SummaryMedical implants are sometimes colonized by biofilm-forming bacteria that are very difficult to treat effectively. The combination of gentamicin and ultrasonic exposure for 24 h was previously shown to reduce the viability of E. coli biofilms in vivo. This article shows that such treatment for 48 h reduced viable E. coli bacteria to nearly undetectable levels. However, when P. aeruginosa biofilms were implanted and treated for 24 and 48 h, no significant ultrasonic-enhanced reduction of viable bacteria was observed. The difference in response of these two organisms is attributed to greater impermeability and stability of the outer membrane of P. aeruginosa.

Escherichia coli biofilms on two polyethylene disks were implanted subcutaneously into rabbits receiving systemic gentamicin. Ultrasound was applied for 24 h to one disk. Both disks were removed, and viable bacteria were counted. Pulsed ultrasound significantly reduced bacterial viability below that of nontreated biofilms without damage to the skin.

The hypothesis that ultrasound increases antibiotic transport through biofilms of Escherichia coli and Pseudomonas aeruginosa was investigated using colony biofilms. Biofilms grown on membrane filters were transferred to nutrient agar containing 50 μg/mL gentamicin. A smaller filter was placed on top of the biofilm and a blank concentration disk was situated atop the filter. Diffusion of antibiotic through the biofilms was allowed for 15, 30, or 45 min at 37°C. Some of these biofilms were exposed to 70 kHz ultrasound and others were not. Each concentration disk was then placed on a nutrient agar plate spread with a lawn of E. coli. The resulting zone of inhibition was used to calculate the amount of gentamicin that was transported through the biofilm into the disk. The E. coli and P. aeruginosa biofilms grown for 13 and 24 h were exposed to two different ultrasonic power densities. Ultrasonication significantly increased the transport of gentamicin through the biofilm. Insonation of biofilms of E. coli for 45 minutes more than doubled the amount of gentamicin compared to their non-insonated counterparts. For P. aeruginosa biofilms, no detectable gentamicin penetrated the biofilm within 45 min without ultrasound; however, when insonated (1.5 W/cm 2 ) for 45 min, the disks collected more than 0.45 μg of antibiotic. Ultrasonication significantly increased transport of gentamicin across biofilms that normally blocked or slowed gentamicin transport when not exposed to ultrasound. This enhanced transport may be partially responsible for the increased killing of biofilm bacteria exposed to combinations of antibiotic and ultrasound.

Biofilm infections are a common complication of prosthetic devices in humans. Previous in vitro research has determined that low-frequency ultrasound combined with aminoglycoside antibiotics is an effective method of killing biofilms. We report the development of an in vivo model to determine if ultrasound enhances antibiotic action. Two 24-h-old Escherichia coli (ATCC 10798) biofilms grown on polyethylene disks were implanted subcutaneously on the backs of New Zealand White female rabbits, one on each side of the spine. Low-frequency (28.48-kHz) and low-power-density (100- and 300-mW/cm2) continuous ultrasound treatment was applied for 24 h with and without systemic administration of gentamicin. The disks were then removed, and the number of viable bacteria on each disk was determined. At the low ultrasonic power used in this study, exposure to ultrasound only (no gentamicin) caused no significant difference in bacterial viability. In the presence of antibiotic, there was a significant reduction due to 300-mW/cm2 ultrasound (P = 0.0485) but no significant reduction due to 100-mW/cm2 ultrasound. Tissue damage to the skin was noted at the 300-mW/cm2 treatment level. Further development of this technique has promise in treatment of clinical implant infections.

SUMMARYInfection of implanted medical devices by Gram-positive organisms such as Staphylococcus ssp. is a serious concern in the biomaterial community. In this research the application of low frequency ultrasound to enhance the activity of vancomycin against implanted Staphylococcus epidermidis biofilms was examined. Polyethylene disks covered with a biofilm of S. epidermidis were implanted subcutaneously in rabbits on both sides of their spine. The rabbits received systemic vancomycin for the duration of the experiment. Following 24 h of recovery, one disk was insonated for 24 or 48 h while the other was a control. Disks were removed and viable bacteria counted. At 24 h of insonation, there was no difference in viable counts between control and insonated biofilms, while at 48 h of insonation there were statistically fewer viable bacteria in the insonated biofilm. The S. epidermidis biofilms responded favorably to combinations of ultrasound and vancomycin, but longer treatment times are required for this Gram-positive organism than was observed previously for a Gram-negative species.

