Interaction between the probiotic Bacillus subtilis and Salmonella Typhimurium in Caco-2 cell culture
,
Abstract
Background and Objectives: Bacillus probiotics have been recently considered in biotechnological researches, and food additives. The present study was aimed to investigate the effects of Bacillus subtilis probiotics (PY79 and ATCC 6633) and their metabolites on Salmonella Typhimurium in Caco-2 cells.
Materials and Methods: Cytotoxicity of B. subtilis ATCC 6633 crude supernatant (CS) was evaluated by 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. S. Typhimurium invasion assay was performed in the presence of the probiotics. Cell viability, apoptosis, and necrosis were evaluated in presence of S. Typhimurium , B. subtilis strains, and CS (4%, 8%) using flow cytometry.
Results: Results showed a significant reduction in the invasive ability of S. Typhimurium to Caco-2 cells by employing B. subtilis probiotics, and CS) p < 0.05). The less invasion was indicated in B. subtilis PY79 and Salmonella co-cultural group. Furthermore, the cell survival rates, and apoptosis/necrosis were respectively increased and decreased in co-culture groups (p < 0.05).
Conclusion: Hence, it seems that B. subtilis strains could be suggested as beneficial candidates to overcome the invasion and cytotoxicity of Salmonella on the intestinal cells. However, additional in vivo models are suggested to validate our results.
1. Coconnier M-H, Bernet M-F, Kernéis S, Chauvière G, Fourniat J, Servin AL. Inhibition of adhesion of enteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion. FEMS Microbiol Lett 1993; 110: 299-305.
2. Golowczyc M, Mobili P, Garrote G, Abraham A, De Antoni G. Protective action of Lactobacillus kefir carrying S-layer protein against Salmonella enterica serovar Enteritidis. Int J Food Microbiol 2007; 118: 264-273.
3. Servin AL, Coconnier M-H. Adhesion of probiotic strains to the intestinal mucosa and interaction with pathogens. Best Pract Res Clin Gastroenterol 2003; 17: 741-754.
4. Sorokulova IB, Pinchuk IV, Denayrolles M, Osipova IG, Huang JM, Cutting SM, et al. The safety of two Bacillus probiotic strains for human use. Dig Dis Sci 2008; 53: 954-963.
5. Cutting SM. Bacillus probiotics. Food Microbiol 2011; 28: 214-220.
6. Huang J-M, La Ragione RM, Nunez A, Cutting SM. Immunostimulatory activity of Bacillus spores. FEMS Immunol Med Microbiol 2008; 53: 195-203.
7. Ye X, Li P, Yu Q, Yang Q. Bacillus subtilis inhibition of enterotoxic Escherichia coli-induced activation of MAPK signaling pathways in Caco-2 cells. Ann Microbiol 2013; 63: 577-581.
8. Sharp RJ, Scawen MD, Atkinson T (1989). Fermentation and downstream processing of Bacillus. In: CR Harwood, editor. In Biotechnology Handbook: Bacillus. New York, Plenum Press; pp. 255-292.
9. Hong HA, Khaneja R, Tam NM, Cazzato A, Tan S, Urdaci M, et al. Bacillus subtilis isolated from the human gastrointestinal tract. Res Microbiol 2009; 160: 134-143.
10. Gálvez A, Abriouel H, López RL, Omar NB. Bacteriocin-based strategies for food biopreservation. Int J Food Microbiol 2007; 120: 51-70.
11. Nithya V, Halami PM. Evaluation of the probiotic characteristics of Bacillus species isolated from different food sources. Ann Microbiol 2013; 63: 129-137.
12. Parisot J, Carey S, Breukink E, Chan WC, Narbad A, Bonev B. Molecular mechanism of target recognition by subtilin, a class I lanthionine antibiotic. Antimicrob Agents Chemother 2008; 52: 612-618.
13. Xie J, Zhang R, Shang C, Guo Y. Isolation and characterization of a bacteriocin produced by an isolated Bacillus subtilis LFB112 that exhibits antimicrobial activity against domestic animal pathogens. Afr J Biotechnol 2009; 8: 5611-5619.
