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

Empirical Studies

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


Preventing wound infection and the development of resistant bacteria are important concerns in wound management. To determine if noncontact, nonthermal, low-frequency ultrasound therapy is effective in controlling wound bacterial colony counts, a series of four related experiments was conducted.

First, ultrasound penetration in both wounded and intact skin was assessed in vitro. Compared to sham, noncontact ultrasound penetrated farther into both wounded (3 mm to 3.5 mm versus 0.35 mm to 0.50 mm) and intact (2.0 mm to 2.5 mm versus 0.05 mm to 0.07 mm, respectively) pig skin. Second, using an in vitro model to stain and count live/dead bacteria, 0% of sham treated and 33% of Pseudomonas aeruginosa, 40% of Escherichia coli and 27% of Enterococcus faecalis were dead after one ultrasound application. Minimal effects on methicillin-resistant Staphylococcus aureus and S. aureus were observed. Third, using an in vivo model, after 1 week, while differences between different bacterial species were observed, overall bacterial quantity decreased with ultrasound treatment (from 7.2 ± 0.79 to 6.7 ± 0.91 colony forming units per gram of tissue [CFU/g]) and silver antimicrobial dressings (from 7.2 ± 0.79 to 5.7 ± 0.6 CFU/g) but increased to 8.6 ± 0.15 CFU/g for sham and 8.6 ± 0.06 CFU/g for water-moistened gauze. Fourth, 11 patients (average age 60 years) with pressure ulcers containing bacterial counts >105 CFU/g of tissue received 2 weeks of noncontact ultrasound therapy. The quantities of seven bacterial organisms were reduced substantially from baseline to 2 weeks post treatment. None of the wounds exhibited signs of a clinical infection during the treatment period and no adverse events were observed. Taken together, these four studies indicate that noncontact ultrasound can be used to reduce bacterial quantity. Controlled clinical studies are warranted to ascertain the efficacy of this treatment and to further elucidate its effects on various Gram-negative and Gram-positive bacteria.

     All chronic wounds are contaminated with bacteria; however, bacteria virulence and quantity, together with the host’s immune response, determine the clinical response and, ultimately, if infection is present.1 A small bioburden in an immune-competent host may have no impact on healing; a large bioburden or an inadequate immune response can manifest clinically as either gross wound infection or as a “stunned,” nonhealing wound.2 Optimal diagnosis and treatment of wound bioburden are subject to debate and no current standard of care exists. Many clinicians have adopted a universal policy of bioburden elimination through the routine use of antibiotics and/or local antiseptics. This approach has led to a widespread increase in antibiotic-resistant organisms and other untoward effects such as an increase in Clostridium difficile infections. 3

     The presence of biofilms within chronic wounds has further reduced the effectiveness of systemic antibiotic use. 4 In addition, there is concern that antimicrobials will not achieve tissue levels in ischemic wounds and wounds with heavily fibrotic granulation tissue adequate to be sufficiently effective, 5 underscoring a pressing need for a locally administered, nontoxic method to reduce bacterial counts within a wound bed that does not increase bacterial resistance.

     Noncontact, nonthermal, low-frequency ultrasound (MIST Therapy® System, Celleration, Inc., Eden Prairie, MN) — hereafter “noncontact ultrasound” — delivers ultrasound energy through a sterile saline mist. This therapy has been shown in randomized, controlled clinical trials to improve both time to healing and the proportion of wounds healed compared with conventional wound care in the treatment of diabetic foot ulcers, lower extremity wounds, and ischemic ulcerations. 6-9 In vitro and animal studies suggest that potential biophysical effects of ultrasound relevant to wound healing include increased angiogenesis, collagen production, cytokine and growth factor release, improved blood flow and vascular permeability, and protection from reperfusion injury. 10-13 The literature supporting the potential biophysical effects of noncontact ultrasound at each stage of the wound healing process was recently reviewed. 14

     To determine if noncontact ultrasound is effective in reducing bacterial counts, a series of controlled experiments was conducted. The initial study set out to determine the depth at which ultrasound could have a physiologic effect, assuming differences in wound bioburden penetration between intact skin (barrier function intact) and wounded skin. To determine the potential depth of penetration for noncontact ultrasound, a pig model was used to measure the transfer of a lipophilic dye into wounded and intact skin treated with noncontact ultrasound or sham ultrasound. Determining the depth of penetration in a wound is important to establish at what depth ultrasound might have an effect on bacteria not only on the surface, but also potentially within the tissue construct itself, which might be of greater clinical significance. A controlled in vitro study then was performed to assess the immediate bactericidal effect of a single noncontact ultrasound treatment. Finally, the effectiveness of noncontact ultrasound in reducing quantitative bacteria counts was assessed both in vivo and in a clinical case study.


