Flow-Dependent CFU Dynamics Reshape Polymicrobial Biofilm with a Pronounced Dominance Shift under Sub-Inhibitory Antibiotic Stress

Catheter-associated urinary tract infections (CAUTIs) are the most prevalent healthcare-associated infections globally, yet the ecological dynamics governing polymicrobial biofilm communities on indwelling catheters remain poorly understood under physiologically relevant conditions. Most prior work uses static in vitro models that fail to capture continuous urine flow and sub-inhibitory (sub-MIC) antibiotic gradients. We investigated how continuous flow and sub-MIC concentrations of ciprofloxacin and gentamicin reshape colony-forming unit (CFU) dynamics across attached biofilm and dispersed effluent fractions, and species dominance in mono- and polymicrobial biofilms of Pseudomonas aeruginosa (Pa), Klebsiella pneumoniae (Kp), and Enterococcus faecium (Ef) using silicone-coated latex catheter segments, volumetric infusion pumps, and ibidi {micro}-slide VI 0.4 microfluidic chambers. Under antibiotic-free conditions, Pa dominated both dual co-cultures (Pa+Kp, Pa+Ef) in static condition, but this dominance was not sustained under flow in the Pa+Ef pairing, where Ef rose to 62.5% relative abundance. Sub-MIC ciprofloxacin under flow promoted Kp dispersal (+15.87 log? fold change in dispersed-cell fraction(filter), cooperative Pa recovery via Ef co-occupancy, and pronounced Ef dominance in the triple-species community (64.71% relative abundance). Ef exhibited enhanced growth under sub-MIC gentamicin in static conditions that was abolished under flow. CLSM imaging revealed ciprofloxacin-induced Kp filamentation under flow, with Ef microcolonies localising at filament termini--a novel architectural interaction providing spatial scaffolding for the gram-positive partner. These findings establish that continuous flow and antibiotic class jointly determine polymicrobial dominance outcomes in ways invisible to static assays, underpinning Ef persistence in mature CAUTI biofilms and highlighting flow as a central ecological variable in infection pathogenesis.