In Vitro Evaluation of Swine-Derived Lactobacillus reuteri: Probiotic Properties and Effects on Intestinal Porcine Epithelial Cells Challenged with Enterotoxigenic Escherichia coli K88 (original) (raw)

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Research article

Food Microbiology and Biotechnology

Zhilin Wang 1, Li Wang 1, Zhuang Chen 1, Xianyong Ma 1, Xuefen Yang 1, Jian Zhang 1 and Zongyong Jiang 1*

State Key Laboratory of Livestock and Poultry Breeding, Ministry of Agriculture Key Laboratory of Animal Nutrition and Feed Science in South China, Guangdong Public Laboratory of Animal Breeding and Nutrition, Guangdong Key Laboratory of Animal Breeding and Nutrition, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou, Guangdong 510640, P.R. China

Received: October 27, 2015; Accepted: February 23, 2016

J. Microbiol. Biotechnol. 2016; 26(6): 1018-1025

Published June 28, 2016 https://doi.org/10.4014/jmb.1510.10089

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

Probiotics are considered as the best effective alternatives to antibiotics. The aim of this study was to characterize the probiotic potential of lactobacilli for use in swine farming by using in vitro evaluation methods. A total of 106 lactic acid bacterial isolates, originating from porcine feces, were first screened for the capacity to survive stresses considered important for putative probiotic strains. Sixteen isolates showed notable acid and bile resistance, antibacterial activity, and adherence to intestinal porcine epithelial cells (IPEC-1). One isolate, LR1, identified as Lactobacillus reuteri, was selected for extensive study of its probiotic and functional properties in IPEC-1 cell models. L. reuteri LR1 exhibited good adhesion to IPEC-1 cells and could inhibit the adhesion of enterotoxigenic Escherichia coli (ETEC) to IPEC-1 cells. L. reuteri LR1 could also modulate transcript and protein expression of cytokines involved in inflammation in IPEC-1 cells; the Lactobacillus strain inhibited the ETEC-induced expression of proinflammatory transcripts (IL-6 and TNF-α) and protein (IL-6), and increased the level of anti-inflammatory cytokine (IL-10). Measurement of the permeation of FD-4 showed that L. reuteri LR1 could maintain barrier integrity in monolayer IPEC-1 cells exposed to ETEC. Immunolocalization experiments showed L. reuteri LR1 could also prevent ETEC-induced tight junction ZO-1 disruption. Together, these results indicate that L. reuteri LR1 exhibits desirable probiotic properties and could be a potential probiotic for use in swine production.

Keywords

Lactobacillus reuteri, in vitro evaluation, probiotic properties, enterotoxigenic Escherichia coli

References

1. Araya M, Morelli L, Reid G, Sanders M, Stanton C, Pineiro M, Ben Embarek P. 2002. Guidelines for the evaluation of probiotics in food. Report of a Joint FAO/WHO working group on drafting guidelines for the evaluation of probiotics in food. London, Ontario, Canada.
2. Cario E, Gerken G, Podolsky DK. 2004. Toll-like receptor 2 enhances ZO-1-associated intestinal epithelial barrier integrity via protein kinase C. Gastroenterology 127: 224-238.
   
3. Casas IA, Dobrogosz WJ. 2000. Validation of the probiotic concept: Lactobacillus reuteri confers broad-spectrum protection against disease in humans and animals. Microbial Ecol. Health Dis. 12: 247-285.
   
4. De Angelis M, Siragusa S, Berloco M, Caputo L, Settanni L, Alfonsi G, et al. 2006. Selection of potential probiotic lactobacilli from pig feces to be used as additives in pelleted feeding. Res. Microbiol. 157: 792-801.
   
5. Dibner J, Richards J. 2005. Antibiotic growth promoters in agriculture: history and mode of action. Poultry Sci. 84: 634-643.
   
6. Fairbrother JM, Nadeau É, Gyles CL. 2005. Escherichia coli in postweaning diarrhea in pigs: an update on bacterial types, pathogenesis, and prevention strategies. Anim. Health Res. Rev. 6: 17-39.
   
7. Foligne B, Nutten S, Grangette C, Dennin V, Goudercourt D, Poiret S, et al. 2007. Correlation between in vitro and in vivo immunomodulatory properties of lactic acid bacteria. World J. Gastroenterol. 13: 236-243.
   