     Four sequential experiments were conducted to assess the effects of noncontact ultrasound on wound bacteria levels. Endpoints included: 1) depth of penetration, 2) in vitro bacteria reduction, 3) bacteria reduction in an animal model, and 4) bacteria reduction in a human model. Animals were housed and cared for in accordance with guidelines published by the National Research Council and approved by the Association for Assessment and Accreditation of Laboratory Animal Care. All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee. The human study protocol was approved by institutional review boards (IRB) at two of the sites and by a central IRB for the third site; facilities were located in northwestern Ohio; Erie, Pennsylvania; and Chicago, Illinois. A signed, IRB-approved consent form was obtained from each study participant before baseline tissue biopsy.

    Equipment. Noncontact ultrasound treatment. The ultrasound therapy system used is a noncontact, nonthermal, low-frequency ultrasound device. The generator converts voltage to high-frequency electrical energy. The electrical energy is transmitted to a piezoelectric transducer (lead zirconate titanate) where it is changed to mechanical energy. The transducer operates at 40 kilohertz (kHz) with a distal displacement of 60 to 70 microns. The mechanical energy is transferred to the transducer horn (titanium alloy), which vibrates longitudinally, creating an acoustic pressure output. The maximum transducer intensity is 1.25 Watts (W)/cm2 at a distal displacement of 65 microns when the leading edge of the applicator tip touches tissue (10 mm). Sterile saline in a disposable reservoir is used to create the vaporized mist, which acts as a conduit for delivery of the ultrasound energy to the wound bed without the need for direct patient contact.

     The atomized saline is directed at the wound bed by holding the device perpendicular to the wound and passing the device horizontally and vertically using multiple passes typically over a 3- to 5-minute time frame. Ultimately, treatment times (previously validated and published7) are determined by overall wound surface area. The leading edge of the disposable applicator is held at a distance of 0.5 cm to 1.5 cm from the wound bed during treatment. The leading edge of the applicator is also 1.0 cm from the transducer’s radiating surface, allowing the treatment range to be 1.5 cm to 2.5 cm from the transducer’s radiating surface. Therefore, at a distal displacement of 65 microns, the nominal treatment intensity within the therapeutic range is 0.1 to 0.5 W/cm2. For all experiments reported here, the applicator was held at a distance of 0.5 cm to 1.5 cm from the wound bed. The application of noncontact ultrasound is simple and reproducible. In the authors’ clinics, it is applied by nursing or physical therapy staff.

     Sham treatment. The sham device was designed to deliver an equivalent volume, flow rate, and pressure of saline mist to the wound bed. In order to reproduce an identical energy (pressure) delivery to the wound bed, the sham device was required to be held 10 cm to 15 cm from the wound bed as compared to the noncontact ultrasound device described above. The treatment time was 4 minutes; the technique for holding the device was the same as that described above for the noncontact ultrasound device. No off-the-shelf device could be located to achieve the goals of this study; therefore, a sham device was designed, developed, and manufactured by the study sponsor. Pressure delivered was analyzed using the measured displacement angle of a standard sheet of cellophane. The only difference between the noncontact ultrasound and sham systems was the distance the devices were held from the wound bed. The distance is required to ensure that equivalent amounts of saline mist are applied to the wounds.

     Depth of penetration study. Full-thickness skin samples were harvested using a scalpel blade from the backs of 12 euthanized Yorkshire pigs weighing between 25 kg and 35 kg. The skin was placed on ice, but not frozen, and shipped overnight from a reference laboratory (Thomas D. Morris, Inc., Reisterstown, MD) to the research facility (BRIDGE PTS, Inc., San Antonio, Texas). The full-thickness tissue samples were removed from the packing and a 2-cm punch biopsy specimen was harvested from the center of the initial full-thickness sample. These samples provided the intact skin specimens for the study. To create wounded skin samples, a 810-micron piece of superficial skin was removed with a dermatome from the original full-thickness skin samples. Harvested samples were randomized to treatment with noncontact ultrasound or sham therapy without regard for which pig provided the samples. Immediately before treatment with either the noncontact ultrasound or sham therapy, Nile red dye was mixed into the saline canister to achieve a final concentration of 0.01%. Nile red dye was selected because of its strong lipophilic properties as well as a strong fluorescence that is resistant to photobleaching. Nile red dye has a long history of use as a marker of both epidermal and dermal penetration in published scientific investigations.15 Two ring stands were arranged opposite one another. The sham or noncontact ultrasound unit was affixed to the first stand. 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. aureus, and Escherichia coli.