14. Finlay BB, Falkow S. Salmonella interactions with polarized human intestinal Caco-2 epithelial cells. J
Infect Dis 1990; 162: 1096-1106.
15. Dunne C, O'Mahony L, Murphy L, Thornton G, Morrissey D, O'Halloran S, et al. In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings. Am J Clin Nutr 2001; 73(2 Suppl):386S-392S.
16. Lehto EM, Salminen SJ. Inhibition of Salmonella Typhimurium adhesion to Caco-2 cell cultures by Lactobacillus strain GG spent culture supernate: only a pH effect? FEMS Immunol Med Microbiol 1997; 18: 125-132.
17. Kawarizadeh A, Nojoomi F, Tabatabaei M, Hosseinzadeh S, Farzaneh M. The effect of Bacillus coagulans on cytotoxicity and apoptosis induced by Salmonella Typhimurium in HT-29 cell culture. Iran J Microbiol 2019; 11: 305-312.
18. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254.
19. Thirabunyanon M, Thongwittaya N. Protection activity of a novel probiotic strain of Bacillus subtilis against Salmonella Enteritidis infection. Res Vet Sci 2012; 93: 74-81.
20. Ozkan AD, Han D, Umu OC, Angun P, Senturk B, Yasa O, et al. Screening and selection of novel animal probiotics isolated from bovine chyme. Ann Microbiol 2013; 63: 1291-1300.
21. Grootaert C, Boon N, Zeka F, Vanhoecke B, Bracke M, Verstraete W, et al. Adherence and viability of intestinal bacteria to differentiated Caco-2 cells quantified by flow cytometry. J Microbiol Methods 2011; 86: 33-41.
22. Poormontaseri M, Hosseinzadeh S, Shekarforoush SS, Kalantari T. The effects of probiotic Bacillus subtilis on the cytotoxicity of Clostridium perfringens type A in Caco-2 cell culture. BMC Microbiol 2017; 17: 150.
23. Castillo NA, de LeBlanc AdM, Galdeano CM, Perdigón G. Probiotics: An alternative strategy for combating salmonellosis: Immune mechanisms involved. Food Res Int 2012; 45: 831-841.
24. Upadhaya S, Hossiendoust A, Kim I. Probiotics in Salmonella-challenged Hy-Line brown layers. Poult Sci 2016;95:1894-1897.
25. Ramarao N, Lereclus D. Adhesion and cytotoxicity of Bacillus cereus and Bacillus thuringiensis to epithelial cells are FlhA and PlcR dependent, respectively. Microbes Infect 2006; 8: 1483-1491.
26. Hong H, Huang JM, Khaneja R, Hiep L, Urdaci M, Cutting S. The safety of Bacillus subtilis and Bacillus indicus as food probiotics. J Appl Microbiol 2008; 105: 510-520.
27. Rowan NJ, Deans K, Anderson JG, Gemmell CG, Hunter IS, Chaithong T. Putative virulence factor expression by clinical and food isolates of Bacillus spp. after growth in reconstituted infant milk formulae. Appl Environ Microbiol 2001; 67: 3873-3881.
28. Abdel-Daim A, Hassouna N, Hafez M, Ashor MSA, Aboulwafa MM. Antagonistic activity of Lactobacillus isolates against Salmonella typhi in vitro. Biomed Res Int 2013; 2013:680605.
29. Kusters J, Mulders-Kremers G, Van Doornik C, Van Der Zeijst B. Effects of multiplicity of infection, bacterial protein synthesis, and growth phase on adhesion to and invasion of human cell lines by Salmonella Typhimurium. Infect Immun 1993; 61: 5013-5020.
30. Lin WH, Yu B, Lin CK, Hwang WZ, Tsen HY. Immune effect of heat-killed multistrain of Lactobacillus acidophilus against Salmonella Typhimurium invasion to mice. J Appl Microbiol 2007; 102: 22-31.
31. Lin C-K, Tsai H-C, Lin P-P, Tsen H-Y, Tsai C-C. Lactobacillus acidophilus LAP5 able to inhibit the Salmonella choleraesuis invasion to the human Caco-2 epithelial cell. Anaerobe 2008; 14: 251-255.
