicles *absolutely must be shown* - in Supplementary is fine. At such resolution, it would also be possible to see hinges/asymmetric unit plaques by EM and this would also be very useful to confirm physiological relevance – as you are showing evidence of damage from high-solute urine, one could argue that this isn’t surprising if the model lacks AUM plaques. The authors should also confirm the presence of the uroplakins by IFA – it is a straightforward thing to check as there are a number of great antibodies. As the authors will know, the uroplakin complex will not form properly unless all components are present and correct – only UP3 RNA was tested. The question of whether intact uroplakin complexes are elaborated on the surface and make AUM is arguably more important for infection studies and differentiation status than is CK20. If you tried to see UP3 or other UPs by IFA and the model does not express them on the surface, this needs to be admitted. We aren’t shown any higher resolution top-down confocals with actin either – do you see the expected enlarged, honeycomb-like pattern? Bottom line, are these apical cells truly terminally differentiated umbrella cells or not?\n5. Regarding the solute status in human and synthetic urine – matching osmolarity seemed to be your only focus. But as I understand it, the kidneys concentrate different solutes differently; for example, urea is subject to disproportionate concentration compared with other chemicals. Just diluting globally to the right osmolarity implies that it is only osmolarity per se that governs any downstream effects, when actually it might be the concentration of one or a few key chemicals, which again are not proportionally concentrated by dehydration. I think getting the proportions right is crucial – which could be found in the literature, or by analyzing the components of the human urine you are using, and making informed choices how to make up the synthetic high and low urines. If my statements are incorrect, perhaps insert a line in the paper about how your choices were made.\n6. In a related vein, pH is known to affect bacterial behavior as well as drug action. Why have you made your synthetic urines so acidic compared with the urine you’ve collected from volunteers?\n7. Limitations. In the discussion, it should be acknowledged that, from what is shown, the mini-bladder might not be uniformly stratified and differentiated in some/many experiments (depending on how often they look like Figure 1), and this state could absolutely affect how easily the tissue could be damaged by high solutes, as well as susceptibility to infection. It should be clear to the reader that you understand the limits of the model, and where there is room for future improvement.\nMinor points\n1. What sex are H-6215 primary human bladder epithelial cells? This should be specified in the Methods.\n2. Line 687. “In case of any tissue defects or failure of stratification, post day 10 the basal media was supplemented with propagation factors...” This is a massive protocol difference between cultures with and without this propagation fudge factor. Experiments with one or other should be noted in the figures as the two types of tissue may not be comparable in all aspects.\n3. What is the rationale for using high solute urine for differentiation? Sorry if I missed it.\n4. MOI describes the probability of initial collision with surface host cells (Poisson distribution). Better to calculate MOI based on the estimated superficial cell number across the accessible surface area. Or at least give both figures so the reader can compare it to conditions in their own infection models.\n5. Fig 1A. Add some direction of flow arrows into schematic just to make schematic clearer.\n6. Fig 1G. ZO-1 is located at cell junctions. Hard to know if ZO-1 is expressed in between cells at this resolution. Could the authors use a higher objective for greater cell resolution and if it is logistically possible, image from top-down?\n7. Fig. 1H & I. Is this from stretched or unstretched samples? And would this change with the physiological difference?\n8. Fig 1F. The assay used to quantify “expression” is not mentioned in results text or figure legends. Readers should not have to trawl though the methods to find out if this is protein level (image quantification) or RNA. I couldn’t find it in the methods either. As there is some RNA work in the paper, and there are no IF images of the parental cell line, perhaps it’s RNA expression? Entirely unclear. I may have just missed it, but figure legends should tell you what sort of assay it is at the bare minimum. “RE units” on the Y axis could be any sort of arbitrary unit including fluorescence intensity so they don’t shed light.\n9. 131. Nit-picky point perhaps, but strong ZO-1 staining does not in fact confirm that the tissue retains a ‘robust barrier’. Only the subsequent functional assay can.\n10. Why didn’t the authors do TEER? If the model is accessible to the probe, that would have been interesting, to compare with data from other models.\n11. Fig 1J. The unstretched bladder EM looks very reminiscent of bladder biopsies. But in the stretched version, I’m not really seeing the flattened umbrella cells I’d expect. The image is very limited (2-3 superficial cells). Could further examples be presented (even if in Supplementary) so we can view the ‘average’ superficial cell morphology? This goes back to major point 4, above.\n12. All your tables are supplementary but in the text they are referred to as Table 1, Table 2 etc., not S1, S2, which sent me on a fruitless hunt.\n13. Figure 2. Given that an infected urothelium will shed cells, can your bulk transcriptomics distinguish between “down-regulation” of proteins vs loss through absence of cells? All your effects are described as downregulation. Line 181-2 – ‘Long-term exposure to high solute SHU led to lower expression of cell-cell adhesion and proliferation gene sets (Fig 2E)’. Sorry if I’m missing something, but if high solute PHU is important for proliferation of immature bladders then how can SHU high /low solute then lead to lower/higher expression cell adhesion and proliferation, respectively?\n14. Line 201-202 – Is there difference to CFT073 if pre-exposed to low and high solute urine?\n15. Line 208 – IBC formation. Could Figure S4E show an orthogonal view to show depth of IBCs?\n16. Figure 3B – Please confirm laser power for confocal microscopy is the same for all images?\n17. Line 215. “but washing did not completely clear the lumen. This is not unexpected as bacteria near the epithelial layer boundary experience lower urine flow rates…” The context around this point could be more nuanced given that flow has actually been shown to increase UPEC adhesion, which isn’t mentioned here.\n18. The word ‘axenic’ is not perhaps the best choice choice, as it is usually used to refence single-species growth in comparison to polymicrobial growth with other bacteria or parasites, which is not studied in this paper. I took a survey on my floor and only about 2 people out of 20 had even ever seen this word before. Most people in the field would say broth culture?\n19. Line 289. Technically speaking, a still image cannot distinguish whether a bacterium is going from coccus to bacillus or vice versa.\n20. Line 292. Here or in the discussion, you should explore the relationship between the CWD spherical E. coli you saw, versus the coccus-shaped E. coli found in mouse IBCs. Do you think these are totally different beasts?\n21. Line 353. Seems to imply that hydrated individuals would not get UTIs. That is likely not true: rephrase. Also begs the question why rodents, with their more concentrated urines, are difficult to give a UTI. Perhaps this should be discussed.\n22. Is there a difference between stretched and unstretched bladder with regard to infection? Maybe more clarity needed in text whether differences are seen with this phenotype.\n23. Figure 4 - Is 2.5 hours enough to show regrowth with ciprofloxacin? For Fosfomycin, in low solute urine would regrowth rebound to higher CFU levels if more time for regrowth were allowed?\n24. Line 281 – Will D-sucrose or BHI affect growth by introduction of new carbon sources, which may explain higher urinal CFUs in figure S6C & D?\n25. Would you find higher CFU counts 29 hpi in tissue associated CFUs as CWD may have invaded into bladder? Only urinal UPEC tested (Figure S6 E & F).\n26. Under what ethical framework were patients consented for urine? Please give IRB/ethics committee and license number in the Methods as is standard, or insert a statement why this is not necessary (perhaps they do things differently in Switzerland than in the rest of the world?)", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "A bioengineered human urothelial organoid model reveals the urine-urothelium interplay in tissue resilience and UPEC recurrence in urinary tract infections\nIn this study Paduthol et al. have developed a urothlial organoid model to study tissue resilience and bacterial recurrence during model UTI.\nBuilding on previous work from this group, and others, in this study the authors have combined various important aspects of the infection cycle from a host perspective – e.g. stratification and dynamics of micturition – in a single system.\nOverall, I find this work to be of technically high standards with complementary approaches used, the biological leaps are perhaps a bit less advanced but would likely be interesting for an audience relevant to Nature Communications.\nApart from a few general issues (outlined below), I think this study is a significant step forward in the infection-on-a-chip model development and could be accepted, pending satisfactory answers to the comments.\nGeneral/model comments:\n• The figures are quite busy, and figure legends not always clear to follow. Could this be looked over throughout.\n• The superficial layer(s?) of cells seem quite uneven (Figure 1C,E,G), how did that correlate with bacterial invasion? Did the authors notice if the green blobs (multiple IBCs? Figure 3B) colocalised with these protrusions of bladder cells?\n• One possibly important issue is that the UPEC strain CFT073 is used, this strain could be viewed as substandard when it comes to study cystitis (common strain would be UTI89), as CFT073 was isolated from a pyelonephritis patient. I believe this study would be significantly stronger if data from au UTI89 strain was to be included. The experiments should at the very least be repeated with a ΔfimH (main adhesion protein in bladder infection) or ΔpapG (main adhesion protein in kidney infection) strain, i.e. CFT073ΔpapG, to show that they system behaves like previously shown in-vivo systems.