     Bacteria reduction animal model. Pathogen-free, female Yorkshire pigs (20 kg to 25 kg) were fed antibiotic-free diets and tap water ad libitum. The animals were acclimatized for 10 days before the study. On the day of surgery, the pigs were premedicated by intramuscular (IM) injection of atropine (0.5 mg/kg Atroject, S.A., Burns Veterinary Supply Inc, Westbury, NY) followed by tiletamine/zolazepam (Telazol, Fort Dodge Animal Health, Fort Dodge, IA), 5mg/kg IM. Intubation and inhalation of 1% to 2% isoflurane USP (Attane, Minrad Inc., Buffalo, NY) were provided. The dorsal and lateral thorax and abdomen of the pig were clipped with a #40 Oster clipper blade and washed with antimicrobial-free soap. Twenty full-thickness wounds (10 per side of the pig, 2 cm in diameter) were created 2 cm apart using a custom-designed 2-cm trephine. Hemostasis was achieved with gauze soaked in epinephrine solution (1:10,000 dilution) for 10 minutes for topical application. P. aeruginosa, Fusobacterium spp, and coagulase-negative Staphylococci were grown overnight before surgery. On the morning of surgery, the organisms were washed with sterile saline and resuspended to a final density of 107 CFU/mL in saline. The bacteria were mixed together in a ratio of 1.0:0.5:1.0, respectively, and inoculated onto the wounds, ensuring that all wounds were wetted with the gauze. An occlusive dressing (Saran Wrap, S.C. Johnson and Sons, Brantford, Ontario, Canada) then was applied for 15 minutes, after which the gauze and occlusive coverings were removed and discarded.

     All four animals received noncontact ultrasound treatment on their left side. On the right side, Pigs 1 and 2 received silver antimicrobial dressing (Acticoat™, Smith and Nephew, Largo, FL), Pig 3 received sham therapy, and Pig 4 was treated with a moist control consisting of water-moistened Telfa gauze (Covidien, Inc., Mansfield, MA). During application of the real and sham treatments, neighboring wounds were covered with DuraSorb (Covidien-Kendall, Mansfield, MA) keeping the occlusive side up and the absorbent side down to minimize risk of fluid leakage from these treatments reaching neighboring wounds. Noncontact ultrasound was applied for 4-minute treatment sessions per wound with the applicator tip held 1 cm from the wound base, every other day, for 7 days. After treatment, the wounds were covered with water-moistened Telfa gauze. Silver dressings were changed every other day to coordinate with the noncontact ultrasound treatments. Sham therapy was administered 10 cm from the wound surface for 4 minutes per session per wound on the same days as the noncontact ultrasound treatments. Punch biopsies were taken on days 0, 1, 3, 5, and 7. Two biopsies were taken once from each wound: a 4-mm punch biopsy was taken from the center of the wound for microbiological sampling and analysis and a 6-mm punch biopsy adjacent to the first was used for histopathological examination. The initial 4-mm biopsy samples were homogenized in phosphate-containing buffered saline and 4 minutes per session per wound on the same days as the noncontact ultrasound treatments. Punch biopsies were taken on days 0, 1, 3, 5, and 7. Two biopsies were taken from each wound: first, a 4-mm punch biopsy from the center of the wound for microbiological sampling and analysis and, second, a 6-mm punch biopsy adjacent to the first for histopathological examination. The initial 4-mm biopsy samples were homogenized in phosphate-containing buffered saline and serially diluted. Tryptic soy agar was used for total bacterial counts and mannitol salt agar was used for Staphylococci identification and Pseudomonas isolation agar for the identification of P. aeruginosa. Bacteria counts were expressed as log10 CFU/g.

