Bactericidal action of 308 nm excimer-laser radiation: An in vitro investigation

0099-2399/98/2412-0781 $03.00/0 JOURNALOF ENDODONTICS

Printedin U.S.A. VOL.24, NO.12, DECEMBER1998

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SCIENTIFIC ARTICLES Bactericidal Action of 308 nm Excimer-Laser Radiation: An In Vitro Investigation Matthias Folwaczny, Dr med dent, Tim Liesenhoff, Dr med, Norbert Lehn, Prof Dr med, and Hans-Henning Horch, Prof Dr med, Dr med dent

in improving endodontic treatment (3-5). Irradiating root canals with Nd:YAG and carbon dioxide laser caused serious side effects (e.g. supraphysiological heating and induction of a wide zone of necrosis at the border of irradiated tissue). In contrast, no or only minor side effects following a 308 nm excimer-laser radiation were observed (6). In comparison with conventional manual techniques of endodontic treatment, the 308 nm excimer laser offers the opportunity to prepare root canals without any smear layer and with open dentin tubuti. In addition to the treatment of dental hard tissues, the potential action of laser radiation on bacteria was a main point of interest. Recent clinical studies using 308 nm excimer-laser radiation demonstrated better success than conventional preparation of the root canal (5), These results were found even if radiation was applied in conventionally prepared root canals (7). According to this observation, and because the wavelength of 308 nm is in the middle ultraviolet region of electromagnetic radiation, it was postulated that XeCI excimer-laser radiation may reduce the bacterial load in addition to the pure ablative tissue effects. The aim of the present study was to investigate whether a 308 nm excimer-laser radiation has antirnicrobial effects. Moreover, information was to be gained about the way these effects depend on the irradiated bacterial strain, radiation time, and the energy density of radiation. Thermal killing of bacteria should be excluded. Reduction of bacteria caused by mechanical ablation should also be excluded by a suitable test procedure.

The aim of the present study was to investigate the influence of 308 nm excimer-laser radiation on bacterial growth. Six different bacterial strains

(Staphylococcus aureus, Escherichia coli, Streptococcus faecalis, Lactococcis lactis, Salmonella typhimurium, and Deinococcus radiodurans) were exposed in vitro to various doses and energy densities of laser radiation. To exclude bacterial killing by supraphysiological heating, the temperature change in the samples during irradiation was measured. Extended antimicrobial effects of XeCl excimer-laser radiation depending on the time of radiation, the energy density of the laser beam, and the irradiated bacterial strain were observed. Reduction of bacterial g r o w t h is independent of temperature and not linked to any ablative tissue removal. In almost all cases, a 99.9% reduction of bacteria was reached by total radiation times <100 ms. The proven antimicrobial effects of 308 nm excimer-laser radiation may be of significant clinical importance in e n d o d o n t i c s and periodontoiogy in the future.

MATERIALS AND METHODS Growth of bacteria in necrotic pulp--and the resulting exposure to toxic substances from bacterial metabolism--is considered to be one of the most important etiological factors in the development of inflammation at the periapical region of the tooth (1). Therefore, the complete removal of soft tissue particles from the root canal wall by mechanical preparation and extensive reduction of pathogenic bacteria (2) by irrigating the root canal with disinfecting chemical solutions is necessary. However, most conventional end0dontic treatment methods are often not sufficient to meet these demands. The special characteristics of laser radiation, such as precise and contactless application mode, seemed to be a promising possibility

The laser device used was a 308 nm excimer-laser (Summit Technology, Waltham, MA) filled with xenon and chloride as laser media. The excited dimers emitted radiation at a wavelength of 308 nm. The maximum radiation energy was 64 mJ, the laser pulse duration was 60 ns, and the pulse frequency was 20 Hz. Outside the laser device, the laser beam was guided through flexible~quartz glass fibers with a core diameter of 600 /xm. Before and after irradiating each sample, the delivered energy density was measured with a joulemeter (Gentec ED 500; Gentec Corp., Ontario, Canada). The subject of irradiation was different bacterial strains from the American Type Culture Collection (ATCC). Selection of

