Korean Journal of Environmental Biology (환경생물)

Korean Society of Environmental Biology (한국환경생물학회)
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Isolation and Characterization of a Marine Bacterium, Pseudomonas sp. YJ-1 with Anti-Methicillin Resistant Staphylococcus aureus Activity

항 Methicillin Resistant Staphylococcus aureus 활성을 가지는 해양미생물 Pseudomonas sp. YJ-1의 분리와 특성

  • Woo, Ye-Ju (Department of Biomedical Science, Daegu Catholic University) ;
  • Jeong, Seong-Yun (Department of Biomedical Science, Daegu Catholic University)
  • 우예주 (대구가톨릭대학교 의생명과학과) ;
  • 정성윤 (대구가톨릭대학교 의생명과학과)
  • Received : 2017.11.26
  • Accepted : 2017.12.14
  • Published : 2017.12.31
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Abstract

The aim of this study was to isolate and identify marine bacterium with anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, and to purify the anti-MRSA compound, as well as to determine its activity and synergistic effects. Among the marine bacteria isolated in this study, the YJ-1 isolate had the strongest anti-MRSA activity. The YJ-1 isolate was identified on the basis of its biochemical characteristics and an analysis of 16S rRNA gene sequences. The YJ-1 isolate showed over 99.2% homology with Pseudomonas stutzeri, and was designated as a Pseudomonas sp. YJ-1. The optimal culture conditions were $25^{\circ}C$ and initial pH 7.0. For the purification of the anti-MRSA compounds, the YJ-1 was cultured in Pa PES-II medium, and the culture filtrates were extracted by ethyl acetate, hexane, and 80% MeOH. The 80% MeOH fraction was separated by a $C_{18}$ ODS column, silica gel chromatography and a reverse phase HPLC, to yield three anti-MRSA agents, the MR1, MR2, and MR3 compounds. When the MR1 compound of $250{\mu}g\;mL^{-1}$ concentration was applied to the MRSA cells, over 95% of bacterial cells was killed within 48 hr. Compared with vancomycin and ampicillin, the MR1 compound showed significant anti-MRSA activity. In addition, the anti-MRSA activity was increased by dose and time dependent manners. Furthermore, the combination of an MR1 compound with vancomycin produced a more rapid decrease in the MRSA cells than did the MR1 compound alone. Taken together, our results suggest that the Pseudomonas sp. YJ-1 and its anti-MRSA compounds could be employed as a natural antibacterial agent in MRSA infections.

본 연구의 목적은 항 methicillin-resistant Staphylococcus aureus (MRSA) 활성을 가지는 해양미생물을 분리, 동정하고 항MRSA 물질을 분리 정제하여 그 물질의 활성과 시너지 효과를 밝히는 것이다. 본 연구에서 분리한 해양미생물 중에서, YJ-1 분리 균주가 가장 강한 항MRSA 활성을 나타내었다. YJ-1 균주는 생화학적 특성과 16S rRNA 유전자 염기서열에 기초하여 분류 동정되었다. YJ-1 균주의 염기서열은 Pseudomonas stutzeri와 99.2%의 상동성을 나타내어, Pseudomonas sp. YJ-1이라 명명하였다. 이 균주의 최적 성장조건은 $25^{\circ}C$와 초기 pH 농도 7.0이었다. 항MRSA 물질들을 분리정제하기 위하여 YJ-1 균주를 PPES-II 배지에 배양하였으며, 배양 상등액을 ethyl acetate, hexane과 80% MeOH로 순차적으로 추출하였다. 활성을 보인 80% MeOH 분획을 $C_{18}$ ODS 칼럼 크로마토그래피, silica gel 크로마토그래피와 역상 HPLC법으로 순차적으로 정제하여 항MRSA 활성을 가지는 3개의 순수물질 MR1, MR2 및 MR3를 얻었다. $250{\mu}g\;mL^{-1}$ 농도의 MR1 물질을 MRSA 세포에 접종했을 때, MRSA 세포의 95%가 48시간 이내에 사멸하였다. 활성을 vancomycin 및 ampicillin과 비교해 보아도, MR1 물질은 보다 우수한 항MRSA 활성을 나타내었다. 또한 항MRSA 활성은 투여량과 시간에 비례하여 증가하였다. 더욱이 MR1 물질과 vancomycin을 조합하여 시너지 효과를 보았을 때, MR1 물질을 단독으로 투여했을 때보다 신속한 MRSA 세포의 감소를 관찰할 수 있었다. 이상의 결과를 종합해보면, MRSA 감염에 있어서 Pseudomonas sp. YJ-1과 이것이 생산하는 항MRSA 물질은 천연 항균제로서 기여할 수 있으리라 판단된다.

