Paduthol, G. | bioRxiv Science

bioRxiv2025

A bioengineered human urothelial organoid model reveals the urine-urothelium interplay in tissue resilience and UPEC recurrence in urinary tract infections

Urine is a dynamic and highly variable biofluid. Urine-urothelium interactions are a critical yet underexplored factor in bladder homeostasis 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 colonization of the bladder by uropathogenic E. 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 contributed 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.

bioengineering↗

bioRxiv2025

Epithelial Reprogramming by OM-89 Enhances Antibiotic Clearance of Uropathogenic E. coli in a Bladder Organoid Model

Recurrent urinary tract infections (UTIs) are a major clinical burden, driven in part by the ability of uropathogenic Escherichia coli (UPEC) to establish intracellular niches within the bladder epithelium, where bacteria withstand antibiotics and host defenses. The oral bacterial lysate OM-89 (Uro-Vaxom(R)), a clinically approved and globally used therapy for the prevention and management of recurrent UTIs for several decades, reduces recurrence rates, but its cellular mechanisms of action remain incompletely understood. Here, we demonstrate that OM-89 strengthens antimicrobial defenses in bladder epithelial cells and, in combination with antibiotic therapy, limits post-treatment regrowth in epithelial infection models. Using bladder organoids together with differentiated epithelial monolayers, OM-89 promotes lysosomal acidification and increases lysosomal protease activity, driving intracellular UPEC toward degradative compartments. In parallel, OM-89 improves intracellular antibiotic efficacy across multiple antibiotic classes, leading to enhanced bacterial clearance and reduced post-treatment bacterial recovery. These effects are conserved across distinct UPEC strains and in both murine and human epithelial models. Our findings position the bladder epithelium from a passive barrier to an active, targetable determinant of treatment outcome and suggest host-directed modulation of epithelial antimicrobial pathways as a promising strategy to enhance intracellular bacterial clearance.

microbiology↗