Robert Pieper

Nonstarch Polysaccharides Modulate Bacterial Microbiota, Pathways for Butyrate Production, and Abundance of Pathogenic Escherichia coli in the Pig Gastrointestinal Tract†

ABSTRACT The impact of nonstarch polysaccharides (NSP) differing in their functional properties on intestinal bacterial community composition, prevalence of butyrate production pathway genes, and occurrence of Escherichia coli virulence factors was studied for eight ileum-cannulated growing pigs by use of terminal restriction fragment length polymorphism (TRFLP) and quantitative PCR. A cornstarch- and casein-based diet was supplemented with low-viscosity, low-fermentability cellulose (CEL), with high-viscosity, low-fermentability carboxymethylcellulose (CMC), with low-viscosity, high-fermentability oat β-glucan (LG), and with high-viscosity, high-fermentability oat β-glucan (HG). Only minor effects of NSP fractions on the ileal bacterial community were observed, but NSP clearly changed the digestion in the small intestine. Compared to what was observed for CMC, more fermentable substrate was transferred into the large intestine with CEL, LG, and HG, resulting in higher levels of postileal dry-matter disappearance. Linear discriminant analysis of NSP and TRFLP profiles and 16S rRNA gene copy numbers for major bacterial groups revealed that CMC resulted in a distinctive bacterial community in comparison to the other NSP, which was characterized by higher gene copy numbers for total bacteria, Bacteroides-Prevotella-Porphyromonas, Clostridium cluster XIVa, and Enterobacteriaceae and increased prevalences of E. coli virulence factors in feces. The numbers of butyryl-coenzyme A (CoA) CoA transferase gene copies were higher than those of butyrate kinase gene copies in feces, and these quantities were affected by NSP. The present results suggest that the NSP fractions clearly and distinctly affected the taxonomic composition and metabolic features of the fecal microbiota. However, the effects were more linked to the individual NSP and to their effect on nutrient flow into the large intestine than to their shared functional properties.

Effect of barley and oat cultivars with different carbohydrate compositions on the intestinal bacterial communities in weaned piglets.

This experiment was aimed at comparing the intestinal microbial community composition in pigs fed hulled common barley supplemented with isolated barley mixed-linked beta-glucan, four hulless barley varieties and breeding lines with mixed-linked beta-glucan contents ranging from 41 to 84 g kg(-1) and different amylose/amylopectin ratios as well as two oat varieties. Seventy-two weaned piglets were allocated to one of nine diets composed of 81.5% cereal, 6% whey, 9% soy protein isolate and 3.5% minerals. After 15 days, pigs were sacrificed and ileum and colon contents were collected for quantitative real-time PCR (qPCR) and denaturing gradient gel electrophoresis to evaluate microbial communities. Shifts in intestinal microbial communities were observed with the hulless barley cultivars with a normal to high beta-glucan content and from normal starch toward either high-amylopectin or high-amylose starch. These hulless barleys had the lowest (P<0.05) microbial diversity, whereas oats had intermediate diversity compared with low-beta-glucan hulless cultivars and hulled varieties. Furthermore, hulless varieties favoured xylan- and beta-glucan-degrading bacteria whereas mixed-linked beta-glucan-supplemented hulled barley favoured lactobacilli. Numbers of lactobacilli decreased in the ileum of pigs fed hulless/high mixed-linked beta-glucan barley-based diets. Thus, cultivar differences in both the form and the quantity of carbohydrates affect gut microbiota in pigs, which provides information for future feeding strategies.

Fermentable Fiber Ameliorates Fermentable Protein-Induced Changes in Microbial Ecology, but Not the Mucosal Response, in the Colon of Piglets