\n• Line 178: RE: transcriptional profiling of mini-bladder tissue exposed to high- and low-solute SHU. A comparison should be made to the qRT-PCR expression profiles from Fig. 1F. Were relative gene expression comparable for specific targets (e.g., P63 and others)? How would expression profiles differ (especially those related to chemokines and TLRs) between high-solute PHU when compared to high-solute SHU?\n• Line 211: Can the authors provide more explanation into the micturition cycling events of the mini-bladder model? Previous models have utilised a urine-flow system which likely also mimics the micturition cycles within UTI, albeit less natural due to the constant flow.\n• The amount of bacteria seems high (5 x 10^8 cell/mL, times 3 perfusions), can the authors comment on why they used this high numbers. To me this do not seem like a physiological bacterial load.\n• There is a clear lack of immune cells in the current model. Addition of immune cells to this model would significantly strengthen the study. But I understand that doing so may require a fair amount of extra experiments. Maybe a consideration for the future, but the very least this should be expanded on in the discussion.\nPHU vs SHU:\n• For flushed bacteria, did the authors find filaments in the SHU experiments? And did they see differences between PHU and SHU (frequency, length, amount ect?), and between high and low solutes? If they saw no filamentation in the SHU, it would be a red flag of the chemical composition as it has been shown in various models (both in-vivo, in-vitro and human) that UPEC undergo morphology changes into a filamentous form during UTI when exposed to real human urine.\nI’d say it is essential to show that morphology change also in SHU.\n• Another critical aspect of this is the growth of UPEC in PHU vs SHU. Others have in the past tried to formulate a SHU that is similar to that of a PHU, with varying success. Notably, if I recall correctly, I don’t believe that ipe et al, did that comparison in their paper (cited by the authors as source for their SHU). It needs to be shown that bacteria grow at a similar rate in the two, otherwise the comparison is not particularly informative.\nIn addition, the pHs are different between the PHU and SHU (table S1), this too has been shown to affect UPEC growth and morphology in the past, but is not commented on (only how varying concentrations affect the bladder cells themselves, Lines 167-176).\n• The initial bacterial burden is higher for high solute SHU, so this is perhaps not very surprising that the final bacterial burden is also higher, by the authors interpreted as lower antibiotic clearance (Figure 4D). Would the conclusions change if you for example normalised these data in order to compare?\n• Line 202: Can the overall density (ρ) of the urine be determined and reported in Table 1? Previous studies have shown that the specific ρ of PHU impacts bacterial cellular fitness and survival within the intracellular bladder environment and may also impact the bladder ecosystem.\n• Line 240: Can the authors give reasoning as to why they have used SHU instead of PHU for the antibiotic mini-bladder model assays? I understand that SHU is more robustly made and consistently produced for both high- and low-solute concentrations, but are additional crosstalks between cipro/fosfo treatment and the PHU that may have been missed?\nCWD:\n• Line 290: Given the fact that CWD UPEC can revert back to normal rod-shaped after antibiotic pressure is removed may suggest changes in gene regulation drive the CWD phenotype in the presence of fosfo. Can the authors consult the literature for transcriptomics studies with acute fosfo treatment in E. coli to better understand this mechanism or alternatively perform mechanistic analyses in their CFT073 model +/- fosfo treatment or in axenic high-solute culture. This will also give more functional insights into the persistence of CWD UPEC within the bladder tissue.\n• Line 318: What is the proportion of CWD and non-CWD UPEC in high-solute SHU given the heterogenous nature of CWD bacteria?\nMethods:\n• Overall the model and biological methods sections are mostly well explained. There are issues with the imaging sections: they should be more detailed.\nIn general: not adequate description of fluorescence imaging, for example:\n“The sample was imaged using Leica SP8 confocal microscope with a white light laser and 25x water immersion objective (NA = 0.95, Leica).” (Lines 803-804).\n“Fixed samples (e.g., Fig 1D, 5E, F, S4E) were imaged using Leica SP8 confocal microscope with a 63x water immersion objective (NA =1.20, Leica)” (Lines 828-830).\nWhat wavelengths were used, at what laser powers, pixel size, acquisition times, etc?\nIt would be impossible to repeat these experiment based on the information given currently.\n• The images in Figures 3B, 4B and 4E are of varying quality. Particularly the fluorescence channel, it is hard to truly assess the data based on these images, was the fluorescence in the full volume of the images superimposed? I’m not questioning that the signal increased over time, looking at the images it does look like that qualitatively. But it is not immediately clear how quantification was done when comparing end point fluorescence.\nThere is no description of how they were acquired nor analyzed (or I couldn’t find it at least. Lines 823-824 is not a well enough description of the analysis). Images like Figure 1D, including bacteria, clearly showing that they are intracellular show be included. Otherwise, how can the authors claim that the schematics only based on this model? This is also an excellent opportunity to show “how deep” the UPEC travels in the stratified layers."}, "author_response": "Point-by-point reply\nWe thank the reviewers for the constructive feedback to improve our manuscript. Guided by this\nfeedback we have performed several new experiments and include additional data, as\nsummarised below. In the manuscript file, the corresponding data and textual changes in the\nmanuscript file are also highlighted in blue.\n1) Figure 1: we have edited panel A to show direction of flow (requested by Reviewer 2/3)\n2) Figure 2: new panels G, H - Immunostaining shows lower expression of ZO-1 in mini-bladders\nexposed to high-solute urine which is corroborated functionally with increased leakage using\nthe dextran diffusion assay, demonstrating the loss of tight junctions (requested by Reviewer\n1).\n3) Figure 3: new panels E, G - New infection data with CFT073ΔfimH strain shows that it\nphenocopies the exogenous addition of D-mannose with reduced tissue-associated\nbacteria, urinal bacterial burden, and tissue permeability (requested by Reviewer 4).\n4) Supplementary Figure 2: High resolution images of the urothelial architecture and tissue\ndifferentiation (requested by Reviewer 1 and 2/3)\n5) Supplementary Figure 3: Additional magnified array tomography SEM images of unstretched\nand stretched tissue cross-sections to visualise the tight junctions, vesicles and lumenal cell\nmorphology in stretched tissue (requested by reviewer 2/3).\n6) Supplementary Figure 4 (previously Supplementary Figure 2): panel B, C - Representative\nimages for DRAQ7 staining in PHU-exposed mini-bladders confirm that outcomes with PHU\nare along the same lines as SHU. Panel D, E show the mini-bladders exposed to low solute\nSHU have a higher proportion of EdU+ proliferating cells (requested by reviewer 1).\n7) Supplementary Figure 5 (previously Supplementary Figure 3): panel C - A comparison of qRT-\nPCR expression profiles in the mini-bladder at D=0 with that in mini-bladders exposed to low\nand high solute urine for 6 days show comparable expression of targets (requested by\nReviewer 4).\n8) Supplementary Figure 6 (previously Supplementary Figure 4): Panel B-F shows data from a\n10-hour infection in the mini-bladder with UTI89 strain with PHU. Panel G shows filamentous\nUPEC collected in the effluent of mini bladders infected in high and low solute SHU\n(requested by Reviewer 4).\nPanel E shows the cross section of an IBC (requested by Reviewer 2/3).\n9) Supplementary Figure 7 (previously Supplementary Figure 5): Panel E - mini-bladders treated\nwith EDTA to weaken tight junctions show an increased burden of tissue-associated bacteria\nwhen infected in low solute SHU (requested by Reviewer 1).\nPanel F - Normalised tissue associated bacterial volume related to Fig 4D shows no\ndifference in the clearance rate or recovery of tissue-associated UPEC after antibiotic\ntreatment, reinforcing the higher tissue-associated bacterial load accumulated prior to\nciprofloxacin treatment as a driver for the differences between high and low solute SHU\ninfection dynamics (requested by Reviewer 4).\n10) Supplementary Figure 8 (previously Supplementary Fig 6): panel C, D - representative images\ncorresponding to the data in panel A, B (requested by Reviewer 1).\nPanel L shows representative images of CWD UPEC generated with Fosfomycin in axenic\nculture in high solute PHU (requested by Reviewer 4).\n11) Supplementary Figure 9 (previously Supplementary Fig 7): panel C - Quantification of the\ndifferential colony counts on LB and LM of axenic UPEC from high solute and low solute SHU\nshows the predominance of CWD UPEC in high solute SHU (requested by Reviewer 4).\n12) Supplementary Figure 10: An illustrated example of the image processing pipeline used to\nquantify tissue -associated bacteria (requested by Reviewer 1 and 4).\n13) Figure 3, D, F, G, Supplementary Figure 4 B, C, Supplementary Figure 7 B, C, D,\nSupplementary Figure 9 panel D, E: subset of data points corresponding to these figures were\nreplicated and replaced to remove data from experiments where a different set of\ndifferentiation protocol was used (requested by Reviewer 1 and 2/3). New data agree with the\nprevious versions of these panels in the original submission.\n14) Materials and Methods: this section has been comprehensively edited to improve clarity and\nprovide additional details where required to assist the reader in evaluating the experimental\nprocedures described (requested by reviewer 2/3 and 4).\n#Reviewer 1 (Remarks to the Author)\nIn this study, Paduthol & Nikolaev et al. developed a mini-bladder model that simulates urine-\nurothelium interactions, incorporating a stratified urothelium and micturition. Using this system,\nthe authors demonstrate that exposure to high-solute urine disrupts tight junctions, alters\nimmune responses, and reduces tissue resilience, increasing susceptibility to uropathogenic\nEscherichia coli (UPEC) colonization. They also show that bacteria evade killing by ciprofloxacin\nand fosfomycin in this environment and that fosfomycin promotes the formation of cell wall-\ndeficient UPEC, which could contribute to recurrent infections. This model is novel and\nrepresents a significant advancement in the field. The results are compelling and of interest to the\nfield.