Appl Environ Microbiol 70(4): 2161-2171

Characterization of <em>Bacillus</em> Probiotics Available for Human Use

School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 OEX, United Kingdom,^{Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2781-901 Oeiras Codex, Portugal}²

^{Corresponding author. Mailing address: School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 OEX, United Kingdom. Phone: 44 1784 443760. Fax: 44 1784 434326. E-mail:}ku.ca.luhr@gnittuc.s.

Received 2003 Jul 24; Accepted 2003 Dec 20.

Abstract

Bacillus species (Bacillus cereus, Bacillus clausii, Bacillus pumilus) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties. Three B. cereus strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize. Spores of one B. cereus strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids. Spores of all strains were immunogenic when they were given orally to mice, but the B. pumilus strain was found to generate particularly high anti-spore immunoglobulin G titers. Spores of B. pumilus and of a laboratory strain of B. subtilis were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of B. pumilus and B. subtilis induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon. The B. pumilus strain and one B. cereus strain (B. cereus var. vietnami) were found to produce a bacteriocin-like activity against other Bacillus species. The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect. However, the three B. cereus strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use.

Abstract

Probiotics are live microbial feed supplements which beneficially affect the host animal by improving its intestinal microbial balance (10, 11). The potential benefits that are claimed include improved nutrition and growth and prevention of various gastrointestinal disorders. Probiotic-containing products are available for human nutrition, as animal feed supplements, and also for aquaculture (35, 36, 41, 43). In some countries probiotics are taken as prophylactic agents (for example, to prevent childhood diarrhea), while in southeast Asia they are also used as therapeutic agents (25). Products containing endospores of members of the genus Bacillus (in single doses of up to 10^{spores/g or 10}^{spores/ml) are used commercially as probiotics, and they offer some advantages over the more common}Lactobacillus products in that they can be stored indefinitely in a desiccated form (25). Originally, many commercial products were sold as products that carry Bacillus subtilis spores, but recent studies have shown that most products are mislabeled and carry other Bacillus species, including Bacillus clausii, Bacillus pumilus, and a variety of Bacillus cereus strains (13, 17). Product mislabeling raises a number of concerns about consumer confidence (15), as well as attendant safety issues, since some of the organisms found were strains of B. cereus, which is a major cause of gastrointestinal infections (12).

Continued ingestion of large quantities of Bacillus spores raises the question of what happens to the spores in the gastrointestinal tract (GIT). While no evidence of colonization has been found, it is possible that a spore can interact with the gut-associated lymphoid tissue (GALT). Recent studies have shown that orally ingested B. subtilis spores are immunogenic and can disseminate to the Peyer's patches and mesenteric lymph nodes (MLN) (5, 6). Additional work has provided compelling evidence that ingested B. subtilis spores can germinate in the small intestine. This conclusion is based on three findings. First, when mice are given an oral inoculum, more spores are excreted than are ingested (18). Second, vegetatively expressed mRNA is detected in the GIT by reverse transcription (RT)-PCR following administration of spores to mice (2). Finally, systemic immunoglobulin G (IgG) responses are generated against vegetative B. subtilis following administration of suspensions carrying only spores to mice (5). Together, these studies show that spores may not be transient passengers in the gut or that if they are, they may still have an intimate interaction with the host cells or microflora that can enhance their potential probiotic effect.

The following three basic mechanisms have been proposed for how orally ingested nonindigenous bacteria can have a probiotic effect in a host: (i) immunomodulation (that is, stimulation of the GALT) (e.g., induction of cytokines), (ii) competitive exclusion of gastrointestinal pathogens (e.g., competition for adhesion sites), and (iii) secretion of antimicrobial compounds which suppress the growth of harmful bacteria (10). Few studies have demonstrated a direct probiotic effect of Bacillus spores, but preliminary studies with poultry have provided evidence that there is competitive exclusion of Escherichia coli 078:K80 by B. subtilis (24) and a number of studies have demonstrated that Vibrio harveyi in shrimp is suppressed by various Bacillus spore formers (34, 42). A recent study has described the characterization of an antibiotic produced by the B. subtilis strain (B. subtilis 3) found in the commercial product Biosporin, which has been shown to inhibit growth of Helicobacter pylori (31).

In this study we examined five commercially available Bacillus probiotic strains whose inoculum is in the spore form. These strains were Bactisubtil (B. cereus IP 5832) (17), Enterogermina (B. clausii) (13, 17, 39), Biosubtyl Nha Trang (referred to here as Biosubtyl^{; a strain of}B. pumilus) (13, 17), Biosubtyl Da Lat (referred to here as Biosubtyl^{; a}B. cereus strain) (17), and Subtyl (a strain similar to B. cereus spp. and designated B. cereus var. vietnami) (17). We looked for evidence of colonization and immune stimulation, and we determined potential pathogenic traits of the B. cereus products. Our results provide some interesting insights into a potential probiotic mechanism, and they also raise further concerns over the potential danger of using poorly characterized strains.

Acknowledgments

We thank Cláudia Serra for help with the bacteriocin-like inhibitory substance assays.