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Folwacznyetal.

bacteria was done on the basis of well-known growth characteristics to exclude interruption of bacterial growth not caused by laser radiation. Six different bacteria--Staphylococcus aureus (ATCC 25923), Eseherichia coli (ATCC 25922), Salmonella typhimurium (ATCC 13311), Streptoeoeeusfaeealis (ATCC 29212), Lactococeis lactis (ATCC 19435), and Deinoeoecus radiodurans (ATCC 13939)--were investigated. All strains were incubated at a constant temperature of 37°C, except D. radiodurans, which was incubated at 30°C. Twenty-four hours before conducting single experiments, the investigated bacteria was incubated on culture medium (Columbia Agar with 5% sheep blood). Afterward, one or two bacterial colonies were placed into nutrient broth (MtillerHinton) and incubated for an additional 6 h. The concentration of bacteria in the cell suspension was then calibrated nephelometrically in accordance with the method of the American Society of Microbiology at a cell density of 1 to 10 × 106 m1-1. For laser irradiation, 100/xl of bacterial suspension was put into microtiter plates. The end of the optical fiber was fixed at a constant distance from the surface of the single samples by a special fiber support. The focus of the laser beam was adjusted in the center of the specimen. During laser exposure, the suspension was constantly mixed at a frequency of 100 min - t . After irradiation, the samples were diluted in sterile saline solution (0.9% NaC1) and spread on culture medium. The samples were incubated for 12 to 16 h, after which the number of colony-forming units was counted. To obtain information about the dependency of antimicrobial effects on the radiation time, bacterial specimens were irradiated with constant energy densities of the laser beam for varying durations. All investigations were conducted with energy density at 0.8 J/cm2. For validation, bacterial reduction was plotted in relation to the number of given laser pulses. The influence of energy density of radiation on antimicrobial effects was investigated on bacteria of the species S. aureus and E. coll. Single measurements were conducted with radiation of constant energy densities that ranged from 0.5 to 2.5 J/cm2 for each sample. The reduction of bacteria was plotted according to the radiation time. In addition, the number of pulses causing a 90% reduction of bacterial load was plotted in relation to the chosen energy density. To obtain information about differences in sensitivity of bacterial strains to the effects of 308 nm laser radiation, the number of pulses for a 90% reduction was plotted for each strain. To exclude bacterial killing by supraphysiological heating during laser radiation, the variation in temperature with increasing number of laser pulses was measured under identical experimental conditions. Measurements were performed to the peak of the plateau phase in the course of temperature. Statistical analysis of the results was performed using the ANOVA with Scheff6 test.

RESULTS

Temperature Measurement The temperature rise in the samples was dependent on the transported energy density of the laser beam. In general, the plateau phase--no change in temperature despite increasing number of pulse s - w a s reached after exposure of the specimen to 4000 laser pulses (Fig. 1). The mean starting temperature value was 23.1°C and minimum starting temperature was 22.8°C. Minimum rise of temperature was measured by applying laser energy densities of 0.5 J/cm2. After reaching 4000 pulses, the average temperature change was 4.5°C. The maximum temperature increase was observed 'after irradiating the

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FIG 2. Effect of 308 nm excimer-laser radiation on six different bacterial strains with increasing radiation time (E = 0.8 J/cm2). D. radiodurans, Deinococcus radiodurans; E. coli, Escherichia coil; L lactis, Lactococcus lactis; Salm. typh., Salmonella typhimurium; Staph. aur., Staphylococcus aureus; Str. faec., Streptococcus fae-

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samples with laser light at an energy density of 2.5 J/cm z. The average temperature rise after exposure to 4000 laser pulses was 16.3°C, with a maximum temperature of 39.9°C.

Dependence of Bacterial Killing on the Radiation Time A rapid decline of viable microorganisms in the curves at smaller pulse numbers is detectable. Furthermore, a different degree of bacterial killing for various strains was observed (Fig. 2). The killing rate for D. radiodurans is lowest and strongest for S. faecalis.