\nAU response: We thank the Reviewer for this positive assessment of the manuscript and\nconstructive suggestions which have improved the manuscript in revision.\nMajor comments:\n1. The term organoid refers to self-organized three-dimensional tissue cultures that are derived\nfrom stem cells. Normally these are grown in 3D dimensions (in ECM – e.g. Matrigel). I find it\nmisleading since the authors use here a primary cell line.\nAU response: We appreciate the Reviewer’s point of view that describes perfectly the canonical\npicture of organoids. However, we do note that there are several instances of organoid cultures\nfrom primary cells, just like the ones we have used in this study. Furthermore, although the model\ndoes incorporate a defined 3D mould, the seeded cells differentiate, stratify, and form a three-\ndimensional tissue that looks completely different from the starting cells. We therefore think that\nthe term organoid is suited to the description of this model, akin to the use of organoid-on-chip\nby several contemporaneous models.\n2. Line 175 “the mini-bladders exposed to low solute SHU continued to support active\nproliferation”: The images show that the mini-bladders are not destroyed by the low solute SHU\nbut they do not show active proliferation (e.g., EdU staining). In fact, if the model accurately\nmimics the bladder, proliferation should be very low after differentiation and in the absence of\nany insult.\nAU response: We thank the Reviewer for this suggestion. We have performed EdU staining which\nshow the mini-bladders exposed to low solute SHU have a higher proportion of EdU+ proliferating\ncells than comparable mini-bladders exposed to high solute SHU, as shown in Supplementary\nFigure 4 D, E in the revised manuscript. For mini-bladders exposed to SHU, the overall\nproliferation rate is low, and comparable to earlier timepoints of differentiation. We acknowledge\nthat the proliferation potential of the mini-bladder might still be higher than that of the human\nbladder, as outlined in the sections on Model limitations.\n3. Line 244: “which can be explained by improved access to deeper layers of the tissue caused by\nthe loss of tight junctions…”. The authors do not demonstrate whether the loss of tight junctions\nis directly related. They could either provide evidence (e.g., loosening tight junctions with EDTA\nin the presence of low or mild solute) or adjust the wording to indicate that it \"could\" be explained\nby this mechanism.\nAU response: We thank the Reviewer for this constructive feedback which has strengthened this\naspect of the manuscript considerably. We have performed the suggested experiment and the\ndata in Supplementary Figure 7E and 2G, H in the revised manuscript. The immunostaining\nshows lower expression of ZO-1 in mini-bladders exposed to high-solute urine which is\ncorroborated functionally with increased leakage of dextran using the dextran diffusion assay\n(Figure 2G, H). Further we show that the weakened junctions in mini-bladders pre-treated with\nEDTA directly correlate with an increased burden of tissue-associated bacteria (Supplementary\nFigure 7E).\n4. In Fig. 4: the authors state that when infecting with high or low solute SHU, 6 hpi is already\nsufficient to observe differences in tissue-associated bacteria. Do tight junctions disrupt that\nquickly? Only nuclei counts are shown in Fig. 2 (for SHU) and Fig. S2 (for PHU). Given the\nimportance the authors place on tight junction integrity throughout the manuscript, it would be\nhelpful to include ZO-1 staining over time to better assess these changes.\nAU response: We thank the Reviewer for this constructive suggestion, a representative 3D image\nof the mini-bladder with ZO-1 labelled via immunofluorescence in high and low solute SHU is\nincluded in Figure 2G in the revised manuscript.\n5. It is unusual that the mini-bladders were grown in different media depending on their\nappearance. The authors mentioned that in about 10% of the chips, they had to supplement the\nmedium with growth factors (Y-27632, A83-01, CHIR99021, B27, FGF7, 10, and 2). This could\nhave significant effects on the tissue or its response, as for example, Y-27632 and A83-01 are\ninhibitors of key host pathways. It may also explain why some replicates appear very different\nfrom others in some experiments. Defective mini-bladders should be excluded from the analysis.\nAU response: We sincerely apologize for the confusion. Model development is a continuous\nprocess, and we inadvertently included data from experiments where a different set of protocols\nwere being tested, which could accelerate differentiation. There was no intention to suggest, nor\nshould it be implied that the protocol used for the vast majority of mini-bladders in the paper\ngenerated inconsistent or defective mini-bladders from which data was used in the paper. To\nclarify this point completely we have replicated and replaced all the data in the original\nsubmission that was generated with these mini-bladders. These datapoints were a small subset\nof datapoints in Supplementary Figure S7B, C, D, Supplementary Figure 9 D, E, Supplementary\nFigure 4B, C and Figure 3 D, F and G in the revised manuscript. Importantly, the new data agree\nwith the previous versions of these panels in the original submission.\nMinor comments:\n6. Fig.1. (C,E,G):A close-up in the figures would improve visualization.\nAU response: We thank the Reviewer for the constructive feedback. The required closeups are\nnow provided in Supplementary Figure 2 in the revised manuscript.\n7. Since the quantification is image-based for tissue-associated bacteria, it would be helpful to\ninclude some close-up images. Were these quantified from images similar to those in Fig. 3, 4,\nS5, and S6?\nAU response: We confirm that the quantification is from images similar to Figures 3, 4,\nSupplementary Fig 6 and Supplementary Fig 7. We show the general workflow for analysis of\ntissue-associated bacteria in Imaris in Supplementary Figure 10 in the revised manuscript.\n8. It would be helpful to see the DRAQ7 staining in the mini-bladders exposed to the PHU (Fig.\n2S) to compare it with those in Fig. 2 with SHU.\nAU response: We have included representative images for DRAQ7 staining in PHU-exposed\nmini-bladders in Supplementary Figure 4 B and C in the revised manuscript; the images confirm\nthat outcomes with PHU are along the same lines as SHU.\n9. I am sure there is a reason for using SHU or PHU in different experiments, but it is not always\nclearly stated in the text. Some clarification would help the reader understand.\nAU response: We apologize for the confusion. Our experimental design has used PHU to\nestablish the chronic infection model. We then used the consistency and reliability of SHU for\nfurther experiments to obtain mechanistic insight and characterisation for different functional\nreadouts. SHU also provides us a means to benchmark the mini-bladder to other in vitro models,\nas well as provides an experimental approach where the bacteria growth rate is controlled and\nthe same across different conditions. To clearly indicate this to the reader, we have included a\nsentence in Line 184-185 of the revised manuscript:\n“The two formulations are termed high solute and low solute SHU respectively and are used\nfor experiments in the rest of the manuscript unless otherwise described.”\n10. Does the permeability of the model change with exposure to high-solute PHU or SHU?\nAU response: We confirm that the exposure to high-solute SHU increased permeability in the\ndextran diffusion assay. Immunofluorescence imaging confirmed that this was accompanied by\na reduction in tight junctions. We have incorporated this data in Figure 2G and H in the revised\nmanuscript.\nFigure R1: Permeability measurements via a Dextran diffusion assay.\nWe observed that high solute PHU also increased permeability significantly relative to low solute PHU\n(see Figure R1 for Reviewers).\n11. It is interesting that while the bacterial doubling time in SHU remains the same between high\nand low solute conditions, in PHU, there is a significant difference between high and low solute\nbut not as much between mild and high. Could this be due to a lack of nutrients in the low (more\ndiluted) solute PHU?\nAU response: We agree with the Reviewer that there is far more heterogeneity of CFT073 growth\nin PHU as compared to SHU. A complete analysis of the difference between the PHUs is beyond\nthe scope of the manuscript. We are not sure if this would be informative, as they come from\ndifferent individuals, with different metabolic statuses etc. In fact, it is to overcome these\ninherent differences in urine composition that we have used SHU as a standard formulation. The\nSHU formulations are devised to maintain relatively the same nutrient content between high and\nlow solute formulations so that the bacterial doubling time is consistent. This helps in\nstandardising experimental protocols in relation to infection across all the conditions.\n12. Representative images corresponding to the measurements in Fig. S6A-B would be good as\nwell.\nAU response: We have provided these in Supplementary Figure 8 C in the revised manuscript.\nAlthough the bacteria were not imaged in high-resolution at the single-cell level, the significantly\nlarger spherical shapes of the CWD are easily appreciated.\n13. Reference 46 is missing.\nAU response: We apologise for this omission; the correct reference has now been provided.\nReviewer #2 and 3 (Remarks to the Author)\nThe paper from Paduthol and colleagues presents an intriguing new human urothelial model,\nwhich is used to explore several interesting biological questions including the effect of urine\nconcentration on tissue damage and antibiotic response. It’s a tremendous achievement to\nmanage to get (mostly) differentiated, urine-tolerant urothelium, flow and stretch all into one\nmodel, which is a great advance from this lab’s previous work with Emulate. Overall, the model\nhas potential for discovering some worthwhile new host/pathogen biology beyond what has been\nexplored here, and is impactful for the field. There are, however, some concerns, both major and\nminor, which we’ve outlined below in the hopes that these comments might prove useful for\nimproving the manuscript.