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Bactericidal Effect of 0.95-mW Helium-Neon and 5-mW Indium-Gallium-Aluminum-Phosphate Laser Irradiation at Exposure Times of 30, 60, and 120 Seconds on Photosensitized Staphylococcus aureus and Pseudomonas aeruginosa In Vitro

Noelle A DeSimone, Cory Christiansen, David Dore

Abstract

Background and Purpose. Studies have demonstrated a bactericidal effect of laser irradiation when lasers with power outputs of ≥6 mW are directed toward pathogenic or opportunistic bacteria previously treated with a photosensitizing agent. The purpose of this study was to determine the bactericidal capabilities of irradiation from lasers with power outputs of less than 6 mW on photosensitized microorganisms. Methods. Two bacteria that commonly infect skin lesions, Staphylococcus aureus and Pseudomonas aeruginosa, were used. The 2 lasers used, the 0.95-mW helium-neon laser and the 5-mW indium-gallium-aluminum-phosphate laser, emit light at a wavelength close to the absorption maxima of the sensitizing agent chosen, toluidine blue O. This agent was used because of its proven effectiveness in sensitizing bacteria. For each bacterial strain, toluidine blue O was added to a 108 cells/mL solution until a 0.01% weight/volume ratio was obtained. These mixtures were spread on agar-coated petri dishes, which were then exposed to 1 of the 2 lasers for 30, 60, and 120 seconds. The cultures were then grown overnight and examined for one or more visible zones of inhibition. The areas surrounding the irradiated zone provided a control for the effects of toluidine blue O alone. To determine the effects of laser irradiation without prior toluidine blue O sensitization, separate plates were established using unsensitized bacteria. Results. Although inconsistencies between plates were noted, both lasers produced at least one zone of inhibition in both bacterial species at all 3 time periods. The 5-mW laser, however, produced a greater number of these zones. Conclusion and Discussion. Laser-induced microbial killing of photosensitized organisms could have clinical applications in the treatment of infected skin lesions, pending in vivo studies.

The use of lasers with power outputs of ≥6mWin conjunction with photosensitizing agents to induce microbial death has been demonstrated in vitro.16 Research regarding the bactericidal capabilities of lasers with lower average power output on photosensitized microorganisms is lacking. Such low-power lasers (<6 mW) have been used in physical therapy practice.7 The identification of a bactericidal effect of these lasers could be useful in the treatment of people with infected wounds. This application of laser irradiation could serve as an adjunct to other uses of lasers such as in promoting wound healing.811

Currently, treatment of wounds includes irrigating, cleansing, debriding, dressing with wet or dry material, and alleviating pressure.10,1214 Other treatments involve the use of ultrasound, electrical stimulation, and ultra-violet and laser irradiation to stimulate tissue healing.10 Infected skin lesions are typically treated with oral and topical antibacterial agents in addition to other treatments.13,14 Studies of infected soft tissue lesions have revealed the presence of a variety of microorganisms, including normal skin flora.15,16 Staphylococcus aureus and Pseudomonas aeruginosa are 2 of the most commonly found bacteria.15,16 The development of antibiotic resistance in the infecting organisms augments the difficulty in disinfecting wounds infected with these bacteria.1,17 Because bacterial growth interferes with tissue healing, other methods of eliminating microorganisms in the infected wounds would be useful in promoting healing.

The use of laser light may prove to be beneficial in inhibiting bacterial growth in wounds. Lasers with average power outputs ranging from 1 to 20 mW have been used outside of the United States for more than a decade in the treatment of patients with a variety of musculoskeletal ailments.7,18 Uses such as reducing pain through pressure point stimulation, accelerating wound healing, activating hematopoiesis, decreasing inflammation, and treating dermatoses have all been researched for these types of lasers.811,1820 In the United States, these lasers are in the investigational stage for use by physical therapists and are not a widely accepted treatment modality.7 Two lasers that fall into this range of average power outputs are the 0.95-mW helium-neon (He-Ne) atomic gas laser* and the 5-mW indium-gallium-aluminum-phosphate (In-Ga-Al-PO4) solid-state semiconductor laser. The He-Ne laser has been the most common laser available for use by physical therapists.7 Both of these lasers, how-ever, could prove to be effective if data support their use and if the devices are approved for use in wound healing by the Food and Drug Administration.

