The objective of the present study was to describe the physiological properties of seven potential probiotic strains of Bacillus spp. Isolates were characterized morphologically, biochemically, and by 16S rRNA sequence analyses for identification. Tolerance to acidic pH, high osmotic concentrations of NaCl, and bile salts were tested. Isolates were also evaluated for their ability to metabolize different carbohydrates sources. The antimicrobial sensitivity profiles were determined. Inhibition of gastrointestinal Salmonella colonization in an avian model was also evaluated. Five strains of Bacillus were tolerant to acidic conditions (pH 2.0) and all strains were tolerant to a high osmotic pressure (NaCl at 6.5%). Moreover, all strains were able to tolerate concentration of 0.037% bile salts after 24？h of incubation. Three strains were able to significantly reduce Salmonella Typhimurium levels in the crop and in the ceca of broiler-type chickens. Among the 12 antibiotics tested for antibiotic resistance, all strains were resistant to bacitracin and susceptible to gentamycin, neomycin, ormethoprim, triple sulfa, and spectinomycin. Bacterial spore formers have been shown to prevent gastrointestinal diseases in animals and humans. The results obtained in this study show important characteristics to be evaluated when selecting Bacillus spp. candidates to be used as probiotics. 1. Introduction Probiotics have been commercialized for both animal and human uses. Probiotics for humans use are subject to minimal restrictions and come in many different forms. Probiotics in animal feed have been used for the prevention of gastrointestinal infections, with a wide use in poultry and aquaculture productions [1–6]. Diarrhea is one of the major side effects of chemotherapy in cancer treatments and has been associated with increased morbidity, mortality, increased treatment costs, and restrictions related to the ability to deliver full doses of chemotherapy [7, 8]. Enterocyte proliferation in the intestinal mucosa and the intestinal microflora can be directly harmed by the effect of chemotherapeutic agents as well as radiation, often causing bacterial translocation, malabsorption, and/or diarrhea [8, 9]. Therefore, in order to reduce systemic bacterial diseases, high doses of broad spectrum antibiotics are usually used in cancer patients undergoing chemotherapy or radiation therapy. The disruption of the beneficial intestinal microflora is a common consequence to this type of treatment, which may lead to the colonization of opportunistic pathogenic bacteria such as
References
[1]
H. A. Hong, H. D. Le, and S. M. Cutting, “The use of bacterial spore formers as probiotics,” FEMS Microbiology Reviews, vol. 29, no. 4, pp. 813–835, 2005.
[2]
A. Jadamus, W. Vahjen, K. Sch？fer, and O. Simon, “Influence of the probiotic strain Bacillus cereus var. toyoi on the development of enterobacterial growth and on selected parameters of bacterial metabolism in digesta samples of piglets,” Journal of Animal Physiology and Animal Nutrition, vol. 86, no. 1-2, pp. 42–54, 2002.
[3]
H. Kasper, “Protection against gastrointestinal diseases—present facts and future developments,” International Journal of Food Microbiology, vol. 41, no. 2, pp. 127–131, 1998.
[4]
R. D. Sleatorand and C. Hill, “New frontiers in probiotic research,” Letters in Applied Microbiology, vol. 46, pp. 143–147, 2008.
[5]
R. D. Rolfe, “The role of probiotic cultures in the control of gastrointestinal health,” Journal of Nutrition, vol. 130, pp. 396S–402S, 2000.
[6]
C. H. Liu, C. H. Chiu, S. W. Wang, and W. Cheng, “Dietary administration of the probiotic, Bacillus subtilis E20, enhances the growth, innate immune responses, and disease resistance of the grouper, Epinephelus coioides,” Fish & Shellfish Immunology, vol. 33, no. 4, pp. 699–706, 2012.
[7]
K. Kobayashi, “Chemotherapy-induced diarrhea,” Cancer & Chemotherapy, vol. 30, no. 6, pp. 765–771, 2003.
[8]
D. M. F. Savarese, G. Savy, L. Vahdat, P. E. Wischmeyer, and B. Corey, “Prevention of chemotheraphy and radiation toxicity with glutamine,” Cancer Treatment Reviews, vol. 29, no. 6, pp. 501–513, 2003.
[9]
C. McGough, C. Baldwin, G. Frost, and H. J. N. Andreyev, “Role of nutritional intervention in patients treated with radiotherapy for pelvic malignancy,” British Journal of Cancer, vol. 90, no. 12, pp. 2278–2287, 2004.
