Antimicrobial Effect of Extract of Glycyrrhiza uralensis on Methicillin-Resistant Staphylococcus aureus

감초 추출물이 항생제 내성균주의 항균활성에 미치는 영향

  • Lee, Ji-Won (Department of Food Science & Technology, Keimyung University) ;
  • Ji, Young-Ju (Department of Food Science & Technology, Keimyung University) ;
  • Yu, Mi-Hee (Department of Food Science & Technology, Keimyung University) ;
  • Im, Hyo-Gwon (Department of Food Science & Technology, Keimyung University) ;
  • HwangBo, Mi-Hyang (Department of Food Science & Technology, Keimyung University) ;
  • Lee, In-Seon (The Center for Traditional Microorganism Resources Center)
  • 이지원 (계명대학교 식품가공학과) ;
  • 지영주 (계명대학교 식품가공학과) ;
  • 유미희 (계명대학교 식품가공학과) ;
  • 임효권 (계명대학교 식품가공학과) ;
  • 황보미향 (계명대학교 식품가공학과) ;
  • 이인선 (계명대학교 전통미생물자원개발 및 산업화연구센터)
  • Published : 2005.06.30

Abstract

Antimicrobial drug-resistance is natural response to antimicrobial stress based on selection, which weakens chemotherapy effect. Introduction of large numbers of chemotherapeutic agents to clinical practice has generated strains of microorganisms that survive and multiply in vivo with high-drug concentrations. Methicillin-resistant Staphylococcus aureus (MRSA), bacteria found in normal daily life, can be easily ingested through milk vegetables, and meats, etc. MRSA emerged in many port of the world, increasing complex clinical problems. Therefore, new agents are needed to treat MRSA. Glycyrrhiza uralensis was extracted using 80% MeOH to investigate its antimicrobial activity against MRSA stains KCCM 11812, 40510, and 40512 through bacterial measurement, disc diffusion, and O.D. methods, MIC values, MRSA gene expression investigation, and scanning electron microscope observation. Results revealed MecA, Mecl, MecRI, and FemA were the most highly manifested MRSA genes. Methanolic extract of G. uralensis significantly inhibited MRSA and thus could be used in development of antibacteria.

본 연구에서는 최근 전세계적으로 문제가 되고 있는 항생제 내성균에 대해 그 항균효과를 천연의 약용식물을 이용하여 분자생물학적 수준에서 비교해보고 천연항균제의 개발에 우선하여 직접 식품에 첨가하여 그 효과를 검색하고자 하였다. 병원내감염의 주된 원인균으로 알려진 황색포도상구균(Staphylococcusaureus)은 여러 항균제에 대하여 내성을 획득함으로써 질병의 치료를 어렵게 한다는 점에서 매우 심각한 문제이다. 이러한 내성균은 다양한 방법으로 동 식물에 빠르게 전파되고 있으므로 소비자가 내성균을 지닌 식품을 섭취해 잠재적인 내성균을 보유하게 되는 결과를 낳게 된다. 이러한 항생제 내성 황색포도상구균(Methicillin Resistant S. aureus, MRSA)에 대해 천연 약용 식물 중 우수한 항균력을 가지는 감초를 순차 분획하여 각 분획물을 이용한 MRSA에 대한 항균 활성을 알아본 결과 hexane, chloroform 분획물에서 항균활성을 나타냈으며, 동일농도의 생육저해비교 실험에서 대부분의 내성균주에 대해 대조군과 유사한 효과를 나타낸 penicillin에 비해 감초분획물이 대수기를 넘는 억제효과를 나타내었으며 이 중 chloroform 분획물이 가장 높은 저해활성을 나타내었다. 또한 RT-PCR을 통하여 표준균주와 내성균주의 내성 정도와 내성 유전자를 관찰해본 결과 KCCM 40510은 내성획득유전자인 mecA, mecRI, mecI, femA에 발현이 높게 나타나 고도내성균임을 알 수 있었으며, 고도내성을 나타내는 균주를 이용하여 감초의 chloroform 분획물을 농도별로($50,\;100,\;250\;{\mu}/mL$) 처리한 결과 농도 의존적으로 유전자 발현이 억제됨을 볼 수 있었다. 특히 고도내성균주인 KCCM 40510은 메티실린 내성을 상승시키는 mecA 유전자의 발현을 억제시키는 mecRI 유전자의 pathway와 상응하는 것으로 보여지므로 감초가 이러한 고도내성균에 효과가 있음을 알 수 있었다. 또한 SEM을 통한 형태학적 관찰을 통해 분획물 처리 시 세포벽이 파괴되는 것을 확인할 수 있었다. 한편 이러한 감초추출물을 직접 식품에 첨가하여 내성 균주에 대한 억제 효과도 확인한 결과 추출물 200mg/100mL 농도에서 포도상구균에 대해 항균효과를 나타내는 것을 볼 수 있었다.

