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Research Collection Research Collection Doctoral Thesis Mechanistic study of bifidobacterium thermophilum RBL67 for Salmonella inhibition using in vitro fermentation and in vivo swine models Author(s): Tanner, Sabine Publication Date: 2014 Permanent Link: https://doi.org/10.3929/ethz-a-010276721 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library DISS. ETH NO. 22115 MECHANISTIC STUDY OF BIFIDOBACTERIUM THERMOPHILUM RBL67 FOR SALMONELLA INHIBITION USING IN VITRO FERMENTATION AND IN VIVO SWINE MODELS A thesis submitted to attain the degree of DOCTOR OF SCIENCES of ETH ZURICH (Dr. sc. ETH Zurich) presented by SABINE Amani TANNER MSc ETH in Food Science born on 13.01.1986 citizen of Basel-Stadt (BS) and Eriswil (BE) accepted on the recommendation of Prof. Dr. Christophe Lacroix, examiner Prof. Dr. Ismaïl Fliss, co-examiner Dr. Christophe Chassard, co-examiner 2014 Contents Abbreviations 4 Summary 7 Zusammenfassung 11 Chapter 1 General introduction Gastrointestinal physiology of the pig 18 Salmonella in pigs 38 Microbial interactions in complex intestinal ecosystems 60 Background and objectives of the thesis 66 Chapter 2 In vitro continuous fermentation model (PolyFermS) of the swine proximal colon for simultaneous testing on the same gut microbiota 69 Chapter 3 Synergistic effects of Bifidobacterium thermophilum RBL67 and selected prebiotics on inhibition of Salmonella colonization in the swine proximal colon PolyFermS model 97 Chapter 4 Unraveling the transcriptome response of Salmonella enterica subsp. enterica serovar Typhimurium N-15 and Bifidobacterium thermophilum RBL67 grown in co-culture 121 Chapter 5 Effect of Bifidobacterium thermophilum RBL67 and fructooligosaccharide on the gut microbiota of Göttingen minipigs 151 Chapter 6 General conclusions and perspectives 175 Bibliography 185 Appendix 207 Acknowledgements 255 Curriculum vitae 257 3 Abbreviations BCFA Branched-chain fatty acid(s) bp base pairs CDC Centers for Disease Control and Prevention cDNA complementary DNA cfu Colony-forming units DC Dendritic cell(s) DGGE Denaturing gradient gel electrophoresis DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid EFSA European Food Safety Authority FAO Food and Agriculture Organization of the United Nations FISH Fluorescent in situ hybridization FOS Fructooligosaccharide GALT Gut-associated lymphoid tissue GIT Gastrointestinal tract GOS Galactooligosaccharide HPLC High performance liquid chromatography H2S Hydrogen sulfide IgA Immunoglobulin A IL- Interleukin MOS Mannanoligosaccharide mRNA Messenger RNA MRS(-C) Man Rogosa Sharpe (cysteine-HCl) broth NCBI National Center for Biotechnology Information OD Optical density ORF Open reading frame OTU Operational taxonomic unit 4 Abbreviations PCR Polymerase chain reaction qPCR Quantitative polymerase chain reaction RDP Ribosomal database project RT Retention time rRNA Ribosomal ribonucleic acid SCFA Short chain fatty acids SD Standard deviation SPI-1/2 Salmonella pathogenicity island 1/2 SRA Sequence read archive TE tris(hydroxymethyl)aniomethane-EDTA TGGE Temperature gradient gel electrophoresis WHO World Health Organization xfp Xylulose-5-phosphate/fructose-6-phosphate phosphoketolase gene YCFA Yeast extract-casein hydrolysate-fatty acid medium 5 Summary 7 Summary There is a high prevalence of Salmonella in swine livestock production that negatively impacts animal health and performance. Pigs are also known to be persistent carriers of Salmonella, which can pose a significant health risk to the consumer due to transmission of the pathogen via the food chain. After the ban of in-feed antibiotics in the European Union and in Switzerland, probiotics and prebiotics are promising alternative strategies to ensure animal gut health, thereby maintaining animal welfare and productivity of the swine livestock production. The human fecal isolate Bifidobacterium thermophilum RBL67 (RBL67) has been previously shown to possess remarkable features, including the production of a bacteriocin-like inhibitory substance (BLIS), antagonism to Salmonella and high adhesion capacity to human intestinal cell lines. B. thermophilum is predominantly encountered in the gastrointestinal tract (GIT) of animals, including pigs. The overall aim of this study was to further investigate B. thermophilum RBL67 and its anti-Salmonella activity for potential application in swine livestock production. To study nutritional additives and microbial interactions in the complex environment of the swine gut, we successfully developed and validated a novel in vitro PolyFermS continuous fermentation model, simulating the swine proximal colon. The porcine PolyFermS model was designed as a two-stage multiple reactors model with