[10]
L. M. Noriega, P. van der Auwera, D. Daneau, F. Meunier, and M. Aoun, “Salmonella infections in a cancer center,” Supportive Care in Cancer, vol. 2, no. 2, pp. 116–122, 1994.
[11]
J. Delaloye, G. Merlani, C. Petignat et al., “Nosocomial nontyphoidal salmonellosis after antineoplastic chemotherapy: reactivation of asymptomatic colonization?” European Journal of Clinical Microbiology and Infectious Diseases, vol. 23, no. 10, pp. 751–758, 2004.
[12]
E. I. Benchimol and D. R. Mack, “Probiotics in relapsing and chronic diarrhea,” Journal of Pediatric Hematology/Oncology, vol. 26, no. 8, pp. 515–517, 2004.
[13]
M. W. Hull and P. L. Beck, “Clostridium difficile—associated colitis,” Canadian Family Physician, vol. 50, pp. 1536–1545, 2004.
[14]
D. Czerucka and P. Rampal, “Experimental effects of Saccharomyces boulardii on diarrheal pathogens,” Microbes and Infection, vol. 4, no. 7, pp. 733–739, 2002.
[15]
D. Czerucka, S. Dahan, B. Mograbi, B. Rossi, and P. Rampal, “Saccharomyces boulardii preserves the barrier function and modulates the signal transduction pathway induced in enteropathogenic Escherichia coli-infected T84 cells,” Infection and Immunity, vol. 68, no. 10, pp. 5998–6004, 2000.
[16]
N. K. Lee, J. S. Park, E. Park, and H. D. Paik, “Adherence and anticarcinogenic effects of Bacillus polyfermenticus SCD in the large intestine,” Letters in Applied Microbiology, vol. 44, no. 3, pp. 274–278, 2007.
[17]
S. V. Malkov, V. V. Markelov, G. Y. Polozov, B. I. Barabanschikov, A. Y. Kozhevnikov, and M. V. Trushin, “Significant delay of lethal outcome in cancer patients due to peroral administration of Bacillus oligonitrophilus KU-1,” TheScientificWorldJournal, vol. 6, pp. 2177–2187, 2006.
[18]
E. Park, G. I. Jeon, J. S. Park, and H. D. Paik, “A probiotic strain of Bacillus polyfermenticus reduces DMH induced precancerous lesions in F344 male rat,” Biological and Pharmaceutical Bulletin, vol. 30, no. 3, pp. 569–574, 2007.
[19]
L. O'Mahony, M. Feeney, S. O'Halloran et al., “Probiotic impact on microbial flora, inflammation and tumour development in IL-10 knockout mice,” Alimentary Pharmacology and Therapeutics, vol. 15, no. 8, pp. 1219–1225, 2001.
[20]
M. C. Urdaci, P. Bressollier, and I. Pinchuk, “Bacillus clausii probiotic strains: antimicrobial and immunomodulatory activities,” Journal of clinical gastroenterology, vol. 38, no. 6, pp. S86–S90, 2004.
[21]
T. M. Barbosa, C. R. Serra, R. M. La Ragione, M. J. Woodward, and A. O. Henriques, “Screening for Bacillus isolates in the broiler gastrointestinal tract,” Applied and Environmental Microbiology, vol. 71, no. 2, pp. 968–978, 2005.
[22]
L. H. Duc, H. A. Hong, T. M. Barbosa, A. O. Henriques, and S. M. Cutting, “Characterization of Bacillus probiotics available for human use,” Applied and Environmental Microbiology, vol. 70, no. 4, pp. 2161–2171, 2004.
[23]
P. Permpoonpattana, H. A. Hong, R. Khaneja, and S. M. Cutting, “Evaluation of Bacillus subtilis strains as probiotics and their potential as a food ingredient,” Beneficial Microbes, vol. 3, no. 2, pp. 127–135, 2012.
[24]
L. H. Duc, H. A. Hong, and S. M. Cutting, “Germination of the spore in the gastrointestinal tract provides a novel route for heterologous antigen delivery,” Vaccine, vol. 21, no. 27–30, pp. 4215–4224, 2003.
[25]
L. H. Duc, H. A. Hong, N. Fairweather, E. Ricca, and S. M. Cutting, “Bacterial spores as vaccine vehicles,” Infection and Immunity, vol. 71, no. 5, pp. 2810–2818, 2003.
