In vitro continuous fermentation model (PolyFermS) of the swine proximal colon for simultaneous testing on the same gut microbiota

Sabine A Tanner, Annina Zihler Berner, Eugenia Rigozzi, Franck Grattepanche, Christophe Chassard, Christophe Lacroix, Sabine A Tanner, Annina Zihler Berner, Eugenia Rigozzi, Franck Grattepanche, Christophe Chassard, Christophe Lacroix

Abstract

In vitro gut modeling provides a useful platform for a fast and reproducible assessment of treatment-related changes. Currently, pig intestinal fermentation models are mainly batch models with important inherent limitations. In this study we developed a novel in vitro continuous fermentation model, mimicking the porcine proximal colon, which we validated during 54 days of fermentation. This model, based on our recent PolyFermS design, allows comparing different treatment effects on the same microbiota. It is composed of a first-stage inoculum reactor seeded with immobilized fecal swine microbiota and used to constantly inoculate (10% v/v) five second-stage reactors, with all reactors fed with fresh nutritive chyme medium and set to mimic the swine proximal colon. Reactor effluents were analyzed for metabolite concentrations and bacterial composition by HPLC and quantitative PCR, and microbial diversity was assessed by 454 pyrosequencing. The novel PolyFermS featured stable microbial composition, diversity and metabolite production, consistent with bacterial activity reported for swine proximal colon in vivo. The constant inoculation provided by the inoculum reactor generated reproducible microbial ecosystems in all second-stage reactors, allowing the simultaneous investigation and direct comparison of different treatments on the same porcine gut microbiota. Our data demonstrate the unique features of this novel PolyFermS design for the swine proximal colon. The model provides a tool for efficient, reproducible and cost-effective screening of environmental factors, such as dietary additives, on pig colonic fermentation.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Experimental reactor set-up and time…
Figure 1. Experimental reactor set-up and time schedule of the swine PolyFermS model.
IR: inoculum reactor, containing immobilized swine feces (30% v/v); CR: control reactor; TR1-TR4: test reactors 1-4; M: fresh nutritive medium supply; S: effluent sampling; F: flow rate; Stab: stabilization period; T: treatment period; W: wash period
Figure 2. Daily main SCFA concentrations in…
Figure 2. Daily main SCFA concentrations in fermentation effluents of IR and CR measured by HPLC.
Initial stabilization: stabilization period in continuous mode to reach pseudo steady-state; closed symbol: IR; open symbol: CR; (▴, Δ) total SCFA, (•, ○) acetate, (♦, ◊) propionate, and (▪, □) butyrate.
Figure 3. Microbial composition in the fecal…
Figure 3. Microbial composition in the fecal inoculum (FI), IR and CR measured by 454 pyrosequencing.
The relative abundance on family level is shown. Values

Figure 4. Main metabolites in CR and…

Figure 4. Main metabolites in CR and TR1-4 on the last day of each stabilization…

Figure 4. Main metabolites in CR and TR1-4 on the last day of each stabilization period.
(◊) total SCFA; (○) acetate; (□) propionate; (Δ) butyrate; (black) CR; (blue) TR1; (red) TR2; (green) TR3; (pink) TR4.
Figure 4. Main metabolites in CR and…
Figure 4. Main metabolites in CR and TR1-4 on the last day of each stabilization period.
(◊) total SCFA; (○) acetate; (□) propionate; (Δ) butyrate; (black) CR; (blue) TR1; (red) TR2; (green) TR3; (pink) TR4.