Dietary inclusion of fermentable carbohydrates (fCHO) is reported to reduce large intestinal formation of putatively toxic metabolites derived from fermentable proteins (fCP). However, the influence of diets high in fCP concentration on epithelial response and interaction with fCHO is still unclear. Thirty-two weaned piglets were fed 4 diets in a 2 × 2 factorial design with low fCP/low fCHO [14.5% crude protein (CP)/14.5% total dietary fiber (TDF)]; low fCP/high fCHO (14.8% CP/16.6% TDF); high fCP low fCHO (19.8% CP/14.5% TDF); and high fCP/high fCHO (20.1% CP/18.0% TDF) as dietary treatments. After 21-23 d, pigs were killed and colon digesta and tissue samples analyzed for indices of microbial ecology, tissue expression of genes for cell turnover, cytokines, mucus genes (MUC), and oxidative stress indices. Pig performance was unaffected by diet. fCP increased (P < 0.05) cell counts of clostridia in the Clostridium leptum group and total short and branched chain fatty acids, ammonia, putrescine, histamine, and spermidine concentrations, whereas high fCHO increased (P < 0.05) cell counts of clostridia in the C. leptum and C. coccoides groups, shifted the acetate to propionate ratio toward acetate (P < 0.05), and reduced ammonia and putrescine (P < 0.05). High dietary fCP increased (P < 0.05) expression of PCNA, IL1β, IL10, TGFβ, MUC1, MUC2, and MUC20, irrespective of fCHO concentration. The ratio of glutathione:glutathione disulfide was reduced (P < 0.05) by fCP and the expression of glutathione transferase was reduced by fCHO (P < 0.05). In conclusion, fermentable fiber ameliorates fermentable protein-induced changes in most measures of luminal microbial ecology but not the mucosal response in the large intestine of pigs.

Increased dietary zinc oxide changes the bacterial core and enterobacterial composition in the ileum of piglets

This study was conducted to investigate the effects of increased dietary ZnO on the bacterial core and enterobacterial composition in the small intestine of piglets that were fed diets containing a total of 124 or 3,042 mg of Zn per kilogram of diet, respectively. Zinc was supplemented to the basal diet as ZnO. Bacterial 16S rRNA genes of ileal DNA extracts were PCR-amplified with 2 bar-coded primer sets and sequenced by 454 pyrosequencing. The bacterial core species were calculated from the relative abundances of reads present in 5 of 6 samples per group and at a minimum of 5 sequences per sample. The reference database SILVA was used to assign sequence reads at an alignment minimum of 200 bases and 100% identity. Lactic acid bacteria dominated the bacterial core, but showed diverse responses to dietary ZnO. Of the dominant Lactobacillus spp., Lactobacillus reuteri was reduced due to increased dietary ZnO (44.7 vs. 17.9%; P=0.042), but L. amylovorus was not influenced. However, the changes of relative abundances of other lactic acid bacteria were more noteworthy; Weissella cibaria (10.7 vs. 23.0%; P=0.006), W. confusa (10.0 vs. 22.4%; P=0.037), Leuconostoc citreum (6.5 vs. 14.8%; P=0.009), Streptococcus equinus (0.14 vs. 1.0%; P=0.044), and S. lutetiensis (0.01 vs. 0.11%; P=0.016) increased in relative abundance. Nonlactic acid bacteria that were influenced by increased dietary ZnO included the strict anaerobic species, Sarcina ventriculi, which showed a strong numerical decrease in relative abundance (14.6 vs. 5.1%). Species of the Enterobacteriaceae increased their relative abundance, as well as species diversity, in the high dietary ZnO experimental group. Bacterial diversity indices were increased due to increased dietary ZnO (P < 0.05), which was traced back to the increase of sequences from subdominant species. Increased dietary ZnO led to an increase of less prominent species and, thus, had a major impact on the bacterial composition and diversity in piglets. This effect may help to stabilize the intestinal microbiota in the sensitive postweaning period.

The broader context of antibiotic resistance: Zinc feed supplementation of piglets increases the proportion of multi-resistant Escherichia coli in vivo

Following the Europe-wide ban of antimicrobial growth promoters, feed supplementation with zinc has increased in livestock breeding. In addition to possible beneficial effects on animal health, feed supplementation with heavy metals is known to influence the gut microbiota and might promote the spread of antimicrobial resistance via co-selection or other mechanisms. As Escherichia coli is among the most important pathogens in pig production and often displays multi-resistant phenotypes, we set out to investigate the influence of zinc feed additives on the composition of the E. coli populations in vivo focusing on phylogenetic diversity and antimicrobial resistance. In a piglet feeding trial, E. coli were isolated from ileum and colon digesta of high dose zinc-supplemented (2500ppm) and background dose (50ppm) piglets (control group). The E. coli population was characterized via pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) for the determination of the phylogenetic background. Phenotypic resistance screening via agar disk diffusion and minimum inhibitory concentration testing was followed by detection of resistance genes for selected clones. We observed a higher diversity of E. coli clones in animals supplemented with zinc compared to the background control group. The proportion of multi-resistant E. coli was significantly increased in the zinc group compared to the control group (18.6% vs. 0%). For several subclones present both in the feeding and the control group we detected up to three additional phenotypic and genotypic resistances in the subclones from the zinc feeding group. Characterization of these subclones suggests an increase in antimicrobial resistance due to influences on plasmid uptake by zinc supplementation, questioning the reasonability of zinc feed additives as a result of the ban of antimicrobial growth promoters.

