미생물학회지 (Korean Journal of Microbiology)

  • 제54권4호
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  • Pages.428-435
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  • 2018
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  • 0440-2413(pISSN)
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  • 2383-9902(eISSN)
한국미생물학회 (The Microbiological Society of Korea)
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Burkholderia sp. OS17의 항균활성 증진을 위한 배양최적화

Antimicrobial activities of Burkholderia sp. strains and optimization of culture conditions

  • 남영호 (국립낙동강생물자원관 배양기술개발부) ;
  • 최아영 (국립낙동강생물자원관 배양기술개발부) ;
  • 황병수 (국립낙동강생물자원관 산업소재화연구부) ;
  • 정유진 (국립낙동강생물자원관 배양기술개발부)
  • Nam, Young Ho (Culture Techniques Research Division, Nakdonggang National Institute of Biological Resources (NNIBR)) ;
  • Choi, Ahyoung (Culture Techniques Research Division, Nakdonggang National Institute of Biological Resources (NNIBR)) ;
  • Hwang, Buyng Su (Bioresources Industrialization Research Division, Nakdonggang National Institute of Biological Resources (NNIBR)) ;
  • Chung, Eu Jin (Culture Techniques Research Division, Nakdonggang National Institute of Biological Resources (NNIBR))
  • 투고 : 2018.06.04
  • 심사 : 2018.10.08
  • 발행 : 2018.12.31
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초록

본 연구는 담수환경에서 항균활성을 보유한 미생물을 발굴하고, 활성 증진을 위해 배양조건을 최적화하는 것이다. 상주시 중동면 오상저수지에서 시료를 채취하여 38종의 미생물을 순수분리하였다. 16S rRNA 염기서열 분석에 근거하여 Proteobacteria강(22종), Actinobacteria강(7종), Bacteroidetes강(6종), Firmicutes강(3종)으로 구성되어있는 것을 확인하였다. 메티실린내성 황색포도상구군 등 10종의 유해미생물에 대한 항균활성을 보유한 Burkholderia sp. OS17 균주를 선발하였다. 항균활성 증진을 위한 상용배지, 온도, 초기 pH별 생육 및 항균활성 비교실험을 수행하였다. OS17 균주는 YPD 배지, $35^{\circ}C$, pH 6.5로 배양했을 때 가장 활성이 높았다. LB, NB, TSB, R2A 배지와 $20^{\circ}C$, $25^{\circ}C$ 배양했을 때는 생장은 가능하나 항균활성이 전혀 없었다. 이전결과를 바탕으로 YPD 배지, $35^{\circ}C$에서 배양하면서 5 L fermenter를 이용하여 생육, 항균활성, pH 확인을 통해 배양 48시간을 최적 배양시간으로 선정하였다. 항균활성을 보유한 미생물의 배양 최적화는 항균물질 생산에 영향을 미치고, 이는 상업적 응용에 이점으로 작용할 수 있다.

In this study, we isolated and identified bacteria from freshwater and soil collected from Osang reservoir, to screen antimicrobial bacteria against various pathogenic bacteria. 38 strains were isolated and assigned to the class Proteobacteria (22 strains), Actinobacteria (7 strains), Bacteroidets (6 strains), and Firmicutes (3 strains) based on 16S rRNA gene sequence analysis. Among them, strain OS17 showed a good growth inhibition against 5 methicillin-resistant Staphylococcus aureus subsp. aureus strains and Bacillus cereus, Bacillus subtilis, Filobasidium neoformans. As a result of the 16S rRNA gene sequence analysis, strain OS17 show the high similarity with Burkholderia ambifaria $AMMD^T$, B. diffusa $AM747629^T$, B. tettitorii $LK023503^T$ 99.8%, 99.7%, 99.6%, respectively. We investigated cell growth and antimicrobial activity according to commercial culture medium, temperature, pH for culture optimization of strain OS17. Optimal conditions for growth and antimicrobial activity in strain OS17 were found to be: YPD medium, $35^{\circ}C$ and pH 6.5. When the strain was cultured in LB, NB, TSB, R2A media at $20^{\circ}C$ and $25^{\circ}C$, the antimicrobial activity did not show. Culture filtrate of strain OS17 showed antimicrobial activity against 5 MRSA strains, Bacillus cereus, Bacillus subtilis, and Filobasidium neoformans with inhibition zones from 2 to 8 mm. Optimal reaction time was 48 h in YPD medium, 100 rpm and 0.3 vvm in 2 L-scale fed-batch fermentation process for antimicrobial activity. Culture optimization of strain OS17 can be improved on antimicrobial activity. Therefore, the antimicrobial activity of Burkholderia sp. OS17 had potential as antibiotics for pathogens including MRSA.