8. Frese SA, Benson AK, Tannock GW, Loach DM, Kim J, Zhang M, et al. 2011. The evolution of host specialization in the vertebrate gut symbiont Lactobacillus reuteri. PLoS Genet. 7: e1001314.
   
9. Gaggìa F, Mattarelli P, Biavati B. 2010. Probiotics and prebiotics in animal feeding for safe food production. Int. J. Food Microbiol. 141: S15-S28.
   
10. Heller KJ. 2001. Probiotic bacteria in fermented foods:product characteristics and starter organisms. Am. J. Clin. Nutr. 73: 374s-379s.
    
11. Juntunen M, Kirjavainen P, Ouwehand A, Salminen S, Isolauri E. 2001. Adherence of probiotic bacteria to human intestinal mucus in healthy infants and during rotavirus infection. Clin. Diagn. Lab. Immunol. 8: 293-296.
    
12. Kaushik JK, Kumar A, Duary RK, Mohanty AK, Grover S, Batish VK. 2009. Functional and probiotic attributes of an indigenous isolate of Lactobacillus plantarum. PLoS One 4: e8099.
    
13. Kim HB, Isaacson RE. 2015. The pig gut microbial diversity:understanding the pig gut microbial ecology through the next-generation high-throughput sequencing. Vet. Microbiol. 177: 242-251.
    
14. Koh SY, George S, Brözel V, Moxley R, Francis D, Kaushik RS. 2008. Porcine intestinal epithelial cell lines as a new in vitro model for studying adherence and pathogenesis of enterotoxigenic Escherichia coli. Vet. Microbiol. 130: 191-197.
    
15. Kotzamanidis C, Kourelis A, Litopoulou-Tzanetaki E, Tzanetakis N, Yiangou M. 2010. Evaluation of adhesion capacity, cell surface traits and immunomodulatory activity of presumptive probiotic Lactobacillus strains. Int. J. Food Microbiol. 140: 154-163.
    
16. Kowapradit J, Opanasopit P, Ngawhiranpat T, Apirakaramwong A, Rojanarata T, Ruktanonchai U, Sajomsang W. 2008. Methylated N-(4-N, N-dimethylaminobenzyl) chitosan, a novel chitosan derivative, enhances paracellular permeability across intestinal epithelial cells (Caco-2). AAPS PharmSciTech 9:1143-1152.
    
17. Lähteinen T, Malinen E, Koort JM, Mertaniemi-Hannus U, Hankimo T, Karikoski N, et al. 2010. Probiotic properties of Lactobacillus isolates originating from porcine intestine and feces. Anaerobe 16: 293-300.
    
18. Lebeer S, Vanderleyden J, De Keersmaecker SC. 2010. Host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens. Nat. Rev. Microbiol. 8: 171-184.
    
19. Lee DY, Seo Y-S, Rayamajhi N, Kang ML, Lee SI, Yoo HS. 2009. Isolation, characterization, and evaluation of wild isolates of Lactobacillus reuteri from pig feces. J. Microbiol. 47:663-672.
    
20. Lee J, Yun HS, Cho KW, Oh S, Kim SH, Chun T, et al. 2011. Evaluation of probiotic characteristics of newly isolated Lactobacillus spp.: immune modulation and longevity. Int. J. Food Microbiol. 148: 80-86.
    
21. Meurens F, Berri M, Auray G, Melo S, Levast B, VirlogeuxPayant I, et al. 2009. Early immune response following Salmonella enterica subspecies enterica serovar Typhimurium infection in porcine jejunal gut loops. Vet. Res. 40: 1.
    
22. Oh PL, Benson AK, Peterson DA, Patil PB, Moriyama EN, Roos S, Walter J. 2010. Diversification of the gut symbiont Lactobacillus reuteri as a result of host-driven evolution. ISME J. 4: 377-387.
    
23. Roselli M, Finamore A, Britti MS, Konstantinov SR, Smidt H, de Vos WM, Mengheri E. 2007. The novel porcine Lactobacillus sobrius strain protects intestinal cells from enterotoxigenic Escherichia coli K88 infection and prevents membrane barrier damage. J. Nutr. 137: 2709-2716.
    
