The Impact of Noncontact, Nonthermal, Low-Frequency Ultrasound on Bacterial Counts in Experimental and Chronic Wounds

Author(s): 
Thomas Serena, MD, FACS; S. Kwon Lee, MS, FACS, FCCWS; Kan Lam, BS, RLAT; Paul Attar, PhD; Patricio Meneses, PhD; and William Ennis, DO

The appropriate skin sample then was attached to a cassette and positioned on the second ring stand. The separation distances, 10 cm for the sham unit and 1 cm for the noncontact ultrasound device, ensured equivalent kinetic energy transfer for both devices as previously described. Three samples each of intact and wounded skin were treated using a 5-minute treatment protocol. This treatment time was selected to simulate treatment time commonly used for smaller wounds in clinical practice. After the treatment, skin samples were embedded in optimal cutting temperature (OCT) and 6-micron frozen sections were created. The sections then were placed on glass using a fluorescence-preserving mounting media and a coverslip was applied. Photographs were taken with a Zeiss fluorescent microscope using a 1.910-second exposure time. The ultimate limit of dye diffusion was determined by applying a long exposure time (>10 seconds) to identify the approximate depth where background illumination was indistinguishable from true fluorescence. Depth measurements were calibrated using the thickness of the epidermis as a standard control of 50 microns (excluding rete pegs).

     In vitro bacteria reduction experiment. During experimental design, it was noted that the saline spray used with noncontact ultrasound might reduce the bacterial count in a petri dish through a simple washout mechanism. Alternative methods for quantifying bacteria reduction in vitro subsequently were explored. It was discovered that bacteria can be trapped using suction to draw the bacteria onto the surface of a sterile 0.2-micron Nuclepore filter. To verify that the integrity of Nuclepore filters would be maintained after extended ultrasound treatment (maximum exposure tested: 10 minutes), aliquots of the fluid that had been sucked through the filter by plating on bacterial growth agar were tested. The Nuclepore filter was applied to the surface of a 150-cc Nalgene analytical filtering unit with a suction unit attached to the canister (see Figure 1). The bacteria then were treated directly on the filter paper itself. For the evaluations of Pseudomonas aeruginosa (ATCC 27317), an inoculum was cultured on nonselective media (trypticase soy agar) and incubated overnight at 37° C. A loopful of the test organism from the agar was transferred into tryptic soy broth and vortexed. This solution was incubated at 37° C for 18 hours and the optical density (OD) was adjusted to 0.16 OD at 625 nm against sterile trypticase soy broth (TSB) as “zero”. Five cc of this solution was transferred to a 495-cc bottle, achieving the final study concentration of 107 colony-forming units (CFU)/mL.

     The noncontact ultrasound unit was attached to a ring stand in a vertical position and the fluid was delivered to the unit through an intravenous catheter of sterile saline. After attaching the noncontact ultrasound or sham units, 100 cc of the bacterial solution was filtered through the treatment membrane. A 5-minute treatment was performed, after which the filter was removed and a strip (0.5 cm x 2.5 cm) from the center of each filter was cut out and stained with live/dead stain. Live-dead percentages were calculated by counting the number of red fluorescent cells (total dead bacteria count) and the number of green fluorescent cells (live bacteria count). Counts were conducted over four microscopic fields for each filter on areas that were exposed to the highest levels of ultrasound (center of the filter). Scanning electromicroscopy was used to provide supporting evidence for the live/dead staining protocol. Similar procedures were carried out with Enterococcus faecalis, methicillin-resistant Staphylococcus aureus (MRSA), S.

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