Keywords

References

  1. Francis AW. Staphylococcus aureus (including toxic shock syndrome). In: Mandell Douglas and Bennetts Priciples and Infections Diseasea. 4: 1754-1755 Churchill Livingstone (1995)
  2. Han SJ, Jung PM, Kim HG, Hwang EH, Seong IW. Multiple intestinal ulcerations and perforations secondary to methicillin-resistant Staphylococcus aureus enteritis in infants. J. Pediatr. Surg. 34:381-386(1999) https://doi.org/10.1016/S0022-3468(99)90481-5
  3. Hiramatsu K, Hanaki H, Ino T, Yabuta K, Oguri T, Tenover FC. Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. J. Antimicrob. Chemother. 40: 135-136(1997) https://doi.org/10.1093/jac/40.1.135
  4. Haley RW, Hightower AW, Khabbaz RF, Thrnsberry C, Martone WJ, Allen JR and Hughes JM. The emergence of methicillin-resistant Staphylococcus aureus infection in united states hospi-tials. Ann. Intern. Med. 97: 297 (1982)
  5. Locksley RM, Cohen ML, Quinn TC, Tompkins LS, Coyle MB, Kirigara JM, Counts GW. Multiple antibiotic-resistant Staphylococcus aureus: Introduction Transmission and evolution of nosocomial infection. Ann. Intern. Med. 97: 317 (1982)
  6. Crowford JJ. New light on the transmissibility of viral hepatitis in dental practice and its control. JADA 91: 829 (1975)
  7. Codino RC, Marshall WE. Control of infection in the dental operatory. Dent. Surv. 42 (1976)
  8. Mirrier RL, Burton WE, Spore RW. Aerosols produced by dental instrumentation. Proc. First Intl Symp. Aerosol, p. 97 (1963)
  9. Codino RC, Marshall WE. Control of infection in the dental operatory. Dent. Surv. 42 (1976)
  10. Barrett FF, McGehee RF, Finland M. Methicillin-resistant Staphylococcus aureus at Boston City hospital. N. Engl. J. Med. 279: 441-448(1968) https://doi.org/10.1056/NEJM196808292790901
  11. Klimek JJ, Marsik FJ, Bartlett RC, Weir B, Shea P, Quintiliani R. Clinical, epidemiologic and bacteriologic observations of an outbreak of methicillin-resistant Staphylococcus aureus at a large community hospital. Am. J. Med. 61: 340-345 (1967) https://doi.org/10.1016/0002-9343(76)90370-3
  12. Dixon RE. Second international conference on nosocomial. Am. J. Med. 70:379, 631, 899(1981)
  13. Cohen SH, Morita MM, Bradfor M. A-seven year experience with methicillin-resistant Staphylococcus aureus. Am. J. Med. 91: 233-237(1991) https://doi.org/10.1016/0002-9343(91)90121-D
  14. Sabath LD. Mechanisms of resistant to beta-lactam antibiotics in strains of Staphylococcus aureus. Ann. Intl. Med. 97: 339-344 (1982) https://doi.org/10.7326/0003-4819-97-3-339
  15. Ubukata, Nonoguchi R, Matsuhashi M, Konno M. Expression and inducibility in Staphylococcus aureus of the mecA gene, which encodes a methicillin-resistant S. aureus-specific penicillin-binding protein. J. Bacteriol. 171: 2882-2885 (1989) https://doi.org/10.1128/jb.171.5.2882-2885.1989
  16. Wyke AW, Ward JB, Hayes MV, Curitis NAC. A role in vivo for penicillin-binding protein-4 of Staphylococcus aureus. Eur. J. Biochem. 119: 389-393(1981) https://doi.org/10.1111/j.1432-1033.1981.tb05620.x
  17. Tesch W, Ryffel C, Strassle A, Kayser FH, Berger-Bachi B. Evidence of a novel staphylococcal mec-encoded element (mecR) controlling expression of penicillin-binding protein 2'. Antimicrob. Agents Chemother. 34: 1703-1706 (1990) https://doi.org/10.1128/AAC.34.9.1703
  18. Hiramatsu K, Asada K, Suzuki E, Okonogi K, Yokota T. Molecular cloning and nucleotide sequence determination of the regulator region of mecA gene in methicillin-resistant Staphylococcus aureus (MRSA). FEBS Lett. 298: 133-136 (1992) https://doi.org/10.1016/0014-5793(92)80039-J
