Biology subjects

Rohn, J. L.

Publications and source records attributed to Rohn, J. L..

7 recordsLinked to original sources

bioRxiv2026

In vitro evolution of uropathogenic Escherichia coli to fosfomycin resistance in a 3D cultured human bladder microtissue model

In vitro studies of antimicrobial resistance (AMR) using laboratory growth media produce important, fundamental information. However, their inability to more closely replicate the in vivo environment limits the translational potential of this work. Here, we used a 3D cultured microtissue model which reflects the human bladder microenvironment to select for resistance to fosfomycin in two uropathogenic strains of Escherichia coli, UTI-34 and UTI-59. To assess the clinical relevance of the mutations produced, we screened the observed mutations in the fosfomycin-selected variants against a curated dataset of 14,163 E. coli genomes isolated from urine. The four independent fosfomycin-selected variants of UTI-34 contained diverse mutations, while the mutations in the five independent fosfomycin-selected variants of UTI-59 were more constrained. All variants contained mutations in glpT, uhpT, uhpA and uhpC, which are commonly linked to fosfomycin-resistance in clinical isolates of E. coli. Screening of the mutations against the 14,163 E. coli genomes from urine confirmed that four of these mutations were found as exact matches in the dataset, while other mutation types were confirmed at a regional and gene level. These mutations did not result in any collateral susceptibility or resistance to other antibiotics recommended for the treatment of urinary tract infections. The use of a human 3D microtissue model, which closely replicates the urothelial microenvironment to study AMR during urinary tract infection treatment, could improve the clinical relevance of in vitro AMR studies. This has the potential to provide a better understanding of how AMR is acquired and expressed, and inform new strategies to combat AMR.

microbiology↗

bioRxiv2025

Effect of human urinary microenvironment and fluid flow on antibiotic and phage therapy efficacy against uropathogenic Escherichia coli

Urinary tract infections (UTI) remain a major global health burden, with high recurrence despite antibiotic treatment. The escalating prevalence of antimicrobial resistance further compromises therapeutic efficacy, contributing to an estimated 260,000 deaths annually. Conventional in vitro susceptibility assays often fail to predict clinical outcomes, underscoring the urgent need for physiologically relevant infection models. Here, we examined how microenvironmental complexity shapes uropathogenic Escherichia coli (UPEC) responses to antibiotics and bacteriophages using: human urine, a three-dimensional urothelial microtissue model (3D-UHU), and a novel mesofluidic system (P-FLO) that introduces physiologically relevant flow dynamics to the 3D-UHU. P-FLO was engineered from cost-effective 3D-printed components compatible with standard Transwell systems. Among the antibiotics tested, nitrofurantoin exhibited the greatest potency in minimum inhibitory concentration assays, but it failed to fully eradicate infection within the more physiological 3D-UHU model. A bacteriophage cocktail (LCPR1) showed markedly reduced activity in urine compared with nutrient-rich media, highlighting the influence of infection-site conditions. In contrast, in 3D-UHU, LCRP1 modulated host responses without reducing bacterial burden. Combination therapy (nitrofurantoin + LCPR1) eliminated planktonic bacteria under static conditions but offered no added benefit against adherent or intracellular populations relative to antibiotic monotherapy. Incorporating flow revealed additional layers of complexity, where shear stress induced bacterial elongation and attachment and altered drug performance, diminishing the efficacy of nitrofurantoin and combination therapy against planktonic populations despite increased drug exposure. Together, these findings demonstrate that the bladder microenvironment and its mechanical forces modulate host-pathogen interactions and profoundly influence UPEC infection dynamics and therapeutic outcomes, emphasizing the need for advanced, physiologically informed models to guide treatment strategies in the post-antibiotic era.

microbiology↗

bioRxiv2025

Modelling catheter-associated bladder mucosal adhesion and microtrauma using a human urothelial microtissue model