     Bacteria reduction human model. This prospective study, conducted at three wound care centers in the US, evaluated the effectiveness of noncontact ultrasound therapy in reducing bacteria quantities in Stage III pressure wounds. Patients in both outpatient and inpatient care settings were included. For the primary analysis, results of quantitative bacterial biopsies at baseline were compared with biopsies taken after 2 weeks of noncontact ultrasound therapy.

     Patient enrollment. Consecutive patients presenting to the investigative sites with Stage III pressure wounds and a wound volume of no more than 160 cm3 were considered for study enrollment. Stage III pressure wounds were defined as ulcerations with full-thickness skin loss extending down to, but not through, underlying fascia, with no exposed muscle or bone. Nonviable tissue could be present if it did not obscure the depth of tissue loss. Eligible patients were at least 18 years of age with no clinical signs of acute wound infection and willing to comply with the study wound care and visit requirements. Potential study patients were excluded if they had a head or neck wound, malignancy in the wound bed, electronic prosthesis near the area to receive ultrasound treatment, or any disorder that, in the judgment of the investigator, might interfere with compliance with study requirements. Patients who were previously taking antibiotics either orally or intravenously had to be off treatment for at least 24 hours before enrollment. For patients who presented with multiple wounds, only the largest wound to meet inclusion criteria was enrolled in the study (eg, the largest Stage III pressure wound measuring no more than 160 cm3).

     Study procedures. At baseline evaluation, the treating clinician documented medical history, index wound dimensions (area and volume), and overall wound assessment. A baseline (pre-treatment) quantitative tissue biopsy was obtained before initiating noncontact ultrasound treatment to assess the initial bacterial load in the wound and confirm quantities >105 CFU/g of tissue, the criterion for inclusion in the efficacy analysis. Before obtaining the tissue biopsy, limited surgical debridement of the enrolled wound was performed to remove only nonviable tissue. Following debridement and irrigation of the wound, a 6-mm punch biopsy device was used to obtain a 0.5-g tissue sample from the center of the wound. If the center of the wound was not the most appropriate biopsy site, the biopsy could be taken from a portion of the wound bed where the quantity of tissue would not require restaging of the wound or inhibit healing.

     Noncontact ultrasound treatments were administered to the wound three times per week for 2 weeks. The duration of each treatment session was dependent on wound area in accordance with the manufacturer’s suggested treatment algorithm (range: 3 minutes for wounds <10 cm2 to 20 minutes for wounds 170 cm2 to 180 cm2). Patients who received at least four of the six required treatments were considered evaluable for the effectiveness analysis. In addition to noncontact ultrasound, patients continued to receive standard wound care as determined by the investigator (ie, appropriate use of therapeutic surfaces, pressure-reduction positioning, ongoing management of nutritional status, and moist wound dressings). Treatment with systemic or topical antibiotics, topical antiseptics, EMLA cream, silver dressings, or any antimicrobial dressing was not permitted at any time during the study.

     After 2 weeks of noncontact ultrasound therapy, patients underwent a second quantitative tissue biopsy (post-treatment biopsy). Pre- and post-treatment tissue biopsies were obtained by following a specified protocol and sent to an independent central laboratory (LabCorp, Burlington, NC) for quantitative analysis.

     Data collection and analysis. Data collection and documentation were performed by investigative site personnel. The study sponsor’s clinical personnel monitored data abstraction, completion of study documentation, and correctness of case report forms. Descriptive statistics were performed to summarize and compare baseline data with outcomes following 2 weeks of noncontact ultrasound therapy. Bacteria quantities were summarized in CFU/g of tissue.


     Depth of penetration. Nile red dye penetrated into the intact porcine skin samples 2.0 mm to 2.5 mm with noncontact ultrasound therapy compared with 0.05 mm to 0.07 mm with the sham therapy. The majority of the dye was concentrated in the epidermis and stratum corneum for both treatments. In wounded skin samples, Nile red dye penetrated 3 mm to 3.5 mm with noncontact ultrasound therapy and 0.35 mm to 0.50 mm with sham therapy. With noncontact ultrasound therapy, the dye penetrated into the reticular dermis but not into the underlying subcutaneous fat (see Table 1).