Keywords

References

  1. Akcam FZ, GB Tinaz, O Kaya, A Tigl, E Ture and S Hosoglu. 2009. Evaluation of methicillin resistance by cefoxitin disk diffusion and PBP2a latex agglutination test in mecApositive Staphylococcus aureus, and comparison of mecA with femA, femB, femX positivities. Microbiol. Res. 164: 400-403. https://doi.org/10.1016/j.micres.2007.02.012
  2. Bassetti M, E Nicco and M Mikulska. 2009. Why is community-associated MRSA spreading across the world and how will it change clinical practice? Int. J. Antimicrob. Agents 34:S15-S19.
  3. Bell JM, JD Tumidge and A Sentry. 2002. High prevalence of oxacillin-resistant Staphylococcus aureus isolates from hospitalized patients in Asia Pacific and South Africa: results from sentry antimicrobial surveillance program, 1998-1999. Antimicrob. Agents Chemother. 46:879-881. https://doi.org/10.1128/AAC.46.3.880-882.2002
  4. Berdy JJ. 2005. Bioactive microbial metabolites. J. Antibiot. 58:1-26. https://doi.org/10.1038/ja.2005.1
  5. Boyle-Vavra S, RB Carey and RS Daum. 2001. Development of vancomycin and lysostaphin resistance in a methicillinresistant Staphylococcus aureus isolate. J. Antimicrob. Chemother. 48:617-625. https://doi.org/10.1093/jac/48.5.617
  6. Cha JD, JH Lee, KM Choi, SM Choi and JH Park. 2014. Synergistic effect between cryptotanshinone and antibiotics against clinic methicillin and vancomycin-resistant Staphylococcus aureus. Evid.-based Complement Altern. 2014:450572.
  7. Crum NF, RU Lee, SA Thornton, OC Stine, MR Wallace, C Barrozo, A Keffer-Norris, S Judd and KL Russell. 2006. Fifteen-year study of the changing epidemiology of methicillin-resistant Staphylococcus aureus. Am. J. Med. 119:943-951. https://doi.org/10.1016/j.amjmed.2006.01.004
  8. Davis SL, MB Perri, SM Donabedian, C Manierski, A Robinson-Dunn, MK Hayden and MJ Zervos. 2007. Epidemiology and outcomes of community-associated methicillin-resistant Staphylococcus aureus infection. J. Clin. Microbiol. 45:1705-1711. https://doi.org/10.1128/JCM.02311-06
  9. Dunbar J, LO Tickno and CR Kuske. 2000. Assessment of microbial diversity in four Southwestern United States soils by 16S rRNA gene terminal restriction fragment analysis. Appl. Environ. Microbiol. 66:2943-2950. https://doi.org/10.1128/AEM.66.7.2943-2950.2000
  10. Faver B, B Hofbauer, KS Hildering and NS Ryder. 2003. Comparison of in vitro activities of 17 antifungal drugs against a panel of 20 dermatophytes by using a microdilution assay. J. Clin. Microbiol. 41:4817-4819. https://doi.org/10.1128/JCM.41.10.4817-4819.2003
  11. Fritza E, A Fekete, J Lintelmann, P Schmitt-Kopplin and RU Meckenstock. 2009. Isolation of two Pseudomonas strains producing pseudomonic acid A. Syst. Appl. Microbiol. 32: 56-64. https://doi.org/10.1016/j.syapm.2008.11.001
  12. Grundmann H, M Aires-de-Sousa, J Boyce and E Tiemersama. 2006. Emergence and resurgence of methicillin-resistant Staphylococcus aureus as a public-health threat. Lancet 368:874-885. https://doi.org/10.1016/S0140-6736(06)68853-3
  13. Gulder TA and BS Moore. 2009. Chasing the treasures of the sea-bacterial marine natural products. Curr. Opin. Microbiol. 12:252-260. https://doi.org/10.1016/j.mib.2009.05.002
  14. Hamann MT and PJ Scheuer. 1993. Kahalalide F: a bioactive depsipeptide from the sacoglossan mollusk Elysia rufescens and the green alga Bryopsis sp. J. Am. Chem. Soc. 115: 5825-1826. https://doi.org/10.1021/ja00066a061
  15. Hong SN, J Kim and HH Sung. 2016. A study on changes in antimicrobial resistant Staphylococcus aureus from wound isolates in a South Korean University hospital for the past 10 years. Korean J. Clin. Lab. Sci. 48:335-342. https://doi.org/10.15324/kjcls.2016.48.4.335
  16. Howden BP, JK Davies, PD Jonhson, TP Stinear and ML Grayson. 2010. Reduced vancomycin susceptibility in Staphylococcus aureus, including vancomycin-intermediate and heterogeneous vancomycin-intermediate strains: resistance mechanisms, laboratory detection, and clinical implications. Clin. Microbiol. Rev. 23:99-139. https://doi.org/10.1128/CMR.00042-09