Animal models have been used to investigate urinary tract catheters, but most do not accurately recapitulate human bladder physiology nor support different types of catheters. Recent advances in three-dimensional models mimicking tissue microenvironments have improved our understanding of human tissue development and allowed us to model many diseases. Here, our objective was to characterise bladder urothelial mucoadhesion and microtrauma associated with intermittent catheterisation. We employed our three- dimensional Urine-tolerant Human Urothelial model (3D-UHU) to investigate two different intermittent catheter types, a polyvinylpyrrolidone (PVP)-coated catheter (PVP-CC), and a coating-free integrated amphiphilic surfactant (IAS) catheter. We showed that pressing catheters onto the models for two minutes caused compression of the 3D-UHU model in contact regions, with disruption of urothelial umbrella cells detected by immunofluorescence staining of surface markers uroplakin III, cytokeratin-20 and chondroitin sulphate, alongside occasional effacement of apical surfaces exposing intermediate layers. PVP-CC had a significantly higher number of urothelial cells adhered to its surface after catheter removal compared with IAS. Moreover, application and removal of PVP-CC caused a decrease in transepithelial electrical resistance, suggesting barrier disruption, whereas IAS did not cause a statistically different effect. However, no change in paracellular permeability rates assessed by FITC-dextran were observed in models after application of catheter pieces. Finally, compression of models induced trauma-triggered inflammatory cytokine responses. Specifically, we observed increased secretion of the pro- inflammatory cytokine IL-1{beta} as well as the cell adhesion molecule CEACAM1 after exposure to PVP-CC compared with IAS. Our findings suggest that the IAS catheter damaged the epithelium less than the PVP-CC catheter. These data demonstrate that the 3D-UHU model holds promise as an alternative to animal models for investigating the effects of urinary catheters on the urothelium.

bioengineering↗

bioRxiv2024

3D-UHU-TU: A Three-Dimensional Bladder Cancer Model in a Healthy Urothelial Environment

Bladder cancer cases and fatalities continue to rise worldwide with treatment outcomes not improving in the last four decades. Poor translation of potential new therapies from pre- clinical studies to the clinic could be one reason behind this. The patient-derived xenograft (PDX) mouse is the gold-standard for testing new bladder cancer therapies, but there are key physiological and molecular differences between mouse and human bladders. Thus, more human cell-based models may improve translation of treatments. Here, we introduce a bladder cancer microtissue model called 3D Urine-tolerant Human Urothelium-Tumour (3D-UHU-TU), which incorporates spheroids derived from human bladder cancer cell lines RT112 (low grade) and T24 (high grade) into the previously published 3D-UHU healthy urothelial model in a 100% urine environment. Both low- and high-grade 3D- UHU-TU models were characterised using immunofluorescence and immunohistochemistry staining with diagnostic markers (CK7, CK20 and GATA3), cadherin markers (E- and N-Cadherin), invasion and migration markers (MMP-2 and MMP-9) and a proliferation marker (Ki-67). Both models expressed the correct markers in the correct spatial areas. We also investigated the utility of both 3D-UHU-TU models as a platform to test treatments, using the conventional chemotherapeutic Mitomycin C as proof of principle. After 2 hours of treatment and 24 hours of recovery, cell lysis and nuclear damage were observed in both low- and high- grade cancer spheroids, with minimal damage to the surrounding healthy urothelium. At higher doses, cancer spheroids either disintegrated or were reduced in size, with the healthy urothelium still intact. Taken together, 3D-UHU-TU is a novel, in vitro model for testing both the safety and efficacy of new treatments. Furthermore, our work lays the foundation for testing treatments on patient-derived tumour spheroids in a personalised medicine approach.

cancer biology↗

bioRxiv2023

A human urothelial microtissue model reveals shared colonization and survival strategies between uropathogens and asymptomatic bacteria

