Digestive Diseases and Sciences

, Volume 53, Issue 4, pp 954–963 | Cite as

The Safety of Two Bacillus Probiotic Strains for Human Use

  • Iryna B. Sorokulova
  • Iryna V. Pinchuk
  • Muriel Denayrolles
  • Irina G. Osipova
  • Jen M. Huang
  • Simon M. Cutting
  • Maria C. Urdaci
Original Paper


Probiotics based on Bacillus strains have been increasingly proposed for prophylactic and therapeutic use against several gastro-intestinal diseases. We studied safety for two Bacillus strains included in a popular East European probiotic. Bacillus subtilis strain that was sensitive to all antibiotics listed by the European Food Safety Authority. Bacillus licheniformis strain was resistant to chloramphenicol and clindamycin. Both were non-hemolytic and did not produce Hbl or Nhe enterotoxins. No bceT and cytK toxin genes were found. Study of acute toxicity in BALB/c mice demonstrated no treatment-related deaths. The oral LD50 for both strains was more than 2 × 1011 CFU. Chronic toxicity studies were performed on mice, rabbits, and pigs and showed no signs of toxicity or histological changes in either organs or tissues. We demonstrated that while certain risks may exist for the B. licheniformis strain considering antibiotic resistance, B. subtilis strain may be considered as non-pathogenic and safe for human consumption.


Probiotics Bacillus Safety Toxicity Antimicrobials resistance 



The authors thank Anne-Marie ELIE for technical assistance.