A laser's capability to produce injury is an important consideration in clinical applications. Diffuse reflection from the 5-mW laser is not considered hazardous, and more than 0.25 second of direct viewing would be required to cause retinal injury.21 The 0.95-mW laser is thought to be hazardous to the eye if the light is directly focused on the retina for a period of seconds.21 Accidental exposure of this type is highly unlikely, making the hazard minimal. The effect of irradiation from either of these lasers on the skin is essentially the same as for nonlaser optical radiation, and the skin's threshold for injury is about 1 to 10 J/cm2.21

The usefulness of lasers with power outputs greater than 5 mW in conjunction with dyeing agents to induce microbial death has been demonstrated.16 In these studies, bacteria were first treated with a dyeing agent that became concentrated in the cells. This process is called photosensitization. Such agents have specific absorption maxima of light, meaning that they absorb light of certain wavelengths preferentially.22,23 If the absorption maxima of the dye is close to the emission wavelength of the laser being applied to the bacteria, more radiation will be absorbed by the cells than if no dye were present.2,6 As a result of the concentrated laser light, cell death occurs.24 The mechanism responsible for causing bacterial death has been reported to involve the formation of singlet oxygen and free radicals at the level of the cell membrane.5,6,25,26 This treatment of irradiating cells after sensitization to the light by a dye has been termed photochemotherapy and photodynamic therapy.2,5,27 The term “photodynamic therapy” is often used to describe an anticancer treatment that involves laser irradiation after the systemic administration of a photosensitizing drug that is preferentially absorbed by the tumor.

Toluidine blue O (TBO), a photosensitizing agent with an absorption maxima (620–638 nm) close to the emission wavelength of both the He-Ne and In-Ga-Al-PO4 lasers, has been proven to be effective in sensitizing several microorganisms.2,3,5,6 In previous in vitro studies,2,3,5,6 the photosensitizing agents were used at low concentrations, and the irradiation periods were short. If these procedures were applied clinically, the chemical would not need to be maintained within the lesion for a long period of time.28 Therefore, the use of these chemicals in wound healing may be clinically feasible.

The fact that studies that have demonstrated effective killing of photosensitized bacteria have used lasers with power outputs greater than 5 mW is important. At high levels of power output, there is the potential for damaging host tissue during in vivo use.21 The level of radiant exposure that is safe for use on open wounds has not been identified. The lower the dose and the lower the average power output of the laser, however, the less possible hazard there is for the human tissue. One in vitro project not using a photosensitizing agent used a laser with a power output of 0.5 mW and demonstrated no bactericidal effect.29 No data on the bactericidal capabilities of lasers with average power outputs of 5 mW or less on photosensitized microorganisms have been reported.

In order to consider low-power lasers as possible bactericidal tools, it must first be determined whether any bactericidal effect occurs when these lasers are directed toward photosensitized microorganisms. Once the minimum irradiation dose required to kill photosensitized bacteria is determined, the use of the photosensitizing agent and level of laser irradiation on human cells can be examined. The purpose of this in vitro study was to identify any possible bactericidal effects of the 0.95-mW He-Ne and 5-mW In-Ga-Al-PO4 lasers on photosensitized S aureus and P aeruginosa.

Method

Sampling Organisms

Staphylococcus aureus and P aeruginosa were the 2 bacteria used in this study. The specific strains of bacteria were S aureus (ATCC 25923) and P aeruginosa (ATCC 27853), as cataloged in the American Type Culture Collection.

Instrumentation and Materials

The lasers used were the 0.95-mW He-Ne gas laser and the 5-mW solid-state In-Ga-Al-PO4 diode laser. These lasers were selected because of their low-power outputs, which make them relevant for use in physical therapy practice.

The 0.95-mW He-Ne atomic gas laser used was the Dynatron 1120 laser.* This laser uses a mixture of helium and neon gases as its active medium. Light emitted from this laser has a wavelength of 632.8 nm (red light). The output beam used was continuous wave for the purposes of this study.

The 5.0-mW In-Ga-Al-PO4 solid-state semiconductor laser used was the Omni Probe 670P laser. This laser uses indium, gallium, aluminum, and phosphate as semiconductor materials for its active medium, with an emitted wavelength of 670 nm (red light). The output beam of this laser is continuous wave.

Toluidine blue O (CI 52040) was used as the photosensitizing agent because of its demonstrated effectiveness in sensitizing bacteria.2,3,5 In addition, the absorption maxima (620–638 nm) of TBO are similar to those of the 2 lasers chosen.2,3,5,6,22 Toluidine blue O is often used as a stain in the study of cells and tissues.23

Sterile saline was used as a diluent for both the bacteria and the dye. The medium used was 5% sheep blood agar (SBA), which was prepoured into petri dishes (plates) and stored at 4°C. An electrically heated burner provided the capability for sterilizing inoculating loops to transfer and spread bacteria. The bacteria were grown in an incubator set at 37°C and stored in a refrigeration unit maintained at 0° to 10°C.

