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Infection is a common complication associated with the use of transcutaneous and implanted medical devices. These infections are generally difficult to treat and frequently require removal of the biomaterial before the infection can be completely eradicated. The presence of a bacterial biofilm recalcitrant to treatment often mediates these infections. We studied the influence of a polycationic protein, protamine sulfate, on the efficacy of the fluoroquinolone ciprofloxacin against a clinical isolate of Pseudomonas aeruginosa. A P. aeruginosa biofilm was developed on 1-cm sections of red rubber catheter material and then treated with various combinations of protamine sulfate and ciprofloxacin. The present work demonstrated that ciprofloxacin in combination with protamine was more effective against biofilms than was ciprofloxacin alone. Protamine sulfate at 50 micrograms/ml combined with antibiotic at 0.5 microgram/ml reduced the number of viable cells by an average of 98.97%, while protamine sulfate at 50 micrograms/ml alone resulted in an average 107.8% increase and antibiotic alone resulted in an average 58.6% reduction after 24 h. Furthermore, protamine sulfate, in combination with ciprofloxacin, inhibited P. aeruginosa in a dose-dependent fashion. It was further observed that treatment with the combination of protamine sulfate and ciprofloxacin had a more drastic effect on planktonic organisms as compared with the P. aeruginosa biofilms; the MBC was reduced to < 0.05 microgram/ml in the presence of 25 micrograms of protamine sulfate per ml. These findings were substantiated by ultrastructure studies of treated cells using scanning and transmission electron microscopy. The synergism between ciprofloxacin and protamine sulfate significantly enhanced the efficacy of ciprofloxacin against planktonic and biofilm P. aeruginosa.