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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 28.48 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.

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.

Bacterial biofilms growing on implanted medical devices are difficult to eradicate, even with aggressive antibiotic therapy. However, application of ultrasound enhances the effectiveness of the antibiotic. The possible mechanisms of this phenomenon were explored in light of the observed influence of various ultrasonic parameters on the enhanced action of gentamicin against biofilms of Pseudomonas aeruginosa. It is postulated that ultrasound increases the transport of gentamicin through the cell membranes, which is the proposed rate determining step in killing by gentamicin. It is possible that the ultrasound perturbs the cell membrane and stimulates active uptake or permits passive uptake by temporarily disrupting the membrane or other structural cell components. The cell membrane disruption could be caused by high pressure, high shear stress, or cavitation. The dependence upon peak power density suggests that acoustic pressure plays a significant role. There is also a strong frequency component that causes the killing effect to decrease as frequency increases. A mathematical analysis of oscillatory shear stress on the cell shows that the magnitude of stress increases with frequency; thus, the hypothesis of oscillatory shear inducing antibiotic uptake is discounted. In addition, the shear displacement caused by shear forces is very small, so the shear disruption caused by oscillatory flow in an acoustic field has minimal impact. The experimental data also rule out the existence of transient cavitation in the bioacoustic effect. It is possible that stable cavitation and the accompanying microstreaming contribute to the bioacoustic effect., (Copyright 1999 John Wiley & Sons, Inc.)

The effect of erythromycin on planktonic cultures of Psedomonas aeruginosa, with and without application of 70 kHz ultrasound, was studied. Ultrasound was applied at levels that had no inhibitory effect on cultures of Ps. aeruginosa. Ultrasound in combination with erythromycin reduced the viability of Ps. aeruginosa by 1-2 orders of magnitude compared with antibiotic alone, even at concentrations below the minimum inhibitory concentration (MIC). Electron-spin resonance studies suggest that ultrasound induces uptake of antibiotic by perturbing or stressing the membrane. This application of ultrasound may be useful for expanding the number of drugs available for treating localized infections by rendering bacteria susceptible to normally ineffective antibiotics.

New studies on the effect of frequency and duration of exposure upon the ultrasonic enhanced action of gentamicin against biofilms of Escherichia coli are presented in this paper. Ultrasound was applied at clinical levels which would not harm human tissue. The study on frequency indicated that lower frequencies produce higher levels of killing. The results of the timed experiment indicate that complete sterilization of a 14-h biofilm can be achieved after 6 h of exposure. These findings are significant because they show that biofilms can be reduced to zero reproductive ability as assessed by plate counting.

The objective of this research was to elucidate the factors effecting the permeability of cell membranes of gram-negative bacteria toward hydrophobic compounds. Ultrasound treatment, cell age, and the phase state of phospholipid membranes were considered. Spin-labeling EPR method was used to quantify the penetration and distribution of a lipophilic spin probe, 16-doxylstearic acid (16-DS), in Pseudomonas aeruginosa cell membranes. This bacterium was chosen because of its reported resistance to the action of hydrophobic antibiotics caused by the low permeability of the outer cell membrane for hydrophobic compounds. EPR spectra were collected from cell pellets and cell lysates. The overall spin probe uptake was measured in 10% SDS-cell lysates. Lysis with 0.6% SDS revealed the fraction of the probe located in membrane sites readily accessible to the surfactant. The results indicated a structural heterogeneity of P. aeruginosa membranes, with the presence of structurally "stronger" and "weaker" sites characterized by different susceptibility to the SDS treatment. The intracellular concentration of 16-DS was higher in insonated cells and increased linearly with the sonication power. EPR spectra indicated that ultrasound enhanced the penetration of the probe into the structurally stronger sites of the inner and outer cell membranes. The effect of ultrasound on the cell membranes was transient in that the initial membrane permeability was restored upon termination of the ultrasound treatment. These results suggest that the resistance of gram-negative bacteria to the action of hydrophobic antibiotics was caused by a low permeability of the outer cell membranes. This resistance may be reduced by the simultaneous application of antibiotic and ultrasound. This hypothesis was confirmed in our experiments with P. aeruginosa exposed to erythromycin.

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 bacteria by the antibiotic. Herein is reported a similar application of antibiotic and ultrasound to sessile bacteria in biofilms of Pseudomonas aeruginosa on a polyethylene substrate. Biofilm viability was measured after exposure to 12 micrograms/ml gentamicin sulfate and 10 mW/cm2 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 effective than higher frequency in reducing bacterial viability within the biofilm. The possible mechanisms of synergistic action are discussed.

The effect of gentamicin upon planktonic cultures of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, and Staphylococcus aureus was measured with and without application of 67-kHz ultrasonic stimulation. The ultrasound was applied at levels that had no inhibitory or bactericidal activity against the bacteria. Measurements of the MIC and bactericidal activity of gentamicin against planktonic cultures of P. aeruginosa and E. coli demonstrated that simultaneous application of 67-kHz ultrasound enhanced the effectiveness of the antibiotic. A synergistic effect was observed and bacterial viability was reduced several orders of magnitude when gentamicin concentrations and ultrasonic levels which by themselves did not reduce viability were combined. As the age of the culture increased, the bacteria became more resistant to the effect of the antibiotic alone. Application of ultrasound appeared to reverse this resistance. The ultrasonic treatment-enhanced activity was evident with cultures of P. aeruginosa and E. coli but was not observed with cultures of gram-positive S. epidermidis and S. aureus. These results may have application in the treatment of bacterial biofilm infections on implant devices, which infections are usually more resistant to antibiotic therapy.