Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Use of Bioluminescent Indicator Acinetobacter Bacterium for Screening and Characterization of Active Antimicrobial Agents

  • Haleem Abd-El (Environmental Biotechnology Department, Genetic Engineering and Biotechnology Research Institute, Mubarak City for Scientific Research and Technology Applications, New Burg-Elarab City) ;
  • A.M. Desouky (Environmental Biotechnology Department, Genetic Engineering and Biotechnology Research Institute, Mubarak City for Scientific Research and Technology Applications, New Burg-Elarab City) ;
  • Zaki Sahar A. (Environmental Biotechnology Department, Genetic Engineering and Biotechnology Research Institute, Mubarak City for Scientific Research and Technology Applications, New Burg-Elarab City)
  • Published : 2006.11.30
Download PDF
⟨ Previous Next ⟩

Abstract

Because of the need for new antimicrobial substances with novel mechanisms of action, we report here the use of an Acinetobacter reporter system for high-throughput screening of active antimicrobial agents. The bioreporter Acinetobacter strain DF4/PUTK2 carrying luciferase genes luxCDABE was chosen because of its ecological importance and it is widespread in nature. This bioreporter is genetically engineered to emit light constitutively that can be measured in real time by luminometry. Hence, this reporter system was employed to determine the bacteriostatic actions of spent-culture supernatants derived from twelve bacterial isolates. Out of the results, the strongest bioluminescence inhibitory effect of the supernatants was recorded with Bacillus cereus strain BAC (S5). Subsequently, ethyl acetate extracts of extracellular products of strain BAC (S5) were separated by a thin-layer chromatography (TLC). Based on the bioluminescence inhibitory assay, three fractions were found to have antimicrobial activity. One fraction (C) having the strongest antimicrobial activity was further purified using TLC and characterized by IR, $^1H$ NMR, mass spectrometry, SDS-PAGE, and amino acid composition analysis. The results predicted the presence of 2-pyrrolidone-S-carboxylic acid (PCA) and the octadeconic-acid-like fatty acid. Fraction C also demonstrated a broad inhibitory activity on several Gram-negative and Gram-positive bacteria. In conclusion, the Acinetobacter reporter system shows great potential to be a reliable, sensitive, and real-time indicator of the bacteriostatic actions of the antimicrobial agents.