24. Saarela M, Mogensen G, Fondén R, Mättö J, MattilaSandholm T. 2000. Probiotic bacteria: safety, functional and technological properties. J. Biotechnol. 84: 197-215.
    
25. Seo BJ, Mun MR, Kim C-J, Lee I, Chang Y-H, Park Y-H. 2010. Bile tolerant Lactobacillus reuteri isolated from pig feces inhibits enteric bacterial pathogens and porcine rotavirus. Vet. Res. Commun. 34: 323-333.
    
26. Talarico T, Casas I, Chung TC, Dobrogosz W. 1988. Production and isolation of reuterin, a growth inhibitor produced by Lactobacillus reuteri. Antimicrob. Agents Chemother. 32: 1854-1858.
    
27. Tsukita S, Furuse M, Itoh M. 2001. Multifunctional strands in tight junctions. Nat. Rev. Mol. Cell Biol. 2: 285-293.
    
28. Tuomola EM, Ouwehand AC, Salminen SJ. 1999. The effect of probiotic bacteria on the adhesion of pathogens to human intestinal mucus. FEMS Immunol. Med. Microbiol. 26: 137-142.
    
29. Vollenweider S, Lacroix C. 2004. 3-Hydroxypropionaldehyde:applications and perspectives of biotechnological production. Appl. Microbiol. Biotechnol. 64: 16-27.
    
30. Walter J, Britton RA, Roos S. 2011. Host-microbial symbiosis in the vertebrate gastrointestinal tract and the Lactobacillus reuteri paradigm. Proc. Natl. Acad. Sci. USA 108: 4645-4652.
    
31. Yu LC, Flynn AN, Turner JR, Buret AG. 2005. SGLT-1mediated glucose uptake protects intestinal epithelial cells against LPS-induced apoptosis and barrier defects: a novel cellular rescue mechanism? FASEB J. 19: 1822-1835.
    
32. Zanello G, Berri M, Dupont J, Sizaret P-Y, D’Inca R, Salmon H, Meurens F. 2011. Saccharomyces cerevisiae modulates immune gene expressions and inhibits ETEC-mediated ERK1/2 and p38 signaling pathways in intestinal epithelial cells. PLoS One 6: e18573.
    

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Article

Research article

In Vitro Evaluation of Swine-Derived Lactobacillus reuteri: Probiotic Properties and Effects on Intestinal Porcine Epithelial Cells Challenged with Enterotoxigenic Escherichia coli K88

Zhilin Wang 1, Li Wang 1, Zhuang Chen 1, Xianyong Ma 1, Xuefen Yang 1, Jian Zhang 1 and Zongyong Jiang 1*

State Key Laboratory of Livestock and Poultry Breeding, Ministry of Agriculture Key Laboratory of Animal Nutrition and Feed Science in South China, Guangdong Public Laboratory of Animal Breeding and Nutrition, Guangdong Key Laboratory of Animal Breeding and Nutrition, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou, Guangdong 510640, P.R. China

Received: October 27, 2015; Accepted: February 23, 2016

Abstract

Probiotics are considered as the best effective alternatives to antibiotics. The aim of this study
was to characterize the probiotic potential of lactobacilli for use in swine farming by using in
vitro evaluation methods. A total of 106 lactic acid bacterial isolates, originating from porcine
feces, were first screened for the capacity to survive stresses considered important for putative
probiotic strains. Sixteen isolates showed notable acid and bile resistance, antibacterial
activity, and adherence to intestinal porcine epithelial cells (IPEC-1). One isolate, LR1,
identified as Lactobacillus reuteri, was selected for extensive study of its probiotic and
functional properties in IPEC-1 cell models. L. reuteri LR1 exhibited good adhesion to IPEC-1
cells and could inhibit the adhesion of enterotoxigenic Escherichia coli (ETEC) to IPEC-1 cells.
L. reuteri LR1 could also modulate transcript and protein expression of cytokines involved in
inflammation in IPEC-1 cells; the Lactobacillus strain inhibited the ETEC-induced expression of
proinflammatory transcripts (IL-6 and TNF-α) and protein (IL-6), and increased the level of
anti-inflammatory cytokine (IL-10). Measurement of the permeation of FD-4 showed that
L. reuteri LR1 could maintain barrier integrity in monolayer IPEC-1 cells exposed to ETEC.
Immunolocalization experiments showed L. reuteri LR1 could also prevent ETEC-induced
tight junction ZO-1 disruption. Together, these results indicate that L. reuteri LR1 exhibits
desirable probiotic properties and could be a potential probiotic for use in swine production.