\nAU response: We thank the Reviewers for this very positive evaluation of this work, highlighting\nthe significant improvement of this model over the previous models established in our lab, and\nsuggestions for improvement.\nMajor concerns\n1. The premise of the work is based on literature which the authors use to lay out as a general fact\nthat high-solute urine damages the urothelium (“gradual tissue death”, tight junction depletion,\napoptosis etc.). I was surprised to see this as I was not familiar with such facts, especially as it\nseems counterintuitive that any sort of urine would gradually kill the very organ that has been\nhighly evolved and equipped to deal with it. Dehydration would have been a common occurrence\nin the human race (and all mammals) over millennia, and still is. I looked up each of the papers\nsupporting the idea that urine degrades the bladder (13,14,15,16 and 43). Three of the studies\nwere in rats, one was in rat cell culture, and the last was in toads. Cell cultures cannot tolerate\nurine, not having the requisite protection, so this may not be the best model system to study\ncytopathic effects of urine. I’m not familiar with the toad urothelium, but it’s well known that\nrodents have more concentrated urine than humans, and of course they have thinner urothelia,\nwhich might make them more delicate. I think it’s misleading to lay out these “facts” without\nacknowledging that they are done in model systems that might not be as relevant to the human\nbladder – the way it is phrased, the reader may assume these are human studies. I certainly did\nbefore checking the references.\nAU response: We thank the Reviewers for this feedback and regret that we have generated\nunnecessary confusion. There was no intention on our part to mislead with regard to references\nto past literature – most research in infectious diseases is performed in animal models as in\nplace of studies on humans and often cited as mechanistic fact. In the revised manuscript, we\nhave edited line 45 to start as follows:\nLine 45: “ In experiments in animal models including rodents, ...”\nSimilarly, we have edited Line 389-390 in the Discussion as follows:\nLine 389: “ ..confirming reports in vivo in animal models “\nOur claim is not that high-solute urine gradually kills the bladder in humans. Rather, we believe\nit has a deleterious effect on bladder resilience, and long-term function particularly in the context\nof certain stress or injury. These observations are exacerbated in the context and timeline of the\nmini-bladder model. We fully acknowledge that our model is not a complete recapitulation of the\nin vivo bladder, nor does it have a similar longevity, as highlighted in the section on ‘Limitations\nof this study.’\n2. Line 117. “The resultant tissue at day 20 recreates the organization of the human bladder\nepithelium.” Authors are entirely silent, both here in the Results and later in the Discussion, on\nthe fact that the normal urothelium does not have stretches of undifferentiated, CK20-negative\ntissue punctuated by blooms of differentiated (possibly hypertrophic?) tissue. The human\nurothelium should have a uniform layer of intermediate cells 5-6 layers deep. In the example\nshown in Fig 1, it looks like this ranges from 0 to 5 intermediate cells depending on location. Don’t\nget me wrong; this model is still an impressive achievement, but its limitations need to be\nacknowledged up front in the results text and not waved away under the guise of a perfect\n“recreation”. The fact that a significant subset of the bladder urothelia is undifferentiated also\nmakes us take the high-solute damage with a grain of salt – no pun intended. Maybe these\nundifferentiated zones are more prone to infection as well.\nGiven the above, please qualify all sentences such as that in line 151-2: “Together, these results\nconfirm that the mini-bladder microtissue model contains a stratified, differentiated urothelium\n[with the implication that this is 100% true across the tissue]…” See also Discussion, line 344.\nAU response: The Reviewers raise several complex points. First, we agree entirely with the\nReviewers that the normal urothelium does not have undifferentiated stretches. We respectfully\ndisagree with the interpretation that this is the case for our mini-bladders. We attempted to show\nan entire 3D volume for the differentiated mini-bladder in one image- capturing a millimetre or\nmore in X and Y, and nearly 300 µm in Z in one image, or a volume of 3 x 108 µm3. This necessitated\ncontrast adjustments that make it seem like areas with relatively lower intensity of CK20 staining\nlook like there have no CK20 staining. In Supplementary Figure 2A in the revised manuscript, we\nhave changed the intensity scaling to show that even in these areas, there is staining for CK20,\nand in a similar manner, we also show that there are no large “gaps” or “holes” in the CK13\nstaining. To further convince the reviewers we have provided magnified images of sections of the\nurothelium with the same resolution and spatial size as the current state-of-art shown with\nTranswells in Supplementary Figures 2C-H in the revised manuscript. In this case, the epithelium\nappears locally flat, and the stratification is clearly visible. Thus, we find no evidence for the\nconclusion that a “significant subset of the bladder urothelial is undifferentiated,” or the\nsubsequent extrapolations of this claim by the Reviewers. Indeed, all the images in subsequent\nfigures clearly show bacteria throughput the bladder, which would be unlikely if infection was\nconcentrated at the “undifferentiated zones” as claimed by the Reviewers.\nWe acknowledge that the epithelial surface is more undulating than similar models based on a\nflat architecture grown on rigid plastic substrates e.g., Transwells. We believe this is likely more\nreflective of the real 3-D mechanoactive organ 1. Evidence for the uniform nature of the\nstratification in Transwell models itself is also only provided in the literature with comparatively\nhigh-resolution images. In the mini-bladder model there are regions with a higher number of\nintermediate cell layers and those with a thinner urothelium, a difference which can be easily\nobscured if one showed higher resolution images. Overall, the limitations and avenues for further\ndevelopment have been outlined in the paragraph on limitations of the study.\n3. Line 162. Experiments are said to be done here with mini-bladders that had “differentiated\nuniformly”: this is interesting, as it confirms that perhaps they often don’t, as demonstrated in\nFigure 1 – but we should be shown an image or two of fully/uniformly differentiated and stratified\nbladders in Figure 1 as well, just so we can see for ourselves. By extension, are we to assume that\nall the other experiments were done with the incompletely developed mini-bladders shown in\nFigure 1?\nAU response: We regret a loosely worded sentence but also the connotations derived from them\nthat have been extrapolated to the rest of the manuscript. What we meant to report was that the\ndevices were differentiated under exactly the same conditions starting with the same batch of\ninitial cells, to minimize batch-to-batch variability. The sentence has now been edited for clarity\nin this regard. No data in the original submission or the revised submission was obtained from\nexperiments performed with incompletely developed mini-bladders.\n4. Fig 1K. Knowing whether fusiform vesicles are present in the model, meaning that the recycling\nthat has been shown to allow bacterial entry during the stretch/relax cycle (e.g. Truschel et al.)\nmight take place, is crucial to understand how physiological your dynamic bladder model is. We\nas readers cannot be satisfied with Figure 1K without seeing any images of these vesicles. There\nis nothing evident in Figure 1J – not surprising at that resolution. Representative images of the\ncorrect resolution that were used to quantify these vesicles *absolutely must be shown* - in\nSupplementary is fine. At such resolution, it would also be possible to see hinges/asymmetric unit\nplaques by EM and this would also be very useful to confirm physiological relevance – as you are\nshowing evidence of damage from high-solute urine, one could argue that this isn’t surprising if\nthe model lacks AUM plaques. The authors should also confirm the presence of the uroplakins\nby IFA – it is a straightforward thing to check as there are a number of great antibodies. As the\nauthors will know, the uroplakin complex will not form properly unless all components are\npresent and correct – only UP3 RNA was tested. The question of whether intact uroplakin\ncomplexes are elaborated on the surface and make AUM is arguably more important for infection\nstudies and differentiation status than is CK20. If you tried to see UP3 or other UPs by IFA and\nthe model does not express them on the surface, this needs to be admitted. We aren’t shown any\nhigher resolution top-down confocals with actin either – do you see the expected enlarged,\nhoneycomb-like pattern? Bottom line, are these apical cells truly terminally differentiated\numbrella cells or not?\nAU response: The Reviewers have raised several interesting points; we thank them for this\nconstructive feedback.\nFusiform vesicles: We now provide higher resolution images of the unstretched and stretched\ntissues in Supplementary Figure 3 in the revised manuscript and representative zooms of the\nvesicles in each case are also shown. Information on how the number of vesicles were counted\nis provided in the section titled ‘Analysis of Vesicles’ in the Materials and Methods.\nHinges/plaques: These phenomena are challenging to see. In the revised manuscript\n(Supplementary Figure 3 C, D), we highlight areas of intense contrast in SEM images of the tissue\nthat is localised to the very apical edge of the junction. This extends for a short distance along the\nsurface of the plasma membrane, providing the characteristic curve on the membrane expected\nwhen viewing the hinges/asymmetric unit plaques side-on. However, we recognize that the mini-\nbladder does not recapitulate this feature to the same level and extent as in vivo, and this is an\narea for further development. This is outlined in the paragraph on limitations of this study in the\nDiscussion.\nUroplakin immunostaining: We provide evidence of strong expression of UP3A and UP1A on the\napical surface of the umbrella cells in the mini-bladder model in Supplementary Figure 2D, E in\nthe revised manuscript.\nUmbrella cell morphology – there are several panels that reflect the umbrella cell morphology.