     In vitro bacteria reduction. Following the noncontact ultrasound treatment, the percentage of dead bacteria was 33%, 40%, and 27% for P. aeruginosa, E. coli, and E. faecalis, respectively (see Figures 2, 3a,b). The sham treatment resulted in no dead organisms (0%). Using this treatment protocol, noncontact ultrasound had little or no effect on MRSA (1% increase of live bacteria) and S. aureus (0% change).

     Scanning electron microscopic images of E. faecalis showed structural changes to the round shape of the bacteria with distinct cell wall punctures or wall destruction after noncontact ultrasound treatment compared with intact cell walls in sham-treated controls (see Figure 4).

     In vivo bacteria reduction. Histologically, no differences were noted between the noncontact ultrasound and sham treatment groups with regard to edema, granulation tissue formation, or the presence of eschar. All animals remained healthy and gained weight during the 7-day study. Both the noncontact ultrasound therapy and silver antimicrobial dressing resulted in an overall reduction of bacterial counts (see Table 2). Overall bacterial colony counts increased consistently over time in the moist control group; whereas, bacterial counts in sham therapy-treated wounds decreased or increased at various treatment times. However, the patterns for P. aeruginosa and S. aureus were different. Although noncontact ultrasound therapy resulted in reduced bacterial counts of both organisms during days 3 to 7, during that time, bacterial counts of the Gram-negative bacteria P. aeruginosa changed from 8 ± 0.73 to 5.8 ± 0.74 in the ultrasound, and from 5.7 ± 2. to 4.7 ± 0.85 in the silver antimicrobial dressing group. Given the small sample sizes (as low as n = 2), statistical comparisons between treatment groups were not performed.

     Clinical bacteria reduction study. Of the 18 patients with Stage III pressure wounds enrolled between November 2006 and April 2007, 11 completed baseline and post-treatment biopsies and were considered evaluable for the effectiveness analysis. Five patients were determined unevaluable because their baseline wound bacterial loads were less than 105 CFU/g of tissue or a baseline biopsy was not available. Two patients discontinued study participation — one due to hospitalization for an event unrelated to the study treatment and one due to difficulty arranging transportation to the treatment visits. Of the remaining 11 patients (average age 60 years), eight were African American, six were men, and all had musculoskeletal comorbid conditions. Most wounds were located in the truncal region (see Table 3). Patients received six noncontact ultrasound treatments for a mean duration of 4 minutes per treatment. At no time during the study did the investigators report clinical signs of infection in the enrolled wounds, despite the presence of bacterial loads >105 CFU/g of tissue.

     The quantity of bacterial organisms was reduced from baseline to post treatment. Overall, the mean pre-treatment bioburden was 4 x107 compared with 2 x107 after 2 weeks of noncontact ultrasound treatment. Notable exceptions were beta-hemolytic Streptococcus G, which was only marginally reduced, and beta-hemolytic Streptococcus A, which increased during the study. Beta-hemolytic Streptococcus G was present in five patients at baseline and a bacterial reduction was noted in four (only one patient’s count increased) (see Table 4). Over the 2-week treatment period, mean wound area decreased by 26% from 13.8 cm2 to 10.8 cm2 and mean wound volume decreased by 20% from 18.5 cm3 to 11.6 cm3.


     Currently available antimicrobial agents have limited clinical application in chronic wound care. Topical antiseptics, such as iodine and hydrogen peroxide, reduce bacterial burden but have been shown to be highly cytotoxic. 16 Topical antibiotics are rarely indicated in chronic wounds because of a limited spectrum of activity. The wound care community has turned to the use of silver preparations, cadexomer iodine, Hydrofera blue (Healthpoint, Fort Worth, TX), and similar antimicrobials that are active against most bacterial species and relatively nontoxic to host cells. However, based on in vitro studies, concern is growing that these antimicrobials may have deleterious effects on healing if used for a prolonged period of time.17

     An in vitro experiment by Kavros and Schenck18 identified a potential bactericidal effect of noncontact ultrasound that may be at least partly responsible for the healing response reported in clinical studies of chronic wounds. 8,9,18 The purpose of this study was to test the hypothesis that noncontact ultrasound energy may reduce wound bacterial burden.