A number 0.5 MacFarland Equivalence Turbidity Standard (METS)§ prepared from suspensions of uniform latex particles was used to determine the concentration of bacteria in the test tubes. The number 0.5 METS is adjusted by spectrophotometry to approximate a bacterial solution cell density of 1.5 × 108 cells/mL.30 This concentration of bacterial solution was sufficient for creating confluent growth of bacteria on the agar.

Procedure

A pure culture of each species of bacteria was kept in a test tube in the refrigeration unit. Using bacteria loops that were sterilized by an electronic heating device, the bacteria were streaked onto the surface of an SBA plate and grown overnight (16 hours) at 37°C. Isolated colonies from the plates were then transferred to test tubes of sterile saline. The bacteria were added to 0.9 mL of saline until the turbidity of the solution was equal to that of the METS when visually compared.

Once the concentration of the bacteria solution was equal to the METS, 0.1 mL of a 0.1% weight/volume solution of TBO was added to the 0.9-mL bacteria solutions. The result was a solution of bacteria with a 0.01% weight/volume concentration of dye. After mixing, the mixture was then spread, using a sterile cotton swab, onto the SBA in 3 separate orientations to completely cover the surface. Control plates, which consisted of a spread plate of bacteria with no dye, were also established using the same technique described. In this way, the effects of laser irradiation on bacteria that were not treated with a photosensitizing agent were also examined. A control plate of each bacterial species was made for every exposure dose. On the plates of bacteria that were treated with dye, the region outside the irradiated areas revealed the effects of TBO alone.

The spread plates of bacteria were then exposed to one of the lasers for 30, 60, and 120 seconds (Fig. 1). Six plates were prepared for each experimental condition (ie, either laser, either bacterium, for one of the time periods, with or without dye). The area of exposure was held constant by keeping the distance from the tip of the laser to the bacteria constant at 1 cm. At this distance, the areas of irradiation were measured to be 0.28 cm2 for the He-Ne laser and 0.06 cm2 for the In-Ga-Al-PO4 laser. The exposure dose for each trial was calculated using the area, time, and average power outputs. The exposure dose for the 0.95-mW (He-Ne) laser was 0.1 J/cm2 at 30 seconds, 0.2 J/cm2 at 60 seconds, and 0.4 J/cm2 at 120 seconds. The exposure dose for the 5-mW (In-Ga-Al-PO4) laser was calculated to be 2.5 J/cm2 at 30 seconds, 5.0 J/cm2 at 60 seconds, and 10.0 J/cm2 at 120 seconds.

Figure 1.

Example of experimental layout drawn to scale. Large circle represents a petri dish. Small circles represent areas of irradiation by the heliumneon laser.

After irradiation, the control and experimental plates of bacteria were incubated overnight (16 hours) at 37°C. The plates were then visually examined for zones of inhibition, which were used to indicate cell death by laser irradiation.

Measurement and Data Analysis

A plate with a zone of cell death at the area of irradiation was recorded as being positive (+) for inhibition of growth. A plate with no zone of cell death was recorded as being negative (−) for inhibition of growth. Any plates that showed some growth inhibition but not a complete zone of cell death were recorded as partially positive (±). Partially positive plates showed decreased growth in the area of irradiation, but still had a film of bacteria on the surface of the agar.

To determine the score (+, ±, or −), the plates were visually and independently examined by 2 researchers. Because the zones of inhibition were visually obvious, the researchers were not blinded to which lasers were used on a particular plate. The researchers, however, were blinded to each other's findings.

Results

The results for each plate are shown in Tables 1 through 4, with each table representing one bacterial species after treatment with one laser type. For all plates, the researchers agreed on the results.

Table 1.

Results for Staphylococcus aureus After 0.95-mW Laser Irradiationa

No growth inhibition zones were revealed in the controls of irradiated bacteria without prior TBO sensitization, which demonstrates that the dyeing agent was required for cell destruction (Fig. 2). Bacteria that were sensitized with TBO but not subsequently irradiated were present on all experimental plates in the areas surrounding the irradiated zones. These areas provided evidence that TBO alone did not have bactericidal capabilities.

Figure 2.

Photograph of zone of inhibition and controls for both bacterial types. Data not shown for Staphylococcus aureus after irradiation with the 0.95-mW laser.

