Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Antibacterial Effects and Cellular Responses of Imipenem-resistant Pseudomonas aeruginosa Exposed to Green Tea Polyphenols

녹차 폴리페놀에 노출된 Imipenem 내성 Pseudomonas aeruginosa의 항균효과 및 세포반응

  • Song, You-Jin (Department of Biotechnology, Soonchunhyang University) ;
  • Cho, Yun-Seok (Department of Biotechnology, Soonchunhyang University) ;
  • Oh, Kye-Heon (Department of Biotechnology, Soonchunhyang University)
  • 송유진 (순천향대학교 생명공학과) ;
  • 조윤석 (순천향대학교 생명공학과) ;
  • 오계헌 (순천향대학교 생명공학과)
  • Received : 2010.04.27
  • Accepted : 2010.06.07
  • Published : 2010.06.28
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Abstract

The aim of this work was to investigate the synergically bactericidal effects and cellular responses of tea polyphenols (TPP) and imipenem on imipenem-resistant Pseudomonas aeruginosa. Imipenem-resistant Ps. aeruginosa was isolated from patient in hospital. The bactericidal effects of TPP and imipenem were evaluated on the basis of its minimum inhibitory concentrations (MIC). The combined use of TPP and imipenem resulted in 16-fold and 8-fold reductions in the MICs of imipenem for the imipenem-susceptible and imipenem-resistant Ps. aeruginosa, respectively. The bactericidal effects of the imipenem and TPP against the Ps. aeruginosa was evaluated using the time-kill assay. The synergetic effects of the combinations of TPP and imipenem against Ps. aeruginosa were confirmed. Western blot using anti-DnaK and anti-GroEL monoclonal antibodies was performed to investigate the expression of stress shock proteins (SSPs) in imipenem-susceptible and imipenem-resistant strains exposed to TPP. The amount of SSPs were induced as the exposure time increased and decreased. The molecular weights of DnaK and GroEL were 70 kDa and 60 kDa, respectively. SDS-PAGE with silver staining revealed that the amount of lipopolysaccharides (LPS) increased or decreased in the strain treated to different concentrations and exposing periods of TPP. Scanning electron microscopic analysis demonstrated the presence of umblicated and wrinkled surfaces for cells treated with TPP or imipenem.

본 연구는 항생제인 imipenem에 내성이 있는 Pseudomonas aeruginosa에 대한 차 폴리페놀(TPP)과 imipenem의 살균 상승효과와 세포반응을 조사하기 위하여 수행되었다. Imipenem 내성 Ps. aeruginosa는 병원의 환자로부터 분리하였다. TPP와 imipenem을 단독으로 처리하였을 때와 병용으로 처리하였을 때의 최소억제농도(MIC)를 측정한 결과, imipenem 감수성과 내성 균주는 TPP와 imipenem을 병용처리 하였을 때, imipenem 농도가 각각 16배, 8배가 감소되는 것을 확인하였다. 또한, time-kill 조사를 통해 TPP와 imipenem의 항균효과를 조사하였으며, 병용처리 하였을 때 낮은 농도의 imipenem에서도 동일한 항균효과를 나타내는 것을 확인하였다. TPP에 의한 imipenem 감수성과 내성 균주의 스트레스 충격 단백질 발현을 조사하기 위하여 anti-DnaK와 anti-GroEL 단일항체를 이용한 Western blot을 통해 관찰하였다. 스트레스 충격 단백질인 DnaK와 GroEL은 TPP의 노출시간이 증가함에 따라 발현양이 증가하다가 감소하는 것을 확인하였으며, 유도된 DnaK와 GroEL의 분자량은 각각 70 kDa과 60 kDa으로 나타났다. TPP의 농도와 시간에 따른 세균의 LPS 증감 변화를 SDS-PAGE와 은 염색을 통하여 확인하였고, TPP와 imipenem에 노출된 세균의 세포 외부 형태변화를 주사전자현미경을 이용하여 관찰한 결과, 움푹 패이고, 주름진 표면을 가지는 것으로 관찰되었다.

