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Synergistic effects of Bifidobacterium thermophilum RBL67 and selected prebiotics on inhibition of Salmonella colonization in the swine proximal colon PolyFermS model.

Tanner SA, Chassard C, Zihler Berner A, Lacroix C - Gut Pathog (2014)

Bottom Line: Our study demonstrates the suitability of the porcine PolyFermS in vitro model to study nutritional effects of pro- and prebiotics on gut microbiota composition and activity.The inhibition effects of FOS and GOS on N-15 colonization are partly due to an increased acetate production, while further antimicrobial mechanisms may contribute to an enhanced inhibition with prebiotic-RBL67 combinations.A future direction of this work could be to understand the anti-Salmonella effects of Bifidobacterium thermophilum RBL67 in the presence of prebiotics to unravel the mechanism of this probiotic:pathogen interaction.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Food Biotechnology, Institute of Food, Nutrition and Health, Department of Health Science and Technology, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich, Switzerland.

ABSTRACT

Background: Probiotics and prebiotics are promising strategies to counteract Salmonella prevalence in swine. In the present study, we investigated the effects of prebiotics (fructo- (FOS), galacto- (GOS) and mannan- (MOS) oligosaccharides) and the bacteriocinogenic Bifidobacterium thermophilum RBL67 (RBL67) on Salmonella enterica subsp. enterica serovar Typhimurium N-15 (N-15) colonization using the PolyFermS in vitro continuous fermentation model simulating the swine proximal colon.

Material and methods: The PolyFermS model was designed with a first-stage reactor containing immobilized fecal pig microbiota. This reactor continuously inoculated five parallel second-stage reactors, a control and four treatment reactors, all operated with proximal colon conditions. FOS and GOS (5.2 g/day), and MOS (half dosage) and RBL67 (10(8) copy numbers/mL applied daily) were tested on the ability of N-15 to colonize reactors, inoculated with the same microbiota. Reactor effluents were collected daily and analyzed for microbial composition (quantitative PCR and 454 pyrosequencing of 16S rRNA gene pool) and main metabolites (HPLC).

Results: RBL67 and N-15 were shown to stably colonize the system. Colonization of N-15 was strongly inhibited by FOS and GOS, whereas addition of RBL67 alone or combined with MOS showed intermediate results. However, the effect of FOS and GOS was enhanced when prebiotics were combined with a daily addition of RBL67. FOS and GOS increased the total short chain fatty acid production, especially acetate and propionate. RBL67 combined with FOS additionally stimulated butyrate production.

Conclusions: Our study demonstrates the suitability of the porcine PolyFermS in vitro model to study nutritional effects of pro- and prebiotics on gut microbiota composition and activity. It can further be used to monitor Salmonella colonization. The inhibition effects of FOS and GOS on N-15 colonization are partly due to an increased acetate production, while further antimicrobial mechanisms may contribute to an enhanced inhibition with prebiotic-RBL67 combinations. A future direction of this work could be to understand the anti-Salmonella effects of Bifidobacterium thermophilum RBL67 in the presence of prebiotics to unravel the mechanism of this probiotic:pathogen interaction.

No MeSH data available.


Microbial composition in reactors during treatment periods 2–4 measured by 454 pyrosequencing on family level. The microbiota profile in reactor effluents during treatment periods was analyzed by 454 pyrosequencing of the entire 16S rRNA gene pool in the V5-V6 region. Reactor effluents were pooled in a ratio 1:1 from two consecutive days of the N-15 challenge period (days 3 and 4) for genomic DNA extraction and subsequent sequencing on a 454 Life Sciences Genome Sequencer GS FLX instrument. Quality-filtered sequencing reads were assigned using the Ribosomal Database Project (RDP) Bayesian classifier (v2.1) and applying a confidence threshold of 80%. CR: control reactor; values <1% are summarized in the group others.
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Fig4: Microbial composition in reactors during treatment periods 2–4 measured by 454 pyrosequencing on family level. The microbiota profile in reactor effluents during treatment periods was analyzed by 454 pyrosequencing of the entire 16S rRNA gene pool in the V5-V6 region. Reactor effluents were pooled in a ratio 1:1 from two consecutive days of the N-15 challenge period (days 3 and 4) for genomic DNA extraction and subsequent sequencing on a 454 Life Sciences Genome Sequencer GS FLX instrument. Quality-filtered sequencing reads were assigned using the Ribosomal Database Project (RDP) Bayesian classifier (v2.1) and applying a confidence threshold of 80%. CR: control reactor; values <1% are summarized in the group others.

