Comparison of NheA toxin production and doubling time between Bacillus cereus and Bacillus thuringiensis


In this study, we compared the toxin gene expression, NheA toxin production, doubling time, and viable cell number for several strains of the food poisoning bacteria Bacillus cereus and the microbial pesticide Bacillus thuringiensis. The two B. cereus and six B. thuringiensis strains evaluated were confirmed to possess and transcribe the nheABC, hblCDA, and cytK genes using polymerase chain reaction (PCR) and reverse-transcription PCR. NheA toxin production was compared based on the absorbance at 414 nm using a Tecra BDE-VIA kit. The NheA-specific production (absorbance/viable cell number) values indicated that the two B. thuringiensis var. kurstaki isolates from microbial pesticide produced the highest amount of toxin (0.66–0.95) than other B. thuringiensis (0.14–0.45) and the B. cereus strains (0.19–0.31). However, the B. thuringiensis strains had longer doubling time (20–26 min) than the B. cereus strains (18–19 min). Interestingly, two B. thuringiensis var. kurstaki isolates produced the highest amount of NheA toxin, and their doubling times (20–22 min) were close to those of the B. cereus strains tested.

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  1. 1.

    Mahler H, Pasi A, Kramer JM, Schulte P, Scoging AC, Bar W, Krähenbühl S (1997) Fulminant liver failure in association with the emetic toxin of Bacillus cereus. N Engl J Med 336:1142–1148

    CAS  Article  Google Scholar 

  2. 2.

    Dierick K, Coillie EV, Swiecicka I, Meyfroidt G, Devlieger H, Meulemans A, Hoedemaekers G, Fourie L, Heydrickx M, Mahillon J (2005) Fatal family outbreak of Bacillus cereus-associated food poisoning. J Clin Microbiol 43:4277–4279

    Article  Google Scholar 

  3. 3.

    Posfay-Barbe KM, Schrenzel J, Frey J, Studer R, Korff C, Belli DC, Parvex P, Rimensberger PC, Schappi MG (2008) Food poisoning as a cause of acute liver failure. Pediatr Infect Dis J 27:846–847

    Article  Google Scholar 

  4. 4.

    Granum PE, Lund T (1997) Bacillus cereus and its food poisoning toxins. FEMS Microbiol Lett 157:223–228

    CAS  Article  Google Scholar 

  5. 5.

    Lund T, Granum PE (1997) Comparison of biological effect of two different enterotoxin complexes isolated from three different strains of Bacillus cereus. Microbiol 143:3329–3336

    CAS  Article  Google Scholar 

  6. 6.

    Beecher DJ, Wong ACL (1997) 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

    CAS  Article  Google Scholar 

  7. 7.

    Beecher DJ, Wong ACL (1994) Improved purification and characterization of hemolysin BL—a hemolytic dermonecrotic vascular permeability factor from Bacillus cereus. Infect Immun 62:980–986

    CAS  Google Scholar 

  8. 8.

    Heinrichs JH, Beecher DJ, Macmillan JD, Zilinskas BA (1993) Molecular cloning and characterization of the hblA gene encoding the B component of hemolysin BL from Bacillus cereus. J Bacteriol 175:6760–6766

    CAS  Article  Google Scholar 

  9. 9.

    Ryan PA, Macmillan JD, Zilinskas BA (1997) Molecular cloning and characterization of the genes encoding the L1 and L2 component of hemolysin BL from Bacillus cereus. J Bacteriol 179:2551–2556

    CAS  Article  Google Scholar 

  10. 10.

    Granum PE, O’sullivan K, Lund T (1999) The sequence of the non-hemolytic enterotoxin operon from Bacillus cereus. FEMS Microbiol Lett 177:225–229

    CAS  Article  Google Scholar 

  11. 11.

    Ngamwongsatit P, Buasri W, Pianariyanon P, Pulsrikarn C, Ohba M, Assavanig A, Panbangred P (2008) Broad distribution of enterotoxin genes (hblCDA, nheABC, cytK, and entFM) among Bacillus thuringiensis and Bacillus cereus as shown by novel primers. Int J Food Micorbiol 121:352–356

    CAS  Article  Google Scholar 

  12. 12.

    Hofre H, Whiteley HR (1989) Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Mol Biol Rev 53:242–255

    Google Scholar 

  13. 13.

    Crickmore N, Zeigler DR, Feitelson J, Schnepe E, Rie VJ, Lereclus D, Baum J, Dean DH (1998) Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol Mol Biol Rev 62:807–813

    CAS  Google Scholar 

  14. 14.

    Bravo A, Likitvivatanavong S, Gill SS, Soberon M (2011) Bacillus thuringiensis: a story of a successful bioinsecticide. Insect Biochem Mol 41:423–431

    CAS  Article  Google Scholar 

  15. 15.

    Ohba M, Yu YM, Aizawa K (1988) Occurrence of non-insecticidal Bacillus thuringiensis flagellar serotype 14 in the soil of Japan. Syst Appl Microbiol 11:85–89

    CAS  Article  Google Scholar 

  16. 16.