32. Tsai C-C, Hsih H-Y, Chiu H-H, Lai Y-Y, Liu J-H, Yu B, et al. Antagonistic activity against Salmonella infection in vitro and in vivo for two Lactobacillus strains from swine and poultry. Int J Food Microbiol 2005; 102: 185-194.
33. Tuomola E, Crittenden R, Playne M, Isolauri E, Salminen S. Quality assurance criteria for probiotic bacteria. Am J Clin Nutr 2001; 73(2 Suppl):393S-398S.
34. La Ragione RM, Casula G, Cutting SM, Woodward MJ. Bacillus subtilis spores competitively exclude Escherichia coli O78: K80 in poultry. Vet Microbiol 2001; 79: 133-142.
35. La Ragione RM, Woodward MJ. Competitive exclusion by Bacillus subtilis spores of Salmonella enterica serotype Enteritidis and Clostridium perfringens in young chickens. Vet Microbiol 2003; 94: 245-256.
36. Hammami I, Rhouma A, Jaouadi B, Rebai A, Nesme X. Optimization and biochemical characterization of a bacteriocin from a newly isolated Bacillus subtilis strain 14B for biocontrol of Agrobacterium spp. strains. Lett Appl Microbiol 2009; 48: 253-260.
37. Shelburne CE, An FY, Dholpe V, Ramamoorthy A, Lopatin DE, Lantz MS. The spectrum of antimicrobial activity of the bacteriocin subtilosin A. J Antimicrob Chemother 2007; 59: 297-300.
38. Fink SL, Cookson BT. Pyroptosis and host cell death responses during Salmonella infection. Cell Microbiol 2007; 9: 2562-2570.
39. Díaz M, Herrero M, García LA, Quirós C. Application of flow cytometry to industrial microbial bioprocesses. Biochem Eng J 2010; 48: 385-407.
40. Schwan WR, Huang X-Z, Hu L, Kopecko DJ. Differential bacterial survival, replication, and apoptosis-inducing ability of Salmonella serovars within human and murine macrophages. Infect Immun 2000; 68: 1005-1013.
41. Monack DM, Raupach B, Hromockyj AE, Falkow S. Salmonella Typhimurium invasion induces apoptosis in infected macrophages. Proc Natl Acad Sci U S A 1996; 93: 9833-9838.
42. De LeBlanc AdM, Castillo NA, Perdigon G. Anti-infective mechanisms induced by a probiotic Lactobacillus strain against Salmonella enterica serovar Typhimurium infection. Int J Food Microbiol 2010; 138: 223-231.
2. Golowczyc M, Mobili P, Garrote G, Abraham A, De Antoni G. Protective action of Lactobacillus kefir carrying S-layer protein against Salmonella enterica serovar Enteritidis. Int J Food Microbiol 2007; 118: 264-273.
3. Servin AL, Coconnier M-H. Adhesion of probiotic strains to the intestinal mucosa and interaction with pathogens. Best Pract Res Clin Gastroenterol 2003; 17: 741-754.
4. Sorokulova IB, Pinchuk IV, Denayrolles M, Osipova IG, Huang JM, Cutting SM, et al. The safety of two Bacillus probiotic strains for human use. Dig Dis Sci 2008; 53: 954-963.
5. Cutting SM. Bacillus probiotics. Food Microbiol 2011; 28: 214-220.
6. Huang J-M, La Ragione RM, Nunez A, Cutting SM. Immunostimulatory activity of Bacillus spores. FEMS Immunol Med Microbiol 2008; 53: 195-203.
7. Ye X, Li P, Yu Q, Yang Q. Bacillus subtilis inhibition of enterotoxic Escherichia coli-induced activation of MAPK signaling pathways in Caco-2 cells. Ann Microbiol 2013; 63: 577-581.
8. Sharp RJ, Scawen MD, Atkinson T (1989). Fermentation and downstream processing of Bacillus. In: CR Harwood, editor. In Biotechnology Handbook: Bacillus. New York, Plenum Press; pp. 255-292.