[26]
T. T. Hoa, L. H. Duc, R. Isticato et al., “Fate and dissemination of Bacillus subtilis spores in a murine model,” Applied and Environmental Microbiology, vol. 67, no. 9, pp. 3819–3823, 2001.
[27]
G. Casula and S. M. Cutting, “Bacillus probiotics: spore germination in the gastrointestinal tract,” Applied and Environmental Microbiology, vol. 68, no. 5, pp. 2344–2352, 2002.
[28]
R. E. Wolfenden, N. R. Pumford, M. J. Morgan et al., “Evaluation of a screening and selection method for bacillus isolates for use as effective direct-fed microbials in commercial poultry,” International Journal of Poultry Science, vol. 9, no. 4, pp. 317–323, 2010.
[29]
S. E. Gilliland, T. E. Staley, and L. J. Bush, “Importance of bile tolerance of Lactobacillus acidophilus used as a dietary adjunct,” Journal of dairy science, vol. 67, no. 12, pp. 3045–3051, 1984.
[30]
G. Tellez, C. E. Dean, D. E. Corrier, J. R. Deloach, L. Jaeger, and B. M. Hargis, “Effect of dietary lactose on cecal morphology, pH, organic acids, and Salmonella enteritidis organ invasion in Leghorn chicks,” Poultry Science, vol. 72, no. 4, pp. 636–642, 1993.
[31]
SAS Institute Inc. 2002-2003, Version 9.1, Carey, NC, USA.
[32]
J. Zar, Biostatistical Analysis, Prentice-Hall, 2nd edition, 1984.
[33]
N. A. Logan and R. C. W. Berkeley, “Identification of Bacillus strains using the API system,” Journal of General Microbiology, vol. 130, no. 7, pp. 1871–1882, 1984.
[34]
R. D. Wagner, D. D. Paine, and C. E. Cerniglia, “Phenotypic and genotypic characterization of competitive exclusion products for use in poultry,” Journal of Applied Microbiology, vol. 94, no. 6, pp. 1098–1107, 2003.
[35]
R. Havenaar, B. T. Brink, and J. H. J. Huis Veld, “Selection of strains for probiotic use,” in Probiotics, the Scientific Basis, R. Fuller, Ed., vol. 1, pp. 209–224, Chapman and Hall, London, UK, 1992.
[36]
S. M. Kristoffersen, S. Ravnum, N. J. Tourasse, O. A. ？kstad, A. B. Kolst？, and W. Davies, “Low concentrations of bile salts induce stress responses and reduce motility in Bacillus cereus ATCC 14570,” Journal of Bacteriology, vol. 189, no. 14, pp. 5302–5313, 2007.
[37]
D. Bakari, N. L. Tatsadjieu, A. Mbawala, and C. M. Mbofung, “Assessment of physiological properties of some lactic acid bacteria isolated from the intestine of chickens use as probiotics and antimicrobial agents against enteropathogenic bacteria,” Innovative Romanian Food Biotechnology, vol. 8, pp. 33–40, 2011.
[38]
M. Begley, C. G. M. Gahan, and C. Hill, “The interaction between bacteria and bile,” FEMS Microbiology Reviews, vol. 29, no. 4, pp. 625–651, 2005.
[39]
S. B. Hernández, I. Cota, A. Ducret, L. Aussel, and J. Casadesús, “Adaptation and preadaptation of Salmonella enterica to bile,” PLoS Genetics, vol. 8, no. 1, pp. 1–15, 2012.
[40]
M. Du Toit, C. M. A. P. Franz, L. M. T. Dicks et al., “Characterisation and selection of probiotic lactobacilli for a preliminary minipig feeding trial and their effect on serum cholesterol levels, faeces pH and faeces moisture content,” International Journal of Food Microbiology, vol. 40, no. 1-2, pp. 93–104, 1998.
[41]
H. Tanaka, K. Doesburg, T. Iwasaki, and I. Mierau, “Screening of lactic acid bacteria for bile salt hydrolase activity,” Journal of Dairy Science, vol. 82, no. 12, pp. 2530–2535, 1999.
[42]
C. Dunne, L. O'Mahony, L. Murphy et al., “In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings,” American Journal of Clinical Nutrition, vol. 73, no. 2, pp. 386S–392S, 2001.