Keywords

References

  1. Abd-El-Haleem, D., S. Zaki, H. Elbery, A. Tawfiek, and G. Abu-Elreesh. 2006. Assessment of heavy metals toxicity in wastewater using a constructed bioluminescent Acinetobacter bioreporter. J. Basic Microbiol. 46(5): 339-247 https://doi.org/10.1002/jobm.200510122
  2. Abd-El-Haleem, D., H. Moawad, E.A. Zaki, and S. Zaki. 2002. Molecular characterization of phenol-degrading bacteria isolated from different Egyptian ecosystems. Microb. Ecol. 43: 217-224 https://doi.org/10.1007/s00248-002-2003-2
  3. Abd-El-Haleem, D., S. Ripp, C. Scott, and G. S. Sayler. 2002. A luxCDABE-based bioluminescent bioreporter for the detection of phenol. J. Ind. Microbiol. 29: 233-237 https://doi.org/10.1038/sj.jim.7000309
  4. Abee, T., T. R. Klaenhammer, and L. Letellier. 1994. Kinetic studies of the action of lactacin F, a bacteriocin produced by Lactobacillus johnsonii that forms poration complexes in the cytoplasmic membrane. Appl. Environ. Microbiol. 60: 1006-1013
  5. AbuElreesh, G. 2005. Genetically modified biosensor to monitor general toxicity of chemical pollutants. Master degree thesis, Alazhar University, Cairo, Egypt
  6. Airaudo, C. B., A. Gayte-Sorbier, and P. Armand. 1987. Stability of glutamine and pyroglutamic acid under model system conditions: Influence of physical and technological factors. J. Food Sci. 52: 1750-1752 https://doi.org/10.1111/j.1365-2621.1987.tb05926.x
  7. Bizani, D. and A. Brandelli. 2002. Characterization of a bacteriocin produced by a newly isolated Bacillus sp. strain 8A. J. Appl. Microbiol. 93: 512-519 https://doi.org/10.1046/j.1365-2672.2002.01720.x
  8. Budde, B. B. and M. Rasch. 2001. A comparative study on the use of flow cytometry and colony forming units for assessment of the antibacterial effect of bacteriocins. Int. J. Food Microbiol. 63: 65-72 https://doi.org/10.1016/S0168-1605(00)00399-8
  9. Burlage, R. S., L. A. Bemis, A. C. Layton, G. S. Sayler, and F. Larimer. 1990. Comparative genetic organization of incompatibility group P degradative plasmids. J. Bacteriol. 172: 6818-6825 https://doi.org/10.1128/jb.172.12.6818-6825.1990
  10. Chen, G. and J. B. Russel. 1989. Transport of glutamine by Streptococcus bovis and conversion of glutamine to pyroglutamic acid and ammonia. J. Bacteriol. 171: 2981-2985 https://doi.org/10.1128/jb.171.6.2981-2985.1989
  11. Engelhardt, U., K. Peters, and H. G. Maier. 1984. Pyroglutaminsaure in Bohnenkaffee. Z. Lebensm. Unters. Forsch. 178: 288-293 https://doi.org/10.1007/BF01851354
  12. Farre, M. and D. Barcelo. 2001. Characterization of wastewater toxicity by means of a whole-cell bacterial biosensor, using Pseudomonas putida, in conjunction with chemical analysis. Fresenius J. Anal. Chem. 371: 467-473 https://doi.org/10.1007/s002160100925
  13. Gerhardt, G. C., C. D. Salisbury, and J. D. MacNeil. 1994. Analysis of streptomycin and dihydrostreptomycin in milk by liquid chromatography. J. AOAC Int. 77: 765-767
  14. Gould, G. W. 1991. Antimicrobial compound, pp. 461-483. In Goldberg, I. and Williams, R. (eds.). Biotechnology and Food Ingredients. Van Nostrand Reinhold, New York
  15. Huttunen, E., K. Noro, and Z. Yang. 1995. Purification and identification of antimicrobial substances produced by two Lactobacillus casei strains. Int. Dairy J. 5: 503-513 https://doi.org/10.1016/0958-6946(95)00030-7
  16. Jacobsen, C. N., V. Rosenfeldt Nielsen, A. E. Hayford, P. L. Moller, K. F. Michaelsen, A. Paerregaard, B. Sandstrom, M. Tvede, and M. Jakobsen. 1999. Screening of probiotic activities of forty-seven strains of Lactobacillus spp. by in vitro techniques and evaluation of the colonization ability of five selected strains in humans. Appl. Environ. Microbiol. 65: 4949-4956
  17. Kabara, J. J. 1993. Medium-chain fatty acids and esters, pp. 307-342. In Davidson, P. M. and Branen, A. L. (eds). Antimicrobials in Foods, 2nd Ed. Marcel Dekker Inc., New York
  18. Kabashima, T., Y. Li, N. Kanada, K. Ito, and T. Yoshimoto. 2001. Enhancement of the thermal stability of pyroglutamyl peptidase I by introduction of an intersubunit disulfide bond. Biochim. Biophys. Acta 1547: 214-220 https://doi.org/10.1016/S0167-4838(01)00185-6
