| Sign In to gain access to subscriptions and/or personal tools. |
Effects of Low-energy Shock Waves on Oral Bacteria
1 Center for Oral Health Research, College of Dentistry, University of Kentucky, 414, Health Sciences Research Building, Lexington, KY 40536-0305, USA; Correspondence: * corresponding author, mjnova2{at}email.uky.edu
We have recently demonstrated that extracorporeal shock-wave therapy (ESWT) is effective in promoting the healing of dermal wounds and in regenerating alveolar bone lost through periodontal disease. The objective of the present study was to determine any antibacterial effect of ESWT on oral bacteria. Monoculture suspensions of 6 bacterial species were treated with 100 to 500 pulses of ESWT at energy flux densities (EFD) of 0.12 mJ/mm2, 0.22 mJ/mm2, and 0.3 mJ/mm2. Following treatment, aliquots were plated for viability determination and compared with untreated controls. ESWT showed a significant microbicidal effect for Streptococcus mutans and an unencapsulated strain of Porphyromonas gingivalis following as few as 100 pulses at 0.3 mJ/mm2 (p  0.001). In addition, a significant disruption of bacterial aggregates was observed at lower EFDs. No significant reduction in viability was observed for all other bacteria at EFDs and pulses tested (p > 0.05). These findings suggest that low-energy ESWT may be bactericidal for selected oral bacteria.
Key Words: shock waves • therapy • oral bacteria
Extracorporeal shock waves are characterized by positive pressures of up to 100 megapascals (MPa), and negative pressures of 5–10 MPa. The pressure waves travel through fluid and soft tissue, and their physical effects occur at interfaces with a change of impedance in their flow (Sturtevant, 1996). Extracorporeal shock-wave therapy (ESWT) has numerous applications in modern medicine; it is commonly used to fragment renal calculi (lithotripsy) (Fuchs and Patel, 1996), as well as to disintegrate calcium deposits in the pancreas, salivary ducts, and the gall bladder (Sauerbruch et al., 1986; Delhaye et al., 1992; Iro et al., 1992). Focused ESWT is also used in orthopedics to facilitate bone healing and in musculo-skeletal conditions such as plantar fasciitis, symptomatic heel spur, lateral elbow pain, lateral epicondylitis, and upper limb hypertonia in people who have had strokes (Buch et al., 2002; Manganotti and Amelio, 2005; Martini et al., 2005; Trebinjac et al., 2005; Buchbinder et al., 2006; Kudo et al., 2006). More recently, we have demonstrated that ESWT, when used at low energy levels, can promote healing of dermal lesions in a rodent skin flap model (Dr. H. Vasconez, personal communication) and promote the regeneration of alveolar bone in a rodent model of periodontitis (Sathishkumar et al., 2008). However, we did not determine if any antibacterial effects of low-energy ESWT contributed to the clinical outcome in either of these models. Several studies have demonstrated antibacterial effects of high-energy shock waves on planktonic microorganisms (Kerfoot et al., 1992; von Eiff et al., 2000; Gollwitzer et al., 2004; Gerdesmeyer et al., 2005) or in association with urinary calculi (Reid et al., 1990; Stoller and Workman, 1990; Prabakharan et al., 1999). However, no studies to date have examined the effects of low-energy shock waves on bacteria, especially Gram-negative and Gram-positive bacteria of oral origin. The structure and rigidity of the cell walls of bacteria vary significantly, and a thin peptidoglycan layer of Gram-negative cells makes them more sensitive to disruption (Diels and Michiels, 2006). Therefore, the goal of this study was to examine the effects of low-energy ESWT on a selection of Gram-positive and Gram-negative bacteria that are frequently associated with dental plaque.
Bacterial Cultures The bacteria used in this study were a selection of Gram-positive and Gram-negative strains that are important components of complex biofilms. Gram-negative bacteria included: Porphyromonas gingivalis 381 (unencapsulated, fimbriated strain), Porphyromonas gingivalis W83 (heavily encapsulated and essentially afimbriate strain), and Fusobacterium nucleatum ATCC 49256. Gram-positive strains included Actinomyces naeslundii ATCC 49340, Streptococcus mutans ATCC 25175, and Staphylococcus aureus ATCC 12600, which was included in this study as a positive control, since previous studies had shown that it was susceptible to high-energy ESWT (Von Eiff et al., 2000; Gerdesmeyer et al., 2005). P. gingivalis was grown in DifcoTM Anaerobe Broth MIC, and F. nucleatum was grown in BactoTM Tryptic Soy Broth (TSB) at 37°C (10% H2, 85% N2, and 5% CO2), and A. naeslundii, S. mutans, and S. aureus were grown in TSB at 37°C (5% CO2, 5% CO2, and air, respectively) (Voigt Global Distributing, Inc., Lawrence, KS, USA). Colonies from blood agar plates were inoculated into 5 mL of appropriate broth and grown to early-stationary phase. Cultures were then transferred to fresh media and grown to mid-logarithmic phase. We used a 0.1-mL aliquot to quantify the concentration of bacteria using a Petroff-Hausser cell counter. The cultures were then serially diluted in phosphate-buffered saline (PBS). Three milliliters of a monoculture suspension of each bacterium (1–3 x 105 bacteria/mL) were individually placed in pouches made from the fingertips of sterile, non-powdered surgical gloves, to simulate the interaction between bacteria and soft-tissue surfaces. Air was removed, and the opening of each pouch was tightly secured just above the level of the bacterial suspension.
