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.

Urinary tract infection (UTI) ranks among the most prevalent bacterial infections worldwide, affecting over 400 million people each year. Uropathogenic Escherichia coli (UPEC), the main aetiological cause of UTI, colonises the intestinal tract, which is thought to serve as a distal reservoir for gut-UTI recurrence. Despite this, the precise role of the gut in UTI recurrence is still not fully defined. Recent research investigating the gut-UTI axis has revealed that reduced abundance of gut commensals producing short-chain fatty acids (SCFAs, namely acetate, butyrate and propionate) is associated with recurrent and chronic UTI. We therefore aimed to investigate the impact of these gut commensal-derived metabolites on a diverse panel of UPEC strains, including well-studied prototypical strains (UTI89, CFT073), a non-pathogenic isolate E. coli K-12, and various clinical UTI isolates (from the urine of both symptomatic and asymptomatic individuals). We observed that SCFAs modulate bacterial growth kinetics in a concentration- and pH-dependent manner, by prolonging the lag phase without affecting final carrying capacity in vitro . These metabolites further suppressed bacterial swimming motility and biased the orientation of fimS , the phase variable switch for T1 fimbriae, under acidic conditions. In a human polarized, mucus-secreting intestinal infection model, SCFA treatment during UPEC challenge altered bacterial localization patterns, favouring planktonic over mucosal-associated populations, and preserved epithelial barrier function. Together, these in vitro findings demonstrate that SCFAs modulate key UPEC colonization-associated phenotypes and influence host-pathogen dynamics at the colonic epithelial interface. These results provide mechanistic insights into how depletion of SCFA-producing gut commensals may alter the intestinal reservoir environment in vitro and warrants further investigation into the role of gut-derived SCFAs in rUTI susceptibility.