Toxin Gene Analysis of Bacillus cereus and Bacillus thuringiensis Isolated from Cooked Rice

쌀밥에서 분리한 Bacillus cereus와 Bacillus thuringiensis의 독소유전자 분석

  • Jeon, Jong-Hyuk (Department of Food Science and Biotechnology, Kyungwon University) ;
  • Park, Jong-Hyun (Department of Food Science and Biotechnology, Kyungwon University)
  • 전종혁 (경원대학교 식품생물공학과) ;
  • 박종현 (경원대학교 식품생물공학과)
  • Received : 2010.04.12
  • Accepted : 2010.05.14
  • Published : 2010.06.30


Bacterial contamination of cooked rice was analyzed to evaluate the microbial safety. Thirty raw rice samples were collected in Korea and cooked in an electric rice cooker. Mesophilic aerobe, food-poisoning Bacillus cereus group, and their toxin genes were determined on cooked rice. The percentage of total mesophilic aerobe based on 1-3 log CFU/g was 27% among the samples. Bacillus spp. in MYP selective medium was similar to the number of mesophilic aerobe, whileas Bacillus spp. was detected in most samples after enrichment. Thirty-seven isolates from 30 cooked rices were identified as B. thuringiensis, B. cereus, B. valismortis, B. pumilus, B. coagulans, B. licheniformis, Geobacillus stearothermophilus, and Brevibacillus laterosporus. Twenty isolates (54%), more than half of the isolates, were B. thuringiensis while nine (27%) were identified as B. cereus. All B. thuringiensis isolates possessed non-hemolytic toxin genes and interestingly, seven B. cereus among nine isolates possessed emetic toxin genes. More B. thuringiensis was present on the cooked rice than B. cereus and most B. cereus possessed emetic toxin genes rather than diarrheal toxin genes. Therefore, food-borne outbreak due to B.cereus on the cooked rice kept at room temperature might be examples of emetic food-poisoning.


