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

Urinary tract infection (UTI) remains a major global health burden, with frequent recurrences and rising antimicrobial resistance compromising treatment efficacy. Conventional susceptibility assays often fail to predict clinical outcomes, highlighting the need for more physiologically relevant infection models. Here, we investigated how microenvironmental complexity influences uropathogenic Escherichia coli (UPEC) responses to antibiotics and bacteriophages using human urine, a three-dimensional human urothelial microtissue model (3D-UHU), and a novel mesofluidic platform (P-FLO) that applies physiologically relevant wall shear stress to 3D-UHU. Nitrofurantoin showed the greatest potency in conventional susceptibility assays but did not eradicate infection in the static 3D-UHU model. A bacteriophage cocktail (LCPR1) inhibited intracellular bacterial communities, preserved urothelial viability and induced inflammatory cytokine and chemokine secretion. Combined LCPR1 + nitrofurantoin treatment eliminated planktonic bacteria and reduced extracellular and intracellular bacterial communities under static conditions but did not further reduce urothelium-associated bacterial burden compared with nitrofurantoin alone. Under flow, shear stress promoted bacterial elongation, attachment and intracellular community formation, while reducing nitrofurantoin and combination therapy efficacy despite increased drug exposure. These findings demonstrate that the bladder microenvironment profoundly influences UPEC infection dynamics and therapeutic outcomes, underscoring the need for advanced models to guide treatment strategies in the antibiotic resistance era.