  19. Berger-Bachi B, Barberis-Maino L, Strassle A, Kayser FH. FemA, a host-mediated factor essential for methicillin resistant in Staphylococcus aureus: molecular cloning and characterization. Mol. Gen. Genet. 219: 263-269 (1989)
  20. Berger-Bachi B, Strassle A, Gustafson JE, Kayser FH. Mapping and characterization of multiple chromosomal factors involved in methicillin resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 36: 1367-1373 (1992) https://doi.org/10.1128/AAC.36.7.1367
  21. Bignari GE, Woodford N, Chapman A, Johnson AP, Speller DCE. Detection of the mecA gene and phenotyptic detection of resistance in Staphylococcus aureus isolates with borderline or low-level methicillin reaiatance. J. Antimicrob. Chemother. 37: 53-63(1996) https://doi.org/10.1093/jac/37.1.53
  22. Mason WJ, Beenken K, Wibowo N, Ojha N, Smeltzer MS. Multiplex PCR protocol for the doagnosis of staphylococcal infection. J. Clin. Microbiol. 39: 3332-3338 (2001) https://doi.org/10.1128/JCM.39.9.3332-3338.2001
  23. Vanuffel P, Gigi J, Ezzedine H, Vandercam B, Delmee M, Wauter G, Gala JL. Specific detection of methicillin reaiatant Staphylococcus species by multiplex PCR. J. Clin. Microbiol. 33: 2864-2867(1995)
  24. Kee CH. The pharmacology of Chinese herbs. CRC Press, Inc., Boca Raton, Fl, USA. pp. 275-278 (1993)
  25. Kumagai A, Nanabosh M, Asanuma Y, Yagur T, Nishino K. Effect of glycyrrhizin on thymolytic and immuno-suppressive action of cortisone. Endocrinol. Jpn. 14: 39-42 (1967) https://doi.org/10.1507/endocrj1954.14.39
  26. Kiso Y, Tohin M, Ino H, Hattori M, Saamoto T, Namba T. Mechanism of antihepatotoxin activity of glycyrrhizin I. Effect on free radical generation and lipid peroxidation. Planta. Medica. 50: 298-302(1984) https://doi.org/10.1055/s-2007-969714
  27. Pompei R, Flore O, Marcialis MA, Pani A, Loddo B. Glycyr-rhizic acid inhibits virus growth and activates virus particles. Nature 281: 689-690 (1979) https://doi.org/10.1038/281689a0
  28. Mori K, Sakai H, Suzuk S. Effects of glycyrrhizin in hemo-phil-iapatients with HIV-1 infection. Tohoku J. Exp. Med. 162: 183-193(1990) https://doi.org/10.1620/tjem.162.183
  29. Ahn EY, Shin DH, Baek Nl, Oh JA. Isolation and identification of antimicrobial active subsstance from Glycyrrhiza uralensis FISCH. Korean J. Food. Sci. Technol. 30: 680-687 (1998)
  30. Lee YC, Oh SW, Hong HD. Antimicrobial characteristics of edible medicinal herbs extracts. Korean J. Food Sci. Technol. 34: 700 (2002)
  31. Kudo T, Saga N. Development of a simple method for antibiotic susceptibility testing in algae using paper disks. Nippon Suisan Gakkaishi 56:455(1990)
  32. Food code. Korea Food Industry Association. Namhyung-Munhwa, Seoul, Korea, pp. 730-743 (1994)
  33. Park CG, Bang KH, Lee SE, Cha MS, Seong JS, Park SU, Seong NS. Antimicrobial effect of various medicinal herb on Staphylococcus aureus. Korean J. Medicinal Crop. Sci. 9: 251-258 (2001)
  34. Ahn EY, Shin DH, Baek NI, Oh JA. Isolation and identification of antimicrobial active substance from Glycyrrhiza uralensis FISCH. Korean J. Food Sci. Technol. 30: 680-687 (1998)
  35. Shin DH, Han JS, Kim MS. Antimicrobial effect of ethanol extract of Sonomenium acutum (Thunb.) Rehd. et wils and Glyyrrhiza glabra L. var. Glanduliferaregel et zucc on Listeria mono-cytogenes. Korean J. Food Sci. Technol. 26: 627-632 (1994)
  36. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing. Fifth informational supplement, Villanova, PA, USA. M100-55 (1994)
  37. National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. NCCLS Document. Villanova, PA, USA. M7-A2(1990)