키워드

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Fig. 1. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationships among isolates belonging to the order Burkholderia sp. OS17 and related taxa.

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Fig. 2. Growth, pH (A) and antimicrobial activities (B) of Burkholderia sp. OS17 according to commercial media.

MSMHBQ_2018_v54n4_428_f0003.png 이미지

Fig. 3. Growth, pH (A) and antimicrobial activities (B) of Burkholderia sp. OS17 according to culture temperature.

MSMHBQ_2018_v54n4_428_f0004.png 이미지

Fig. 4. Growth, pH (A) and antimicrobial activities (B) of Burkholderia sp. OS17 according to initial pH.

MSMHBQ_2018_v54n4_428_f0005.png 이미지

Fig. 6. HPLC profiles of Burkholderia sp. OS17 according to commercial media.

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Fig. 5. Growth, pH and antimicrobial activities of Burkholderia sp. OS17 according to culture time by 5 L fermenter

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Fig. 7. HPLC profiles of Burkholderia sp. OS17 according to culture temperature.

Table 1. Strains obtained from Osang reservoir

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참고문헌

  1. Abdel-Mawgoud AM, Abouwafa MM, and Hassouna NA. 2008. Optimization of surfactin production by Bacillus subtilis isolate BS5. Appl. Biochem. Biotechnol. 150, 305-325. https://doi.org/10.1007/s12010-008-8155-x
  2. Cartwright DK, Chilton WS, and Benson DM. 1995. Pyrrolnitrin and phenazine production by Pseudomonas cepacia, strain 5.5B, a biocontrol agent of Rhizoctonia solani. Appl. Microbiol. Biotechnol. 43, 211-216. https://doi.org/10.1007/BF00172814
  3. Farh Mel A, Kim YJ, Van An H, Sukweenadhi J, Singh P, Huq MA, and Yang DC. 2015. Burkholderia ginsengiterrae sp. nov. and Burkholderia panaciterrae sp. nov., antagonistic bacteria against root rot pathogen Cylindrocarpon destructans, isolated from ginseng soil. Arch. Microbiol. 197, 439-447. https://doi.org/10.1007/s00203-014-1075-y
  4. Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783-791. https://doi.org/10.1111/j.1558-5646.1985.tb00420.x
  5. Fitch WM. 1971. Toward defining the course of evolution: Minimum change for a specific tree topology. Syst. Zool. 20, 406-416. https://doi.org/10.2307/2412116
  6. Guerra-Santos LH, Kappeli O, and Fiechter A. 1986. Dependence of Pseudomonas aeruginosa continous culture biosurfactant production on nutritional and environmental factors. Appl. Microbiol. Biotechnol. 24, 443-448.
  7. Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95-98.
  8. Homma Y, Sato Z, Hirayama F, Konno K, Shirahama H, and Suzui T. 1989. Production of antibiotics by Pseudomonas cepacia as an agent for biological control of soilborne plant pathogens. Soil Biol. Biochem. 21, 723-728. https://doi.org/10.1016/0038-0717(89)90070-9
  9. Jiao Y, Yoshihara T, Ishikuri S, Uchino H, and Ichihara A. 1996. Structural identification of cepaciamide A, a novel fungitoxic compound from Pseudomonas cepacia D-202. Tetrahedron Lett. 37, 1039-1042. https://doi.org/10.1016/0040-4039(95)02342-9
  10. Kang Y, Carlson R, Tharpe W, and Schell MA. 1998. Characterization of genes involved in biosynthesis of a novel antibiotic from Burkholderia cepacia BC11 and their role in biological control of Rhizoctonia solani. Appl. Environ. Microbiol. 64, 3939-3947.
  11. Kirinuki T, Iwanuma K, Suzuki N, Fukami H, and Ueno T. 1977. Altericidins, a complex polypeptide antibiotic, produced by Pseudomonas sp. and their effect for the control of black spot of pear caused by Alternaria kikuchiana Tanaka. Sci. Rep. Fac. Agric.-Kobe Univ. (Japan) 12, 223-230.
  12. Kumar S, Stecher G, and Tamura K. 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870-1874. https://doi.org/10.1093/molbev/msw054
