Richard D. Sagers

Ultrasonic enhancement of antibiotic action on gram-negative bacteria.

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.

The effect of ultrasonic frequency upon enhanced killing of P. aeruginosa biofilms.

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 ofPseudomonas aeruginosa on a polyethylene substrate. Biofilm viability was measured after exposure to 12 μg/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.

Bacterial adhesion to orthopedic implant polymers

The degradable polymers poly(orthoester) (POE), poly(L-lactic acid) (PLA), and the nondegradable polymers polysulfone (PSF), polyethylene (PE), and poly(ether ether ketone) (PEEK) were exposed to cultures of Staphylococcus epidermidis, Pseudomonas aeruginosa, or Escherichia coli. Bacteria washed and resuspended in phosphate buffered saline (PBS) adhered to polymers in amounts nearly twice those of bacteria that were left in their growth medium, tryptic soy broth (TSB). In TSB, there was variation in adhesion from species to species, but no significant variation from polymer to polymer within one species. In PBS there were significant differences in the amounts of bacteria adhering to the various polymers with the exception, of S. epidermidis, which had similar adhesion to all polymers. As a whole, P. aeruginosa was the most adherent while S. epidermidis was the least adherent. The estimated values of the free energy of adhesion (delta Fadh) correlated with the amount of adherent P. aeruginosa. When POE, PLA, and PSF were exposed to hyaluronic acid (HA) before exposure to the bacteria, there was 50% more adhesion of E. coli and P. aeruginosa on POE and PLA. With respect to bacterial adhesion, the biodegradable polymers (POE and PLA) in general were not significantly different from the nondegradable polymers.

Investigation of the mechanism of the bioacoustic effect.

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.

Air-water interface displaces adsorbed bacteria

Video microscopy was employed to observed the spatial distribution of Staphylococcus epidermidis and Pseudomonas aeruginosa adherent to glass and polymer substrates. During rinsing procedures the bacteria remained in their original positions when the surfaces were rinsed with saline for 3 min followed by ethanol for 3 min before exposure to air. When the surfaces were rinsed with saline only, the air-liquid interface disrupted the spatial distribution of the bacteria, removing and redepositing the bacteria in clumps. A moving air-liquid interface of a gas bubble on substrate also displaced bacteria. Such artefacts produced by air-water interfaces should be avoided during bacterial adhesion experiments.

Measurement of bacterial growth rates on polymers

A video microscope system and a mathematical model were developed to observe and model the early stage of bacterial growth on polymer surfaces. Glass slides were coated with polyorthoester, poly(L-lactic acid), and polysulfone, and inserted into a laminar flow cell to expose them to bacterial cultures of Staphylococcus epidermidis, Pseudomonas aeruginosa, or Escherichia coli. The free energy of adhesion (delta Fadh) was determined from contact-angle measurements. The microscopic observations along with the mathematical model allowed measurement of the rates of adhesion, release, and growth. The growth rate of P. aeruginosa on the various surfaces correlated to the delta Fadh. The growth rates of all species on all of the surfaces were slower than the growth rates of the bacteria in suspension. The mathematical model is valid for early growth before the bacteria form a complete monolayer, and is useful in predicting and modeling early growth of bacteria on implanted biomaterials.

Bacterial Adhesion to Protein-Coated Hydrogels

Extended wear soft contact lenses have been implicated in the increased occurrence of corneal bacterial infections. This research investigated the effects of polymer chemistry, water content, and pre-sorbed proteins upon the adherence of Pseudomonas aeruginosa to model hydrogels with chemistries similar to those of extended wear soft contact lenses. The hydrogels were exposed to washed suspensions of R aeruginosa in a laminar flow cell. Albumin, fibrinogen, desialylated fibrinogen, or mucin were deposited on the hydrogels before exposure to the bacteria. Results showed that with or without protein pre-exposure, bacterial adhesion decreased as water content increased. In the presence of the sorbed protein, the number of adherent bacteria increased by about 45%, and all four proteins caused similar increases in adhesion. Bacterial adhesion was not significantly influenced by the presence of sialic acid residues in the pre-sorbed protein.