Annals of Biomedical Engineering, Vol. 25, pp. 69-76, 1997 0090--6964/97 $10.50 + .00 Printed in the USA. All rights reserved. Copyright 9 1997 Biomedical Engineering Society The Effect of Ultrasonic Frequency upon Enhanced Killing of P. aeruginosa Biofilms ZHEN QIAN,* RICHARD D. SAGERS,'~ and WILLIAM G. PITT* Departments of *Chemical Engineering and tMicrobiology, Brigham Young University, Provo, UT Abstract--It is widely recognized that the bacteria sequestered in a biofilm on a medical implant are much more resistant to antibiotics than their planktonic counterparts. Recent studies have shown that application of antibiotic along with low power ultrasound significantly increases the killing of planktonic bac- teria by the antibiotic. Herein is reported a similar application of antibiotic and ultrasound to sessile bacteria in biofilms of Pseu- domonas aeruginosa on a polyethylene substrate. Biofilm viabil- ity was measured after exposure to 12 I-~g/mlgentamicin sulfate and 10 mW/cm 2 ultrasound at frequencies of 70 kHz, 500 kHz, 2.25 MHz, and 10 MHz. The results indicate that a significantly greater fraction of the bacteria was killed by gentamicin when they were subjected to ultrasound. However, ultrasound by itself did not have any deleterious effect on the biofilm viability. In addition, lower-frequency insonation is significantly more effec- tive than higher frequency in reducing bacterial viability within the biofilm. The possible mechanisms of synergistic action are discussed. Keywords--Ultrasound, Antibiotic, Bacteria, Pseudomonas ae- ruginosa, Gentamicin INTRODUCTION Bacterial attachment and biofilm formation on implants and chronic transdermal medical devices are a serious con- cern to the medical community (1,10). The biofilm infec- tions are typically difficult to resolve with conventional antibiotic therapy. In situ studies have revealed that bio- film bacteria can withstand many times the dosage of an- tibiotic sufficient to completely eradicate the same strain of planktonic bacteria (16). The mechanism of this biofilm resistance is not fully understood, but it appears to depend on both diffusion limitations (1,18,19,22,25) and physi- ological properties of biofilm bacteria (5,6,11-13). The biofilm environment might protect the bacteria in such a manner that not all of the bacteria in the biofilm are killed by the antibiotic. Therefore, the infection returns when the Acknowledgment--The authors would like to gratefully acknowledge funding from the Whitaker Foundation and the National Institutes of Health (R01 HL52216). We also appreciate the helpfulness of Dr. D. A. Christensen in calibrating the ultrasonic transducers. Address correspondence to Dr. William G. Pitt, 350 Clyde Bldg., Brigham Young University, Provo, UT 84602, U.S.A. (Received 15Aug95, Revised 12Mar96, Accepted 25Mar96) antibiotic therapy is stopped, and the implanted device must be removed or replaced to eliminate the infection. Recent research has shown that an applied electric field dramatically enhances the efficacy of antibiotics in killing biofilm bacteria (4,9,15,16). In some cases, 100% killing was reported. It was postulated that the electric field in- creases the effectiveness of the antibiotic by overcoming the permeability problem by driving charged antibiotic molecules through the biofilm matrix by electrophoresis. Although this bioelectric effect appears promising, it may not be applicable to all device-related infections, since not all devices have metallic or conductive surfaces. In a similar approach, Benson et al. reported that the number of viable Pseudomonas aeruginosa adherent to a polyurethane substrate was decreased significantly after combined exposure to a magnetic field and gentamicin antibiotic (3). The mechanism by which magnetic fields increase gentamicin activity against P. aeruginosa biofilm was not clear. It was postulated to result from the produc- tion of radical intermediates and up-regulation of mem- brane porin. Using the magnetic field approach, the maxi- mum reduction in the viable bacteria was only 86.5% (compared to a control biofilm). Previous studies in our laboratory have shown that planktonic P. aeruginosa and Escherichia coli bacteria are significantly more sensitive to low concentrations of gen- tamicin antibiotic when they are subjected to 70-kHz ul- trasound (21). It is postulated that ultrasound enhances the transport of antibiotic into the cells and results in much higher effective antibiotic concentration acting on the bac- teria. In this report we extend the study of this synergistic effect of ultrasound and antibiotics to the killing of bac- teria in a 24-hr-old biofilm on polyethylene. Killing at four frequencies was studied, and the effect of the frequency of ultrasonic irradiation (hereafter referred to as insonation) is reported. EXPERIMENTAL Bacteria Preparation Five milliliters of tryptic soy broth (TSB) without glu- cose (DIFCO, Detroit, MI) were inoculated from a frozen 69