Vol. 24, No. 12, December 1998

Antimicrobial Effects of the XeCI Excimer-Laser

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To compare the numbers of laser pulses necessary to kill 90% of bacteria, we irradiated bacteria with laser light at an energy density of 0.8 J/cm 2. The number of pulses required for a 90% reduction of bacterial cells for the six targeted bacterial strains showed a wide variety. The mean number of laser pulses killing 90% of the microorganisms ranged from 93 pulses for E. coli to 1107 pulses for D. r a d i o d u r a n s (Fig. 6).

DISCUSSION

Dependence of Bacterial Killing on the Energy Density of Radiation To investigate the dependence of bacterial killing effects of a 308 nm excimer-laser on the energy density of radiation, S. a u r e u s and E. coli were exposed to laser radiation of varying energy density. Graphical depiction demonstrates a stronger gradient at higher energy densities, and more microorganisms were killed within less time (Figs. 3 and 4). With regard to the energy density of irradiation, no minimum limit was found to define antimicrobial effects. Bacterial reduction was observed within the entire investigated interval of energy densities ranging from 0.5 to 2.5 J/cm ~. Populations of the two compared bacteria (S. aureus and E. coli) were reduced by >99.9% with <500 pulses of laser radiation, with energy densities >0.8 J/cm 2. This value equals a total radiation time of 30 ms. The number of laser pulses causing a 90% bacterial reduction (D9o) was compared graphically as well. Curves for E. coli and S. aureus have a nearly symmetric but parallel shift form (Fig. 5). In general, higher numbers of laser pulses to attain a 90% bacterial reduction were needed for S. a u r e u s than for E. coli. Both curves demonstrate a significantly increased gradient at energy densities of --1.5 J/cm 2.

Temperature rise was measured during irradiation with five different energy densities of laser beana. Irradiation with the highest energy density of 2.5 J/cm 2 induces a maximum temperature of 39.9°C after delivering 4000 pulses. For all bacterial strains used in this study, their value is in the temperature interval in which growth and propagation of bacteria takes place. It is significantly below the critical temperature value of 60°C at which thermal damage and the resultant killing of bacterial cells is possible (8). However, temperature measurement of the suspension cannot detect the intracytoplasmatic rise of temperature in bacterial cells itself. The extent of bacterial reduction depends on the radiation time, as well as on the energy density of excimer laser radiation, This observation is in accordance with results of former investigations of bacterial growth after exposure to 308 nm excimer laser radiation (9). Graphical depiction in Fig. 2 demonstrates a nearly semilogarithmic dependence of bacterial killing on the number of applied laser pulses, which is similar to the total radiation time. Killing of bacteria starts immediately, because there was no minimum radiation time for the release of antimicrobial effects observed. Regarding the density of laser energy, all investigated

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bacterial strain FIG 6. Comparison of the number of laser pulses necessary to kill 90% of bacteria for six different bacterial strains. Str. faecalis, Streptococcus faecalis; Staph. aureus, Staphylococcus aureus; Salm. typh., Salmonella typhimurium; Lac. lactis, Lactococcis lactis; D. radiod., Deinococcus radiodurans.

bacterial strains showed a nonlinear relationship to cell number reduction. As expected, the efficacy of bacterial killing increased with higher energy densities. Comparable bacterial reduction was achieved with radiation of higher energy density in less time. In this correlation, no minimum energy density could be found, below which value bacterial killing was not detectable. All strains of S. a u r e u s and E. coli undergo a significant reduction of viable cells by irradiation with 308 nm excimer laser light at an energy density of 0.5 J/cm 2. The hyperproportional increasing killing rate with energy densities between 1.0 and 2.0 J/cm 2 is remarkable. A nearly symmetric but parallel shift position of the graphs for S. aureus and E. coli reveals a different sensitivity of various bacteria to application of 308 nm excimer-laser radiation. Comparison of the number of pulses for a reduction of 90% of viable cells for the six investigated bacterial strains results in the same conclusion. Corresponding observation was made after irradiating bacterial suspensions with ultraviolet light from a conventional source and ionizing radiation. There are obviously different factors influencing this reaction. As described later, alteration of procaryotic DNA by absorption of radiation energy is primary. Sutherland and Griffin (10) postulate that the strength of absorption of ultraviolet radiation depends on the content of guanine and adenosine of the DNA, which is characteristic for each bacterial strain. A conclusive answer about mechanisms of bacterial killing cannot be given on the basis of the present study. Interaction between the genetic information (DNA) and ultraviolet radiation seems to be very likely, even if the caused alteration is different, depending on the wavelength of radiation. A former investigation by Peak et al, (11) reported that absorption of ultraviolet B-radiation most frequently leads to pyrimidine dimers in the DNA molecule. According to Colella et al. (12), the development of pyrimidine dimers is not a sufficient explanation of the effect of ultraviolet light on the