[43]
A. C. Ouwehand, S. Salminen, and E. Isolauri, “Probiotics: an overview of beneficial effects,” Antonie van Leeuwenhoek, vol. 82, no. 1–4, pp. 279–289, 2002.
[44]
C. Ibourahema, R. D. Dauphin, D. Jacqueline, and P. Thonart, “Characterization of lactic acid bacteria isolated from poultry farms in Senegal,” African Journal of Biotechnology, vol. 7, no. 12, pp. 2006–2012, 2008.
[45]
P. M. Bennett, “Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria,” British Journal of Pharmacology, vol. 153, no. 1, pp. S347–S357, 2008.
[46]
J. B. Dodgson and M. N. Romanov, “Use of chicken models for the analysis of human disease,” Current Protocols in Human Genetics, vol. 15, unit 15.5, pp. 15.5.1–15.5.12, 2004.
[47]
R. M. La Ragione and M. J. Woodward, “Competitive exclusion by Bacillus subtilis spores of Salmonella enterica serotype Enteritidis and Clostridium perfringens in young chickens,” Veterinary Microbiology, vol. 94, no. 3, pp. 245–256, 2003.
[48]
B. Vilà, A. Fontgibell, I. Badiola et al., “Reduction of Salmonella enterica var. Enteritidis colonization and invasion by Bacillus cereus var. toyoi inclusion in poultry feeds,” Poultry Science, vol. 88, no. 5, pp. 975–979, 2009.
[49]
J. A. Patterson and K. M. Burkholder, “Application of prebiotics and probiotics in poultry production,” Poultry Science, vol. 82, no. 4, pp. 627–631, 2003.
[50]
T. D. Leser, A. Knarreborg, and J. Worm, “Germination and outgrowth of Bacillus subtilis and Bacillus licheniformis spores in the gastrointestinal tract of pigs,” Journal of Applied Microbiology, vol. 104, no. 4, pp. 1025–1033, 2008.
[51]
S. C. Ng, A. L. Hart, M. A. Kamm, A. J. Stagg, and S. C. Knight, “Mechanisms of action of probiotics: recent advances,” Inflammatory Bowel Diseases, vol. 15, no. 2, pp. 300–310, 2009.
[52]
P. Rupa and Y. Mine, “Recent advances in the role of probiotics in human inflammation and gut health,” Journal of Agricultural and Food Chemistry, vol. 60, pp. 8249–8256, 2012.
[53]
S. C. Corr, Y. Li, C. U. Riedel, P. W. O’Toole, C. Hill, and C. G. M. Gahan, “Bacteriocin production as a mechanism for the antiinfective activity of Lactobacillus salivarius UCC118,” Proceedings of the National Academy of Sciences of the United States of America, vol. 104, pp. 7617–7621, 2007.
[54]
A. Anadón, M. R. Martinez-Larranaga, and M. Aranzazu Martinez, “Probiotics for animal nutrition in the european union regulation and safety assessment,” Regulatory Toxicology and Pharmacology, vol. 45, no. 1, pp. 91–95, 2006.
[55]
H. A. Hong, J. M. Huang, R. Khaneja, L. V. Hiep, M. C. Urdaci, and S. M. Cutting, “The safety of Bacillus subtilis and Bacillus indicus as food probiotics,” Journal of Applied Microbiology, vol. 105, no. 2, pp. 510–520, 2008.
[56]
C. A. McNulty, P. Boyle, T. Nichols, P. Clappison, and P. Davey, “The public's attitudes to and compliance with antibiotics,” The Journal of Antimicrobial Chemotherapy, vol. 60, supplement 1, pp. i63–i68, 2007.
[57]
I. G. Osipova, N. A. Makhailova, I. B. Sorokulova, E. A. Vasil'eva, and A. A. Gaiderov, “Spore probiotics,” Zhurnal Mikrobiologii Epidemiologii i Immunobiologii, vol. 3, no. 3, pp. 113–119, 2003.
[58]
P. Williams, “Bacillus subtilis: a shocking message from a probiotic,” Cell Host and Microbe, vol. 1, no. 4, pp. 248–249, 2007.
[59]
W. A. M. Wolken, J. Tramper, and M. J. Van Der Werf, “What can spores do for us?” Trends in Biotechnology, vol. 21, no. 8, pp. 338–345, 2003.