\nThis is evident in the immunostaining in Figure 1D, which shows binucleate larger cells facing the\nlumen of the mini-bladder. Additional examples of binucleated cells are shown in Supplementary\nFigure 2B where the honeycomb-like Actin pattern is also evident. Lastly, the binucleated cell in\nFigure 5J is also larger than the cells below in in the same Z-stack.\n5. Regarding the solute status in human and synthetic urine – matching osmolarity seemed to be\nyour only focus. But as I understand it, the kidneys concentrate different solutes differently; for\nexample, urea is subject to disproportionate concentration compared with other chemicals. Just\ndiluting globally to the right osmolarity implies that it is only osmolarity per se that governs any\ndownstream effects, when actually it might be the concentration of one or a few key chemicals,\nwhich again are not proportionally concentrated by dehydration. I think getting the proportions\nright is crucial – which could be found in the literature, or by analyzing the components of the\nhuman urine you are using, and making informed choices how to make up the synthetic high and\nlow urines. If my statements are incorrect, perhaps insert a line in the paper about how your\nchoices were made.\nAU response: We thank the Reviewers for this comment. Our goal in this study was not to invent\na better formulation of synthetic urine; instead, we relied on the published literature that has\nestablished a formulation that has been used by several labs in a clinical setting and specifically\nfor urinary tract infections (e.g., Abbot et al 2). The media has shown to support the growth of both\ngram-positives and gram-negatives; thus, it was important to demonstrate that our model is\ncompatible with this media when administered in the formulation reported by Ipe et al. 2016 3.\nWe have edited the sentence at line 173 to further clarify our intent.\nLine 173: Ipe et al developed a protocol for synthetic human urine (SHU) that closely mimics\nhuman urine in nutritional content but with well-defined ingredients. This formulation has been\nshown to support the growth of several uropathogens and has been used as a surrogate to pooled\nhuman urine to model antibiotic responses in urinary tract infections.\nIn response to query 8 from Reviewer 4, we also provide evidence of the formation of filaments in\nSHU in the conditions used for the experiments. This is a further validation of this formulation.\nWe agree that the exact solute compositions that might contribute to osmolarity driven changes\nto a greater or lesser degree is a matter for further investigation as is the development of the next-\ngeneration of synthetic urines.\n6. In a related vein, pH is known to affect bacterial behavior as well as drug action. Why have\nyou made your synthetic urines so acidic compared with the urine you’ve collected from\nvolunteers?\nAU response: We thank the Reviewers for raising this important point. We regret that in the first\nsubmission, a crucial methodological detail was inadvertently omitted from the description in\nthe Materials and Methods. While we do generate SHU formulations at pH = 5.6, this is to follow\nthe established protocol. The acidic pH ensures long-term stability as the formulation tends to\ngenerate precipitates over time. However, when introduced into the mini-bladder, the pH of the\nSHU was adjusted by addition of 20 mM of HEPES buffer. This effectively changes the pH of the\nSHU to above 6, which is closer to that of the PHUs collected (updated in Supplementary Table\n1). Thus, we do not think the pH mismatch is as severe as it might appear. The Materials and\nMethods and Supplementary Table 2 (SHU formulation) have been updated to reflect this.\n7. Limitations. In the discussion, it should be acknowledged that, from what is shown, the mini-\nbladder might not be uniformly stratified and differentiated in some/many experiments\n(depending on how often they look like Figure 1), and this state could absolutely affect how easily\nthe tissue could be damaged by high solutes, as well as susceptibility to infection. It should be\nclear to the reader that you understand the limits of the model, and where there is room for future\nimprovement.\nAU response: We disagree with the assessment that the images in Figure 1 represent mini-\nbladders without a very high degree of differentiation, and stratification (albeit variable).\nNevertheless, we fully agree that our model still has limitations, and room for future\nimprovement, including related to stratification and differentiation. We have elaborated on both\npoints in the section on “Limitations of this Study” in the Discussion, and we hope that the\nReviewers will find this satisfactory.\nMinor points\n1. What sex are H-6215 primary human bladder epithelial cells? This should be specified in the\nMethods.\nAU response: The necessary details have been added to the revised manuscript.\n2. Line 687. “In case of any tissue defects or failure of stratification, post day 10 the basal media\nwas supplemented with propagation factors...” This is a massive protocol difference between\ncultures with and without this propagation fudge factor. Experiments with one or other should\nbe noted in the figures as the two types of tissue may not be comparable in all aspects.\nAU response: We are grateful to the Reviewers for pointing out this difference and apologise for\nthe confusion. We draw the attention of the Reviewers to the Response to point 5 by Reviewer 1\nwho raises the same issue. In the revised manuscript, the sentences have been removed as all\nmini-bladders were differentiated with the same protocol. Experiments have been repeated\nwhere needed to provide the necessary datapoints. In no case have any of the conclusions\nchanged.\n3. What is the rationale for using high solute urine for differentiation? Sorry if I missed it.\nAU response: We chose to use high solute SHU as it is the published formulation for consistency\nof protocol and to generate a formulation that could be reproducible across labs. The inability to\nuse undiluted urine was also a clear limiting factor in our earlier bladder-chip model (\nSharma et al. 4) and so this motivated us to demonstrate the ability to generate a fully stratified\ntissue in\nundiluted urine as we do in this study.\n4. MOI describes the probability of initial collision with surface host cells (Poisson distribution).\nBetter to calculate MOI based on the estimated superficial cell number across the accessible\nsurface area. Or at least give both figures so the reader can compare it to conditions in their own\ninfection models.\nAU response: The MOI is now mentioned at Line 222 in the Main Text of the revised manuscript.\nOne caveat we would like to highlight is that it is more difficult to accurately determine the\nnumber of host cells available for attachment which would be the superficial layer and whether\nall parts of the 3D tissue would be equally exposed.\n5. Fig 1A. Add some direction of flow arrows into schematic just to make schematic clearer.\nAU response: We thank the Reviewers for this suggestion; this has been implemented in Figure\n1A in the revised manuscript.\n6. Fig 1G. ZO-1 is located at cell junctions. Hard to know if ZO-1 is expressed in between cells at\nthis resolution. Could the authors use a higher objective for greater cell resolution and if it is\nlogistically possible, image from top-down?\nAU response: High resolution imaging is more challenging in this model, and if the Reviewers\nmean from top-down that we should use an upright microscope, then this is not experimentally\nfeasible. Nevertheless, we have generated higher-resolution images of sections of mini-bladder\nthat were accessible via the inverted microscopy, and this is shown in Supplementary Figure 2 in\nthe revised manuscript. The presence of ZO-1 in the junctions is visible in Supplementary Figure\n2H.\n7. Fig. 1H & I. Is this from stretched or unstretched samples? And would this change with the\nphysiological difference?\nAU response: The data in these figures is from the unstretched conditions. Carattino et al 2013\n4 showed that the stretching of ex-vivo rabbit urothelium mounted in Ussing chambers reduced\nTEER but not the permeability of fluorescein, concluding that stretching increased ion\nconduction without creating a leaky tissue. We therefore would expect no change in our model\nwith the dextran assay due to its functional similarity to the fluorescein assay. However, it was\nnot possible to perform these measurements at the frequency and consistency needed for\nincorporation into a regular protocol.\n8. Fig 1F. The assay used to quantify “expression” is not mentioned in results text or figure\nlegends. Readers should not have to trawl though the methods to find out if this is protein level\n(image quantification) or RNA. I couldn’t find it in the methods either. As there is some RNA\nwork in the paper, and there are no IF images of the parental cell line, perhaps it’s RNA\nexpression? Entirely unclear. I may have just missed it, but figure legends should tell you what\nsort of assay it is at the bare minimum. “RE units” on the Y axis could be any sort of arbitrary\nunit including fluorescence intensity so they don’t shed light.\nAU response: The Figure Legend has been updated to state that RE refers to relative\nexpression and that the assay used is qRT-PCR.\n9. 131. Nit-picky point perhaps, but strong ZO-1 staining does not in fact confirm that the tissue\nretains a ‘robust barrier’. Only the subsequent functional assay can.\nAU response: We agree with the Reviewers on this point and therefore have provided functional\nreadout on the dextran diffusion assay wherever relevant including in Figure 1H, I ,Figure 3F, G\nand now additionally in Figure 2H in the revised manuscript.\n10. Why didn’t the authors do TEER? If the model is accessible to the probe, that would have\nbeen interesting, to compare with data from other models.\nAU response: TEER is easier to implement in more accessible geometries such as the Transwell\nbut more difficult to implement on a consistent basis in the mini-bladder. We feel the dextran\ndiffusion assay is a highly relevant alternative assay used by several publications.\n11. Fig 1J. The unstretched bladder EM looks very reminiscent of bladder biopsies. But in the\nstretched version, I’m not really seeing the flattened umbrella cells I’d expect. The image is very\nlimited (2-3 superficial cells). Could further examples be presented (even if in Supplementary)\nso we can view the ‘average’ superficial cell morphology? This goes back to major point 4, above.