This work was supported by a grant from The Wellcome Trust to S.M.C. and by EU 5th Framework grant QLK5-CT-2001-01729 to S.M.C. and A.O.H.

Acknowledgments

REFERENCES

References

1. Andersson, A., P. E. Granum, and U. Ronner. 1998. The adhesion of Bacillus cereus spores to epithelial cells might be an additional virulence mechanism. Int. J. Food Microbiol.39:93-99. [[PubMed]
2. Casula, G., and S. M. Cutting. 2002. Bacillus probiotics: spore germination in the gastrointestinal tract. Appl. Environ. Microbiol.68:2344-2352.
3. de Simone, C., R. Veseley, B. Salvadori, and E. Jirillo. 1993. The role of probiotics in modulation of the immune system in man and animals. Int. J. Immunother.9:23-28. [PubMed]
4. Dornand, J., A. Gross, V. Lafont, J. Liautard, J. Oliaro, and J.-P. Liautard. 2002. The innate immune response against Brucella in humans. Vet. Microbiol.90:383-394. [[PubMed]
5. Duc, L. H., H. A. Hong, and S. M. Cutting. 2003. Germination of the spore in the gastrointestinal tract provides a novel route for heterologous antigen presentation. Vaccine21:4215-4224. [[PubMed]
6. Duc, L. H., H. A. Hong, N. Fairweather, E. Ricca, and S. M. Cutting. 2003. Bacterial spores as vaccine vehicles. Infect. Immun.71:2810-2818.
7. Erdile, L. F., D. A. Smith, and D. Berd. 2001. Whole cell ELISA for detection of tumor antigen expression in tumor samples. J. Immunol. Methods258:47-53. [[PubMed]
8. Faille, C., J. Membre, M. Kubaczka, and F. Gavini. 2002. Altered ability of Bacillus cereus spores to grow under unfavorable conditions (presence of nisin, low temperature, acidic pH, presence of NaCl) following heat treatment during sporulation. J. Food Prot.65:1930-1936. [[PubMed]
9. Faye, T., D. A. Brede, T. Langsrud, I. F. Nes, and H. Holo. 2002. An antimicrobial peptide is produced by extracellular processing of a protein from Propionibacterium jensenii.J. Bacteriol.184:3649-3656.
10. Fuller, R. 1991. Probiotics in human medicine. Gut32:439-442.
11. Fuller, R. 1989. Probiotics in man and animals. J. Appl. Bacteriol.66:365-378. [[PubMed]
12. Granum, P. E., and T. Lund. 1997. Bacillus cereus and its food poisoning toxins. FEMS Microbiol. Lett.157:223-228. [[PubMed]
13. Green, D. H., P. R. Wakeley, A. Page, A. Barnes, L. Baccigalupi, E. Ricca, and S. M. Cutting. 1999. Characterization of two Bacillus probiotics. Appl. Environ. Microbiol.65:4288-4291.
14. Guinebretiere, M.-H., V. Broussolle, and C. Nguyen-The. 2002. Enterotoxigenic profiles of food-poisoning and food-borne Bacillus cereus strains. J. Clin. Microbiol.40:3053-3056.
15. Hamilton-Miller, J. M. T., S. Shah, and J. T. Winkler. 1999. Public health issues arising from microbiological and labelling quality of foods and supplements containing probiotic microorganisms. Public Health Nutr.2:223-229. [[PubMed]
16. Henriques, A. O., and C. P. Moran. 2000. Structure and assembly of the bacterial endospore coat. Methods Compan. Methods Enzymol.20:95-110. [[PubMed]
17. Hoa, N. T., L. Baccigalupi, A. Huxham, A. Smertenko, P. H. Van, S. Ammendola, E. Ricca, and A. S. Cutting. 2000. Characterization of Bacillus species used for oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders. Appl. Environ. Microbiol.66:5241-5247.
18. Hoa, T. T., L. H. Duc, R. Isticato, L. Baccigalupi, E. Ricca, P. H. Van, and S. M. Cutting. 2001. Fate and dissemination of Bacillus subtilis spores in a murine model. Appl. Environ. Microbiol.67:3819-3823.
19. Isolauri, E., J. Joensuu, H. Suomalainen, M. Luomala, and T. Vesikari. 1995. Improved immunogenicity of oral DxRRV reassortant rotavirus vaccine by Lactobacillus casei GG. Vaccine13:310-312. [[PubMed]
20. Jeong, H.-S., S.-K. Yoo, and E.-J. Kim. 2001. Cell surface display of salmobin, a thrombin-like enzyme from Agkistrodon halys venom on Escherichia coli using ice nucleation protein. Enzyme Microb. Technol.28:155-160. [[PubMed]
21. Jia, Y. H., L. P. Li, Q. M. Hou, and S. H. Pan. 2002. An Agrobacterium gene involved in tumorigenesis encodes an outer membrane protein exposed on the bacterial cell surface. Gene284:113-124. [[PubMed]
22. Keynan, A., and ZEvenchik. 1969. Activation, p. 359-396. In G. W. Gould and A. Hurst (ed.), The bacterial spore. Academic Press, London, United Kingdom.[Google Scholar]