References

    1. Lamendella R, Domingo JW, Ghosh S, Martinson J, Oerther DB (2011) Comparative fecal metagenomics unveils unique functional capacity of the swine gut. BMC Microbiol 11: 103.
    1. Macfarlane GT, Macfarlane S (2007) Models for intestinal fermentation: association between food components, delivery systems, bioavailability and functional interactions in the gut. Curr Opin Biotechnol 18: 156–162.
    1. Russell WR, Hoyles L, Flint HJ, Dumas ME (2013) Colonic bacterial metabolites and human health. Curr Opin Microbiol 16: 246–254.
    1. Payne AN, Zihler A, Chassard C, Lacroix C (2012) Advances and perspectives in in vitro human gut fermentation modeling. Trends Biotechnol 30: 17–25.
    1. Jha R, Bindelle J, Van Kessel A, Leterme P (2011) In vitro fibre fermentation of feed ingredients with varying fermentable carbohydrate and protein levels and protein synthesis by colonic bacteria isolated from pigs. Anim Feed Sci Technol 165: 191–200.
    1. Jonathan MC, van den Borne JJGC, van Wiechen P, da Silva CS, Schols HA, et al. (2012) In vitro fermentation of 12 dietary fibres by faecal inoculum from pigs and humans. Food Chem 133: 889–897.
    1. Lin B, Gong J, Wang Q, Cui S, Yu H, et al. (2011) In-vitro assessment of the effects of dietary fibers on microbial fermentation and communities from large intestinal digesta of pigs. Food Hydrocolloid 25: 180–188.
    1. Zhu WY, Williams BA, Konstantinov SR, Tamminga S, De Vos WM, et al. (2003) Analysis of 16S rDNA reveals bacterial shift during in vitro fermentation of fermentable carbohydrate using piglet faeces as inoculum. Anaerobe 9: 175–180.
    1. Williams BA, Bosch MW, Awati A, Konstantinov SR, Smidt H, et al. (2005) In vitro assessment of gastrointestinal tract (GIT) fermentation in pigs: Fermentable substrates and microbial activity. Anim Res 54: 191–201.
    1. Ricca DM, Ziemer CJ, Kerr BJ (2010) Changes in bacterial communities from swine feces during continuous culture with starch. Anaerobe 16: 516–521.
    1. Messens W, Goris J, Dierick N, Herman L, Heyndrickx M (2010) Inhibition of Salmonella typhimurium by medium-chain fatty acids in an in vitro simulation of the porcine cecum. Vet Microbiol 141: 73–80.
    1. Pinloche E, Williams M, D'Inca R, Auclair E, Newbold CJ (2012) Use of a colon simulation technique to assess the effect of live yeast on fermentation parameters and microbiota of the colon of pig. J Anim Sci 90: 353–355.
    1. Cinquin C, Le Blay G, Fliss I, Lacroix C (2004) Immobilization of infant fecal microbiota and utilization in an in vitro colonic fermentation model. Microb Ecol 48: 128–138.
    1. Cinquin C, Le Blay G, Fliss I, Lacroix C (2006) New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota. FEMS Microbiol Ecol 57: 324–336.
    1. Le Blay G, Chassard C, Baltzer S, Lacroix C (2010) Set up of a new in vitro model to study dietary fructans fermentation in formula-fed babies. Br J Nutr 103: 403–411.
    1. Zihler Berner A, Fuentes S, Dostal A, Payne AN, Vazquez Gutierrez P, et al. (2013) Novel Polyfermentor Intestinal Model (PolyFermS) for controlled ecological studies: validation and effect of pH. PLoS One 8: e77772.
    1. Macfarlane GT, Macfarlane S, Gibson GR (1998) Validation of a three-stage compound continuous culture system for investigating the effect of retention time on the ecology and metabolism of bacteria in the human colon. Microb Ecol 35: 180–187.
    1. Nyannor EK, Williams P, Bedford MR, Adeola O (2007) Corn expressing an Escherichia coli-derived phytase gene: a proof-of-concept nutritional study in pigs. J Anim Sci 85: 1946–1952.
    1. Lee TT, Huang YF, Chiang CC, Chung TK, Chiou PW, et al. (2011) Starch characteristics and their influences on in vitro and pig prececal starch digestion. J Agric Food Chem 59: 7353–7359.