Urinary tract infection is among the most common infections worldwide, and is typically studied in animals and cell lines with limited uropathogenic strains. Here, we assessed diverse bacterial pathogens and asymptomatic bacteria (ASB) in a human urothelial microtissue model including full stratification/differentiation and urine tolerance. Several uropathogens and ASB-like E. coli invaded intracellularly, suggesting invasion is a shared survival strategy, instead of a virulence hallmark. The E. coli adhesin FimH was required for intracellular community formation, but not for invasion. Other shared lifestyles included filamentation (Gram-negatives), chaining (Gram-positives) and hijacking of exfoliating cells, while biofilm-like aggregates formed mainly with Pseudomonas and Proteus. Urothelial cells expelled invasive bacteria in Rab-/LC3-decorated structures, while highly cytotoxic/invasive uropathogens, but not ASB, disrupted host barrier function and strongly induced exfoliation and cytokine production. Overall, this work highlights diverse species-/strain-specific infection strategies and corresponding host responses in a human urothelial microenvironment, providing insights at the tissue, cell and molecular level. One-Sentence SummaryA human urothelial model revealed shared colonization strategies between uropathogens and asymptomatic bacteria, and pathogen-specific innate immune responses

microbiology↗

bioRxiv2022

An immunoresponsive three-dimensional urine-tolerant human urothelial (3D-UHU) model to study urinary tract infection

Murine models of urinary tract infection (UTI) have significantly improved our understanding of host-pathogen interactions. However, given some differences between the rodent and human bladder which may modulate bacterial response, including certain biomarkers, urothelial thickness and the concentration of urine, the development of new human-based models is important to complement mouse studies and to provide a more complete picture of UTI in patients. We originally developed a human urothelial three-dimensional (3D) model which was urine tolerant and demonstrated several biomarkers of the urothelium, but it only achieved human thickness in heterogenous, multi-layered zones and did not demonstrate the comprehensive differentiation status needed to achieve barrier function. Here, we report an improved 3D urine-tolerant human urothelial (3D-UHU) model, which after 18-20 days of growth, stratifies uniformly to 7 layers. We confirmed by flow-cytometric analysis that the model is comprised of the three expected, distinct human cell types. The apical surface differentiated into large, CD227+ umbrella-like cells expressing uroplakin-1A, II, III, and cytokeratin 20, all of which are important terminal differentiation markers, and a glycosaminoglycan layer. Below this layer, several layers of intermediate cells were present, with a single underlying layer of CD271+ basal cells. The apical surface also expressed E-cadherin, ZO-1, claudin-1 and -3, necessary for barrier formation; barrier integrity was confirmed by transepithelial electrical resistance and FITC-dextran permeability assay. Infection with both Gram-negative and Gram-positive bacterial classes elicited elevated levels of pro-inflammatory cytokines and chemokines characteristic of urinary tract infection in humans, and caused a decrease in barrier function, suggesting that the 3D-UHU model holds promise for studying host-pathogen interactions and host innate immune response.

microbiology↗

bioRxiv2017

A urine-dependent human urothelial organoid offers a viable alternative to rodent models of infection

Murine models describe a defined host/pathogen interaction for urinary tract infection, but human cell studies are scant. Although recent human urothelial organoid models are promising, none demonstrate long-term tolerance to urine, the natural substrate of the tissue and of the uropathogens that live there. We developed a novel human organoid from progenitor cells which demonstrates key structural hallmarks and biomarkers of the urothelium. After three weeks of transwell culture with 100% urine at the apical interface, the organoid stratified into multiple layers. The apical surface differentiated into enlarged and flattened umbrella-like cells bearing characteristic tight junctions, structures resembling asymmetric unit membrane plaques, and a glycosaminoglycan layer. The apical cells also expressed apical cytokeratin-20, a spatial feature of the mammalian urothelium. Urine itself was necessary for full development, and undifferentiated cells were urine-tolerant despite the lack of membrane plaques and a glycosaminoglycan layer. Infection with Enterococcus faecalis revealed the expected invasive outcome, including urothelial sloughing and the formation of intracellular colonies similar to those previously observed in patient cells. This new biomimetic model could help illuminate invasive behaviours of uropathogens, and serve as a reproducible test bed for disease formation, treatment and resolution in patients.

cell biology↗