Abstract 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 Summary A human urothelial model revealed shared colonization strategies between uropathogens and asymptomatic bacteria, and pathogen-specific innate immune responses