Mentions: Using 454 pyrosequencing of the entire 16S rRNA gene pool, a mean value of 6259 ± 3730 quality-filtered reads per sample was obtained with an average read length of 256 ± 1 bp. All samples revealed the predominance of the 3 phyla, Firmicutes, Bacteroidetes and Proteobacteria (Additional file 1: Figure S1). Additionally, Actinobacteria were detected at low levels (<1%; except for R-FOS in period 2 with 1.9%). Firmicutes and Bacteroidetes accounted for more than 80% of assigned reads in all samples for periods 2 and 3. However, during period 4, Proteobacteria increased to up to 30% while Firmicutes and Bacteroidetes decreased to approximately 70% of all reads. The phylum Proteobacteria displayed a steady increase in all reactors during the fermentation, including in CR where no treatment was applied. In general, pro- and prebiotic treatments and N-15 infection did not markedly impact microbiota composition. At the phylum level, Bacteroidetes increased and Firmicutes decreased in the N-15 (alone) and RBL67 treatments compared to CR (period 3). On the family level a consistent increase of Erysipelotrichaceae was observed with prebiotics, alone (FOS, GOS) or in combination with RBL67 (R-FOS, R-FOS, R-MOS), compared to CR, with highest effect for R-FOS (6.4% compared to 0.3% in CR, period 2 and 4.7% compared to 0.1% in CR, period 4) (Figure 4). Changes observed at the genus level (Additional file 2: Figure S2) were consistent with observations at the family level. The genus Sharpea, a member of the family Erysipelotrichaceae, was highly abundant in the TRs after FOS or GOS treatments and the combined treatments of RBL67 with prebiotics (R-FOS, R-GOS, R-MOS) compared to CR. The genus increased to 6% (period 2) and 5% (period 4) of total reads after the R-FOS treatment with values < 0.1% in CR.Figure 4


Synergistic effects of Bifidobacterium thermophilum RBL67 and selected prebiotics on inhibition of Salmonella colonization in the swine proximal colon PolyFermS model.

Tanner SA, Chassard C, Zihler Berner A, Lacroix C - Gut Pathog (2014)

Microbial composition in reactors during treatment periods 2–4 measured by 454 pyrosequencing on family level. The microbiota profile in reactor effluents during treatment periods was analyzed by 454 pyrosequencing of the entire 16S rRNA gene pool in the V5-V6 region. Reactor effluents were pooled in a ratio 1:1 from two consecutive days of the N-15 challenge period (days 3 and 4) for genomic DNA extraction and subsequent sequencing on a 454 Life Sciences Genome Sequencer GS FLX instrument. Quality-filtered sequencing reads were assigned using the Ribosomal Database Project (RDP) Bayesian classifier (v2.1) and applying a confidence threshold of 80%. CR: control reactor; values <1% are summarized in the group others.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4215022&req=5