Keywords

References

  1. Beveridge, T. J. 1999. Structures of gram-negative cell walls and their derived membrane vesicles. J. Bacteriol. 181: 4725-4733.
  2. Blanco, A. R., A. Sudano-Roccaro, G. C. Spoto, A. Nostro, and D. Rusciano. 2005. Epigallocatechin gallate inhibits biofilm formation by ocular staphylococcal isolates. Antimicrob. Agents Chemother. 49: 4339-4343. https://doi.org/10.1128/AAC.49.10.4339-4343.2005
  3. Bollag. D. M., M. D. Rozycki, and S. J. Edelstein. 1996. Protein methods. 2nd ed., New York, Wiley-Liss, USA.
  4. Cho, Y. S., N. L. Schiller, H. Y. Kahng, and K. H. Oh. 2007. Cellular responses and proteomic analysis of Escherichia coli exposed to green tea polyphenols. Curr. Microbiol. 53: 501-506.
  5. Cho, Y. S., N. L. Schiller, and K. H. Oh. 2008. Antibacterial effects of green tea polyphenols on clinical isolates of methicillin-resistant Staphylococcus aureus. Curr. Microbiol. 57: 542-546. https://doi.org/10.1007/s00284-008-9239-0
  6. National Committee for Clinical Laboratory Standards. 2007. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. 7th ed. Approved standard M7-A5. CLSI, Wayne, PA, USA.
  7. Flattery-O'Brien, J., L. P. Collinson, and I. W. Daews. 1993. Saccharomyces cerevisiae has an inducible response to menadione which differs from that to hydrogen peroxide. J. Gen. Microbiol. 139: 501-507. https://doi.org/10.1099/00221287-139-3-501
  8. Hitchcock, P. J. and T. M. Brown. 1983. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J. Bacteriol. 154: 269-277.
  9. Ikigai, H., T. Nakae, Y. Hara, and T. Shimamura. 1993. Bactericidal catechins damage the lipid bilayer. Biochim. Biophys. Acta. 1147: 132-136. https://doi.org/10.1016/0005-2736(93)90323-R
  10. Karlowsky, J. A., D. C. Draghi, M. E. Jones, C. Thornsberry, I. R. Friedland, and D. F. Sahm. 2003. Surveillance for antimicrobial susceptibility among clinical isolates of Pseudomonas aeruginosa and Acinetobacter baumannii from hospitalized patients in the United States, 1998 to 2001. Antimicrob. Agents Chemother. 47: 1681-1688. https://doi.org/10.1128/AAC.47.5.1681-1688.2003
  11. Koyama, S., Y. Yamaguchi, S. Tanaka, and J. Motoyoshiya. 1997. A new substance (Yoshixol) with an interesting antibiotic mechanism from wood oil of Japanese traditional tree (Kiso-Hinoki), Chamaecyparis obtusa. Gen. Pharmacol. 28: 797-804. https://doi.org/10.1016/S0306-3623(96)00370-9
  12. Lim, Y. J., Y. S. Cho, and K. H. Oh. 2008. Antibacterial synergic effect and cellular responses of nalidixic acidresistant Salmonella typhimurium exposed to tea polyphenols and nalidixic acid. Kor. J. Microbiol. 44: 122-129.
  13. Lyczak, J. B., C. L. Cannon, and G. B. Pier. 2000. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect. 2: 1051-1060. https://doi.org/10.1016/S1286-4579(00)01259-4
  14. Mogens, K., S. Jacobsen, K. Hammer, and F. K. Vogensen. 1997. Induction of heat shock proteins DnaK, GroEL, and GroES by salt stress in Lactococcus lactis. Appl. Environ. Microbiol. 63: 1826-1837.
  15. Park, S. H., K. H. Oh, and C. K. Kim. 2001. Adaptive and cross-protective responses of Pseudomonas sp. DJ-12 to several aromatics and other stress shock. Curr. Microbiol. 43: 176-181. https://doi.org/10.1007/s002840010283
  16. Sambrook, J. K., E. F. Fritsch, and T. Maniatis. 2001. Molecular cloning. 3rd ed., Cold Spring Harbor Laboratory, New York, USA.
  17. Shimamura, T., W. H. Zhao, and Z. Q. Hu. 2007. Mechanism of action and potential for use of tea catechin as an antiinfective agent. Med. Chem. 6: 57-62.
  18. Song, Y. J., Y. S. Cho, and K. H. Oh. 2009. Antibacterial activity against Salmonella enteritidis JK-15 and LPS changes caused by rose flower extracts. Kor. J. Microbiol. 45: 318-323.
  19. Vaara, M. 1992. Agents that increase the permeability of the outer membrane. Microbiol. Rev. 56: 395-411.
  20. Wiedemann, B., and M. T. Zuhlsdorf. 1989. Brief report: resistance development to fluoroquinolones in Europe. Am. J. Med. 87: 9-11. https://doi.org/10.1016/0002-9343(89)90011-9
  21. Yehuda, C., N. Troillet, G. M. Eliopoulos, and M. H. Samore. 1999. Emergence of antibiotic-resistant Pseudomonas aeruginosa: comparison of risks associated with different antipseudomonal agents. Antimicrob. Agents Chemother. 43: 1379-1382.