  19. Kawai, Y., T. Saito, J. Uemura, and T. Itoh. 1997. Rapid detection method for bacteriocin and distribution of bacteriocin-producing strains in Lactobacillus acidophilus group lactic acid bacteria isolated from human feces. Biosci. Biotechnol. Biochem. 61: 179-182 https://doi.org/10.1271/bbb.61.179
  20. Klaenhammer, T. R. 1988. Bacteriocins of lactic acid bacteria. Biochimie 70: 337-349 https://doi.org/10.1016/0300-9084(88)90206-4
  21. Loimaranta, V., J. Tenovuo, L. Koivisto, and M. Karp. 1998. Generation of bioluminescent Streptococcus mutans and its usage in rapid analysis of the efficacy of antimicrobial compounds. Antimicrob. Agents Chemother. 42: 1906-1910
  22. Looveren, V. and H. Goossens. 2004. Antimicrobial resistance of Acinetobacter spp. in Europe. Clin. Microbiol. Infect. 10: 684-704 https://doi.org/10.1111/j.1469-0691.2004.00942.x
  23. Lyytikainen, O., S. Koljalg, M. Harma, and J. Vuopio-Varkila. 1995. Outbreak caused by two multi-resistant Acinetobacter baumannii clones in a burns unit: Emergence of resistance to imipenem. J. Hosp. Infect. 31: 41-54 https://doi.org/10.1016/0195-6701(95)90082-9
  24. Masaaki, N., S. Akio, I. Manabu, and Y. Hitomi. 1992. Comparison of organic acid patterns in miso. Shishu Miso Kenkyosho Kenkyu Hokoku 33: 58-63
  25. National Committee for Clinical Laboratory Standards. 1993. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, 3rd Ed. Approved Standards NCCLS Document M7-AE, NCCLS, Villanova, PA, U.S.A
  26. Paik, H. D., S. S. Bae, S. H. Park, and J. G. Pan. 1997. Identification and partial characterization of tochicin, a bacteriocin produced by Bacillus thuringiensis subsp tochigiensis. J. Ind. Microbiol. 19: 294-298 https://doi.org/10.1038/sj.jim.2900462
  27. Paton, R. H., R. S. Miles, J. Hood, and S. G. B. Amyes. 1993. ARI-1: Beta-lactamase-mediated imipenem resistance in Acinetobacter baumannii. Int. J. Antimicrob. Agents 2: 81-88 https://doi.org/10.1016/0924-8579(93)90045-7
  28. Rao, D. R. and J. C. Reddy. 1984. Effect of lactic fermentation of milk on milk lipids. J. Food Sci. 49: 748-750 https://doi.org/10.1111/j.1365-2621.1984.tb13201.x
  29. Salisbury, V., A. Pfoestl, H. Wiesinger-Mayr, R. Lewis, K. E. Bowker, and A. P. MacGowan. 1999. Use of a clinical Escherichia coli isolate expressing lux genes to study the antimicrobial pharmacodynamics of moxifloxacin. J. Antimicrob. Chemother. 43: 829-832 https://doi.org/10.1093/jac/43.6.829
  30. Sanz, B., D. Selgas, I. Parejo, and J. A. Ordonez. 1988. Characteristics of lactobacilli isolated from dry fermented sausages. Int. J. Food Microbiol. 6: 199-205 https://doi.org/10.1016/0168-1605(88)90012-8
  31. Schillinger, U. and F. K. Lucke. 1989. Antibacterial activity of Lactobacillus sake isolated from meat. Appl. Environ. Microbiol. 55: 1901-1906
  32. Syuhei, K., T. Hiroshi, and I. Hiroshi. 1994. The improvement and development of Chinese soy sauce: 1. The chemical composition of Chinese soy sauce. Nippon Shoyu Kenkyusho Zasshi 20: 1-6
  33. Todoriki, K., T. Mukai, S. Sato, and T. Toba. 2001. Inhibition of adhesion of food-borne pathogens to Caco-2 cells by Lactobacillus strains. J. Appl. Microbiol. 91: 154-159 https://doi.org/10.1046/j.1365-2672.2001.01371.x
  34. Vesterlund, S., J. Paltta, A. Laukova, M. Karp, and A. C. Ouwehand. 2004. Rapid screening method for the detection of antimicrobial substances. J. Microbiol. Methods 57: 23-31 https://doi.org/10.1016/j.mimet.2003.11.014
  35. Virta, M., M. Karp, and P. Vuorinen. 1994. Nitric oxide donor-mediated killing of bioluminescent Escherichia coli. Antimicrob. Agents Chemother. 38: 2775-2779 https://doi.org/10.1128/AAC.38.12.2775
  36. von Dohren, H. 1995. Peptides. Biotechnology 28: 129-171
  37. Zheng, G., L. Z. Yan, J. C. Vederas, and P. Zuber. 1999. Genes of the sbo-alb locus of Bacillus subtilis are required for production of the antilisterial bacteriocin, subtilosin, and are regulated by abrB. J. Bacteriol. 181: 7346-7455