Aims: The aim of this study is to investigate whether pulsed ultrasound (US) in combination with gentamicin yields a decreased viability of bacteria in biofilms on bone cements in vivo. Methods and Results: Bacterial survival on bone cement in the presence and absence of ultrasound was compared in a rabbit model. Two bone cement samples with an Escherichia coli ATCC 10798 biofilm were implanted in a total of nine rabbits. In two groups bone cement discs loaded with gentamicin, freshly prepared and aged were used, and in one group unloaded bone cement discs in combination with systemically administered gentamicin. Pulsed ultrasound with a frequency of 28AE48 kHz and a maximum acoustic intensity of 500 mW cm )2 was applied continuously from 24 h till 72 h postsurgery on one of the two implanted discs.After euthanization and removal of the bacteria from the discs, the number of viable bacteria were quantified and skin samples were analysed for histopathological examination. Application of ultrasound, combined with gentamicin, reduced the viability of the biofilms in all three groups varying between 58 and 69% compared with the negative control. Histopathological examinations showed no skin lesions. Conclusions: Ultrasound resulted in a tendency of improved efficacy of gentamicin, either applied locally or systemically. Usage of ultrasound in this model proved to be safe. Significance and Impact of the Study: This study implies that ultrasound could improve the prevention of infection immediately after surgery, especially because the biomaterials, gentamicin and ultrasound used in this model are all in clinical usage, but not yet combined in clinical practice.

Low-frequency ultrasound has been investigated as an adjuvant to antimicrobial therapy, targeted at both planktonic and biofilm (sessile) organisms. Our previous work showed that ultrasound (US) effectively enhances the bactericidal activity of certain antibiotics against planktonic cultures (Pitt et al., 1994;Rediske et al., 1999) and in vitro biofilms (Johnson et al., 1998;Qian et al., 1999) and in vivo biofilms (Carmen et al., 2004b(Carmen et al., , 2005Rediske et al., 2000) of gram-positive and gram-negative bacteria. Ultrasound was shown to increase the transport of antibiotics through biofilms (Carmen et al., 2004a) which could account for some (or all) of the enhanced antibiotic activity against insonated biofilms; but such a mechanism could not account for US-enhanced antibiotic activity in planktonic cultures which have no extensive exopolymer matrix to retard antibiotic transport.Because this ultrasonic enhancement of antibiotic activity operates on both planktonic and sessile bacteria, we posit that US does more than simply increase the transport of antibiotic to the cells; ultrasound is postulated to increase uptake of antibiotic into the cells by rendering the cell membrane more permeable to the antibiotic. To examine this postulate, we must first review how ultrasound interacts with cells.Bacterial cells are fairly transparent to ultrasound; that is, ultrasonic waves go right through cells with little absorption, scattering or other interaction. However, the pressure oscillations of ultrasound produce size oscillations in any gas bubbles in the liquid (Brennen, 1995). These bubbles range in size from approximately 1 mm to 100 mm in diameter (Brennen, 1995). The oscillations of bubbles, called cavitation, are generally divided into "stable" and "collapse" types of cavitation. Stable cavitation is the low intensity oscillation of the bubbles without complete collapse of the bubble, while collapse cavitation occurs at higher intensity levels and lower frequencies wherein these bubbles collapse and violently accelerate the fluid around them. During bubble collapse, adiabatic heating of the gas produces very high temperature, produces free radicals, generates very high liquid shear force, and generates a shock wave as the collapsing spherical wall slams into itself (Brennen, 1995). With a sufficient number of collapse cavitation events, cell membranes

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