    Tamez-guerra P, Iracheta MM, Pereyra-Alferez B, Galán-Wong LJ, Gomez-Flores R, Tamez-guerra RS, Rodriquez-Padilla C (2004) Characterization of Mexican Bacillus thuringiensis strains toxic for lepidopteran and coleopteran larvae. J Invertebr Pathol 86:7–18

    Article  Google Scholar 

  17. 17.

    Feitelson JS, Payne J, Kim L (1992) Bacillus thuringiensis: insect and beyond. Nat Biotechnol 10:271–275

    Article  Google Scholar 

  18. 18.

    Olson S, Ranade A, Kurkjy N, Pang K, Hazekamp C (2013) Green dreams or growth opportunities: assessing the market potential for “greener” agricultural technologies. Lux Research Inc, Boston

    Google Scholar 

  19. 19.

    Fisher R, Rosner L (1959) Toxicology of the microbial insecticide, thuricide. J Agric Food Chem 7:686–688

    CAS  Article  Google Scholar 

  20. 20.

    Elliott LJ, Sokolow R, Heumann M, Elefant SL (1988) An exposure characterization of a large scale application of a biological insecticide, Bacillus thuringiensis. Appl Ind Hyg 3:119–122

    CAS  Article  Google Scholar 

  21. 21.

    Noble MA, Riben PD, Cook GJ (1992) Microbiological and epidemiological surveillance programme to monitor the health effects of Foray 48B BTK spray. Ministry of Forests, Cambridge

    Google Scholar 

  22. 22.

    Siegel JP, Shadduck JA (1987) Safety of the entomopathogen Bacillus thuringiensis var. israelensis for mammals. J Econ Entomol 80:717–723

    CAS  Article  Google Scholar 

  23. 23.

    Hernandez E, Ramisse F, Cruel T, Vagueresse RL, Cavallo JD (1999) Bacillus thuringiensis serotype H34 isolated from human and insecticidal strains serotypes 3a3b and H14 can lead to death of immunocompetent mice after pulmonary infection. FEMS Immunol Med Microbiol 24:43–47

    CAS  Article  Google Scholar 

  24. 24.

    Ignoffo CM (1973) Effects of entomopathogens on vertebrates. Ann N Y Acad Sci 217:141–164

    CAS  Article  Google Scholar 

  25. 25.

    Bishop AH, Johnson C, Perani M (1999) The safety of Bacillus thuringiensis to mammals investigated by oral and subcutaneous dosage. World J Microbiol Biotechnol 15:375–380

    Article  Google Scholar 

  26. 26.

    Tsai SF, Liao JW, Wang SC (1997) Clearance and effects of intratracheal instillation to spores of Bacillus thuringiensis or Metarhizium anisopliae in rats. J Chin Soc Vet Sci 23:515–522

    Google Scholar 

  27. 27.

    Siegel JP, Shadduck JA (1990) Clearance of Bacillus sphaericus and Bacillus thuringiensis ssp. israelensis from mammals. J Econ Entomol 83:347–355

    CAS  Article  Google Scholar 

  28. 28.

    Hadley WM, Burchiel SW, Mcdowell TD, Thilsted JP, Hibbs CM, Whorton JA, Day PW, Friedman MB, Stoll RE (1987) Five-month oral (diet) toxicity/infectivity study of Bacillus thuringiensis insecticides in sheep. Fund Appl Toxicol 8:236–242

    CAS  Article  Google Scholar 

  29. 29.

    Amorim GVD, Whittome B, Shore B, Levin DB (2001) Identification of Bacillus thuringiensis subsp. Kurstaki strain HD1-like bacteria from environmental and human samples after aerial spraying of Victoria, British Columbia, Canada, with Foray 48B. Appl Environ Microbiol 67:1035–1043

    Article  Google Scholar 

  30. 30.

    Jensen GB, Larsen P, Jacobsen BL, Madsen B, Wilcks A, Smidt L, Andrup L (2002) Isolation and characterization of Bacillus cereus-like bacteria from fecal samples from greenhouse workers who are using Bacillus thuringiensis-based insecticides. Int Arch Occup Environ Health 75:191–196

    CAS  Article  Google Scholar 

  31. 31.

    Damgaard PH, Larsen HD, Hansen BM, Bresciani J, Jorgensen K (1996) Enterotoxin-producing strains of Bacillus thuringiensis isolated from food. Lett Appl Microbiol 23:146–150

    CAS  Article  Google Scholar 

  32. 32.

    Damgaard PH, Granum PE, Bresciani J, Torregrossa MV, Eilenber J, Valentino L (1997) Characterization of Bacillus thuringiensis isolated from infections in burn wounds. FEMS Immunol Med Microbiol 18:47–53

    CAS  Article  Google Scholar 

  33. 33.