\nAU response: We thank the Reviewers for the positive evaluation of the EM data from the\nunstretched bladder, particularly in that they are reminiscent of bladder biopsies. Additional EM\nimages of the mini-bladder in unstretched and stretched conditions are included in\nSupplementary Figure 3A,B in the revised manuscript and show that the umbrella layer has a\nflattened morphology.\n12. All your tables are supplementary but in the text they are referred to as Table 1, Table 2\netc., not S1, S2, which sent me on a fruitless hunt.\nAU response: This has been corrected in the revised manuscript.\n13. Figure 2. Given that an infected urothelium will shed cells, can your bulk transcriptomics\ndistinguish between “down-regulation” of proteins vs loss through absence of cells? All your\neffects are described as downregulation. Line 181-2 – ‘Long-term exposure to high solute SHU\nled to lower expression of cell-cell adhesion and proliferation gene sets (Fig 2E)’. Sorry if I’m\nmissing something, but if high solute PHU is important for proliferation of immature bladders\nthen how can SHU high /low solute then lead to lower/higher expression cell adhesion and\nproliferation, respectively?\nAU response: We thank the Reviewers for raising several interesting points. The RNAseq was\ndone in the absence of any infection, therefore we do not expect a large incidence of shedding.\nNevertheless, we cannot rule out that the effect we see at the transcriptional level is indicative\nof the loss of certain cell types. This is likely to be particularly the case for cell-type specific\nmarkers mentioned at Line 197. In contrast, changes in more general markers of tight junctions\nare likely reflective of overall changes in this tissue. We have therefore nuanced the sentence on\ncell-type specific markers in the revised manuscript as follows:\nLine 197: In particular, genes related to urothelial differentiation such as KRT8, KRT5, KRT20,\nKRT13 and UPK2 had lower expression in conditions with long-term exposure to high solute\nSHU (Fig 2F)...\nOur protocol relies on urine for differentiation, and to maintain a consistent protocol we used the\nSHU formulation developed by Ipe et al., which has a high osmolarity. We do not claim that high\nsolute PHU is a necessity for bladder differentiation. Further studies will evaluate the optimal\nosmolarity conditions for bladder differentiation.\n14. Line 201-202 – Is there difference to CFT073 if pre-exposed to low and high solute urine?\nAU response: We thank the Reviewers for raising an interesting point. As we outline in\nSupplementary Table 1, growth in urine of different compositions is varied and so therefore for\nease of experimentation and for compatibility with previous protocols we did not pre-expose the\nbacteria to urine. Additionally, growth in static LB to stationary phase has been shown to enhance\ntype I pili production, and was used in the bladder organoid and bladder-chip models developed\nin the lab, thus we retained this protocol to be able to compare the performance of this model\nwith those. Nevertheless, while this may change the kinetics of the very early stages of infection,\nit is unlikely to impact the overall time course or outcomes of the experiment. This may be\nobserved in the chronic micturition experiments where the bacteria that reseed the infection in\nthe bladder after micturition are those that have been exposed to urine in the mini-bladder for\nseveral hours. Overall, this is a useful methodological innovation that will be examined in future\nstudies.\n15. Line 208 – IBC formation. Could Figure S4E show an orthogonal view to show depth of\nIBCs?\nAU response: We have included an orthogonal view for this panel; it is now in Supplementary\nFigure 6E in the revised manuscript.\n16. Figure 3B – Please confirm laser power for confocal microscopy is the same for all images?\nAU response: We confirm that the laser power used was the same for all images in this panel.\nFurther details on image acquisition and analysis workflow are incorporated in the Methods\nsection and the new Supplementary Figure 10 in the revised manuscript.\n17. Line 215. “but washing did not completely clear the lumen. This is not unexpected as\nbacteria near the epithelial layer boundary experience lower urine flow rates…” The context\naround this point could be more nuanced given that flow has actually been shown to increase\nUPEC adhesion, which isn’t mentioned here.\nAU response: We thank the Reviewers for this comment and agree that there is the possibility of\nadhesion from type I pili activity that was not mentioned. We have rephrased the sentence as\nfollows in the Revised Manuscript:\nLine 236 - This is not unexpected as bacteria near the epithelial layer boundary experience\nlower urine flow rates, and may also adhere better to the umbrella cells because of the catch-\nbond mechanism of type I pili adhesion.\n18. The word ‘axenic’ is not perhaps the best choice, as it is usually used to refence single-species\ngrowth in comparison to polymicrobial growth with other bacteria or parasites, which is not\nstudied in this paper. I took a survey on my floor and only about 2 people out of 20 had even ever\nseen this word before. Most people in the field would say broth culture?\nAU response: We thank the Reviewers for this feedback. In this manuscript we culture UPEC in\nLB broth, in synthetic urine in a flask, and in urine in the mini-bladders. The use of this term allows\nus to refer to conditions of culture of microbe only, separate from the co-culture with mammalian\ncells. This fits with the definition of the term and we therefore will retain it.\n19. Line 289. Technically speaking, a still image cannot distinguish whether a bacterium is going\nfrom coccus to bacillus or vice versa.\nAU response: We agree with the Reviewers, but in this case we do know that the bacteria were\nrod-shaped before the Fosfomycin treatment and so we can infer the direction of change. We\nfurther wish to clarify in case there is a doubt that in these experiments, the rod-shaped bacteria\nare transitioning to a cell-wall-deficient form (the delaminating cell wall is clearly visible) and not\ncoccoid shapes.\n20. Line 292. Here or in the discussion, you should explore the relationship between the CWD\nspherical E. coli you saw, versus the coccus-shaped E. coli found in mouse IBCs. Do you think\nthese are totally different beasts?\nAU response: We thank the Reviewers for raising this point. The CWD bacteria are very different\nfrom the coccoid shaped bacteria in IBCs, which clearly retain their cell wall. Notably, the CWD\nbacteria are swollen and larger than rod-shaped bacteria, whereas the coccoid shaped bacteria\nin IBCs are usually smaller in size. To clarify this, we have edited the sentence accordingly in the\nrevised manuscript:\nLine 329: “The latter population had cell sizes consistent with the morphology of CWD UPEC\nin the urine, and not the densely-packed smaller coccoid bacteria observed in IBCs.”\n21. Line 353. Seems to imply that hydrated individuals would not get UTIs. That is likely not true:\nrephrase. Also begs the question why rodents, with their more concentrated urines, are difficult\nto give a UTI. Perhaps this should be discussed.\nAU response: We disagree with this interpretation. Rather, we think that prolonged exposure to\nconcentrated urine or dehydrated conditions weakens bladder resilience, which could\ncontribute to susceptibility to chronic infection. Rodent urine differs significantly from that of\nhumans and so we do not think these results can be extrapolated to rodents. Interspecies\ndifferences such as these are well-documented but difficult to disentangle given that rodents are\noften used as models for humans. Further work with models such as the mini-bladder can\ndisentangle these.\n22. Is there a difference between stretched and unstretched bladder with regard to infection?\nMaybe more clarity needed in text whether differences are seen with this phenotype.\nAU response: The authors raise an interesting question that we are pursuing in follow-up studies.\nIn this manuscript, the stretching experiments were performed to showcase the capabilities of\nthis model, but the long-term impact of stretch in the context of infection was not the focus of\nthis work. However, we do point to earlier work from our lab (Sharma et al., eLife 2021 5) which\nshowed that urothelial bacterial burden was higher in bladder-chips undergoing periodic\nmechanical stretch which indicates that mechanical forces are contributing factors to infection.\n23. Figure 4 - Is 2.5 hours enough to show regrowth with ciprofloxacin? For Fosfomycin, in low\nsolute urine would regrowth rebound to higher CFU levels if more time for regrowth were\nallowed?\nAU response: We are somewhat puzzled by the comment regarding Ciprofloxacin, as in Figure 4\nwe measure regrowth for 9 hours after withdrawal of antibiotic. Regarding Fosfomycin, if\nmaintained long enough growth in low solute urine would be expected to rebound as rod shaped\nbacteria proliferate to the same extent in the mini-bladder in both urine formulations.\n24. Line 281 – Will D-sucrose or BHI affect growth by introduction of new carbon sources, which\nmay explain higher urinal CFUs in figure S6C & D?\nAU response: We do not observe an appreciable difference in CFU for exponentially growing\nUPEC in axenic culture plated on LM or LB, indicating that the different nutritive content between\nthe two formulations is not the reason for higher recoverable. This data is shown in\nSupplementary Fig 8D in the revised manuscript.\n25. Would you find higher CFU counts 29 hpi in tissue associated CFUs as CWD may have invaded\ninto bladder? Only urinal UPEC tested (Figure S6 E & F).\nAU response: We do not measure tissue-associated CFUs in this manuscript as this is an\nendpoint measurement and is not suited to capturing the kinetics of growth and invasion\nsimultaneously. However, the data in Figure 4G shows clearly an increased in the tissue\nassociated bacteria by 29 hpi when quantified by bacterial fluorescence.\n26. Under what ethical framework were patients consented for urine? Please give IRB/ethics\ncommittee and license number in the Methods as is standard, or insert a statement why this is not\nnecessary (perhaps they do things differently in Switzerland than in the rest of the world?\nAU response: We apologise for the omission of this information in the original submission; this\nis now included in appropriate section in the Methods in the Revised manuscript.\nReviewer #4 (Remarks to the Author)\nIn this study Paduthol et al. have developed a urothlial organoid model to study tissue resilience\nand bacterial recurrence during model UTI. Building on previous work from this group, and\nothers, in this study the authors have combined various important aspects of the infection cycle\nfrom a host perspective – e.g. stratification and dynamics of micturition – in a single system.