  1. 1.
    Hammerman C, Bin-Nun A, Kaplan M (2006) Safety of probiotics: comparison of two popular strains. BMJ 333:1006–1008PubMedCrossRefGoogle Scholar
  2. 2.
    Saarela M, Mogensen G, Fonden R, Matto J, Mattila-Sandholm T (2000) Probiotic bacteria: safety, functional and technological properties. J Biotechnol 84:197–215PubMedCrossRefGoogle Scholar
  3. 3.
    Hong HA, Duc LH, Cutting SM (2005) The use of bacterial spore formers as probiotics. FEMS Microbiol Rev 29:813–835PubMedCrossRefGoogle Scholar
  4. 4.
    Mazza P (1994) The use of Bacillus subtilis as an antidiarrhoeal microorganism. Boll Chim Farm 133:3–118PubMedGoogle Scholar
  5. 5.
    Rolfe RD (2000) The role of probiotic cultures in the control of gastrointestinal health. J Nutr 130:396S–402SPubMedGoogle Scholar
  6. 6.
    Rowland I (1999) Probiotics and benefits to human health—the evidence in favour. Environ Microbiol 1:375–376PubMedCrossRefGoogle Scholar
  7. 7.
    Urdaci MC, Pinchuk I (2004) Antimicrobial activity of Bacillus probiotics In: Ricca E, Henriques AO, Cutting SM (eds) Bacterial spore formers: probiotics and emerging applications. Horizon Bioscience, Norfolk, UK, pp 171–182Google Scholar
  8. 8.
    Logan NA (2004) Safety of aerobic endospore-forming bacteria. In: Ricca E, Henriques AO, Cutting SM (eds) Bacterial spore formers: probiotics and emerging applications. Horizon Bioscience, Norfolk, UK, pp 93–106Google Scholar
  9. 9.
    Workowski KA, Flaherty JP (1992) Systemic Bacillus species infection mimicking listeriosis of pregnancy. Clin Infect Dis 14:694–966PubMedGoogle Scholar
  10. 10.
    Wu YJ, Hong TC, Hou CJ, Chou YS, Tsai CH, Yang DI (1999) Bacillus popilliae endocarditis with prolonged complete heart block. Am J Med Sci 317:263–265PubMedCrossRefGoogle Scholar
  11. 11.
    Drobniewski FA (1993) Bacillus cereus and related species. Clin Microbiol Rev 6:324–338PubMedGoogle Scholar
  12. 12.
    Salkinoja-Salonen MS, Vuorio R, Andersson MA, Kampfer P, Andersson MC, Honkanen-Buzalski T, Scoging AC (1999) Toxigenic strains of Bacillus licheniformis related to food poisoning. Appl Environ Microbiol 65:4637–4645PubMedGoogle Scholar
  13. 13.
    Duc LH, Hong HA, Barbosa TM, Henriques AO, Cutting SM (2004) Characterization of Bacillus probiotics available for human use. Appl Environ Microbiol 70:2161–2171CrossRefGoogle Scholar
  14. 14.
    From C, Pukall R, Schumann P, Hormazabal V, Granum PE (2005) Toxin-producing ability among Bacillus spp. outside the Bacillus cereus group. Appl Environ Microbiol 71:1178–1183PubMedCrossRefGoogle Scholar
  15. 15.
    Phelps RJ, McKillip JL (2002) Enterotoxin production in natural isolates of Bacillaceae outside the Bacillus cereus group. Appl Environ Microbiol 68:3147–3151PubMedCrossRefGoogle Scholar
  16. 16.
    Rowan NJ, Caldow G, Gemmell CG, Hunter IS (2003) Production of diarrheal enterotoxins and other potential virulence factors by veterinary isolates of bacillus species associated with nongastrointestinal infections. Appl Environ Microbiol 69:2372–2376PubMedCrossRefGoogle Scholar
  17. 17.
    FAO/WHO (2002) Joint FAO/WHO (Food and Agriculture Organization/World Health Organization) working group report on drafting guidelines for the evaluation of probiotics in food. London, Ontario, CanadaGoogle Scholar
  18. 18.
    Sanders ME (2003) Probiotics: considerations for human health. Nutr Rev 61:91–99PubMedCrossRefGoogle Scholar
  19. 19.
    European Food Safety Authority (2005) Opinion of the Scientific Committee on a request from EFSA related to a generic approach to the safety assessment by EFSA of microorganisms used in food/feed and the production of food/feed additives. EFSA J 226:1–12Google Scholar
  20. 20.
    Gracheva NM, Gavrilov AF, Solov’eva AI, Smirnov VV, Sorokulova IB, Reznik SR, Chudnovskaia NV (1996) The efficacy of the new bacterial preparation biosporin in treating acute intestinal infections. Zh Mikrobiol Epidemiol Immunobiol 1:75–77PubMedGoogle Scholar
  21. 21.
    Smirnov VV, Reznik SR, V’iunitskaia VA, Sorokulova IB, Samgorodskaia NV, Tofan AV (1993) The current concepts of the mechanisms of the therapeutic-prophylactic action of probiotics from bacteria in the genus Bacillus. Mikrobiol Z 55:92–112PubMedGoogle Scholar
  22. 22.
    Sorokulova IB (1996) Outlook for using bacteria of the genus Bacillus for the design of new biopreparations. Antibiot Khimioter 41:13–15PubMedGoogle Scholar
  23. 23.
    Sorokulova IB (1998) The safety and reactogenicity of the new probiotic subalin for volunteers. Mikrobiol Z 60:43–46PubMedGoogle Scholar
  24. 24.
    Youngman P, Perkins J, Losick R (1984) Construction of a cloning site near one end of Tn917 into which foreign DNA may be inserted without affecting transposition in Bacillus subtilis or expression of the transposon-borne erm gene. Plasmid 12:1–9PubMedCrossRefGoogle Scholar
  25. 25.
    Hoa NT, Baccigalupi L, Huxham A, Smertenko A, Van PH, Ammendola S, Ricca E, Cutting SM (2000) Characterization of Bacillus species used for oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders. Appl Environ Microbiol 66:5241–5247PubMedCrossRefGoogle Scholar
  26. 26.