Keywords: Lactobacillus reuteri, in vitro evaluation, probiotic properties, enterotoxigenic Escherichia coli

• Abstract

References

1. Araya M, Morelli L, Reid G, Sanders M, Stanton C, Pineiro M, Ben Embarek P. 2002. Guidelines for the evaluation of probiotics in food. Report of a Joint FAO/WHO working group on drafting guidelines for the evaluation of probiotics in food. London, Ontario, Canada.
2. Cario E, Gerken G, Podolsky DK. 2004. Toll-like receptor 2 enhances ZO-1-associated intestinal epithelial barrier integrity via protein kinase C. Gastroenterology 127: 224-238.
   
3. Casas IA, Dobrogosz WJ. 2000. Validation of the probiotic concept: Lactobacillus reuteri confers broad-spectrum protection against disease in humans and animals. Microbial Ecol. Health Dis. 12: 247-285.
   
4. De Angelis M, Siragusa S, Berloco M, Caputo L, Settanni L, Alfonsi G, et al. 2006. Selection of potential probiotic lactobacilli from pig feces to be used as additives in pelleted feeding. Res. Microbiol. 157: 792-801.
   
5. Dibner J, Richards J. 2005. Antibiotic growth promoters in agriculture: history and mode of action. Poultry Sci. 84: 634-643.
   
6. Fairbrother JM, Nadeau É, Gyles CL. 2005. Escherichia coli in postweaning diarrhea in pigs: an update on bacterial types, pathogenesis, and prevention strategies. Anim. Health Res. Rev. 6: 17-39.
   
7. Foligne B, Nutten S, Grangette C, Dennin V, Goudercourt D, Poiret S, et al. 2007. Correlation between in vitro and in vivo immunomodulatory properties of lactic acid bacteria. World J. Gastroenterol. 13: 236-243.
   
8. Frese SA, Benson AK, Tannock GW, Loach DM, Kim J, Zhang M, et al. 2011. The evolution of host specialization in the vertebrate gut symbiont Lactobacillus reuteri. PLoS Genet. 7: e1001314.
   
9. Gaggìa F, Mattarelli P, Biavati B. 2010. Probiotics and prebiotics in animal feeding for safe food production. Int. J. Food Microbiol. 141: S15-S28.
   
10. Heller KJ. 2001. Probiotic bacteria in fermented foods:product characteristics and starter organisms. Am. J. Clin. Nutr. 73: 374s-379s.
    
11. Juntunen M, Kirjavainen P, Ouwehand A, Salminen S, Isolauri E. 2001. Adherence of probiotic bacteria to human intestinal mucus in healthy infants and during rotavirus infection. Clin. Diagn. Lab. Immunol. 8: 293-296.
    
12. Kaushik JK, Kumar A, Duary RK, Mohanty AK, Grover S, Batish VK. 2009. Functional and probiotic attributes of an indigenous isolate of Lactobacillus plantarum. PLoS One 4: e8099.
    
13. Kim HB, Isaacson RE. 2015. The pig gut microbial diversity:understanding the pig gut microbial ecology through the next-generation high-throughput sequencing. Vet. Microbiol. 177: 242-251.
    
14. Koh SY, George S, Brözel V, Moxley R, Francis D, Kaushik RS. 2008. Porcine intestinal epithelial cell lines as a new in vitro model for studying adherence and pathogenesis of enterotoxigenic Escherichia coli. Vet. Microbiol. 130: 191-197.
    
15. Kotzamanidis C, Kourelis A, Litopoulou-Tzanetaki E, Tzanetakis N, Yiangou M. 2010. Evaluation of adhesion capacity, cell surface traits and immunomodulatory activity of presumptive probiotic Lactobacillus strains. Int. J. Food Microbiol. 140: 154-163.
    
16. Kowapradit J, Opanasopit P, Ngawhiranpat T, Apirakaramwong A, Rojanarata T, Ruktanonchai U, Sajomsang W. 2008. Methylated N-(4-N, N-dimethylaminobenzyl) chitosan, a novel chitosan derivative, enhances paracellular permeability across intestinal epithelial cells (Caco-2). AAPS PharmSciTech 9:1143-1152.
    
17. Lähteinen T, Malinen E, Koort JM, Mertaniemi-Hannus U, Hankimo T, Karikoski N, et al. 2010. Probiotic properties of Lactobacillus isolates originating from porcine intestine and feces. Anaerobe 16: 293-300.
    
18. Lebeer S, Vanderleyden J, De Keersmaecker SC. 2010. Host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens. Nat. Rev. Microbiol. 8: 171-184.
    
19. Lee DY, Seo Y-S, Rayamajhi N, Kang ML, Lee SI, Yoo HS. 2009. Isolation, characterization, and evaluation of wild isolates of Lactobacillus reuteri from pig feces. J. Microbiol. 47:663-672.
    
20. Lee J, Yun HS, Cho KW, Oh S, Kim SH, Chun T, et al. 2011. Evaluation of probiotic characteristics of newly isolated Lactobacillus spp.: immune modulation and longevity. Int. J. Food Microbiol. 148: 80-86.
    
21. Meurens F, Berri M, Auray G, Melo S, Levast B, VirlogeuxPayant I, et al. 2009. Early immune response following Salmonella enterica subspecies enterica serovar Typhimurium infection in porcine jejunal gut loops. Vet. Res. 40: 1.
    
22. Oh PL, Benson AK, Peterson DA, Patil PB, Moriyama EN, Roos S, Walter J. 2010. Diversification of the gut symbiont Lactobacillus reuteri as a result of host-driven evolution. ISME J. 4: 377-387.
    
23. Roselli M, Finamore A, Britti MS, Konstantinov SR, Smidt H, de Vos WM, Mengheri E. 2007. The novel porcine Lactobacillus sobrius strain protects intestinal cells from enterotoxigenic Escherichia coli K88 infection and prevents membrane barrier damage. J. Nutr. 137: 2709-2716.
    
24. Saarela M, Mogensen G, Fondén R, Mättö J, MattilaSandholm T. 2000. Probiotic bacteria: safety, functional and technological properties. J. Biotechnol. 84: 197-215.
    
25. Seo BJ, Mun MR, Kim C-J, Lee I, Chang Y-H, Park Y-H. 2010. Bile tolerant Lactobacillus reuteri isolated from pig feces inhibits enteric bacterial pathogens and porcine rotavirus. Vet. Res. Commun. 34: 323-333.
    
26. Talarico T, Casas I, Chung TC, Dobrogosz W. 1988. Production and isolation of reuterin, a growth inhibitor produced by Lactobacillus reuteri. Antimicrob. Agents Chemother. 32: 1854-1858.
    
27. Tsukita S, Furuse M, Itoh M. 2001. Multifunctional strands in tight junctions. Nat. Rev. Mol. Cell Biol. 2: 285-293.
    
28. Tuomola EM, Ouwehand AC, Salminen SJ. 1999. The effect of probiotic bacteria on the adhesion of pathogens to human intestinal mucus. FEMS Immunol. Med. Microbiol. 26: 137-142.
    
29. Vollenweider S, Lacroix C. 2004. 3-Hydroxypropionaldehyde:applications and perspectives of biotechnological production. Appl. Microbiol. Biotechnol. 64: 16-27.
    
30. Walter J, Britton RA, Roos S. 2011. Host-microbial symbiosis in the vertebrate gastrointestinal tract and the Lactobacillus reuteri paradigm. Proc. Natl. Acad. Sci. USA 108: 4645-4652.
    
31. Yu LC, Flynn AN, Turner JR, Buret AG. 2005. SGLT-1mediated glucose uptake protects intestinal epithelial cells against LPS-induced apoptosis and barrier defects: a novel cellular rescue mechanism? FASEB J. 19: 1822-1835.
    
32. Zanello G, Berri M, Dupont J, Sizaret P-Y, D’Inca R, Salmon H, Meurens F. 2011. Saccharomyces cerevisiae modulates immune gene expressions and inhibits ETEC-mediated ERK1/2 and p38 signaling pathways in intestinal epithelial cells. PLoS One 6: e18573.