DNA. Chilbert et al. (13) are postulating the induction of single strand brakes for direct irradiation of extracted DNA with high energetic 308 nm laser radiation. Similar results were described in other studies, in which single strand brakes were observed after delivering extremely short laser pulses with very high energy density. It must be further noted that the maximum performance peaks of applied radiation ranged between 1.0 • l 0 9 and 1.0 • l 0 1 3 W / m e. Calkins et al. (14) suspected radiation with performance ranging between 8.0.10 9 t O 5 . 0 " 1 0 1 1 W / m 2 t o trigger the so-called "two-photon effects." Comparisons of our results with those of the aforementioned studies must be limited because of dissimilar applied radiation doses. Summarizing the comparison of the numbers of laser pulses necessary for achieving a 90% reduction of bacterial cells with a 308 nm excimer laser radiation, two mechanisms of action are suspected. On the one hand, development of pyrimidine dimers in the region of energy densities up to 1.0 J/cm 2 seems to be possible. On the other hand, the hyperproportional increase of bacteria reduction beyond this limit is caused by a second mechanism. An energy dose of 1.0 J/cm 2 per laser pulse corresponds to a maximum performance density of 1.66 • 10 ~t W/m 2. This value is in the interval of performance densities given by Calkins et al. (14) in which "two-photon processes" can be triggered. Induction of single strand brakes by "two photon processes" with a 308 nm excimer-laser radiation with energy > 1.0 J/cm 2 seems to be possible. Radiation in this region must, therefore, also lead to the emergence of pyrimidine dimers, as well as to single strand brakes. The flatter gradient for energy densities >2.0 J/cm 2 possibly points to saturation of induction of single strand brakes. Comparison of our results with other studies confirms the peculiarity of UV laser interaction with regard to laser systems of different wavelengths. According to the study of Dederich et al. (15), one may emphasize that carbon dioxide laser obtains comparable effects, only at significantly higher energy density. Furthermore, sensitivity of varying bacterial strains to irradiation with carbon dioxide laser is, in contrast to the results with excimer-laser, quite equal. This seems to indicate a predominantly thermal bactericidal action. In contrast, Byrne et al. (16) are postulating dissimilar sensitivity of bacteria for the thermal effects of this laser system. Irradiation of bacteria with the Nd:YAG laser is bactericidal only at energies > 1.0 J/cm 2 (17). Bacterial killing was caused, at least partially, by thermal side effects of radiation. Maker and Kaplan (18) obtained significant bacterial reduction with Nd:YAG laser system as well, although they did not report details about temperature changes during irradiation. Stabholz et al. (19) observed antimicrobial effects after irradiating S. m u t a n s with 308 nm excimer laser for > 2 s. However, information about the energy density of radiation used in these experiments was not provided. Jahn et al. (20) also examined the effects of 308 nm excimer-laser light on bacteria, but could not observe unambiguous bacterial reduction in all cases. Although in this study bacteria were exposed to radiation of very low energy density, well-balanced exposure to radiation in all parts of specimen was not ensured. Using XeC1 laser radiation only with an energy density of 0.48 J/cm 2 caused bacterial reducing effects as well. In summary, it was ascertained that 308 nm excimer-laser radiation causes very intensive antimicrobial effects. The degree of bacterial reduction depends on time and energy density of radiation and the irradiated strain of bacteria. Even with energy densities far below ablation threshold for human tissue ranking at 1.0 J/cm 2 (6), a significant reduction of microorganisms was obtained. Critical