    Sneath PHA (1986) Endospore-forming gram-positive rods and cocci. In: Mair NS, Sharpe ME, Holt JG (eds) Bergey’s manual of systematic bacteriology 2. Lippincott Williams &Wilkins, Baltimore, pp 1104–1207Google Scholar
  27. 27.
    Reva ON, Sorokulova IB, Smirnov VV (2001) Simplified technique for identification of the aerobic spore-forming bacteria by phenotype. Int J Syst Evol Microbiol 51:1361–1371PubMedGoogle Scholar
  28. 28.
    Hyronimus B, Le Marrec C, Urdaci MC (1998) Coagulin, a bacteriocin-like inhibitory substance produced by Bacillus coagulans I4. J Appl Microbiol. 85:42–50PubMedCrossRefGoogle Scholar
  29. 29.
    Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703PubMedGoogle Scholar
  30. 30.
    Pinchuk IV, Bressollier P, Sorokulova IB, Verneuil B, Urdaci MC (2002) Amicoumacin antibiotic production and genetic diversity of Bacillus subtilis strains isolated from different habitats. Res Microbiol 153:269–276PubMedCrossRefGoogle Scholar
  31. 31.
    Voskuil MI, Chambliss GH (1993) Rapid isolation and sequencing of purified plasmid DNA from Bacillus subtilis. Appl Environ Microbiol 59:1138–1142PubMedGoogle Scholar
  32. 32.
    National Committee for Clinical Laboratory Standards (NCCLS) (1997) Performance standards for antimicrobial disk susceptibility tests, 6th edn. Approved standard M2-A5. National Committee for Clinical Laboratory standards, Wayne, PAGoogle Scholar
  33. 33.
    Guinebretiere MH, Broussolle V, Nguyen-The C (2002) Enterotoxigenic profiles of food-poisoning and food-borne Bacillus cereus strains. J Clin Microbiol 40:3053–3056PubMedCrossRefGoogle Scholar
  34. 34.
    Tallon R, Arias S, Bressollier P, Urdaci MC (2007) Strain- and matrix-dependent adhesion of Lactobacillus plantarum is mediated by proteinaceous bacterial compounds. J Appl Microbiol 102:442–451PubMedCrossRefGoogle Scholar
  35. 35.
    Pinchuk I, Starcher BC, Livingston B, Tvninnereim A, Wu S, Appella E, Sidney J, Sette A, Wizel B (2005) A CD8+ T cell heptaepitope minigene vaccine induces protective immunity against Chlamydia pneumoniae. J Immunol 174:5729–5739PubMedGoogle Scholar
  36. 36.
    Duc LH, Dong TC, Logan NA, Sutherland AD, Taylor J, Cutting SM (2005) Cases of emesis associated with bacterial contamination of an infant breakfast cereal product. Int J Food Microbiol 102:245–251CrossRefGoogle Scholar
  37. 37.
    Scientific Committee on Animal Nutrition (2000) SCAN opinion on the safety of use of Bacillus species in animal nutrition
  38. 38.
    Green DH, Wakeley PR, Page A, Barnes A, Baccigalupi L, Ricca E, Cutting SM (1999) Characterization of two Bacillus probiotics. Appl Environ Microbiol 65:4288–4291PubMedGoogle Scholar
  39. 39.
    Senesi S, Celandroni F, Tavanti A, Ghelardi E (2001) Molecular characterization and identification of Bacillus clausii strains marketed for use in oral bacteriotherapy. Appl.Environ.Microbiol 67:834–839PubMedCrossRefGoogle Scholar
  40. 40.
    Silley P (2006) Do bacteria need to be regulated? J Appl Microbiol 101:607–615PubMedCrossRefGoogle Scholar
  41. 41.
    von Wright A (2005) Regulating the safety of probiotics—the European approach. Curr Pharm Des 11:17–23CrossRefGoogle Scholar
  42. 42.
    Ivanova EP, Vysotskii MV, Svetashev VI, Nedashkovskaya OI, Gorshkova NM, Mikhailov VV, Yumoto N, Shigeri Y, Taguchi T, Yoshikawa S (1999) Characterization of Bacillus strains of marine origin. Int Microbiol 2:267–271PubMedGoogle Scholar
  43. 43.
    Reva ON, Vyunitskaya VA, Reznik SR, Kozachko IA, Smirnov VV (1995) Antibiotic susceptibility as a taxonomic characteristic of the genus Bacillus. Int J Syst Bacteriol 45:409–411PubMedCrossRefGoogle Scholar
  44. 44.
    Pinchuk IV, Bressollier P, Verneuil B, Fenet B, Sorokulova IB, Megraud F, Urdaci MC (2001) In vitro anti-Helicobacter pylori activity of the probiotic strain Bacillus subtilis 3 is due to secretion of antibiotics. Antimicrob Agents Chemother 45:3156–3161PubMedCrossRefGoogle Scholar
  45. 45.
    Donohue DC (2006) Safety of probiotics. Asia Pac J Clin Nutr 15:563–569PubMedGoogle Scholar
  46. 46.
    Zhou JS, Shu Q, Rutherfurd KJ, Prasad J, Birtles MJ, Gopal PK, Gill HS (2000) Safety assessment of potential probiotic lactic acid bacterial strains Lactobacillus rhamnosus HN001, Lb. acidophilus HN017, and Bifidobacterium lactis HN019 in BALB/c mice. Int J Food Microbiol 56:87–96PubMedCrossRefGoogle Scholar
  47. 47.
    Osipova IG, Sorokulova IB, Tereshkina NV, Grigor’eva LV (1998) Safety of bacteria of the genus Bacillus, forming the base of some probiotics. Zh Mikrobiol Epidemiol Immunobiol 6:68–70PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Iryna B. Sorokulova
    • 1
  • Iryna V. Pinchuk
    • 1
  • Muriel Denayrolles
    • 1
  • Irina G. Osipova
    • 2
  • Jen M. Huang
    • 3
  • Simon M. Cutting
    • 3
  • Maria C. Urdaci
    • 1
  1. 1.Laboratoire de Microbiologie, UMR5248ENITA de BordeauxGradignanFrance
  2. 2.Tarasevich State Research Institute for Standardization and Control of Medical Biological PreparationsMoscowRussia
  3. 3.School of Biological Sciences, Royal HollowayUniversity of LondonEghamUK

Personalised recommendations