23. Kotiranta, A., K. Lounatmaa, and M. Haapasalo. 2000. Epidemiology and pathogenesis of Bacillus cereus infections. Microbes Infect.2:189-198. [[PubMed]
24. La Ragione, R. M., G. Casula, S. M. Cutting, and S. M. Woodward. 2001. Bacillus subtilis spores competitively exclude Escherichia coli 070:K80 in poultry. Vet. Microbiol.79:133-142. [[PubMed]
25. Mazza, P. 1994. The use of Bacillus subtilis as an antidiarrhoeal microorganism. Boll. Chim. Farm.133:3-18. [[PubMed]
26. Melby, P., F. J. Andrade-Narvaez, B. J. Darnell, G. Valencia-Pacheco, V. V. Tryon, and A. Palomo-Cetina. 1994. Increased expression of proinflammatory cytokines in chronic lesions of human cutaneous leishmaniasis. Infect. Immun.62:837-842.
27. Moore, J. C., P. J. Muckett, and M. J. G. Hughes. 2001. Age-dependent presence of antibodies in rat dams, capable of conferring protection against group B Streptococcus infection in neonates. FEMS Microbiol. Lett.202:125-127. [[PubMed]
28. Nicholson, W. L., and P. Setlow. 1990. Sporulation, germination and outgrowth, p. 391-450. In C. R. Harwood and S. M. Cutting (ed.), Molecular biological methods for Bacillus. John Wiley & Sons Ltd., Chichester, United Kingdom.
29. Oggioni, M. R., A. Ciabattini, A. M. Cuppone, and G. Pozzi. 2003. Bacillus spores for vaccine delivery. Vaccine21:96-101. [[PubMed]
30. Phelps, R. J., and J. L. McKillip. 2002. Enterotoxin production in natural isolates of Bacillaceae outside the Bacillus cereus group. Appl. Environ. Microbiol.68:3147-3151.
31. Pinchuk, I. V., P. Bressollier, B. Verneuil, B. Fenet, I. B. Sorokulova, F. Megraud, and M. C. Urdaci. 2001. In vitro anti-Helicobacter pylori activity of the probiotic strain Bacillus subtilis 3 is due to secretion of antibiotics. Antimicrob. Agents Chemother.45:3156-3161.
32. Platzer, C., G. Richter, K. Uberla, W. Muller, H. Blocker, T. Diamantstein, and T. Blankenstein. 1992. Analysis of cytokine mRNA levels in interleukin-4-transgenic mice by quantitative polymerase chain reaction. Eur. J. Immunol.22:1179-1184. [[PubMed]
33. Pugsley, AP. 1985. Escherichia coli K12 strains for use in the identification and characterization of colicins. J. Gen. Microbiol.131:369-376. [[PubMed][Google Scholar]
34. Rengpipat, S., W. Phianphak, S. Piyatiratitivorakul, and P. Menasveta. 1998. Effects of a probiotic bacterium on black tiger shrimp Penaeus monodon survival and growth. Aquaculture167:301-313. [PubMed]
35. Rolfe, RD. 2000. The role of probiotic cultures in the control of gastrointestinal health. J. Nutr.130:396S-402S. [[PubMed][Google Scholar]
36. Rowland, I. 1999. Probiotics and benefits to human health—the evidence in favour. Environ. Microbiol.1:375-382. [[PubMed]
37. Sartor, RB. 1996. Cytokine regulation of experimental intestinal inflammation in genetically engineered and T-lymphocyte reconstituted rodents. Aliment. Pharmacol. Therap.10:36-42. [[PubMed][Google Scholar]
38. Schiffrin, E. J., F. Rochat, H. L. Link-Amster, J. M. Aeschlimann, and A. Donnet-Hughes. 1995. Immunomodulation of human blood cells following the ingestion of lactic acid bacteria. J. Dairy Sci.78:491-497. [[PubMed]
39. Senesi, S., F. Celandroni, A. Tavanti, and E. Ghelardi. 2001. Molecular characterization and identification of Bacillus clausii strains marketed for use in oral bacteriotherapy. Appl. Environ. Microbiol.67:834-839.
40. Spinosa, M. R., T. Braccini, E. Ricca, M. De Felice, L. Morelli, G. Pozzi, and M. R. Oggioni. 2000. On the fate of ingested Bacillus spores. Res. Microbiol.151:361-368. [[PubMed]
41. Tournot, J. 1989. Applications of probiotics to animal husbandry. Rev. Sci. Tech. O.I.E. (Off. Int. Epizoot.)8:551-566. [PubMed]
42. Vaseeharan, B., and PRamasamy. 2003. Control of pathogenic Vibrio spp. by Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon.Lett. Appl. Microbiol.36:83-87. [[PubMed][Google Scholar]
43. Verschuere, L., G. Rombaut, P. Sorgeloos, and W. Verstraete. 2000. Probiotic bacteria as biological control agents in aquaculture. Microbiol. Mol. Biol. Rev.64:655-671.
44. Youngman, P., J. Perkins, and R. Losick. 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. Plasmid12:1-9. [[PubMed]