  17. Hiramatsu K. 2001. Vancomycin-resistant Staphylococcus aureus: a new model of antibiotic resistance. Lancet Infect. Dis. 1:147-155. https://doi.org/10.1016/S1473-3099(01)00091-3
  18. Jung JH and HC Chang. 2009. Bacillus polufermenticus CJ9, isolated from Jeju, showing antifugal and antibacterial activities. Korean J. Microbiol. Biotechnol. 37:340-349.
  19. Kim ES, SI Jeong, JH Kim, C Park, SM Kim, JK Kim, KM Lee, SH Lee, H So and R Park. 2009. Synergistic effects of the combination of 20-hydroxyecdysone with ampicillin and gentamicin against methicillin-resistant Staphylococcus aureus. J. Microbiol. Biotechnol. 19:1576-1581. https://doi.org/10.4014/jmb.0903.03015
  20. Kitai S, A Shimizu, J Kawano, E Sato, C Nakano, T Uji and H Kitagawa. 2005. Characterization of methicillin-resistant Staphylococcus aureus isolated from retail raw chicken meat in Japan. J. Vet. Med. Sci. 67:107-110. https://doi.org/10.1292/jvms.67.107
  21. Koehn FE and GT Carter. 2005. The evolving role of natural products in drug discovery. Nat. Rev. Drug Discov. 4:206-220. https://doi.org/10.1038/nrd1657
  22. Lee K, SJ Jang, HJ Lee, N Ryoo, M Kim, SG Hong and Y Chong. 2004. Increasing prevalence of vancomycin-resistant Enterococcus faecium, expanded-spectrum cephalosporin-resistant Klebsiella pnuemoniae, and imipenem-resistant Pseudomonas aeruginosa in Korea: Konsar study in 2001. J. Korean Med. Sci. 19:8-14. https://doi.org/10.3346/jkms.2004.19.1.8
  23. MacFaddin JF. 1980. Biochemical tests for identification of medical bacteria. 2nd ed. Williams and Wilkins Co., Baltimore.
  24. Nagai K, K Kanigiri, N Arao, K Suzumura, Y Kawano, M Yamaoka, H Zhang, H Zhang, M Watanabe and K Suzuki. 2003. Novel thiopeptide antibiotics produced by Bacillus cereus isolated from a marine sponge. I. Taxonomy, fermentation, isolation, physicochemical properties and biological properties. J. Antibiot. 56:123-128. https://doi.org/10.7164/antibiotics.56.123
  25. National Committee for Clinical Laboratory Standards. 2009. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard M07-A8. 8th ed. National Committee for Clinical Laboratory Standards, Wayne, PA.
  26. Park YJ, JS Jeong, ES Park, ES Shin, SH Kim and YS Lee. 2007. Survey on the infection control of multidrug-resistant microorganisms in general hospitals in Korea. Korean J. Nosocomial Infect. Control 12:112-121.
  27. Proksch P, RA Edrada and R Ebel. 2003. Drugs from the seaopportunities and obstacles. Mar. Drugs 1:5-17. https://doi.org/10.3390/md101005
  28. Salomon CE, NA Magarvey and DH Sherman. 2004. Merging the potential of microbial genetics with biological and chemical diversity: an even brighter future for marine natural product drug discovery. Nat. Prod. Rep. 21:105-121. https://doi.org/10.1039/b301384g
  29. Sambrook J, EF Fritsch and T Maniatis. 1989. Molecular cloning, a laboratory manual. 2nd ed. Cold Spring Harbor Laboratory, New York.
  30. Shiota S, M Shimizu, J Sugiyam, Y Morita, T Mizushima and T Tsuchiya. 2004. Mechanisms of action of corilagin and tellimagrandin I that remarkably potentiate the activity of beta-lactams against methicillin-resistant Staphylococcus aureus. Microbiol. Immunol. 48:67-73. https://doi.org/10.1111/j.1348-0421.2004.tb03489.x
  31. Taga N. 1968. Some ecological aspects of marine bacteria in the KuroShio current. Bull. Misaki Mar. Biol. Inst. Kyoto Univ. 12:65-76.
  32. Tamura K, D Peterson, N Peterson, G Stecher, M Nei and S Kumar. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28: 2731-2739. https://doi.org/10.1093/molbev/msr121
  33. Watve MG, R Tickoo, MM Jog and BD Bhole. 2001. How many antibiotics are produced by the genus Streptomyces? Arch. Microbiol. 176:386-390. https://doi.org/10.1007/s002030100345
  34. Yee C, D Biek, K Karause and G Williams. 2011. Ceftarolin: a cephalosporin with anti-MRSA activity. Clin. Microbiol. Newsl. 33:161-169. https://doi.org/10.1016/j.clinmicnews.2011.10.001
  35. Yucel N, S Citak and M Onder. 2005. Prevalence and antibiotic resistance of Listeria species in meat products in Ankara, Turkey. Food Microbiol. 22:241-245. https://doi.org/10.1016/j.fm.2004.03.007