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Pseudomonas aeruginosa BCNU 1204의 항균활성과 활성 물질

Antimicrobial Activity of Pseudomonas aeruginosa BCNU 1204 and Its Active Compound

  • 신화진 (창원대학교 생물학화학융합학부) ;
  • 주우홍 (창원대학교 생물학화학융합학부)
  • Shin, Hwa Jin (Department of Biology and Chemistry, Changwon National University) ;
  • Joo, Woo Hong (Department of Biology and Chemistry, Changwon National University)
  • 투고 : 2018.11.12
  • 심사 : 2018.12.03
  • 발행 : 2019.01.30

초록

신규 항세균물질을 탐색하는 사전조사에서 몇몇 분리균주들이 그람양성 세균과 그람음성 세균 모두에 항균활성을 보이며, 심지어 methicillin내성 Staphylococcus aureus (MRSA)에도 항균활성을 나타내었다. 이들 균주 중에서 한 균주가 표현형과 계통분석을 이용하여 특히 16S 리보좀 RNA 유전자 염기서열에 기초하여 Pseudomonas aeruginosa로 동정되었다. BCNU 1204 균주의 항균물질은 King's medium B (pH 7.0)에서 $35^{\circ}C$의 온도 조건으로 4일 배양 후 가장 최대로 생산되었다. 항균물질을 각종 유기용매로 분획한 결과, P. aeruginosa BCNU 1204의 dichloromethane (DCM)분획과 ethylacetate (EA) 분획이 그람 양성 세균에 강력한 항균활성을 보였으며, 특히 ethylacetate (EA) 분획이 methicillin내성 Staphylococcus aureus (MRSA)에 대하여 강한 항균활성을 나타내었다. Recycling preparative LC와 preparative TLC 로 활성물질 하나(분획 5-2)를 분리하여 GC-MS 분석한 결과 phenazine 화합물에 속하는 phenazine-1-carboxylic acid 로 동정하였다. 그리고 MRSA 균주에 대한 최소저해농도(minimum inhibitory concentration, MIC)가 MRSA균주인 CCARM 3089, 3090, 3091 그리고 3095 균주에 대하여 각각 $25{\mu}g/ml$, $50{\mu}g/ml$, ${\geq}25{\mu}g/ml$ 그리고 ${\geq}50{\mu}g/ml$ 임을 확인하였다. 그러므로 P. aeruginosa BCNU 1204 분리균주는 항 MRSA 항생물질을 개발하기 위한 잠재 가치가 높은 생물자원으로 기대되며, P. aeruginosa BCNU 1204 균주로부터 리더 화합물을 획득하기 위한 보다 많은 연구가 요구된다.

Previous screening of novel antibacterial agents revealed that some bacterial isolates exhibited antibiotic activity against both gram-positive and gram-negative bacteria and that they showed antibacterial activity, even against methicillin-resistant Staphylococcus aureus (MRSA). Among these isolates, one bacterial strain, BCNU 1204, was identified as Pseudomonas aeruginosa using phenetic and phylogenetic analysis, based on 16S ribosomal RNA gene sequences. The maximum productivities of antimicrobial substances of BCNU 1204 were obtained after being cultured at $35^{\circ}C$ and pH 7.0 for 4 d in King's medium B (KMB). Dichloromethane (DCM) and ethylacetate (EA) extracts of P. aeruginosa BCNU 1204 exhibited strong antimicrobial activity, particularly against gram-positive bacteria. The EA extracts exhibited broad-spectrum activity against antibiotic resistant strains. Fraction 5-2, was obtained by recycling preparative liquid chromatography (LC) and preparative thin-layer chromatography (TLC) and was identified as phenazine-1-carboxylic acid belonging to phenazines using gas chromatography and mass spectrometry (GC/MS). Its minimum inhibitory concentration (MIC) values were $25{\mu}g/ml$, $50{\mu}g/ml$, ${\geq}25{\mu}g/ml$, and ${\geq}50{\mu}g/ml$ for MRSA CCARM 3089, 3090, 3091, and 3095 strains, respectively. P. aeruginosa BCNU 1204 may be a potential resource for the development of anti-MRSA antibiotics. Additional research is required to identify the active substance from P. aeruginosa BCNU 1204.

키워드

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Fig. 1. Phylogenetic position of Pseudomonas sp. BCNU 1204 based on 16S ribosomal RNA gene sequences.

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Fig. 2. Cell growth of Pseudomonas aeruginosa BCNU 1204 cultured in a King's medium B and antibiotic activity of its crude extract. ●: cell growth of BCNU 1204, ■: pH of culture medium, ◇: its inhibitory activity against S. aureus CCARM 3090 , △: its inhibitory activity against S. aureus CCARM 3091, ○: its inhibitory activity against S. aureus CCARM 3115, □: its inhibitory activity against S. aureus CCARM 3561.

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Fig. 3. Gas chromatographic and gas chromatographic-mass spectrometric (GC-MS) analysis of purified phenazine-1-carboxylic acid. (A) Gas chromatogram of purified phenazine-1-carboxylic acid, (B) GC-MS chromatogram of purified phenazine-1-carboxylic acid.