\nOverall, I find this work to be of technically high standards with complementary approaches\nused, the biological leaps are perhaps a bit less advanced but would likely be interesting for an\naudience relevant to Nature Communications. Apart from a few general issues (outlined below),\nI think this study is a significant step forward in the infection-on-a-chip model development and\ncould be accepted, pending satisfactory answers to the comments.\nAU Response: We thank the Reviewer for the very positive assessment and recommendation for\nacceptance and constructive feedback that has improved the manuscript and increased its\nimpact.\nGeneral/model comments:\n1. The figures are quite busy, and figure legends not always clear to follow. Could this be looked\nover throughout.\nAU response: We thank the Reviewer for this constructive feedback. We have edited the figure\nlegends throughput to make them easier to follow and to add more information where needed.\n2. The superficial layer(s?) of cells seem quite uneven (Figure 1C,E,G), how did that correlate\nwith bacterial invasion? Did the authors notice if the green blobs (multiple IBCs? Figure 3B)\ncolocalised with these protrusions of bladder cells?\nAU response: The Reviewer raises an interesting point. It is true that there are sections of the\nmini-bladder that have greater stratification than others, however as discussed in our response\nto query 2 from Reviewers 2 and 3, the epithelium is still locally flat. We did not observe a\ncorrelation between the protrusions and increased infection.\n3. One possibly important issue is that the UPEC strain CFT073 is used, this strain could be\nviewed as substandard when it comes to study cystitis (common strain would be UTI89), as\nCFT073 was isolated from a pyelonephritis patient. I believe this study would be significantly\nstronger if data from au UTI89 strain was to be included. The experiments should at the very\nleast be repeated with a ΔfimH (main adhesion protein in bladder infection) or ΔpapG or ΔpapG (main\nadhesion protein in kidney infection) strain, i.e. CFT073ΔpapG, to show that they system behaves\nlike previously shown in-vivo systems.\nAU response: We thank the Reviewer for this constructive feedback. We have performed key\nexperiments with the UTI89 strain with PHU in the mini-bladder. Our results in Supplementary\nFigure 6 B, C, D and F in the revised manuscript show that this strain also proliferates in the mini-\nbladder similar to CFT 073, albeit slower. This is consistent with the greater urothelial damage\ncaused by CFT073 reported in in vitro models 6. A more detailed characterization of the\ndifferences between species is beyond the scope of this paper. Data for the ΔfimH strain is\nincluded in Figure 3 E, G in the revised manuscript which shows that it phenocopies the\nexogenous addition of D-mannose with reduced tissue-associated bacteria, urinal bacterial\nburden, and tissue permeability.\n4. Line 178: RE: transcriptional profiling of mini-bladder tissue exposed to high- and low-solute SHU.\nA comparison should be made to the qRT-PCR expression profiles from Fig. 1F. Were relative gene\nexpression comparable for specific targets (e.g., P63 and others)? How would expression profiles\ndiffer (especially those related to chemokines and TLRs) between high-solute PHU when compared\nto high-solute SHU?\nAU response: We have included a comparison for qRT-PCR expression profiles in the mini-\nbladder at D=0 with expression in mini-bladders exposed to low and high solute urine for 6 days\nSupplementary Figure 5C. The expressions are comparable or in line with the trends discussed.\nThe goal of our study was not to characterise different urine formulations and so the\nimmunological differences between PHU and SHU have not been explored, however in Figure R2\nbelow expression profiles for TLRs expressed in the human bladder 7(TLR2, TLR4, TLR5, TLR9) and\nchemokines like CXCL1, CXCL2 and CCL2 that drive monocyte and neutrophil recruitment are\nshown. These show no statistically significant difference between high solute PHU and high\nsolute SHU exposure.\nFigure R2: qRT-PCR data for selected genes from mini-bladders under specified experimental\nconditions (Statistical significance calculated using unpaired t-test)\n5. Line 211: Can the authors provide more explanation into the micturition cycling events of the\nmini-bladder model? Previous models have utilised a urine-flow system which likely also mimics\nthe micturition cycles within UTI, albeit less natural due to the constant flow.\nAU response: We apologise that this important feature of the model was not clearly described.\nWe have included additional details in the Materials and Methods of the revised manuscript.\nEssentially, the lumen of the mini-bladder is accessible via inlet and outlet channels to large\nreservoirs which can be accessed by a laboratory micropipette. The withdrawal of urine from one\nreservoir and the addition of urine to the other causes a flow of urine through the mini-bladder\nuntil the volumes equalise. These “washes” mimic the voiding and refilling of the bladder with\nfresh urine.\n6.The amount of bacteria seems high (5 x 10^8 cell/mL, times 3 perfusions), can the authors\ncomment on why they used this high numbers. To me this do not seem like a physiological\nbacterial load.\nAU response: We apologise for the confusion on this point. In our infection protocol for CFT073,\nca. 100 µL of a 5 x 108 cells/mL is added to one reservoir, flowed through the channel, collected\nfrom the reservoir, and re-introduced into the original reservoir. After two such rounds, the mini-\nbladder is considered infected and the infection monitored thereafter. Thus, the mini-bladder is\nexposed to a total of 5 x 107 bacteria.\n7. There is a clear lack of immune cells in the current model. Addition of immune cells to this\nmodel would significantly strengthen the study. But I understand that doing so may require a fair\namount of extra experiments. Maybe a consideration for the future, but the very least this should\nbe expanded on in the discussion.\nAU response: We agree with the Reviewer that the lack of immune cells is a limitation, but also\nthat this is a goal for future studies. We have incorporated this in the section on Limitations of\nthis Study in the Discussion in the revised manuscript.\nPHU vs SHU:\n8. For flushed bacteria, did the authors find filaments in the SHU experiments? And did they see\ndifferences between PHU and SHU (frequency, length, amount ect?), and between high and low\nsolutes? If they saw no filamentation in the SHU, it would be a red flag of the chemical composition\nas it has been shown in various models (both in-vivo, in-vitro and human) that UPEC undergo\nmorphology changes into a filamentous form during UTI when exposed to real human urine. I’d say\nit is essential to show that morphology change also in SHU.\nAU response: We thank the Reviewer for this important suggestion. In the revised manuscript,\nwe show evidence of filamented urinal UPEC amongst the flushed bacteria in the high and low\nsolute SHU conditions in Supplementary Figure 6G in the revised manuscript. We observed no\nsignificant differences between high and low solute SHU conditions. Further characterisation of\ndifferences in filamentation between SHU and PHU is beyond the scope of this manuscript.\n9. Another critical aspect of this is the growth of UPEC in PHU vs SHU. Others have in the past tried\nto formulate a SHU that is similar to that of a PHU, with varying success. Notably, if I recall correctly,\nI don’t believe that Ipe et al, did that comparison in their paper (cited by the authors as source for\ntheir SHU). It needs to be shown that bacteria grow at a similar rate in the two, otherwise the\ncomparison is not particularly informative.\nIn addition, the pHs are different between the PHU and SHU (table S1), this too has been shown to\naffect UPEC growth and morphology in the past, but is not commented on (only how varying\nconcentrations affect the bladder cells themselves, Lines 167-176).\nAU response: We thank the Reviewer for raising important points. In the revised manuscript,\nwe have included data on doubling time for UPEC in Supplementary Table 1 for the different urine\nformulations used in this study. The doubling time in high solute SHU is consistent with high\nsolute PHU. There are differences between the doubling times in the lower solute conditions.\nThis is partly by design, as the SHU formulations retain the same nutritive content and remove\nbacterial growth rates as a variable between the conditions. If anything, we expect the lower\ngrowth rates in low solute PHU to further accentuate the differences seen between the\nconditions.\nWith regards to pH, we apologise for an omission on our part. The SHU formulations are\ngenerated as described and stored at pH=5.6 for long-term stability. However, in the\nexperimental setup, the pH was increased to above 6 by the addition of HEPES buffer. This\nreduces the gap in pH between PHU and SHU formulations quite significantly. The Materials and\nMethods and Supplementary Table 2 (SHU formulation) have been updated to reflect this.\n10. The initial bacterial burden is higher for high solute SHU, so this is perhaps not very surprising\nthat the final bacterial burden is also higher, by the authors interpreted as lower antibiotic clearance\n(Figure 4D). Would the conclusions change if you for example normalised these data in order to\ncompare?\nAU response: The Reviewer has raised a very valid point. We have reanalysed the data as\nsuggested, and the conclusions are unchanged. The plot with normalization is included in\nSupplementary Figure 7F in the revised manuscript.\n11. Line 202: Can the overall density (ρ) of the urine be determined and reported in Table 1?\nPrevious studies have shown that the specific ρ of PHU impacts bacterial cellular fitness and\nsurvival within the intracellular bladder environment and may also impact the bladder\necosystem.\nAU response: We have incorporated this data in Supplementary Table 1 in the revised\nmanuscript. We see very similar density between all conditions of SHU and PHU.\n12. Line 240: Can the authors give reasoning as to why they have used SHU instead of PHU for the\nantibiotic mini-bladder model assays? I understand that SHU is more robustly made and consistently\nproduced for both high- and low-solute concentrations, but are additional crosstalks between\ncipro/fosfo treatment and the PHU that may have been missed?