Fig4: Microbial composition in reactors during treatment periods 2–4 measured by 454 pyrosequencing on family level. The microbiota profile in reactor effluents during treatment periods was analyzed by 454 pyrosequencing of the entire 16S rRNA gene pool in the V5-V6 region. Reactor effluents were pooled in a ratio 1:1 from two consecutive days of the N-15 challenge period (days 3 and 4) for genomic DNA extraction and subsequent sequencing on a 454 Life Sciences Genome Sequencer GS FLX instrument. Quality-filtered sequencing reads were assigned using the Ribosomal Database Project (RDP) Bayesian classifier (v2.1) and applying a confidence threshold of 80%. CR: control reactor; values <1% are summarized in the group others.
Mentions: Using 454 pyrosequencing of the entire 16S rRNA gene pool, a mean value of 6259 ± 3730 quality-filtered reads per sample was obtained with an average read length of 256 ± 1 bp. All samples revealed the predominance of the 3 phyla, Firmicutes, Bacteroidetes and Proteobacteria (Additional file 1: Figure S1). Additionally, Actinobacteria were detected at low levels (<1%; except for R-FOS in period 2 with 1.9%). Firmicutes and Bacteroidetes accounted for more than 80% of assigned reads in all samples for periods 2 and 3. However, during period 4, Proteobacteria increased to up to 30% while Firmicutes and Bacteroidetes decreased to approximately 70% of all reads. The phylum Proteobacteria displayed a steady increase in all reactors during the fermentation, including in CR where no treatment was applied. In general, pro- and prebiotic treatments and N-15 infection did not markedly impact microbiota composition. At the phylum level, Bacteroidetes increased and Firmicutes decreased in the N-15 (alone) and RBL67 treatments compared to CR (period 3). On the family level a consistent increase of Erysipelotrichaceae was observed with prebiotics, alone (FOS, GOS) or in combination with RBL67 (R-FOS, R-FOS, R-MOS), compared to CR, with highest effect for R-FOS (6.4% compared to 0.3% in CR, period 2 and 4.7% compared to 0.1% in CR, period 4) (Figure 4). Changes observed at the genus level (Additional file 2: Figure S2) were consistent with observations at the family level. The genus Sharpea, a member of the family Erysipelotrichaceae, was highly abundant in the TRs after FOS or GOS treatments and the combined treatments of RBL67 with prebiotics (R-FOS, R-GOS, R-MOS) compared to CR. The genus increased to 6% (period 2) and 5% (period 4) of total reads after the R-FOS treatment with values < 0.1% in CR.Figure 4

Bottom Line: Our study demonstrates the suitability of the porcine PolyFermS in vitro model to study nutritional effects of pro- and prebiotics on gut microbiota composition and activity.The inhibition effects of FOS and GOS on N-15 colonization are partly due to an increased acetate production, while further antimicrobial mechanisms may contribute to an enhanced inhibition with prebiotic-RBL67 combinations.A future direction of this work could be to understand the anti-Salmonella effects of Bifidobacterium thermophilum RBL67 in the presence of prebiotics to unravel the mechanism of this probiotic:pathogen interaction.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Food Biotechnology, Institute of Food, Nutrition and Health, Department of Health Science and Technology, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich, Switzerland.

ABSTRACT

Background: Probiotics and prebiotics are promising strategies to counteract Salmonella prevalence in swine. In the present study, we investigated the effects of prebiotics (fructo- (FOS), galacto- (GOS) and mannan- (MOS) oligosaccharides) and the bacteriocinogenic Bifidobacterium thermophilum RBL67 (RBL67) on Salmonella enterica subsp. enterica serovar Typhimurium N-15 (N-15) colonization using the PolyFermS in vitro continuous fermentation model simulating the swine proximal colon.

Material and methods: The PolyFermS model was designed with a first-stage reactor containing immobilized fecal pig microbiota. This reactor continuously inoculated five parallel second-stage reactors, a control and four treatment reactors, all operated with proximal colon conditions. FOS and GOS (5.2 g/day), and MOS (half dosage) and RBL67 (10(8) copy numbers/mL applied daily) were tested on the ability of N-15 to colonize reactors, inoculated with the same microbiota. Reactor effluents were collected daily and analyzed for microbial composition (quantitative PCR and 454 pyrosequencing of 16S rRNA gene pool) and main metabolites (HPLC).

Results: RBL67 and N-15 were shown to stably colonize the system. Colonization of N-15 was strongly inhibited by FOS and GOS, whereas addition of RBL67 alone or combined with MOS showed intermediate results. However, the effect of FOS and GOS was enhanced when prebiotics were combined with a daily addition of RBL67. FOS and GOS increased the total short chain fatty acid production, especially acetate and propionate. RBL67 combined with FOS additionally stimulated butyrate production.

Conclusions: Our study demonstrates the suitability of the porcine PolyFermS in vitro model to study nutritional effects of pro- and prebiotics on gut microbiota composition and activity. It can further be used to monitor Salmonella colonization. The inhibition effects of FOS and GOS on N-15 colonization are partly due to an increased acetate production, while further antimicrobial mechanisms may contribute to an enhanced inhibition with prebiotic-RBL67 combinations. A future direction of this work could be to understand the anti-Salmonella effects of Bifidobacterium thermophilum RBL67 in the presence of prebiotics to unravel the mechanism of this probiotic:pathogen interaction.

No MeSH data available.