    1. Wang JP, Hong SM, Yan L, Cho JH, Lee HS, et al. (2011) The evaluation of soybean meals from 3 major soybean-producing countries on productive performance and feeding value of pig diets. J Anim Sci 89: 2768–2773.
    1. Kararli TT (1995) Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals. Biopharm Drug Dispos 16: 351–380.
    1. Michel C, Kravtchenko TP, David A, Gueneau S, Kozlowski F, et al. (1998) In vitro prebiotic effects of Acacia gums onto the human intestinal microbiota depends on both botanical origin and environmental pH. Anaerobe 4: 257–266.
    1. Sakata T, Kojima T, Fujieda M, Takahashi M, Michibata T (2003) Influences of probiotic bacteria on organic acid production by pig caecal bacteria in vitro . Proc Nutr Soc 62: 73–80.
    1. Bindelle J, Buldgen A, Boudry C, Leterme P (2007) Effect of inoculum and pepsin-pancreatin hydrolysis on fibre fermentation mecasured by the gas production technique in pigs. Anim Feed Sci Technol 132: 111–122.
    1. von Heimendahl E, Breves G, Abel HJ (2010) Fiber-related digestive processes in three different breeds of pigs. J Anim Sci 88: 972–981.
    1. Dostal A, Fehlbaum S, Chassard C, Zimmermann MB, Lacroix C (2013) Low iron availability in continuous in vitro colonic fermentations induces strong dysbiosis of the child gut microbial consortium and a decrease in main metabolites. FEMS Microbiol Ecol 83: 161–175.
    1. Jost T, Lacroix C, Braegger CP, Chassard C (2012) New insights in gut microbiota establishment in healthy breast fed neonates. PLoS One 7: e44595.
    1. Pluske JR (2013) Feed- and feed additives-related aspects of gut health and development in weanling pigs. J Anim Sci Biotechnol 4: 1.
    1. Guarner F, Malagelada JR (2003) Gut flora in health and disease. Lancet 361: 512–519.
    1. Grieshop CM, Reese DE, Fahey Jr GC (2000) Nonstarch polysaccharides and oligosaccharides in swine nutrition. In: Lewis AJ and Southern LL, editors. Swine Nutrition. Boca Raton, FL: CRC Press. pp. 107–130.
    1. Bird AR, Vuaran M, Crittenden R, Hayakawa T, Playne MJ, et al. (2009) Comparative effects of a high-amylose starch and a fructooligosaccharide on fecal bifidobacteria numbers and short-chain fatty acids in pigs fed Bifidobacterium animalis . Dig Dis Sci 54: 947–954.
    1. Mozzetti V, Grattepanche F, Moine D, Berger B, Rezzonico E, et al. (2012) Transcriptome analysis and physiology of Bifidobacterium longum NCC2705 cells under continuous culture conditions. Benef Microbes 3: 261–272.
    1. Hippe H, Hagelstein A, Kramer I, Swiderski J, Stackebrandt E (1999) Phylogenetic analysis of Formivibrio citricus, Propionivibrio dicarboxylicus, Anaerobiospirillum thomasii, Succinimonas amylolytica and Succinivibrio dextrinosolvens and proposal of Succinivibrionaceae fam. nov. Int J Syst Bacteriol 49 Pt 2: 779–782.
    1. Kim HB, Borewicz K, White BA, Singer RS, Sreevatsan S, et al. (2012) Microbial shifts in the swine distal gut in response to the treatment with antimicrobial growth promoter, tylosin. Proc Natl Acad Sci U S A 109: 15485–15490.
    1. Li RW, Wu S, Li W, Navarro K, Couch RD, et al. (2012) Alterations in the porcine colon microbiota induced by the gastrointestinal nematode Trichuris suis . Infect Immun 80: 2150–2157.
    1. Buzoianu SG, Walsh MC, Rea MC, O'Sullivan O, Cotter PD, et al. (2012) High-throughput sequence-based analysis of the intestinal microbiota of weanling pigs fed genetically modified MON810 maize expressing Bacillus thuringiensis Cry1Ab (Bt maize) for 31 days. Appl Environ Microbiol 78: 4217–4224.