Table 1. Antimicrobial activity of Pseudomonas aeruginosa BCNU 1204 against test bacteria

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Table 2. Antimicrobial activity of HA, DCM and EA extract of Pseudomonas aeruginosa BCNU 1204 against test bac-teria

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Table 3. Minimal inhibitory concentration of purified 5-2 com-pound

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참고문헌

  1. Borrero, N. V., Bai, F., Perez, C., Duong, B. Q., Rocca, J. R., Jin, S. and Huigens III, R. W. 2014. Phenazine antibiotic inspired discovery of potent bromophenazine antibacterial agents against Staphylococcus aureus and Staphylococcus epidermidis. Org. Biomol. Chem. 12, 881-886. https://doi.org/10.1039/C3OB42416B
  2. Brisbane, P. G. and Rovira, A. D. 1988. Mechanism of inhibition of Gaeumannomyces graminis var. tritici by Fluorescent Pseudomonads. Plant Pathol. 37, 104-111. https://doi.org/10.1111/j.1365-3059.1988.tb02201.x
  3. Cardozo, V. F., Oliveira, A. G., Nishio, E. K., Perugini, M. R., Andrade, C. G., Silveira, W. D., Duran, N., Andrade, G., Kobayashi, R. K. T. and Nakazato, G. 2013. Antibacterial activity of extracellular compounds produced by a Pseudomonas strain against methicillin-resistant Staphylococcus aureus (MRSA) strains. Ann. Clin. Microb. Anti. 12, 12. https://doi.org/10.1186/1476-0711-12-12
  4. Chebbi, A., Hentati, D., Zaghden, H., Baccar, N., Rezgui, F., Chalbi, M., Sayadi, S. and Chamkha, M. 2017. Polycyclic aromatic hydrocarbon degradation and biosurfactant production by a newly isolated Pseudomonas sp. strain from used motor oil-contaminated soil. Int. Biodeter. Biodegr. 122, 128-140. https://doi.org/10.1016/j.ibiod.2017.05.006
  5. Chin-A-Woeng, T. F. C., Bloemberg, G. V., van der Bij, A. J., van der Drift, K. M. G. M., Schripsema, J., Kroon, B., Scheffer, R. J., Keel, C., Bakker, P. A. H. M., Tichy, H. V., de Bruijin, F. J., Thomas-Oates, J. and Lugtenberg, B. 1998. Biocontrol by phenazine-1-carboxamide-producing Pseudomonas chlororaphis PCL1391 of tomato root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. Mol. Plant Microbe Interact. 11, 1069-1077. https://doi.org/10.1094/MPMI.1998.11.11.1069
  6. Harney, A. 2000. Strategies for discovering drugs from previously unexplored natural products. Drug Discov. Today 5, 294-300. https://doi.org/10.1016/S1359-6446(00)01511-7
  7. Hasan, R., Acharjee, M. and Noor, R. 2016. Prevalence of vancomycin resistant Staphylococcus aureus (VRSA) in methicillin resistant S. aureus (MRSA) strains isolated from burn wound infections. Tzu. Chi. Med. J. 28, 49-53. https://doi.org/10.1016/j.tcmj.2016.03.002
  8. Jain, R. and Pandey, A. 2016. A phenazine-1-carboxylic acid producing polyextremophilic Pseudomonas chlororaphis (MCC 2693) strain, isolated from mountain ecosystem, possesses biocontrol and plant growth promotion abilities. Microbiol. Res. 190, 63-71. https://doi.org/10.1016/j.micres.2016.04.017
  9. Leisinger, T. and Margraff, R. 1979. Sencondary metabolites of the fluorescent Pseudomonads. Microbiol. Rev. 43, 422-442. https://doi.org/10.1128/MMBR.43.3.422-442.1979
  10. Lee, A. J., Suh, H. S., Jeon, C. H. and Kim, S. G. 2011. Prevalence and clinical characteristics of mupirocin-resistant Staphylococcus aureus. Kor. J. Clin. Microbiol. 14, 18-23. https://doi.org/10.5145/KJCM.2011.14.1.18
  11. Mishra, J. and Arora, N. K. 2018. Secondary metabolites of fluorescent pseudomonads in biocontrol of phytopathogens for sustainable agriculture. Appl. Soil Ecol. 125, 35-45. https://doi.org/10.1016/j.apsoil.2017.12.004
  12. Murray P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C. and Yolke, R. H. 1999. Manual of Clinical Microbiology, pp. 1527-1539, 7th ed., ASM: Washington, DC, USA.
  13. Shoji, J., Hinoo, H., Kato, T., Hattori, T., Hirooka, K., Tawara, K., Shiratori, O. and Terui, Y. 1989. Isolation of cepafungins I, II and III from Pseudomonas species. J. Antibiot. 23, 783-787.
  14. Thomashow, L. S. and Weller, D. M. 1988. Role of phenazine antibiotic from Pseudomonas fluorescens in biological control of Gaeumannomyces graminis var. tritici. J. Bacteriol. 170, 3499-3508. https://doi.org/10.1128/jb.170.8.3499-3508.1988
  15. Saito, N. and Nei, M. 1987. The neighbor-joining method, a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 79, 426-434.
  16. Shanmugaiah, V., Mathivanan, N. and Varghese, B. 2009. Purification, crystal structure and antimicrobial activity of phenazine-1-carboxamide produced by a growth-promoting biocontrol bacterium, Pseudomonas aeruginosa MML2212. J. Appl. Microbiol. 108, 703-711. https://doi.org/10.1111/j.1365-2672.2009.04466.x
  17. Sutter, V. L., Kwok, Y. Y. and Finegold, S. M. 1973. Susceptibility of Bacteroides fragilis to six antibiotics determined by standardized antimicrobial disc susceptibility testing. Antimicrob. Agents Chemother. 3, 188-193. https://doi.org/10.1128/AAC.3.2.188
  18. Upadhyay, A. and Srivastava, S. 2011. Phenazine-1-carboxylic acid is a more important contributor to biocontrol Fusarium oxysporum than pyrrolnitrin in Pseudomonas fluorescens strain Psd. Microbiol. Res. 166, 323-335. https://doi.org/10.1016/j.micres.2010.06.001