    Perani M, Bishop AH, Vaid A (1998) Prevalence of β-exotoxin, diarrhoeal toxin and specific δ-endotoxin in natural isolates of Bacillus thuringiensis. FEMS Microbiol Lett 160:55–60

    CAS  Google Scholar 

  34. 34.

    Rivera AMG, Granum PE, Priest FG (2000) Common occurrence of enterotoxin genes and enterotoxicity in Bacillus thuringiensis. FEMS Microbiol Lett 190:151–155

    Article  Google Scholar 

  35. 35.

    Samples JR, Buettner H (1983) Ocular infection caused by a biological insecticide. J Infect Dis 148:614

    CAS  Article  Google Scholar 

  36. 36.

    Hernandez E, Ramisse F, Ducoureau JP, Cruel T, Cavallo JD (1998) Bacillus thuringiensis subsp. Konkukian (serotype H34) superinfection: case report and experimental evidence of pathogenicity in immunosuppressed mice. J Clin Microbiol 36:2138–2139

    CAS  Google Scholar 

  37. 37.

    Zhou G, Liu H, He J, Yuana Y, Yuan Z (2008) The occurrence of Bacillus cereus, B. thuringiensis and B. mycoides in Chinese pasteurized full fat milk. Int J Food Microbiol 121:195–200

    CAS  Article  Google Scholar 

  38. 38.

    Molva C, Sudagidanb M, Okuklua B (2009) Extracellular enzyme production and enterotoxigenic gene profiles of Bacillus cereus and Bacillus thuringiensis strains isolated from cheese in Turkey. Food Control 20:829–834

    CAS  Article  Google Scholar 

  39. 39.

    Tallent SM, Hait JM, Bennett RW (2015) Analysis of Bacillus cereus toxicity using PCR, ELISA and a lateral flow device. J Appl Microbiol 118:1068–1075

    CAS  Article  Google Scholar 

  40. 40.

    Chang YH, Shangkuan YH, Lin HC, Liu HW (2003) PCR Assay of the groEL gene for detection and differentiation of Bacillus cereus group cells. Appl Environ Microbiol 69:4502–4510

    CAS  Article  Google Scholar 

  41. 41.

    Thammasittirong A, Attathom T (2008) PCR-based method for the detection of cry genes in local isolates of Bacillus thuringiensis from Thailand. J Invertebr Pathol 98:121–126

    CAS  Article  Google Scholar 

  42. 42.

    Sharif FA, Alaeddinoglu NG (1988) A rapid and simple method for staining of the crystal protein of Bacillus thuringiensis. J Ind Microbiol 3:227–229

    Article  Google Scholar 

  43. 43.

    Siegel JP (2001) The mammalian safety of Bacillus thuringiensis-based insecticides. J Invertebr Pathol 77:13–21

    CAS  Article  Google Scholar 

  44. 44.

    Buchanan RL, Schultz FJ (1994) Comparison of the Tecra VIA kit, oxoid BCET-RPLA kit and CHO cell culture assay for the detection of Bacillus cereus diarrhoeal enterotoxin. Lett Appl Microbiol 19:353–356

    CAS  Article  Google Scholar 

  45. 45.

    Day TL, Tatani SR, Notermans S, Bennett RW (1994) A comparison of ELISA and RPLA for detection of Bacillus cereus diarrhoeal enterotoxin. J Appl Microbiol 77:9–13

    CAS  Google Scholar 

  46. 46.

    Arnesen LPS, Fagerlund A, Granum PE (2008) From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol Rev 32:579–606

    Article  Google Scholar 

  47. 47.

    Seo JH, Bailey JE (1985) Effects of recombinant plasmid content on growth properties and cloned gene product formation in Escherichia coli. Biotechnol Bioeng 27:1668–1674

    CAS  Article  Google Scholar 

  48. 48.

    Rasko DA, Altherr MR, Han CS, Ravel J (2005) Genomics of the Bacillus cereus group of organisms. FEMS Microbiol Rev 29:303–329

    CAS  Google Scholar 

  49. 49.

    Yang IC, Shih DYC, Huang TP, Huang YP, Wang JY, Pan TM (2005) Establishment of a novel multiplex PCR assay and detection of toxigenic strains of the species in the Bacillus cereus group. J Food Protect 68:2123–2130

    CAS  Article  Google Scholar 

  50. 50.

    Lim JS, Kim MR, Kim W, Hong KW (2011) Detection and differentiation of non-emetic and emetic Bacillus cereus strains in food by real-time PCR. J Korean Soc Appl Biol Chem 54:105–111

    Article  Google Scholar 

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Correspondence to Kwang Won Hong.

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Choi, H.J., Kang, S.J. & Hong, K.W. Comparison of NheA toxin production and doubling time between Bacillus cereus and Bacillus thuringiensis . Appl Biol Chem 60, 545–551 (2017).

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  • B. cereus
  • B. thuringiensis
  • Doubling time
  • Microbial pesticide
  • NheA toxin