Use of a 3D urine-tolerant human urothelial model (3D-UHU), which recapitulates the crucial features of human urothelium, has enabled characterization and improved understanding of colonization and survival strategies used by uropathogens and commensal bacteria. The results of this study provide insights into strain-specific and species-specific infection strategies and the reciprocal host responses at the microtissue, cellular and molecular level in a human-relevant urothelial microenvironment.