cooked rice;mesophilic aerobe;B. thuringiensis;B. cereus;emetic toxin gene


Supported by : 율촌재단, 경원대학교


  1. Glatz BA, Goepfert JM. Defined conditions for synthesis of Bacillus cereus enterotoxin by fermentor-grown cultures. Appl. Environ. Microbiol. 32: 400-404 (1976)
  2. Kim SH, Kim JS, Choi JP, Park JH. Prevalence and frequency of food-borne pathogens on unprocessed agricultural and marine products. Korean J. Food Sci. Technol. 38: 594-598 (2006)
  3. Chang TE. Moon SY, Lee KW, Park JM, Han JS, Song OJ, Shin IS. Microflora of manufacturing process and final products of saengshik. Korean J. Food Sci. Technol. 36: 501-506 (2004)
  4. Korea Food and Drug Administration. accessed on Dec. 18, 2009.
  5. Lechner S. Mayr R. Francis KP. Bacillus weihenstephanensis sp. nov. is a new psychrotolerant species of the Bacillus cereus group. Int. J. Syst. Bacteriol. 48: 1373-1382 (1998)
  6. Nakamura LK. Bacillus pseudomycoides sp. nov. Int. J. Syst. Bacteriol. 48: 1031-1035 (1998)
  7. Granum PE. Bacillus cereus and its toxins. J. Appl. Bacteriol. 23(Suppl.): 61S-66S (1994)
  8. Hansen BM, Hoiby PE, Jensen GB, Hendriksen NB. The Bacillus cereus bceT enterotoxin sequence reappraised. FEMS Microbiol. Lett. 223: 21-24 (2003)
  9. Beecher DJ, Wong ACL. Tripartite hemolysin BL from Bacillus cereus. Hemolytic analysis of component interaction and a model for its characteristic paradoxical zone phenomenon. J. Biol. Chem. 272: 233-239 (1997)
  10. Schoeni JL, Wong AC. Heterogeneity observed in the components of hemolysin BL, an enterotoxin produced by Bacillus cereus. Int. J. Food Microbiol. 53: 159-167 (1999)
  11. Guinebretiere MH, Broussolle V, Nguyen-The C. Enterotoxigenic profiles of food-poisoning and food-born Bacillus cereus strains. J. Clin. Microbiol. 40: 3053-3056 (2002)
  12. Agata N, Mori M, Ohta M, Suwan S, Ohtani I, Isobe M. A novel dodecadepsipeptide, cereulide, isolated from Bacillus cereus causes vacuole formation in HEp-2 cells. FEMS Microbiol. Lett. 121: 31-34 (1994)
  13. Agata N, Ohta M, Yokoyama K. Production of Bacillus cereus emetic toxin (cereulide) in various foods. Int. J. Food Microbiol. 73: 23-27 (2002)
  14. Mikkola R, Saris NE, Grigoriev PA, Andersson MA, Salkinoja-Salonen MS. Ionophoretic properties and mitochondrial effects of cereulide: The emetic toxin of B. cereus. Eur. J. Biochem. 263: 112-117 (1999)
  15. Pirhonen T, Andersson MA, Jskelinen EL, Salkinoja-Salonen MS, Honkanen-Buzalski T, Johansson TM. Biochemical and toxic diversity of Bacillus cereus in a pasta and meat dish associated with a food-poisoning case. Food Microbiol. 22: 87-91 (2005)
  16. Agata N, Ohta M, Mori M. Production of an emetic toxin, cereulide, is associated with a specific class of Bacillus cereus. Curr. Microb. 33: 67-69 (1996)
  17. Turnbull PCB. Bacillus cereus toxins. pp. 397-448. In: Pharmacology of Bacterial Toxins. Dorner F, Drews J (eds). Pergamon Press, Oxford, England (1986)
  18. Jackson SG, Goodbrand RB, Ahmed R, Kasatiya S. Bacillus cereus and Bacillus thuringiensis isolated in a gastroenteritis outbreak investigation. Lett. Appl. Microbiol. 21: 103-105 (1995)
  19. Noguchi H. Development of Bacillus thuringiensis in Japen. pp. 283-291. In: Advanced engineered pesticides. Kim L. (eds). Marcel Dekker, New York, NY (1993)
  20. Perani M, Bishop AH, Vaid A. Prevalence of $\beta$-exotoxin, diarrhoeal toxin and specific $\delta$-endotoxin in natural isolates of Bacillus thuringiensis. FEMS Microbiol. Lett. 160: 55-60 (1998)
  21. Damgaard PH, Larsen HD, Hansen BM, Bresciani J, Jorgensen K. Enterotoxin-producing strains of Bacillus thuringiensis isolated from food. Lett. Appl. Microbiol. 23: 146-150 (1996)
  22. Prss BM, Dietrich R, Nibler B, Mrtlbauer E, Scherer S. The hemolytic enterotoxin HBL is broadly distributed among species of the Bacillus cereus group. Appl. Environ. Microb. 65: 5436-5442 (1999)
  23. Hwang JH. Biochemical characteristics and enterotoxin gene distribution of food-borne Bacillus cereus. MS thesis, Kyungwon University, Gyeonggi, Korea (2009)
  24. Yamada S, Ohashi E, Agata N, Venkateswaran K. Cloning and nucleotide sequence analysis of gyr B of Bacillus cereus, B. thuringiensis, B. mycoides, and B. anthracis and their application to the detection of B. cereus in rice. Appl. Environ. Microb. 65: 1483-1490 (1999)
  25. Kuo W-S, Chak K-F. Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA. Appl. Environ. Microbiol. 62: 1369-1377 (1996)
  26. Rowan NJ, Caldow G, Gemmell CG, Hunter IS. Production of diarrheal enterotoxins and other potential virulence factors by veterinary isolates of Bacillus species associated with nongastrointestinal infections. Appl. Environ. Microb. 69: 2372-2376 (2003)
  27. Celandroni EG, Salvetti SF, Barsotti C, Baggiani A, Senesi S. Identification and characterization of toxigenic Bacillus cereus isolates responsible for two food-poisoning outbreaks. FEMS Microbiol. Lett. 208: 129-134 (2002)
  28. Lund T, De Buyser ML, Granum PE. A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Mol. Microbiol. 38: 254-261 (2000)
  29. Kim YS, Oh BC, Shin DH. The extension of the shelf life of cooked rice by the treament with the plant extract and their volatile constituents. Food Sci. Biotechnol. 13: 519-522 (2004)
  30. Park SK, Cho YS, Shon MY, Seo KJ. Occurence and repression of off-odor in cooked rice during storage under low temperature warming condition of electric rice cooker. Korean J. Food Sci. Technol. 29: 919-924 (1997)
  31. Roh HJ, Shin YS, Lee KS, Shin MK. Antimicrobial activity of water extract of green tea against cooked rice putrefactive microorganism. Korean J. Food Sci. Technol. 28: 66-71 (1996)
  32. Oh MH, Cox JM. Development and application of a centrifuagation-plate method to study the biodiversity of Bacillus species in rice products. Food Control 21: 7-12 (2010)
  33. Lee MS, Chang DS. Distribution and physiological characteristic of Bacillus cereus in rice and rice products. Bull. Korean Fish. Soc. 13: 163-172 (1980)
  34. From C, Hormazabal V, Granum PE. Food poisoning associated with pumilacidin-producing Bacillus pumilus in rice. Int. J. Food Microbiol. 115: 319-324 (2007)
  35. Ankolekar C, Rahmati T, Labbe' RG. Detection of toxigenic Bacillus cereus and Bacillus thuringiensis spores in U.S. rice. Int. J. Food Microbiol. 128: 460-466 (2009)
  36. Rosenquist H, Smidt L, Anderson SR, Jensen GB, Wilcks A. Occurrence and significance of Bacillus cereus and Bacillus thuringiensis in ready-to-eat food. FEMS Microbiol. Lett. 250: 129-136 (2005)
  37. Cronin UP, Wilkinson MG. The growth, physiology and potential of Bacillus cereus in cooked rice during storage temperature abuse. Food Control 20: 822-282 (2009)
  38. Svensson B, Monthan A, Shaheen R, Ansesson MA, Salkinoja-Salonen M, Christiansson A. Occurence of emetic toxin producing Bacillus cereus in the dairy production chain. Iut. Dairy J. 16: 740-749 (2006)
  39. Carlin F, Fricker M, Pielaat A, Heisterkamp S, Shaheen R, Salonen MS, Svensson B, Nguyen-the C, Ehling-Schulz. Emetic toxin-producing strains of B.cereus show distinct characteristics within the Bacillus cereus group. Int. J. Food Microbiol. 109: 132-138 (2006)
  40. Rivera AMG, Granum PE, Priest FG. Common occurrence of enterotoxin genes and enterotoxicity in Bacillus thuringiensis. FEMS Microbiol. Lett. 190: 151-155 (2000)
  41. Ehling-Schulz M, Vukov N, Schulz A, Shaheen R, Andersson M, Mrtlbauer E, Scherer S. Identification and partial characterization of the nonribosomal peptide synthetase gene responsible for cereulide production in emetic Bacillus cereus. Appl. Environ. Microb. 71: 105-113 (2005)
  42. Apetroaie-Constantin C, Shaheen R, Andrup L, Smidt L, Rita H, Salkinja-Salonen. Environment driven cereulide production by emetic strains of Bacillus cereus. Int. J. Food Microbiol. 127: 60-67 (2008)