Vol. 24, No. 12, December 1998

evaluation of temperature measurement during radiation excludes bacterial killing by supraphysiological heating. Drs. Folwaczny, Liesenhoff, and Horch are affiliated with the Klinik und Poliklinik for Mund-Kiefer-Gesichtschirurgie, Klinikum rechts der Isar, Technische Universit~t M0nchen, Munich, Germany. Dr. Lehn is affiliated with the Institut fur Medizinische Mikrobiologie und Hygiene, Universit~t Regensburg, Regensburg, Germany. Address requests for reprints to Dr. Matthias Folwaczny, Department of Operative Dentistry, Ludwig-Maximilians University, Goethestr. 70, 80336 Munich, Germany.

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8. Sorhaug T. Temperature control. In: Lederberg J, ed. Encyclopedia of Microbiology, VoL 4, 1st Ed. AufL San Diego: Academic Press, 1992:201-11. 9. Uesenhoff T, Folwaczny M, Lehn N. Wirkung von 308 nm ExcimerLaserstrahlung auf Bakteden-eine in vitro Untersuchung. Lasermedizin 1994; 10:52- 8. 10. Sutherland JC, Griffin KP. Absorption spectrum of DNA for wavelengths greater than 300 rim. Radiat Res 1981 ;86:399-409. 11. Peak M J, Peak MP, Moehring MP, Webb RE. Ultraviolet action spectra for DNA dimer induction, lethality, and mutagenesis in Escherichia coil with emphasis on the UV8 region. Photochem Photobiol 1984;40:613-20. 12. Colella CM, Bogani P, Agati G, Fusi F. Genetic effects of UV-B: Mutagenicity of 308 nm light in Chinese hamster V79 cells. Photochem Photobiol 1986;43:437-42. 13. Chilbert MA, Peak M J, Peak JG, Pellin MJ, Gruen DM, Williams GA. Effects of intensity and fluence upon DNA single-strand breaks induced by excimer laser radiation. Photochem Photobiol 1988;47:523-5. 14. Calkins J, Colley E, Wheeler J. Spectral dependence of some UV-B and UV-C responses of Tetrahymena pyriformis irradiated with dye laser generated UV. Photochem Photobiol 1987;45:389-98. 15. Dederich ND, Picckard MA, Vaughn AS, Tulip J, Zakariasen KL. Comparative bactericidal exposures for selected oral bacteria using carbon-dioxide laser radiation. Lasers Surg Med 1990;10:591-4. 16. Byrne PO, Sisson PR, Oliver PD, Ingham HR. Carbon dioxide laser irradiation of bacterial targets in vitro. J Hosp Infec 1987;9:265-73. 17. Schultz RJ, Harvey GP, Fernandez-Beros ME, Krishnamurthy S, Rodriguez JE, Cabello F. Bactedcida( effects of the neodymium:YAG laser: in vitro study. Lasers Surg Med 1986;6:445-8. 18. Maker VK, Kaplan RL. Contact neodynium-yttrium-aluminium garnet laser acts as a sterilizing scalpel. Surg Gyn Obst 1990;170:17-20. 19. Stabholz A, Kettering J, Neev J, Torabinejad M. Effects of the XeCI excimer laser on Streptococcus mutans. J Endodon 1993;19:232-5. 20. Jahn R, Schumacher AK, Hillrichs G, Kaulfers PM, Neu W, Jungbluth KH. In-vitro-Reaktion von 8akterien nach 308 nm- und 2940 nm Laserstrahlung Tell 1: Keimreduktion in Bakteriensuspensionen. Lasermedizin 1994;10: 169-75.

You Might Be Interested Our campaign to acquaint you with diseases with appealing names continues with "orf." Orf is a viral cutaneous pox of sheep and goats, which is transmitable to humans. This most commonly occurs when the hand has had prolonged contact with a new born sheep's muzzle for example when bottle-feeding an orphan lamb (Br J Dermatol 97:447).

Sad that even so benign and laudable an activity has risks. Emma Yeomans