Effect of Phytoncide on Porphyromonas gingivalis

P. gingivalis에 대한 피톤치드의 항균효과

  • Kim, Sun-Q (Department of Oral Medicine, School of Dentistry, Kyung Hee University) ;
  • Shin, Mi-Kyoung (Institute of Oral biology, School of Dentistry, Kyung Hee University) ;
  • Auh, Q-Schick (Department of Oral Medicine, School of Dentistry, Kyung Hee University) ;
  • Lee, Jin-Yong (Institute of Oral biology, School of Dentistry, Kyung Hee University) ;
  • Hong, Jung-Pyo (Department of Oral Medicine, School of Dentistry, Kyung Hee University) ;
  • Chun, Yang-Hyun (Department of Oral Medicine, School of Dentistry, Kyung Hee University)
  • 김선규 (경희대학교 치과대학 구강내과학교실) ;
  • 신미경 (경희대학교 치과대학 구강생물학연구소) ;
  • 어규식 (경희대학교 치과대학 구강내과학교실) ;
  • 이진용 (경희대학교 치과대학 구강생물학연구소) ;
  • 홍정표 (경희대학교 치과대학 구강내과학교실) ;
  • 전양현 (경희대학교 치과대학 구강내과학교실)
  • Published : 2007.06.30

Abstract

Trees emit phytoncide into atmosphere to protect them from predation. Phytoncide from different trees has its own unique fragrance that is referred to as forest bath. Phytoncide, which is essential oil of trees, has microbicidal, insecticidal, acaricidal, and deodorizing effect. The present study was performed to examine the effect of phytoncide on Porphyromonas gingivalis, which is one of the most important causative agents of periodontitis and halitosis. P. gingivalis 2561 was incubated with or without phytoncide extracted from Hinoki (Chamaecyparis obtusa Sieb. et Zucc.; Japanese cypress) and then changes were observed in its cell viability, antibiotic sensitivity, morphology, and biochemical/molecular biological pattern. The results were as follows: 1. The phytoncide appeared to have a strong antibacterial effect on P. gingivalis. MIC of phytoncide for the bacterium was determined to be 0.008%. The antibacterial effect was attributed to bactericidal activity against P. gingivalis. It almost completely suppressed the bacterial cell viability (>99.9%) at the concentration of 0.01%, which is the MBC for the bacterium. 2. The phytoncide failed to enhance the bacterial susceptibility to ampicillin, cefotaxime, penicillin, and tetracycline but did increase the susceptibility to amoxicillin. 3. Numbers of electron dense granules, ghost cell, and vesicles increased with increasing concentration of the phytoncide, 4. RT-PCR analysis revealed that expression of superoxide dismutase was increased in the bacterium incubated with the phytoncide. 5. No distinct difference in protein profile between the bacterium incubated with or without the phytoncide was observed as determined by SDS-PAGE and immunoblot. Overall results suggest that the phytoncide is a strong antibacterial agent that has a bactericidal action against P. gingivalis. The phytoncide does not seem to affect much the profile of the major outer membrane proteins but interferes with antioxidant activity of the bacterium. Along with this, yet unknown mechanism may cause changes in cell morphology and eventually cell death.

피톤치드란 '산림향' 이라고 부르는, 나무가 갖는 특유의 향을 발산하는 휘발성 화학물질로서, 우리 몸을 쾌적하게 해 줄 뿐만 아니라 항균, 방충, 소취 등 다양한 기능을 가지고 있다. 치주질환과 구취를 유발시키는 중요한 원인균인 P. gingivalis에 대한 피톤치드의 항균효과와 항균작용을 연구하기 위하여, 편백 피톤치드와 함께 P. gingivalis 2561을 배양한 후 P. gingivalis 2561의 성장정도, 생존력 및 형태적, 분자생물학적 변화를 관찰하여 다음과 같은 결과를 얻었다. 1. 피톤치드는 P. gingivalis에 매우 강한 항균력을 보였고, 이 항균력은 살균작용에 의한 것으로 나타났다. P. gingivalis에 대한 피톤치드의 최소억제농도는 0.008%, 최소살균농도는 0.01%로 결정되었다. 2. 피톤치드와 함께 배양된 P. gingivalis는 ampicillin, cefatoxime, penicillin, tetracycline에 대한 감수성이 변하지 않았으나 amoxicillin에 대한 감수성은 증가하였다. 3. 피톤치드와 같이 배양된 P. gingivalis를 투과전자현미경으로 관찰한 결과, 핵이 뚜렷해지고 전자밀도가 높은 과립이 증가하였고 리보솜이 세포질 가장 자리로 분포하였으며, 피톤치드 양이 증가할 수록 유령세포, 특히 소포가 특징적으로 크게 증가하였다. 4. RT-PCR 분석 결과, 피톤치드는 P. gingivalis의 superoxide dismutase의 발현을 억제하는 것으로 나타났다. 5. SDS-PAGE와 immunoblot 분석 결과, 피톤치드는 P. gingivalis의 단백질 발현에 큰 영향을 미치지 않는 것으로 나타났다. 이상의 결과로 미루어 피톤치드는 P. gingivalis에 대해 강한 항균효과를 갖고 있으며 이것은 살균작용에 의한 것으로 판단된다. 즉, 피톤치드는 단백질 발현에는 영향을 미치지 못하지만 P. gingivalis의 항산화물질 생산능력을 감소시키거나 아직 밝혀지지 않은 기전을 통해 스트레스 상황을 유도하여 생존능력을 억제하여 결과적으로 세균세포의 구조적 형태 변화와 함께 사멸을 유도하는 것으로 사료된다.