9. Hong HA, Khaneja R, Tam NM, Cazzato A, Tan S, Urdaci M, et al. Bacillus subtilis isolated from the human gastrointestinal tract. Res Microbiol 2009; 160: 134-143.
10. Gálvez A, Abriouel H, López RL, Omar NB. Bacteriocin-based strategies for food biopreservation. Int J Food Microbiol 2007; 120: 51-70.
11. Nithya V, Halami PM. Evaluation of the probiotic characteristics of Bacillus species isolated from different food sources. Ann Microbiol 2013; 63: 129-137.
12. Parisot J, Carey S, Breukink E, Chan WC, Narbad A, Bonev B. Molecular mechanism of target recognition by subtilin, a class I lanthionine antibiotic. Antimicrob Agents Chemother 2008; 52: 612-618.
13. Xie J, Zhang R, Shang C, Guo Y. Isolation and characterization of a bacteriocin produced by an isolated Bacillus subtilis LFB112 that exhibits antimicrobial activity against domestic animal pathogens. Afr J Biotechnol 2009; 8: 5611-5619.
14. Finlay BB, Falkow S. Salmonella interactions with polarized human intestinal Caco-2 epithelial cells. J
Infect Dis 1990; 162: 1096-1106.
15. Dunne C, O'Mahony L, Murphy L, Thornton G, Morrissey D, O'Halloran S, et al. In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings. Am J Clin Nutr 2001; 73(2 Suppl):386S-392S.
16. Lehto EM, Salminen SJ. Inhibition of Salmonella Typhimurium adhesion to Caco-2 cell cultures by Lactobacillus strain GG spent culture supernate: only a pH effect? FEMS Immunol Med Microbiol 1997; 18: 125-132.
17. Kawarizadeh A, Nojoomi F, Tabatabaei M, Hosseinzadeh S, Farzaneh M. The effect of Bacillus coagulans on cytotoxicity and apoptosis induced by Salmonella Typhimurium in HT-29 cell culture. Iran J Microbiol 2019; 11: 305-312.
18. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254.
19. Thirabunyanon M, Thongwittaya N. Protection activity of a novel probiotic strain of Bacillus subtilis against Salmonella Enteritidis infection. Res Vet Sci 2012; 93: 74-81.
20. Ozkan AD, Han D, Umu OC, Angun P, Senturk B, Yasa O, et al. Screening and selection of novel animal probiotics isolated from bovine chyme. Ann Microbiol 2013; 63: 1291-1300.
21. Grootaert C, Boon N, Zeka F, Vanhoecke B, Bracke M, Verstraete W, et al. Adherence and viability of intestinal bacteria to differentiated Caco-2 cells quantified by flow cytometry. J Microbiol Methods 2011; 86: 33-41.
22. Poormontaseri M, Hosseinzadeh S, Shekarforoush SS, Kalantari T. The effects of probiotic Bacillus subtilis on the cytotoxicity of Clostridium perfringens type A in Caco-2 cell culture. BMC Microbiol 2017; 17: 150.
23. Castillo NA, de LeBlanc AdM, Galdeano CM, Perdigón G. Probiotics: An alternative strategy for combating salmonellosis: Immune mechanisms involved. Food Res Int 2012; 45: 831-841.
24. Upadhaya S, Hossiendoust A, Kim I. Probiotics in Salmonella-challenged Hy-Line brown layers. Poult Sci 2016;95:1894-1897.
25. Ramarao N, Lereclus D. Adhesion and cytotoxicity of Bacillus cereus and Bacillus thuringiensis to epithelial cells are FlhA and PlcR dependent, respectively. Microbes Infect 2006; 8: 1483-1491.
26. Hong H, Huang JM, Khaneja R, Hiep L, Urdaci M, Cutting S. The safety of Bacillus subtilis and Bacillus indicus as food probiotics. J Appl Microbiol 2008; 105: 510-520.
27. Rowan NJ, Deans K, Anderson JG, Gemmell CG, Hunter IS, Chaithong T. Putative virulence factor expression by clinical and food isolates of Bacillus spp. after growth in reconstituted infant milk formulae. Appl Environ Microbiol 2001; 67: 3873-3881.