  13. Mahenthiralingam E, Song L, Sass A, White J, Wilmot C, Marchbank A, Boaisha O, Paine J, Knight D, and Challis GL. 2011. Enacyloxins are products of an unusual hybrid modular polyketide synthase encoded by a cryptic Burkholderia ambifaria genomic island. Chem. Biol. 18, 665-677. https://doi.org/10.1016/j.chembiol.2011.01.020
  14. Meyers E, Bisacchi GS, Dean L, Liu WC, Minassian B, Slusarchyk DS, Sykes RB, Tanaka SK, and Trejo W. 1987. Xylocandin: a new complex of antifungal peptides. I. Taxonomy, isolation and biological activity. J. Antibiot. 40, 1515-1519. https://doi.org/10.7164/antibiotics.40.1515
  15. Moon SS, Kang PM, Park KS, and Kim CH. 1996. Plant growth promoting and fungicidal 4-quinolinones from Pseudomonas cepacia. Phytochemistry 42, 365-368. https://doi.org/10.1016/0031-9422(95)00897-7
  16. Parker WL, Rathnum ML, Seiner V, Trejo WH, Principe PA, and Sykes RB. 1984. Cepacin A and cepacin B, two new antibiotics produced by Pseudomonas cepacia. J. Antibiot (Tokyo). 37, 431-440. https://doi.org/10.7164/antibiotics.37.431
  17. Parra-Cota Fl, Pena-Cabriales JJ, de Los Santos-Villalobos S, Martinez-Gallardo NA, and Delano-Frier JP. 2014. Burkholderia ambifaria and B. caribensis promote growth and increase yield in grain amaranth (Amaranthus cruentus and A. hypochondriacus) by improving plant nitrogen uptake. PLoS One 9, e88094. https://doi.org/10.1371/journal.pone.0088094
  18. Quan CS, Zheng W, Liu Q, Ohta Y, and Fan SD. 2006. Isolation and characterization of a novel Burkholderia cepacia with strong antifungal activity against Rhizoctonia solani. Appl. Microbiol. Biotechnol. 72, 1276-1284. https://doi.org/10.1007/s00253-006-0425-3
  19. Saga T and Yamaguchi K. 2009. History of antimicrobial agents and resistant bacteria. JMAJ 52, 103-108.
  20. Saitou N and Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406-425.
  21. Suarez-Moreno ZR, Coutinho BG, Mendonca-Previato L, Previato L, James EK, and Venturi V. 2012. Common features of environmental and potentially beneficial plant-associated Burkholderia. Microb. Ecol. 63, 249-266. https://doi.org/10.1007/s00248-011-9929-1
  22. Tawfik KA, Jeffs P, Bray B, Dubay G, Falkinham JO, Mesbah M, Youssef D, Khalifa S, and Schmidt EW. 2010. Burkholdines 1097 and 1229, potent antifungal peptides from Burkholderia ambifaria 2.2N. Org. Lett. 12, 664-666. https://doi.org/10.1021/ol9029269
  23. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, and Higgins DG. 1997. The CLUSTAL_X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876-4882. https://doi.org/10.1093/nar/25.24.4876
  24. Vial L, Lepine F, Milot S, Groleau MC, Dekimpe V, Woods DE, and Deziel E. 2008. Burkholderia pseudomallei, B. thailandensis, and B. ambifaria produce 4-hydroxy-2-alkylquinoline analogues with a methyl group at the 3 position that is required for quorumsensing regulation. J. Bacteriol. 190, 5339-5352. https://doi.org/10.1128/JB.00400-08
  25. Vandamme P, Opelt K, Knochel N, Berg C, Schonmann S, Brandt E, Eberl L, Falsen E, and Berg G. 2007. Burkholderia bryophila sp. nov. and Burkholderia megapolitana sp. nov., moss-associated species with antifungal and plant-growth-promoting properties. Syst. Evol. Microbiol. 57, 2228-2235. https://doi.org/10.1099/ijs.0.65142-0
  26. Yabuuchi E, Kosako Y, Oyaizu H, Yano I, Hotta H, Hashimoto Y, Ezaki T, and Arakawa M. 1992. Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes, 1981) comb. nov. Microbiol. Immunol. 36, 1251-1275. https://doi.org/10.1111/j.1348-0421.1992.tb02129.x
  27. Yoon S, Ha S, Kwon S, Lim J, Kim Y, Seo H, and Chun J. 2017. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int. J. Syst. Evol. Microbiol. 67, 1613-1617. https://doi.org/10.1099/ijsem.0.001755