\nAU response: The Reviewer raises an important point. As noted in Supplementary Table 1, the\ngrowth rate of UPEC is variable between the different PHUs, reflective of the donor-to-donor\nvariability. This would make it challenging to perform antibiotic time-kill assays across different\nurine formulations. The SHU formulations allow for this. While the kinetics of bacterial clearance\nmay well differ in PHU infections, we do not think there is additional crosstalk that might have\nbeen missed. Of note, we do see L-forms generated in UPEC treated with Fosfomycin in axenic\nculture in high solute PHU. This is shown in Supplementary Figure 8L in the revised manuscript.\nCWD:\n13. Line 290: Given the fact that CWD UPEC can revert back to normal rod-shaped after antibiotic\npressure is removed may suggest changes in gene regulation drive the CWD phenotype in the\npresence of fosfo. Can the authors consult the literature for transcriptomics studies with acute fosfo\ntreatment in E. coli to better understand this mechanism or alternatively perform mechanistic\nanalyses in their CFT073 model +/- fosfo treatment or in axenic high-solute culture. This will also\ngive more functional insights into the persistence of CWD UPEC within the bladder tissue.\nAU response: We thank the Reviewer for raising this important point. The CWD phenotype is a\nspecific response to the mechanism of action of Fosfomycin, which is known to target the very\nearly stages of peptidoglycan synthesis. CWD bacteria, by virtue of the loss of cell wall are no\nlonger targeted by the antibiotic. Work in our lab (PhD thesis of Dr Frédéric Normandeau 8 ) has\nanalysed this response and shown a role for colonic acid biosynthesis pathway (see Figure R3\nbelow). Further work in this regard is ongoing to understand the link between this pathway and\nosmolarity driven changes, but this comprehensive study will be the subject of a future report.\n[Figure Redacted]\n14. Line 318: What is the proportion of CWD and non-CWD UPEC in high-solute SHU given the\nheterogenous nature of CWD bacteria?\nAU response: We have quantified this through the images shown in Supplementary Figure 9B in\nthe revised manuscript, and this suggests a proportion of CWD: non-CWD of 9:1. We have also\nquantified the differential colony counts on LB and LM in Supplementary Figure 9C in the revised\nmanuscript that shows the predominance of CWD UPEC in high solute SHU.\nMethods:\n15. Overall the model and biological methods sections are mostly well explained. There are issues\nwith the imaging sections: they should be more detailed.\nIn general: not adequate description of fluorescence imaging, for example:\n“The sample was imaged using Leica SP8 confocal microscope with a white light laser and 25x\nwater immersion objective (NA = 0.95, Leica).” (Lines 803-804). “Fixed samples (e.g., Fig 1D, 5E, F,\nS4E) were imaged using Leica SP8 confocal microscope with a 63x water immersion objective (NA\n=1.20, Leica)” (Lines 828-830). What wavelengths were used, at what laser powers, pixel size,\nacquisition times, etc? It would be impossible to repeat these experiment based on the\ninformation given currently.\nAU response: We apologize for the lack of information; this has been duly corrected in the\nrevised manuscript. The relevant details are now included in the Materials and Methods section\nof the revised manuscript.\n16. The images in Figures 3B, 4B and 4E are of varying quality. Particularly the fluorescence channel,\nit is hard to truly assess the data based on these images, was the fluorescence in the full volume of\nthe images superimposed? I’m not questioning that the signal increased over time, looking at the\nimages it does look like that qualitatively. But it is not immediately clear how quantification was\ndone when comparing end point fluorescence.\nThere is no description of how they were acquired nor analyzed (or I couldn’t find it at least. Lines\n823-824 is not a well enough description of the analysis). Images like Figure 1D, including bacteria,\nclearly showing that they are intracellular show be included. Otherwise, how can the authors claim\nthat the schematics only based on this model? This is also an excellent opportunity to show “how\ndeep” the UPEC travels in the stratified layers.\nAU response: We regret that the original submission did not provide sufficient methodological\ndetails for the image analysis. This has been corrected in the revised manuscript. The revised\nmanuscript also includes a Supplementary Figure 10 outlining the image analysis pipeline\nfollowed in Imaris. In each case, we analysed a central volume of 650 µm x 650 µm x 100 µm\ncuboid in the mini-bladder, and quantified parameters based on 3D analysis. While we\nappreciate that this is not easy to grasp in the images in the Main Figures due to the lack of space,\nwe hope that the higher resolution images of intracellular bacteria in Supplementary Figure 6E,\nF, the new images of IBCs in Supplementary Figure 6E, the new data on the ΔfimH mutants and\nthe D-mannose experiments in Figure 3, both of which show clearly visible reductions in bacteria\nand the volumetric EM in Figure 5 are convincing as to the intracellular nature of the bacteria.\nReferences:\n1. Zhou, H., Guo, C. C. & Ro, J. Y. Urinary Bladder Pathology.\n2. Abbott, I. J. et al. Evaluation of pooled human urine and synthetic alternatives in a dynamic\nbladder infection in vitro model simulating oral fosfomycin therapy. J Microbiol Methods 171,\n(2020).\n3. Ipe, D. S. & Ulett, G. C. Evaluation of the in vitro growth of urinary tract infection-causing\ngram-negative and gram-positive bacteria in a proposed synthetic human urine (SHU)\nmedium. J Microbiol Methods 127, 164–171 (2016).\n4. Carattino, M. D. et al. Bladder filling and voiding affect umbrella cell tight junction\norganization and function. Am J Physiol Renal Physiol 305, 1158–1168 (2013).\n5. Sharma, K. et al. Dynamic persistence of intracellular bacterial communities of uropathogenic\nescherichia coli in a human bladder-chip model of urinary tract infections. Elife 10, (2021).\n6. Flores, C. et al. A Human Urothelial Microtissue Model Reveals Shared Colonization and\nSurvival Strategies between Uropathogens and Commensals. https://www.science.org (2023).\n7. Jafari, N. V. & Rohn, J. L. The urothelium: a multi-faceted barrier against a harsh environment.\nMucosal Immunol https://doi.org/10.1038/s41385-022-00565-0 (2022) doi:10.1038/s41385-\n022-00565-0.\n8. Normandeau Frédéric. Exploring the Role of Unstable L-Forms in the Survival of\nUropathogenic Escherichia coli Under Fosfomycin Exposure Frédéric NORMANDEAU. (EPFL,\nLausanne, 2025).\nPoint-by-point reply\nWe thank the reviewers for their positive comments on and enthusiasm for the revised\nmanuscript and are pleased that they all recommend publication. Below we address the\nremaining outstanding points raised by Reviewer 1.\nReviewer #1 (Remarks to the Author):\nThe authors have made a significant effort to address the comments, and the manuscript has\nimproved substantially. Most of my comments have been addressed appropriately, although I still\nhave concerns with the following points:\n1. Related to Major Comment 1: I continue to disagree with the authors. The fact that other studies\nhave used the term organoid incorrectly does not justify its use here. While organoids can indeed\nbe derived from primary cells, these organoids are grown in extracellular matrix in a three-\ndimensional context, which was not done here. Instead, the authors describe culture conditions\nthat rely on standard cell culture medium and passaging with TrypLE Express. This is consistent\nwith conventional primary cell culture rather than organoid culture. Therefore, the terminology\nshould be corrected, and the term organoid should be removed from the title.\nAU response: We politely disagree with this restricted interpretation of the term organoid and\ncharacterization of the model as an extension of standard cell culture. In our model, cells are\nalso grown in a three-dimensional extracellular matrix architecture. Nevertheless, we have\nedited the title for brevity and removed the term organoid.\n2. Related to Major Comment 2: Supplementary Figure 4D does not show a representative image\nof the control (D0), which is the condition shown in the corresponding graph (nice data btw).\nAU response: Thank you for the positive feedback, the necessary representative image has been\nadded to Supplementary Fig. 4D in the revised manuscript.\n3. Related to Major Comments 3 and 4: ZO1 staining is expected to localize at cell-to-cell junctions,\nnot across the entire apical surface. In the new figure panels (Figure 2G and Supplementary\nFigure 2H), the ZO1 signal appears to extend across the whole apical membrane. Image resolution\ndoes not explain this observation, since the resolution should be comparable to that of the\nphalloidin images (Supplementary Figure 2B). The authors should clarify how they interpret this\nstaining pattern.\nAU response: The image shown for ZO-1 is a maximum intensity projection of the entire stack.\nAt the lumenal surface, we observe some ZO-1 signal on the entire apical membrane, consistent\nwith the images observed by Jafari and Rohn https://doi.org/10.3389/fcimb.2023.1128132. Deeper into\nthe tissue, the ZO-1 staining is at cell junctions as expected. Data for different Z-slices at\npositions deeper into the tissue are shown in Figure R1. The ZO-1 signal (grayscale) is clearly\nevident in the cellular junctions only.\nFigure R1 – Representative Z-slices within the tissue shown in Supplementary Fig 2H. ZO-1 is shown in grayscale.\n4. Additional Comment: In the new Supplementary Figure 2A and E, mature umbrella cells would\ntypically appear as large polygonal cells positive for both UP3A and UP1A. In the new images,\nUP1A does not appear restricted to the superficial layer, and the expected large umbrella cells\nare not evident in panel D. Although some polygonal cells are visible in panel B, they do not seem\nlarge enough to represent fully mature umbrella cells. This is acceptable as no model is perfect,\nbut it should be acknowledged as a limitation that the umbrella cell layer does not appear fully\ndifferentiated.\nAU response: We have modified the sentence in the Discussion pertaining to the limitations of\nthe model to address these concerns of the reviewer.\nLine 425 - ‘These include a variability in the number of cell layers and umbrella cell size, the absence\nof widespread AUM plaques and uroplakin staining uniformly lining the lumen, and the relatively\nhigher cellular proliferation rate.’"}]

Decision letter