Microorganisms from a patient or their environment may colonize indwelling urinary catheters, forming biofilm communities on catheter surfaces and increasing patient morbidity and mortality. This study investigated the effect of pretreating hydrogelcoated silicone catheters with mixtures of Pseudomonas aeruginosa and Proteus mirabilis bacteriophages on the development of single-and two-species biofilms in a multiday continuous-flow in vitro model using artificial urine. Novel phages were purified from sewage, characterized, and screened for their abilities to reduce biofilm development by clinical isolates of their respective hosts. Our screening data showed that artificial urine medium (AUM) is a valid substitute for human urine for the purpose of evaluating uropathogen biofilm control by these bacteriophages. Defined phage cocktails targeting P. aeruginosa and P. mirabilis were designed based on the biofilm inhibition screens. Hydrogel-coated catheters were pretreated with one or both cocktails and challenged with approximately 1 ؋ 10 3 CFU/ml of the corresponding pathogen(s). The biofilm growth on the catheter surfaces in AUM was monitored over 72 to 96 h. Phage pretreatment reduced P. aeruginosa biofilm counts by 4 log 10 CFU/cm 2 (P < 0.01) and P. mirabilis biofilm counts by >2 log 10 CFU/cm 2 (P < 0.01) over 48 h. The presence of P. mirabilis was always associated with an increase in lumen pH from 7.5 to 9.5 and with eventual blockage of the reactor lines. The results of this study suggest that pretreatment of a hydrogel urinary catheter with a phage cocktail can significantly reduce mixed-species biofilm formation by clinically relevant bacteria.A recently reported survey of 183 acute care hospitals in 2011 found that approximately 9% of the health care-associated infections were catheter-associated urinary tract infections (CAUTIs) (1). The increases in patient morbidity, hospital stays, and costs of care for patients with CAUTIs are substantial (2, 3).Microorganisms may colonize indwelling urinary catheters and form extensive and often multispecies biofilm (4-8). The exact role of catheter-associated biofilms in CAUTI pathogenesis is poorly understood, but there is evidence that such biofilms play an important role as stable reservoirs of uropathogenic microorganisms that are resistant to antimicrobials (9-12) and difficult to eliminate even if the catheter is removed (13-15). Large reductions in CAUTI rates can be achieved by limiting catheterized patient days and by implementing good catheter care practices (16), but there is still substantial interest in developing urinary catheters that are highly resistant to bacterial colonization by virtue of an inherent property of the material itself or by impregnation or coating of the structural material with an antimicrobial or biological agent. Proposed strategies have included surface patterning (17, 18), novel polymers (19), instillation of catheter retention balloons with bactericidal chemicals (20, 21), bacterial interference (22, 23), and catheter coat...