28. Abdel-Daim A, Hassouna N, Hafez M, Ashor MSA, Aboulwafa MM. Antagonistic activity of Lactobacillus isolates against Salmonella typhi in vitro. Biomed Res Int 2013; 2013:680605.
29. Kusters J, Mulders-Kremers G, Van Doornik C, Van Der Zeijst B. Effects of multiplicity of infection, bacterial protein synthesis, and growth phase on adhesion to and invasion of human cell lines by Salmonella Typhimurium. Infect Immun 1993; 61: 5013-5020.
30. Lin WH, Yu B, Lin CK, Hwang WZ, Tsen HY. Immune effect of heat-killed multistrain of Lactobacillus acidophilus against Salmonella Typhimurium invasion to mice. J Appl Microbiol 2007; 102: 22-31.
31. Lin C-K, Tsai H-C, Lin P-P, Tsen H-Y, Tsai C-C. Lactobacillus acidophilus LAP5 able to inhibit the Salmonella choleraesuis invasion to the human Caco-2 epithelial cell. Anaerobe 2008; 14: 251-255.
32. Tsai C-C, Hsih H-Y, Chiu H-H, Lai Y-Y, Liu J-H, Yu B, et al. Antagonistic activity against Salmonella infection in vitro and in vivo for two Lactobacillus strains from swine and poultry. Int J Food Microbiol 2005; 102: 185-194.
33. Tuomola E, Crittenden R, Playne M, Isolauri E, Salminen S. Quality assurance criteria for probiotic bacteria. Am J Clin Nutr 2001; 73(2 Suppl):393S-398S.
34. La Ragione RM, Casula G, Cutting SM, Woodward MJ. Bacillus subtilis spores competitively exclude Escherichia coli O78: K80 in poultry. Vet Microbiol 2001; 79: 133-142.
35. La Ragione RM, Woodward MJ. Competitive exclusion by Bacillus subtilis spores of Salmonella enterica serotype Enteritidis and Clostridium perfringens in young chickens. Vet Microbiol 2003; 94: 245-256.
36. Hammami I, Rhouma A, Jaouadi B, Rebai A, Nesme X. Optimization and biochemical characterization of a bacteriocin from a newly isolated Bacillus subtilis strain 14B for biocontrol of Agrobacterium spp. strains. Lett Appl Microbiol 2009; 48: 253-260.
37. Shelburne CE, An FY, Dholpe V, Ramamoorthy A, Lopatin DE, Lantz MS. The spectrum of antimicrobial activity of the bacteriocin subtilosin A. J Antimicrob Chemother 2007; 59: 297-300.
38. Fink SL, Cookson BT. Pyroptosis and host cell death responses during Salmonella infection. Cell Microbiol 2007; 9: 2562-2570.
39. Díaz M, Herrero M, García LA, Quirós C. Application of flow cytometry to industrial microbial bioprocesses. Biochem Eng J 2010; 48: 385-407.
40. Schwan WR, Huang X-Z, Hu L, Kopecko DJ. Differential bacterial survival, replication, and apoptosis-inducing ability of Salmonella serovars within human and murine macrophages. Infect Immun 2000; 68: 1005-1013.
41. Monack DM, Raupach B, Hromockyj AE, Falkow S. Salmonella Typhimurium invasion induces apoptosis in infected macrophages. Proc Natl Acad Sci U S A 1996; 93: 9833-9838.
42. De LeBlanc AdM, Castillo NA, Perdigon G. Anti-infective mechanisms induced by a probiotic Lactobacillus strain against Salmonella enterica serovar Typhimurium infection. Int J Food Microbiol 2010; 138: 223-231.
| Files | ||
| Issue | Vol 13 No 1 (2021) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v13i1.5497 | |
| Keywords | ||
| Probiotics; Salmonella Typhimurium; Caco-2 cells; Cytotoxicity tests; Immunologic; Flow cytometry | ||
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How to Cite
1.
Ostovan R, Pourmontaseri M, Hosseinzadeh S, Shekarforoush SS. Interaction between the probiotic Bacillus subtilis and Salmonella Typhimurium in Caco-2 cell culture. Iran J Microbiol. 2021;13(1):91-97.