The actual zones of inhibition were slightly smaller than the areas of irradiation, which could be explained by overgrowth of the surrounding bacteria. Despite this decrease in size, the zones were clearly visible. In Figure 2, photographs of the zones of inhibition are shown for P aeruginosa after treatment with the 0.95-mW He-Ne laser and for both bacteria after irradiation with the 5-mW In-Ga-Al-PO4 laser. Data are not shown for S aureus after irradiation with the 0.95-mW He-Ne laser. Both controls, the area around the irradiated zone and the irradiated bacteria without prior TBO, are shown.

As shown in Tables 1 through 4, both lasers produced a zone of inhibition in both bacterial species, although the results were inconsistent. Table 1 shows that the 0.95-mW laser produced a positive result only after 120 seconds of irradiation in S aureus, but caused at least a partial zone of inhibition in two thirds of the 30- and 60-second trials. When directed against P aeruginosa, this same laser was able to produce clear zones of inhibition in each of the 3 time periods, as shown in Table 3. This result, however, was reproducible only during one other 60-second trial and one other 120-second trial. Tables 2 and 4 show that the greatest number of both positive and partially positive results was afforded by the 5-mW laser in both bacteria.

Table 2.

Results for Staphylococcus aureus After 5-mW Laser Irradiationa

Table 3.

Results for Pseudomonas aeruginosa After 0.95-mW Laser Irradiationa

Table 4.

Results for Pseudomonas aeruginosa After 5-mW Laser Irradiationa

Discussion

Several researchers2,3,5 have reported that TBO sensitization and subsequent low-power laser irradiation of various bacteria result in destruction of the organisms. Wilson and Pratten1 used a gallium-aluminum-arsenide diode laser against aluminum disulphonated phthalocyanine-sensitized S aureus, with a resultant decrease in colony-forming units. Wilson and colleagues2,3 successfully sensitized various oral bacteria to the 7.3-mW He-Ne laser using a variety of photosensitizers. Wilson and Yianni5 sensitized S aureus with TBO and subsequently irradiated it with a 35-mW He-Ne laser. Our results support their findings that low-power laser irradiation in conjunction with TBO can kill microorganisms and further this field of study by showing that even lower-power outputs have this capacity. The 0.95-mW He-Ne laser and the 5-mW solid-state In-Ga-Al-PO4 diode laser demonstrated the ability to inhibit bacterial growth.

As shown in Tables 1 through 4, the number of trial runs that were positive (+) or partially positive (±) varied with respect to exposure time, particularly for the 0.95-mW He-Ne laser. Each exposure time represents a radiant energy dose; the longer the time of irradiation, the higher the dose. Therefore, it is reasonable that the lower success rates were congruent with the lower exposure dose.

The 0.95-mW laser was more effective in producing a combination of both positive (+) and partially positive (±) results in S aureus than in P aeruginosa. This finding may be due to the structural differences of these 2 bacteria. Staphylococcus aureus has a thick cell wall, whereas P aeruginosa has a thin cell wall surrounded by a semipermeable outer membrane.31 More positive (+) results alone, however, occurred for P aeruginosa than for S aureus when using either the 0.95-mW laser or the 5.0-mW laser. The reason for these results is unknown.

The 5-mW In-Ga-Al-PO4 laser clearly produced growth inhibition zones more frequently than did the 0.95-mW He-Ne laser. Based on the absorption maxima of TBO (620–638 nm) and the wavelength emitted by the 2 lasers (632.8 nm for the He-Ne laser and 670 nm for In-Ga-Al-PO4 laser), greater success would be expected from the He-Ne laser. Considering that the He-Ne laser in this study had a lower power output and, therefore, provided a much lower energy dose per exposure time, more success with the 5-mW In-Ga-Al-PO4 laser is understandable.

Although we cannot provide a precise explanation for the inconsistencies in our data, it is important to note that consistencies were revealed on a plate. In most cases, a given plate showed all positive results, all partially positive results, or all negative results. For instance, trials 2 and 5 in Table 1 were negative across all exposure times, whereas all remaining trials were partially positive except for one positive result. This pattern suggests that a procedural error may be responsible for the negative results. The method for quantifying the bacteria before streaking the plate involved a visual comparison of METS and sample. Because this method uses the human eye, some error is inevitable. The differences between trial runs, therefore, may partially be the result of a difference in quantity of bacteria on the plate prior to irradiation. For instance, on the plates that did not show zones of inhibition, we may have had more bacteria on that particular plate. Thus, even though the lasers may have inhibited the growth of the same quantity of bacteria as on the plates with clearly positive zones, it was not visually obvious.