a first reactor (inoculum reactor; IR) containing immobilized fecal swine microbiota. IR was used to constantly inoculate five subsequent second-stage reactors, one control and four test reactors, with 10 % effluent, while the remaining 90 % of inflow was fresh medium, formulated to mimic the swine ileal chyme. The novel porcine PolyFermS was validated during 54 days continuous fermentation. A high and stable bacterial composition, diversity and metabolite production was measured in the effluents, akin to in vivo conditions, with the same microbiota in all second-stage reactors. We assume that the swine PolyFermS allows the simultaneous testing of different treatments in parallel and compared to a control. We then used the porcine PolyFermS model to investigate the effect of B. thermophilum RBL67 alone and combined with fructooligosaccharide (FOS), galactooligosaccharide (GOS) and mannanoligosaccharide (MOS) on Salmonella enterica subsp. enterica serovar Typhimurium N-15 (N-15). FOS and GOS increased total short chain fatty acid (SCFA) 8 Summary production, mainly acetate and propionate, and inhibited N-15 colonization. The antagonistic effect on Salmonella was enhanced when FOS and GOS were combined with RBL67, while the increase in SCFA production was similar to the prebiotics alone. Our data suggest that increased SCFA production contributes to the inhibition of N-15 colonization in our in vitro model, but that additional antimicrobial effects are present when prebiotics are combined with RBL67. Furthermore, RBL67 combined with FOS stimulated butyrate production, while with FOS alone no butyrate stimulation was observed. This could be of interest for gut health preservation due to the numerous health-related properties attributed to butyrate. RBL67 alone and combined with MOS increased acetate and propionate production, respectively, but showed only limited effect on Salmonella inhibition compared to the control. In a third step we studied the transcriptome responses when RBL67 and N-15 are co-cultured to gain insights into the mechanisms of probiotic-pathogen interaction on molecular level. RBL67 and N-15 were cultured single and in mixture under pH-controlled (6.0) conditions. Sampling for RNA extraction was done after 4 and 5 hours of growth for N-15 and RBL67 mono- and co-cultures, respectively. Transcriptome analysis was performed by RNA- sequencing and mapping of generated reads (>17 Mio per sample) to the genomes of RBL67 and S. Typhimurium LT2. The growth of RBL67 was stimulated in presence of N-15, but this effect could not be explained. In contrast, growth of N-15 was decreased in presence of RBL67, yielding significantly lower cell numbers of N-15 in co- compared to mono-culture. Transcriptome analysis revealed genes associated with Salmonella virulence (type III secretion systems encoding, fimbrial adherence determinants) higher regulated in the presence of RBL67, while flagellar genes were repressed. Salmonella virulence is subjected to a complex regulatory network, tightly controlling the expression of virulence factors. We hypothesize that RBL67 triggers virulence gene expression of Salmonella N-15 prematurely, which leads to redundant energy expenditure and consequently reduced growth. In the competitive environment of the gut, reduced growth could lead to a loss of competition and enhanced clearance of the pathogen from the gut. Our study provided first insights into probiotic-pathogen interaction under model conditions and suggests a mechanism for probiotic protection of Salmonella infection. 9 Summary At last we performed a first swine in vivo study to investigate the impact of RBL67 alone and combined with FOS on gut microbiota composition and activity in Göttingen minipigs. Eight minipigs were randomly allocated into two treatments groups and a cross-sectional study design was applied. Minipigs (four per group) were fed with a basal diet supplemented with 8 g/day probiotic powder (1 x 109 cfu g-1; PRO), 8 g probiotic powder plus 8 g/day FOS (SYN) or 8 g/d skim milk powder in the control group (CON). RBL67 was consistently detected at 105 – 106 copies g-1 in feces, cecum and the colon when feeding with PRO or SYN diets. Compared to the PRO, the SYN diet significantly increased Bifidobacterium numbers in the colon. The highest numerical values of B. thermophilum were measured in the cecum of pigs fed with SYN, which suggests a promotion effect of FOS. Experimental diets did not induce large shifts in gut microbiota composition, supporting the safety of RBL67. Furthermore, our data indicate that the Göttingen minipig holds promise for a novel model organism in gut microbiota research in pigs, but there is need for a
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