Keywords

References

  1. Gharbia SE, Shah HN. Interactions between blackpigmented Gram-negative anaerobes and other species which may be important in disease development. FEMS Immunol Med Microbiol 1993;6:173-178 https://doi.org/10.1111/j.1574-695X.1993.tb00321.x
  2. Ximenez-Fyvie LA, Haffajee, AD, Socransky SS. Comparison of the microbiota of supra- and subgingival plaque in health and periodontitis. J Clin Periodontol 2000;27:648-657 https://doi.org/10.1034/j.1600-051x.2000.027009648.x
  3. Lamont RJ, Jenkinson HF. Life below the gum line: pathogenic mechanisms of Porphyromonas gingivalis. Microbiol Mol Biol Rev 1998;62:1244-1263
  4. Tanner AA, Socransky SS, Goodson JM. Microbiota of periodontal pockets losing crestal alveolar bone. J Periodontal Res 1984;11:279-291
  5. Tamai R, Asai Y, Ogawa T. Requirement for intercellular adhesion molecule 1 and caveolae in invasion of human oral epithelial cells by Porphyromonas gingivalis. Infect Immun 2005;73:6290-6298 https://doi.org/10.1128/IAI.73.10.6290-6298.2005
  6. Christersson LA, Rosling BG, Dunford RG et al. monitoring of subgingival Bacteroides gingivalis and Actinobacillus actinomycetemcomitans in the management of advanced periodontitis. Adv Dent Res 1988;2:382-388 https://doi.org/10.1177/08959374880020023301
  7. Ding Y, Haapasalo M, Kerosuo E et al. Release and activation of human neutrophil matrix metallo- and serine proteinases during phagocytosis of Fusobacterium nucleatum, Porphyromonas gingivalis and Treponema denticola. J Clin Periodontol 1997;24: 237-248 https://doi.org/10.1111/j.1600-051X.1997.tb01837.x
  8. DeCarlo AA Jr, Windsor LJ, Bodden MK et al. Activation and novel processing of matrix metalloproteinases by a thiol-proteinase from the oral anaerobe Porphyromonas gingivalis. J Dent Res, 1997;76:1260-1270 https://doi.org/10.1177/00220345970760060501
  9. Tonzetich J. Production and origin of oral malodor. A review of mechanisms and methods of analysis. J Periodontol 1997;48:13-20 https://doi.org/10.1902/jop.1977.48.1.13
  10. Bosy A. Oral Malodor: philosophical and practical aspects. J Can Dent Assoc 1997;63:196-201
  11. Nakano Y, Yoshimura M, Koga T. Correlation between oral malodor and periodontal bacteria. Microbes Infect 2002;4:679-683 https://doi.org/10.1016/S1286-4579(02)01586-1
  12. Welsh C. Complementary therapies in hospice care: touch with oils- a pertinent part of holistic care. Am J hospice Palliat Care 1997;14:42-44 https://doi.org/10.1177/104990919701400114
  13. Lis-Balchin M. Essential oils and aromatherapy: their modern role in healing. J R Soc Health 1977;117: 324-329 https://doi.org/10.1177/146642409711700511
  14. 강하영, 오종환. 침엽수 침엽 정유의 방향성 이용적성. 임업연보 1994;49:177-179
  15. 강하영, 이성숙, 최인규. 침엽수 수엽 정유의 항균성에 관한 연구. 한국임산에너지학회지 1993;13:71-79
  16. Whittaker RH, Feeny PP. Alleochemics: chemical interactions between species. Science 1971;171:757-770 https://doi.org/10.1126/science.171.3973.757
  17. Muller CH. Allelopathy as a factor in ecological processes. Vegetation 1969;18:348-357 https://doi.org/10.1007/BF00332847
  18. 이현옥, 백승화, 한동민. 편백정유의 항균효과. Kor J Appl Microbiol Biotechnol 2001;29:253-257
  19. Takarada K, Kimizuka R, Takahashi N et al. A comparison of the antibacterial efficacies of essential oils against oral pathogens. Oral Microbiol Immunol 2004;19:61-64 https://doi.org/10.1046/j.0902-0055.2003.00111.x
  20. Shapiro S, Meier A, Guggenheim B. The antimicrobial activity of essential oils and essential oil components towards oral bacteria. Oral Microbiol Immunol 1994;9:202-208 https://doi.org/10.1111/j.1399-302X.1994.tb00059.x
  21. Schoenknecht FD, Sabath LD, Thornsberry C. Manual of Clinical Microbiology. 4th ed. Washington DC, 1985, American Society for Microbiology, pp. 1000-1008