The mechanism of laser-induced cell destruction has important implications in clinical therapy. According to Karu,25 exposing a cell to laser light causes acceleration of electron transfer in some areas of the respiratory chain. At higher doses, this excitation energy is transferred to oxygen to form singlet oxygen.25 When cells are exposed without dye, the flavins and cytochromes of the electron transport chain serve as photosensitizers. The dyeing agents, which can absorb the radiation, bind to components of the cell and thereby enable more laser light to be absorbed.25 Photodyes produce their cytotoxic effect at the cell membrane level in bacteria because their respiratory chain resides there.26 The interaction of laser light with eucaryotic cells is not well-elucidated but is suspected to be more complicated.25 However, the respiratory chain within the mitochondrial membrane would still be affected, and free radical and singlet oxygen production would cause death.25 Therefore, information regarding the relative susceptibilities of the human and bacterial cell membrane to the binding or absorption of the dye is needed to determine whether concomitant host cell killing would result. The results of the in vivo study by Meyer et al,27 which involved laser irradiation in rabbits, suggest that adjacent host tissue damage may not be a cause for concern. In this experiment, small ulcers formed where the tissue had been treated, but they healed in approximately 2 weeks. One approach that could avoid human cell destruction involves linking TBO to antibodies against the various bacteria.5,27 Friedberg et al26 effectively targeted P aeruginosa in the presence of S aureus using this method.

Although the 3 exposure doses from the 5-mW laser are all within the range of the threshold for skin injury, this is the power output of the lasers, not the actual energy absorbed by the cells. It is also noteworthy that the exposure doses of the 0.95-mW laser are all below this threshold for skin injury. The smallest dose used, 0.1 J/cm2 (30 seconds by the 0.95-mW laser), killed bacteria.

There could be many benefits in using this method of disinfecting pressure ulcers and wounds. First, killing is fast; therefore, both dye and laser exposure times would be minimal.1,3,32 Concern regarding the prospect of negative effects from dye itself would be minimized by the requirement of only small amounts of the dye.3 Second, because free radical and singlet oxygen production are responsible for cell death, the development of resistant strains is highly unlikely.1,32 Finally, with this topical method, only the microbes in those areas exposed to dye and laser light would be subjected to killing.1,32 Thus, unlike with systemic treatments, normal flora elsewhere in the body would be spared.

Further in vitro studies would be beneficial. The use of a spectrophotometer before streaking would ensure a consistent quantity of bacterial in each trial run. Alternatively, performing colony counts, as has been done previously with higher-powered lasers,1,4,5 would provide quantification of bacterial cell death. An essential in vitro study would determine the effect of TBO alone and in combination with laser irradiation on human cells. Methods of dye application and removal are requisite to clinical trials. Finally, in vivo studies are necessary to demonstrate clinical feasibility.

Conclusion

We have shown that both the 5-mW In-Ga-Al-PO4 laser and the 0.95-mW He-Ne laser can destroy photosensitized S aureus and P aeruginosa in vitro, although the best results were afforded by the 5-mW laser. Further studies are needed to determine clinical suitability. Once this method of killing bacteria is approved for human use, it could be invaluable to the physical therapy management of infected skin lesions.

Acknowledgments

We thank Dr Lizzie Harrel for her enthusiasm and Dr Jan Gwyer for assistance with the organization of this project.

Footnotes

  • DeSimone, Christiansen, and Dore provided concept and research design, with contributions from Lizzie J Harrell, PhD; writing, with contributions from Daniel Kleven; data analysis, with advice on statistical analysis from Jan Gwyer, PhD, PT; project management; and institutional liaisons. The authors provided facilities and equipment with the assistance of Harrell, who provided materials and guidance in laboratory techniques; Kleven, who also provided advice on laboratory techniques; Marc Castel of Medelco, who provided the 5-mW In-Ga-Al-PO4 laser; and Linda Lawrence, who provided instruction on the use of camera equipment. Data collection and clerical/secretarial support were provided by DeSimone and Christiansen. Consultation (including review of the manuscript prior to submission) was provided by DeSimone and Kleven.

    This study was completed in partial fulfillment of the requirements for Ms DeSimone's and Mr Christiansen's Master of Science degree, Department of Physical and Occupational Therapy, Duke University, Durham, NC.

    This study was presented at the American Physical Therapy Association Combined Sections Meeting; February 14–18, 1996; Atlanta, Ga.

  • * Dynatronics Corp, 7030 Park Centre Dr, Salt Lake City, UT 84121.

  • Medelco, 55 Queens Plate Dr, Suite 1, Rexdale, Ontario, Canada M9W 6P2.

  • American Type Culture Collection, 12301 Parklawn Dr, Rockville, MD 20852.

  • § Remel Inc, 12076 Santa Fe Dr, Lenexa, KS 66215.

  • Received June 25, 1998.
  • Accepted June 9, 1999.

References

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