    1. Hosseini E, Grootaert C, Verstraete W, Van de Wiele T (2011) Propionate as a health-promoting microbial metabolite in the human gut. Nutr Rev 69: 245–258.
    1. Possemiers S, Verthe K, Uyttendaele S, Verstraete W (2004) PCR-DGGE-based quantification of stability of the microbial community in a simulator of the human intestinal microbial ecosystem. FEMS Microbiol Ecol 49: 495–507.
    1. Van den Abbeele P, Grootaert C, Marzorati M, Possemiers S, Verstraete W, et al. (2010) Microbial community development in a dynamic gut model is reproducible, colon region specific, and selective for Bacteroidetes and Clostridium cluster IX. Appl Environ Microbiol 76: 5237–5246.
    1. Rajilic-Stojanovic M, Maathuis A, Heilig HG, Venema K, de Vos WM, et al. (2010) Evaluating the microbial diversity of an in vitro model of the human large intestine by phylogenetic microarray analysis. Microbiology 156: 3270–3281.
    1. Looft T, Johnson TA, Allen HK, Bayles DO, Alt DP, et al. (2012) In-feed antibiotic effects on the swine intestinal microbiome. Proc Natl Acad Sci U S A 109: 1691–1696.
    1. Riboulet-Bisson E, Sturme MH, Jeffery IB, O'Donnell MM, Neville BA, et al. (2012) Effect of Lactobacillus salivarius bacteriocin Abp118 on the mouse and pig intestinal microbiota. PLoS One 7: e31113.
    1. Poroyko V, White JR, Wang M, Donovan S, Alverdy J, et al. (2010) Gut microbial gene expression in mother-fed and formula-fed piglets. PLoS One 5: e12459.
    1. Kim HB, Borewicz K, White BA, Singer RS, Sreevatsan S, et al. (2011) Longitudinal investigation of the age-related bacterial diversity in the feces of commercial pigs. Vet Microbiol 153: 124–133.
    1. Haenen D, Zhang J, da Silva CS, Bosch G, van der Meer IM, et al. (2013) A diet high in resistant starch modulates microbiota composition, SCFA concentrations, and gene expression in pig intestine. J Nutr 143: 274–283.
    1. Leser TD, Amenuvor JZ, Jensen TK, Lindecrona RH, Boye M, et al. (2002) Culture-independent analysis of gut bacteria: the pig gastrointestinal tract microbiota revisited. Appl Environ Microbiol 68: 673–690.
    1. Chassard C, Lacroix C (2013) Carbohydrates and the human gut microbiota. Curr Opin Clin Nutr Metab Care 16: 453–460.
    1. Guo X, Xia X, Tang R, Zhou J, Zhao H, et al. (2008) Development of a real-time PCR method for Firmicutes and Bacteroidetes in faeces and its application to quantify intestinal population of obese and lean pigs. Lett Appl Microbiol 47: 367–373.
    1. Ramirez-Farias C, Slezak K, Fuller Z, Duncan A, Holtrop G, et al. (2009) Effect of inulin on the human gut microbiota: stimulation of Bifidobacterium adolescentis and Faecalibacterium prausnitzii . Br J Nutr 101: 541–550.
    1. Furet JP, Firmesse O, Gourmelon M, Bridonneau C, Tap J, et al. (2009) Comparative assessment of human and farm animal faecal microbiota using real-time quantitative PCR. FEMS Microbiol Ecol 68: 351–362.
    1. Bartosch S, Fite A, Macfarlane GT, McMurdo ME (2004) Characterization of bacterial communities in feces from healthy elderly volunteers and hospitalized elderly patients by using real-time PCR and effects of antibiotic treatment on the fecal microbiota. Appl Environ Microbiol 70: 3575–3581.
    1. Cleusix V, Lacroix C, Dasen G, Leo M, Le Blay G (2010) Comparative study of a new quantitative real-time PCR targeting the xylulose-5-phosphate/fructose-6-phosphate phosphoketolase bifidobacterial gene (xfp) in faecal samples with two fluorescence in situ hybridization methods. J Appl Microbiol 108: 181–193.
    1. Stevenson DM, Weimer PJ (2007) Dominance of Prevotella and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR. Appl Microbiol Biotechnol 75: 165–174.

Source: PubMed

Sponsors and Collaborators

Medical Conditions

Drug Interventions

3
Subscribe