  22. Dickinson DP, Kubiniec MA, Yoshimura F, Genco RJ. Molecular cloning and sequencing of the gene encoding the fimbrial subunit protein of Bacteroides gingivalis. J Bacteriol 1988;170:1658-1665 https://doi.org/10.1128/jb.170.4.1658-1665.1988
  23. Nakayama K. The superoxide dismutase-encoding gene of the obligately anaerobic bacterium Bacteroides gingivalis. Gene 1990;96:149-150 https://doi.org/10.1016/0378-1119(90)90357-W
  24. Nelson KE, Fleischmann RD, DeBoy RT et al. Complete genome sequence of the oral pathogenic Bacterium Porphyromonas gingivalis strain W83. J Bacteriol 2003;185:5591-5601 https://doi.org/10.1128/JB.185.18.5591-5601.2003
  25. Schnaubelt K. Advanced aromatherapy. Vermont, 1995, Healing Arts Press, pp. 255-286
  26. Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev 1999;12:564-582
  27. Ayres H, Furr JR, Russell AD. A rapid method of evaluating permeabilizing activity against Pseudomonas aeruginosa. Lett Appl Microbiol 1993;17: 149-151 https://doi.org/10.1111/j.1472-765X.1993.tb00381.x
  28. Xajigeorgiou C, Sakellari D, Slini T et al. Clinical and microbiological effects of different antimicrobials on generalized aggressive periodontitis. J Clin Periodontol 2006;33:254-264 https://doi.org/10.1111/j.1600-051X.2006.00905.x
  29. Vaara M, Jaakkola J. Sodium hexametaphosphate sensitizes Pseudomonas aeruginosa, several other species of Pseudomonas, and Escherichia coli to hydrophobic drugs. Antimicrob Agents Chemother 1989;33:1741-1747 https://doi.org/10.1128/AAC.33.10.1741
  30. Nguefack J, Budde BB, Jakobsen M. Five essential oils from aromatic plants of Cameroon: their antibacterial activity and ability to permeabilize the cytoplasmic membrane of Listeria innocua examined by flow cytometry. Lett Appl Microbiol 2004;39: 395-400 https://doi.org/10.1111/j.1472-765X.2004.01587.x
  31. Cox SD, Mann CM, Markham JL et al. The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil). J Appl Microbiol 2000;88: 170-175 https://doi.org/10.1046/j.1365-2672.2000.00943.x
  32. Carson CF, Mee BJ, Riley TV. Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined by time-kill, lysis, leakage, and salt tolerance assay and electron microscopy. Antimicrob Agents Chemother 2002;46:1914-1920 https://doi.org/10.1128/AAC.46.6.1914-1920.2002
  33. Oussalah M, Caillet S, Lacroix M. Mechanism of action of Spanish oregano, Chinese cinnamon, and savory essential oils against cell membranes and walls of Escherichia coli O157:H7 and Listeria monocytogenes. J Food Prot 2006;69:1046-1055 https://doi.org/10.4315/0362-028X-69.5.1046
  34. 최인식, 박병래, 김홍렬 등. Porphyromonas gingivalis 에 대한 polyphosphate의 항균효과. 대한미생물학회지 1999;34:285-301
  35. Rao NN, Kornberg A. Inorganic polyphosphate supports resistance and survival of stationary-phase Escherichia coli. J Bacteriol 1996;178:1394-1400 https://doi.org/10.1128/jb.178.5.1394-1400.1996
  36. Mayrand D, Holt SC. Biology of asaccharolytic black-pigmented Bacteroides species. Microbiol Rev 1988;52:134-152
  37. Fine DH, Furgang D, Lieb R et al. Effects of sublethal exposure to an antiseptic mouthrinse on representative plaque bacteria. J Clin Periodontol 1996;23:444-451 https://doi.org/10.1111/j.1600-051X.1996.tb00572.x
  38. Nakayama KJ. Rapid viability loss on exposure to air in a superoxide dismutase-deficient mutant of Porphyromonas gingivalis. J Bacteriol 1994;176: 1939-1943 https://doi.org/10.1128/jb.176.7.1939-1943.1994
  39. Murakami Y, Masuda T, Imai M et al. Analysis of major virulence factors in Porphyromonas gingivalis under various culture temperatures using specific antibodies. Microbiol Immunol 2004;48:561-569 https://doi.org/10.1111/j.1348-0421.2004.tb03552.x
  40. Masuda T, Murakami Y, Noguchi T, Yoshimura F. Effects of various growth conditions in a chemostat on expression of virulence factors in Porphyromonas gingivalis. Appl Environ Microbiol 2006;72:3458-3467 https://doi.org/10.1128/AEM.72.5.3458-3467.2006