Shock-wave Application
Statistical Analysis Descriptive statistics of the viable bacteria were expressed as a mean ± standard error of the mean (SEM). Comparisons of the colony-forming units (CFU) in control vs. treatment conditions were made by a Kruskal-Wallis one-way analysis of variance (ANOVA), followed by the Mann-Whitney Rank Sum test to determine specific differences between individual treatment conditions and the control cultures. A p < 0.05 was accepted as a statistically significant effect of the treatment.
We undertook this study to determine if low-energy shock waves could effectively decrease the viable counts of monoculture bacterial suspensions in vitro. We specifically targeted oral bacteria that constitute components of the plaque biofilm. Development of the plaque biofilm, as with other complex microbial biofilms, is a sequential process, with oral streptococci being the primary early colonizers. This initial colonization by streptococci is followed by increasing proportions of Actinomyces, Veillonella, Fusobacterium spp., and selected other species (middle colonizers), with subsequent colonization dominated by Gram-negative, anaerobic bacteria (late colonizers) (Rosan and Lamont, 2000; Kolenbrander et al., 2002). We purposefully selected bacterial species representing these various stages of oral biofilm development for study, to determine their individual susceptibility prior to future studies of biofilms. In addition, we compared an encapsulated P. gingivalis strain W83 with the non-encapsulated P. gingivalis strain 381, to determine if there was a protective effect afforded by the capsule (Aduse-Opoku et al., 2006). S. aureus was included as a positive control and to determine if the effect of low-energy ESWT was similar to that seen in earlier studies with higher-energy ESWT (von Eiff et al., 2000).
Effect of ESWT on Gram-positive Bacteria: S. mutans
No significant changes in bacterial viability were observed at any EFD and pulse combination for any of the other Gram-positive strains tested, including A. naeslundii and S. aureus.
Effect of ESWT on Gram-negative Bacteria: P. gingivalis 381
No significant changes in bacterial viability were observed at any EFD and pulse combination for any other Gram-negative strain tested, including the encapsulated strain, P. gingivalis W83, and F. nucleatum.
Previous studies have shown that the delivery of energy, through ultrasound, to bacteria in suspension results in a two-phase response (Joyce et al., 2003). At low power, there is an initial rise in cell numbers as a result of disaggregation of bacterial agglomerates, whereas at high power, there may be an initial rise in numbers from disaggregation, followed by falls in viable cell numbers as the disaggregation finishes and bacterial killing takes over. Ultrasound can inactivate bacteria and disaggregate bacterial clusters through several mechanisms arising from acoustic cavitation (Joyce et al., 2003). Energy released from cavitation bubbles may affect bacterial viability through direct effects on cell membranes, shear forces, and possible chemical attack through the formation of oxygen radicals. The effects of shock waves occur primarily at sites of impedance change in their flow. High-energy ESWT, at EFDs as high as 0.96 mJ/mm2, has been effectively used for the in vitro killing of bacteria (von Eiff et al., 2000; Gollwitzer et al., 2004; Gerdesmeyer et al., 2005). It is clear that bacterial killing is a function of energy level and impulse frequency, and that these variables may differ for different bacteria (Joyce et al., 2003).
In the current study, the impact of low-energy ESWT, at EFDs of In addition to the use of low-energy ESWT in this study vs. the high-energy ESWT in previous studies, other differences in study design should be noted. In this study, both the sample volume and the material holding the sample differed from those used in evaluation of the impact of high-energy ESWT on bacterial viability. This change in study design was intentional, since we attempted to simulate a soft-tissue/fluid interface. Also, the volume used allowed us to withdraw a 0.1-mL aliquot following every 100 pulses without significantly altering the total volume remaining for treatment. In addition, the highest EFD used in this study was 0.3 mJ/mm2 vs. the 0.9 mJ/mm2 used in previous studies of ESWT. This difference is based upon the capabilities of the Dermagold® shock-wave generator, which is specifically designed to generate low-energy ESWT to stimulate the resolution of inflammation, tissue repair, and regeneration. We also limited the number of pulses to 500, since the study was designed to approximate treatment that may be provided in a clinical setting. While it appears from this initial study that low-energy ESWT may not predictably lead to the efficient killing of the majority of Gram-negative or Gram-positive microorganisms in the sample volumes tested in this study, it has the potential to disrupt bacterial aggregates found in dental/medical pathogenic biofilms. This exciting potential for the non-invasive mechanical disruption of biofilms could facilitate the use of other adjunctive antibacterial treatments, such as antimicrobials and/or antibiotics, or even provide enhanced access to the biofilm components by endogenous antimicrobial peptides and/or specific antibodies (Otto, 2006). To assess this potential, additional in vitro experiments in complex microbial biofilms and in vivo studies in animal models will need to be conducted. In summary, these studies demonstrated that low-energy ESWT is capable of disaggregating the Gram-positive and Gram-negative bacteria that are important in biofilm formation, and of selectively killing two of the primary pathogens associated with oral and systemic infections. The limited killing observed in this study may have been due to the low levels of energy and the relatively high volume of target planktonic bacteria. Future studies will focus on the application of this low-energy, non-invasive technology, to complex biofilm formation on solid surfaces.
This study was funded by a grant from Tissue Regeneration Technologies (Woodstock, GA, USA) to Dr. K.F. Novak. Drs Schaden and House are employees of Tissue Regeneration Technologies. No other consultant fees or financial arrangements exist between the authors and the sponsors. Received for publication February 18, 2008. Revision received June 5, 2008. Accepted for publication June 6, 2008.
Journal of Dental Research, Vol. 87, No. 10,
928-931 (2008)
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
|
 |
|
Sign In to gain access to subscriptions and/or personal tools.
TOP
ABSTRACT
INTRODUCTION
indicates a significant (p = 0.025) decrease in CFU vs. corresponding pulse point at EFD 0.12 mJ/mm2.