     The in vitro study using live/dead staining demonstrated a bactericidal effect with a single treatment of noncontact ultrasound for several organisms. The sham treatment did not reduce bacterial counts. This finding suggests that ultrasound is capable of killing bacteria at least in a laboratory setting. The scanning electron micrographs demonstrated damage to the bacterial cell wall, suggesting that, as observed by Kavros and Schenck,18 cell wall damage may account for the bactericidal effect of ultrasound. However, clinically relevant antimicrobial therapy must reduce more than surface bacteria — it also must penetrate into the deeper layers of the wound surface and ideally also adversely affect biofilms. In the depth of penetration experiment, noncontact ultrasound penetrated tissue to a depth that theoretically could allow for an effect on topical micro-organisms as well as those within 3 mm of the surface. It is important to note that biofilms have been found to be present at this depth, although biofilms were not investigated in these studies.

     An investigation into whether the observed antimicrobial action in vitro could be effected in a porcine wound model showed a reduction in the overall quantity of bacteria present in a wound after a single week (four treatments) of noncontact ultrasound therapy. Noncontact ultrasound produced a similar, although less pronounced, reduction in total bioburden compared to a silver-impregnated dressing; whereas, bioburden in sham-treated and control animals increased during a similar time frame. In brief, these preclinical studies demonstrated that noncontact ultrasound can produce an antimicrobial effect.

     Similar to the in vitro results, in the clinical study a difference was noted in the response of Gram-negative and Gram-positive bacteria to administration of noncontact ultrasound. Quantitative biopsy values for Staphylococcus species, including MRSA, decreased substantially between pre- to post-procedure sampling. However, Streptococcus G showed only a modest reduction and Streptococcus A counts increased in a single individual. Ultimately, healing progression was evident, with 26% reduction in area and 20% reduction in volume during the 2-week study.


     Like most early-stage research, this series of experiments to elucidate the effects of noncontact ultrasound on bacterial counts has limitations and raises as many questions as it answers. Most notably, without a sham control in the human trial, it is impossible to discern whether the observed healing was the result of a reduction in bacteria alone or some other mechanisms. Larger human trials utilizing a sham control are planned to further investigate bacteria reduction. Additionally, although the treatment times (minutes) and frequencies (treatments per week) used in the laboratory studies were based on typical clinical application, it is not known whether these treatment parameters are in fact comparable for in vitro and animal application. Finally, further research is needed to investigate the differential effects of noncontact ultrasound on the various bacterial organisms observed in the animal and human models.


     When considered together, the results of this four-part study to assess the effect of noncontact low-frequency ultrasound on wound bacterial counts suggest this therapy may reduce bioburden in the wound bed and have prompted additional research into the effectiveness of noncontact ultrasound therapy on the mechanical disruption of a biofilm. Chronic wounds are frequently characterized by high bioburden, the presence of senescent cells, nonviable slough, and scar tissue. 19

     Noncontact ultrasound is currently used in the treatment of chronic wounds and indicated for debridement of yellow slough, fibrin, tissue exudates, and bacteria. The bacteria reduction observed in this series of studies suggests that noncontact ultrasound may play a role in reducing overall bacterial burden. Moreover, there is evidence that wounds with >105 CFU/g of tissue may not exhibit clinical signs of infection.20 As in most biological systems, the goal is balance, not necessarily extreme wound sterility that is achieved through antisepsis. 21 Future studies will need to focus not only on quantitative biopsy results, but also on the fingerprint of specific bacteria present in the wound. Techniques such as rapid polymerase chain reaction (PCR) analysis should facilitate this type of investigation. Certainly, nontoxic, effective therapy without the risk of developing bacterial resistance would be a promising option for preventing infection and healing infected wounds.

Dr. Serena is the Medical Director, Serena Group Wound, Hyperbaric, and Research Centers, Warren, PA. Dr. Lee is Medical Director, University Hospitals Bedford Medical Center Wound Care and Hyperbaric Medicine Center; and Chief Medical Officer, Northeast Surgical Associates of Ohio Wound Management Service, Cleveland, OH. Kan Lam and Dr. Attar are affiliated with BRIDGE PTS, San Antonio, TX. Dr. Meneses is Director of Research, Center for Comprehensive Wound and Disease Management, St. James Hospital, Olympia Fields, IL. Dr. Ennis is Professor of Clinical Surgery, Chief Section Tissue Repair and Wound Healing, University of Illinois Chicago, Chicago, IL. Please address correspondence to: Thomas Serena, MD, FACS, Penn North Centers for Advanced Wound Care, 552 Quaker Hill Road, Warren, PA: email: