A balanced microbiota of the gastrointestinal tract (GIT) is a prerequisite for a healthy host. The GIT microbiota in preterm infants is determined by the method of delivery and nutrition. Probiotics can improve the GIT microbiota balance and suitable animal models are required to verify their harmlessness. Preterm gnotobiotic piglets were colonized with Lactobacillus rhamnosus GG (LGG) to evaluate its safety and possible protective action against infection with an enteric pathogen, Salmonella Typhimurium (ST). Clinical signs (anorexia, somnolence, fever and diarrhea), bacterial interference and translocation, intestinal histopathology, transcriptions of claudin-1, occludin and interferon (IFN)-γ, intestinal and systemic protein levels of interleukin (IL)-8, IL-12/23 p40 and IFN-γ were compared among (i) germ-free, (ii) LGG-colonized, (iii) ST-infected and (iv) LGG-colonized and subsequently ST-infected piglets for 24 h. Both LGG and ST-colonized the GIT; LGG translocated in some cases into mesenteric lymph nodes and the spleen but did not cause bacteremia and clinical changes. ST caused clinical signs of gastroenteritis, translocated into mesenteric lymph nodes, the spleen, liver and blood, increased claudin-1 and IFN-γ transcriptions, but decreased occludin transcription and increased local and systemic levels of IL-8 and IL-12/23 p40. Previous colonization with LGG reduced ST colonization in the jejunum and translocation into the liver, spleen and blood. It partially ameliorated histopathological changes in the intestine, reduced IL-8 levels in the jejunum and plasma and IL-12/23 p40 in the jejunum. The preterm gnotobiotic piglet model of the vulnerable preterm immunocompromised infant is useful to verify the safety of probiotics and evaluate their protective effect.

Laboratory of Gnotobiology Institute of Microbiology of the Academy of Sciences of the Czech Republic Novy Hradek Czech Republic

Zobrazit více v PubMed

Glass HC, Costarino AT, Stayer SA, Brett CM, Cladis F, Davis PJ. Outcomes for extremely premature infants. Anesth Analg 2015; 120:1337–51. PubMed PMC

Backhed F, Roswall J, Peng Y et al Dynamics and stabilization of the human gut microbiome during the first year of life. Cell Host Microbe 2015; 17:690–703. PubMed

Marques TM, Wall R, Ross RP, Fitzgerald GF, Ryan CA, Stanton C. Programming infant gut microbiota: influence of dietary and environmental factors. Curr Opin Biotechnol 2010; 21:149–56. PubMed

Brooks B, Firek BA, Miller CS et al Microbes in the neonatal intensive care unit resemble those found in the gut of premature infants. Microbiome 2014; 2:1. PubMed PMC

Ramasethu J. Prevention and treatment of neonatal nosocomial infections. Matern Health Neonatol Perinatol 2017; 3:5. PubMed PMC

Bedford Russell AR. Neonatal sepsis. Paediatr Child Health 2015; 25:271–5.

Ghazal P, Dickinson P, Smith CL. Early life response to infection. Curr Opin Infect Dis 2013; 26:213–8. PubMed

Hill C, Guarner F, Reid G et al Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol 2014; 11:506–14. PubMed

Uberos J, Aguilera‐Rodriguez E, Jerez‐Calero A, Molina‐Oya M, Molina‐Carballo A, Narbona‐Lopez E. Probiotics to prevent necrotising enterocolitis and nosocomial infection in very low birth weight preterm infants. Br J Nutr 2017; 117:994–1000. PubMed

Pace F, Pace M, Quartarone G. Probiotics in digestive diseases: focus on Lactobacillus GG. Minerva Gastroenterol Dietol 2015; 61:273–92. PubMed

Gorbach SL, Chang TW, Goldin B. Successful treatment of relapsing Clostridium difficile colitis with Lactobacillus GG. Lancet 1987; 2:1519. PubMed

Meyer MP, Alexander T. Reduction in necrotizing enterocolitis and improved outcomes in preterm infants following routine supplementation with Lactobacillus GG in combination with bovine lactoferrin. J Neonatal Perinatal Med 2017; 10:249–55. PubMed

Neu J. Routine probiotics for premature infants: let’s be careful! J Pediatr 2011; 158:672–4. PubMed PMC

Lunney JK. Advances in swine biomedical model genomics. Int J Biol Sci 2007; 3:179–84. PubMed PMC

Zhang Q, Widmer G, Tzipori S. A pig model of the human gastrointestinal tract. Gut Microbes 2013; 4:193–200. PubMed PMC

Meurens F, Summerfield A, Nauwynck H, Saif L, Gerdts V. The pig: a model for human infectious diseases. Trends Microbiol 2012; 20:50–7. PubMed PMC

Sangild PT, Thymann T, Schmidt M, Stoll B, Burrin DG, Buddington RK. Invited review: the preterm pig as a model in pediatric gastroenterology. J Animal Sci 2013; 91:4713–29. PubMed PMC

Nguyen DN, Jiang P, Frokiaer H, Heegaard PM, Thymann T, Sangild PT. Delayed development of systemic immunity in preterm pigs as a model for preterm infants. Sci Rep 2016; 6:36816. PubMed PMC

Splichalova A, Slavikova V, Splichalova Z, Splichal I. Preterm life in sterile conditions: a study on preterm, germ‐free piglets. Front Immunol 2018; 9:220. PubMed PMC

Zhang S, Kingsley RA, Santos RL et al Molecular pathogenesis of Salmonella enterica serotype Typhimurium‐induced diarrhea. Infect Immun 2003; 71:1–12. PubMed PMC

Keestra‐Gounder AM, Tsolis RM, Baumler AJ. Now you see me, now you don’t: the interaction of Salmonella with innate immune receptors. Nat Rev Microbiol 2015; 13:206–16. PubMed

Santos RL, Tsolis RM, Baumler AJ, Adams LG. Pathogenesis of Salmonella‐induced enteritis. Braz J Med Biol Res 2003; 36:3–12. PubMed

Mandel L, Travnicek J. The minipig as a model in gnotobiology. Nahrung 1987; 31:613–8. PubMed

Foster N, Lovell MA, Marston KL et al Rapid protection of gnotobiotic pigs against experimental salmonellosis following induction of polymorphonuclear leukocytes by avirulent Salmonella enterica . Infect Immun 2003; 71:2182–91. PubMed PMC

Castellheim A, Thorgersen EB, Hellerud BC et al New biomarkers in an acute model of live Escherichia coli‐induced sepsis in pigs. Scand J Immunol 2008; 68:75–84. PubMed

Ingram JP, Brodsky IE, Balachandran S. Interferon‐gamma in Salmonella pathogenesis: new tricks for an old dog. Cytokine 2017; 98:27–32. PubMed PMC

Deshpande G, Jape G, Rao S, Patole S. Benefits of probiotics in preterm neonates in low‐income and medium‐income countries: a systematic review of randomised controlled trials. BMJ Open 2017; 7:e017638. PubMed PMC

Doron S, Snydman DR. Risk and safety of probiotics. Clin Infect Dis 2015; 60 (Suppl 2):S129–34. PubMed PMC

Dani C, Coviello CC, Corsini I, Arena I, Antonelli I, Rossolini GM. Lactobacillus sepsis and probiotic therapy in newborns: two new cases and literature review. AJP Rep 2016; 6:e25–9. PubMed PMC

Salmon H, Berri M, Gerdts V, Meurens F. Humoral and cellular factors of maternal immunity in swine. Dev Comp Immunol 2009; 33:384–93. PubMed

Butler JE, Lager KM, Splichal I et al The piglet as a model for B cell and immune system development. Vet Immunol Immunopathol 2009; 128:147–70. PubMed PMC

Sangild PT. Gut responses to enteral nutrition in preterm infants and animals. Exp Biol Med (Maywood) 2006; 231:1695–711. PubMed

Mandel L, Travnicek J. Haematology of conventional and germfree miniature Minnesota piglets. I. Blood picture. Z Versuchstierkd 1982; 24:299–307. PubMed

Melville JM, Moss TJ. The immune consequences of preterm birth. Front Neurosci 2013; 7:79. PubMed PMC

Hurley D, McCusker MP, Fanning S, Martins M. Salmonella‐host interactions – modulation of the host innate immune system. Front Immunol 2014; 5:481. PubMed PMC

Petrova MI, Imholz NC, Verhoeven TL et al Lectin‐like molecules of Lactobacillus rhamnosus GG inhibit pathogenic Escherichia coli and Salmonella biofilm formation. PLOS ONE 2016; 11:e0161337. PubMed PMC

De Keersmaecker SC, Verhoeven TL, Desair J, Marchal K, Vanderleyden J, Nagy I. Strong antimicrobial activity of Lactobacillus rhamnosus GG against Salmonella typhimurium is due to accumulation of lactic acid. FEMS Microbiol Lett 2006; 259:89–96. PubMed

Vlasova AN, Chattha KS, Kandasamy S et al Lactobacilli and bifidobacteria promote immune homeostasis by modulating innate immune responses to human rotavirus in neonatal gnotobiotic pigs. PLOS ONE 2013; 8:e76962. PubMed PMC

Wen K, Liu F, Li G et al Lactobacillus rhamnosus GG dosage affects the adjuvanticity and protection against rotavirus diarrhea in gnotobiotic pigs. J Pediatr Gastroenterol Nutr 2015; 60:834–43. PubMed

Wu S, Yuan L, Zhang Y et al Probiotic Lactobacillus rhamnosus GG mono‐association suppresses human rotavirus‐induced autophagy in the gnotobiotic piglet intestine. Gut Pathog 2013; 5:22. PubMed PMC

Mileti E, Matteoli G, Iliev ID, Rescigno M. Comparison of the immunomodulatory properties of three probiotic strains of Lactobacilli using complex culture systems: prediction for in vivo efficacy. PLOS ONE 2009; 4:e7056. PubMed PMC

Levy M, Kolodziejczyk AA, Thaiss CA, Elinav E. Dysbiosis and the immune system. Nat Rev Immunol 2017; 17:219–32. PubMed

Knoop KA, McDonald KG, Kulkarni DH, Newberry RD. Antibiotics promote inflammation through the translocation of native commensal colonic bacteria. Gut 2016; 65:1100–9. PubMed PMC

Kandasamy S, Vlasova AN, Fischer D et al Differential effects of Escherichia coli Nissle and Lactobacillus rhamnosus strain GG on human rotavirus binding, infection, and B cell immunity. J Immunol 2016; 196:1780–9. PubMed PMC

Haraga A, Ohlson MB, Miller SI. Salmonellae interplay with host cells. Nat Rev Microbiol 2008; 6:53–66. PubMed

Jones BD, Ghori N, Falkow S. Salmonella typhimurium initiates murine infection by penetrating and destroying the specialized epithelial M cells of the Peyer’s patches. J Exp Med 1994; 180:15–23. PubMed PMC

Francis CL, Starnbach MN, Falkow S. Morphological and cytoskeletal changes in epithelial cells occur immediately upon interaction with Salmonella typhimurium grown under low‐oxygen conditions. Mol Microbiol 1992; 6:3077–87. PubMed

Vazquez‐Torres A, Jones‐Carson J, Baumler AJ et al Extraintestinal dissemination of Salmonella by CD18‐expressing phagocytes. Nature 1999; 401:804–8. PubMed

Niess JH, Brand S, Gu X et al CX3CR49‐mediated dendritic cell access to the intestinal lumen and bacterial clearance. Science 2005; 307:254–8. PubMed

Jepson MA, Schlecht HB, Collares‐Buzato CB. Localization of dysfunctional tight junctions in Salmonella enterica serovar Typhimurium‐infected epithelial layers. Infect Immun 2000; 68:7202–8. PubMed PMC

Splichal I, Trebichavsky I, Splichalova A, Barrow PA. Protection of gnotobiotic pigs against Salmonella enterica serotype typhimurium by rough mutant of the same serotype is accompanied by the change of local and systemic cytokine response. Vet Immunol Immunopathol 2005; 103:155–61. PubMed

Splichalova A, Splichal I, Chmelarova P, Trebichavsky I. Alarmin HMGB1 is released in the small intestine of gnotobiotic piglets infected with enteric pathogens and its level in plasma reflects severity of sepsis. J Clin Immunol 2011; 31:488–97. PubMed

Splichalova A, Trebichavsky I, Rada V, Vlkova E, Sonnenborn U, Splichal I. Interference of Bifidobacterium choerinum or Escherichia coli Nissle 1917 with Salmonella typhimurium in gnotobiotic piglets correlates with cytokine patterns in blood and intestine. Clin Exp Immunol 2011; 163:242–9. PubMed PMC

Hou C, Liu H, Zhang J et al Intestinal microbiota succession and immunomodulatory consequences after introduction of Lactobacillus reuteri I5007 in neonatal piglets. PLOS ONE 2015; 10:e0119505. PubMed PMC

Shirkey TW, Siggers RH, Goldade BG et al Effects of commensal bacteria on intestinal morphology and expression of proinflammatory cytokines in the gnotobiotic pig. Exp Biol Med (Maywood) 2006; 231:1333–45. PubMed

Gunzel D. Claudins: vital partners in transcellular and paracellular transport coupling. Pflugers Arch 2017; 469:35–44. PubMed

Edelblum KL, Shen L, Weber CR et al Dynamic migration of gammadelta intraepithelial lymphocytes requires occludin. Proc Natl Acad Sci U A 2012; 109:7097–102. PubMed PMC

Kohler H, Sakaguchi T, Hurley BP, Kase BA, Reinecker HC, McCormick BA. Salmonella enterica serovar Typhimurium regulates intercellular junction proteins and facilitates transepithelial neutrophil and bacterial passage. Am J Physiol Gastrointest Liver Physiol 2007; 293:G178–G187. PubMed

Galipeau HJ, Verdu EF. The complex task of measuring intestinal permeability in basic and clinical science. Neurogastroenterol Motil 2016; 28:957–65. PubMed

Zamora IJ, Stoll B, Ethun CG et al Low abdominal NIRS values and elevated plasma intestinal fatty acid‐binding protein in a premature piglet model of necrotizing enterocolitis. PLOS ONE 2015; 10:e0125437. PubMed PMC

Basha S, Surendran N, Pichichero M. Immune responses in neonates. Expert Rev Clin Immunol 2014; 10:1171–84. PubMed PMC

Chousterman BG, Swirski FK, Weber GF. Cytokine storm and sepsis disease pathogenesis. Semin Immunopathol 2017; 39:517–28. PubMed

Baggiolini M, Walz A, Kunkel SL. Neutrophil‐activating peptide‐1/interleukin 8, a novel cytokine that activates neutrophils. J Clin Invest 1989; 84:1045–9. PubMed PMC

Maheshwari A, Lu W, Lacson A et al Effects of interleukin‐8 on the developing human intestine. Cytokine 2002; 20:256–67. PubMed

Maheshwari A, Voitenok NN, Akalovich S et al Developmental changes in circulating IL‐8/CXCL8 isoforms in neonates. Cytokine 2009; 46:12–6. PubMed PMC

Nguyen DN, Sangild PT, Ostergaard MV, Bering SB, Chatterton DE. Transforming growth factor‐beta2 and endotoxin interact to regulate homeostasis via interleukin‐8 levels in the immature intestine. Am J Physiol Gastrointest Liver Physiol 2014; 307:G689–G699. PubMed

Maheshwari A, Schelonka RL, Dimmitt RA et al Cytokines associated with necrotizing enterocolitis in extremely‐low‐birth‐weight infants. Pediatr Res 2014; 76:100–8. PubMed PMC

Stoy ACF, Heegaard PMH, Skovgaard K, Bering SB, Bjerre M, Sangild PT. Increased intestinal inflammation and digestive dysfunction in preterm pigs with severe necrotizing enterocolitis. Neonatology 2017; 111:289–96. PubMed

Chauhan N, Tiwari S, Jain U. Potential biomarkers for effective screening of neonatal sepsis infections: an overview. Microb Pathog 2017; 107:234–42. PubMed

Collado‐Romero M, Arce C, Ramirez‐Boo M, Carvajal A, Garrido JJ. Quantitative analysis of the immune response upon Salmonella typhimurium infection along the porcine intestinal gut. Vet Res 2010; 41:23. PubMed PMC

Uribe JH, Collado‐Romero M, Zaldivar‐Lopez S et al Transcriptional analysis of porcine intestinal mucosa infected with Salmonella typhimurium revealed a massive inflammatory response and disruption of bile acid absorption in ileum. Vet Res 2016; 47:11. PubMed PMC

Henard CA, Vazquez‐Torres A. Nitric oxide and Salmonella pathogenesis. Front Microbiol 2011; 2:84. PubMed PMC

Meurens F, Berri M, Auray G et al Early immune response following Salmonella enterica subspecies enterica serovar Typhimurium infection in porcine jejunal gut loops. Vet Res 2009; 40:5. PubMed PMC

Bette M, Jin SC, Germann T et al Differential expression of mRNA encoding interleukin‐12 p35 and p40 subunits in situ. Eur J Immunol 1994; 24:2435–40. PubMed

Croxford AL, Kulig P, Becher B. IL‐12‐and IL‐23 in health and disease. Cytokine Growth Factor Rev 2014; 25:415–21. PubMed

Ling P, Gately MK, Gubler U et al Human IL‐12 p40 homodimer binds to the IL‐12 receptor but does not mediate biologic activity. J Immunol 1995; 154:116–27. PubMed

Awoniyi M, Miller SI, Wilson CB, Hajjar AM, Smith KD. Homeostatic regulation of Salmonella‐induced mucosal inflammation and injury by IL‐23. PLOS ONE 2012; 7:e37311. PubMed PMC

Trinchieri G. Interleukin‐12: a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen‐specific adaptive immunity. Annu Rev Immunol 1995; 13:251–76. PubMed

Thiemann S, Smit N, Roy U et al Enhancement of IFN‐gamma production by distinct commensals ameliorates Salmonella‐induced disease. Cell Host Microbe 2017; 21:682–94. PubMed

Jeong KI, Zhang Q, Nunnari J, Tzipori S. A piglet model of acute gastroenteritis induced by Shigella dysenteriae type 1. J Infect Dis 2010; 201:903–11. PubMed PMC

Splichal I, Trebichavsky I, Muneta Y, Mori Y. Early cytokine response of gnotobiotic piglets to Salmonella enterica serotype typhimurium . Vet Res 2002; 33:291–7. PubMed

Knetter SM, Bearson SM, Huang TH et al Salmonella enterica serovar typhimurium‐infected pigs with different shedding levels exhibit distinct clinical, peripheral cytokine and transcriptomic immune response phenotypes. Innate Immun 2015; 21:227–41. PubMed

Defined Pig Microbiota Mixture as Promising Strategy against Salmonellosis in Gnotobiotic Piglets

Commensal Bacteria Impact on Intestinal Toll-like Receptor Signaling in Salmonella-Challenged Gnotobiotic Piglets

Defined Pig Microbiota with a Potential Protective Effect against Infection with Salmonella Typhimurium

Monoassociation of Preterm Germ-Free Piglets with Bifidobacterium animalis Subsp. lactis BB-12 and Its Impact on Infection with Salmonella Typhimurium

High Mobility Group Box 1 and TLR4 Signaling Pathway in Gnotobiotic Piglets Colonized/Infected with L. amylovorus, L. mucosae, E. coli Nissle 1917 and S. Typhimurium

Impact of the Lipopolysaccharide Chemotype of Salmonella Enterica Serovar Typhimurium on Virulence in Gnotobiotic Piglets

Colonization of Germ-Free Piglets with Commensal Lactobacillus amylovorus, Lactobacillus mucosae, and Probiotic E. coli Nissle 1917 and Their Interference with Salmonella Typhimurium