Bar-Coded Pyrosequencing of 16S rRNA Gene Amplicons Reveals Changes in Ileal Porcine Bacterial Communities Due to High Dietary Zinc Intake

ABSTRACT Feeding high levels of zinc oxide to piglets significantly increased the relative abundance of ileal Weissella spp., Leuconostoc spp., and Streptococcus spp., reduced the occurrence of Sarcina spp. and Neisseria spp., and led to numerical increases of all Gram-negative facultative anaerobic genera. High dietary zinc oxide intake has a major impact on the porcine ileal bacterial composition.

Nutritional and physiological role of medium-chain triglycerides and medium-chain fatty acids in piglets

Abstract Medium-chain fatty acids (MCFAs) are found at higher levels in milk lipids of many animal species and in the oil fraction of several plants, including coconuts, palm kernels and certain Cuphea species. Medium-chain triglycerides (MCTs) and fatty acids are efficiently absorbed and metabolized and are therefore used for piglet nutrition. They may provide instant energy and also have physiological benefits beyond their energetic value contributing to several findings of improved performance in piglet-feeding trials. MCTs are effectively hydrolyzed by gastric and pancreatic lipases in the newborn and suckling young, allowing rapid provision of energy for both enterocytes and intermediary hepatic metabolism. MCFAs affect the composition of the intestinal microbiota and have inhibitory effects on bacterial concentrations in the digesta, mainly on Salmonella and coliforms. However, most studies have been performed in vitro up to now and in vivo data in pigs are still scarce. Effects on the gut-associated and general immune function have been described in several animal species, but they have been less studied in pigs. The addition of up to 8% of a non-esterified MCFA mixture in feed has been described, but due to the sensory properties this can have a negative impact on feed intake. This may be overcome by using MCTs, allowing dietary inclusion rates up to 15%. Feeding sows with diets containing 15% MCTs resulted in a lower mortality of newborns and better development, particularly of underweight piglets. In conclusion, MCFAs and MCTs offer advantages for the improvement of energy supply and performance of piglets and may stabilize the intestinal microbiota, expanding the spectrum of feed additives supporting piglet health in the post-weaning period.

Effect of alginate and inulin on intestinal microbial ecology of weanling pigs reared under different husbandry conditions

The effects of inulin and alginate on intestinal microbial ecophysiology were investigated in piglets fed a diet (C) with 0.1% alginate (C+A) or 1.5% inulin (C+I) from weaning at day 28. The experiment was performed at an experimental farm (EF) and a commercial farm (CF). Digesta was collected from the ileum, caecum and colon of four piglets from each group on days 29, 30, 33 and 39. The metabolite concentrations changed with age. Colonic and caecal metabolites were affected by prebiotic treatment. Changes in microbiota composition were assessed by cultivation and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Enterococci increased in C+A at EF and decreased in C+I at both farms. Lactobacilli decreased in all segments in the experimental groups on days 30 and 33. Yeasts in C+I were five times lower at CF than at EF on day 39. The richness and diversity of DGGE profiles increased in the experimental groups. The evenness of colon digesta-derived DGGE profiles was higher in the experimental groups than in C and this situation was reversed in the distal small intestine. Multivariate redundancy analysis confirmed the recorded effects. In summary, both prebiotics affected the intestinal microbiota, and the changes were more pronounced at the CF.

Barley and oat cultivars with diverse carbohydrate composition alter ileal and total tract nutrient digestibility and fermentation metabolites in weaned piglets.

An experiment was conducted to evaluate the effects of cereal carbohydrate form (isolated v. cereal matrix) and level, especially mixed-linked β-glucan (hereafter referred to as β-glucan) and starch amylase/amylopectin ratio on nutrient digestibility and fermentation parameters in the intestines of weaned pigs. Four hulless barley cultivars containing varying β-glucan levels (41 to 84 g/kg) were compared with hulled barley, supplemented or not with a β-glucan concentrate (BBG; 270 g/kg β-glucan) and two oat cultivars for digestibility and fermentation metabolites. Seventy-two weaned piglets (BW = 12.8 ± 1.9 kg) were assigned to one of nine diets composed of 815 g/kg cereal, 60 g/kg whey, 90 g/kg soy protein isolate and 35 g/kg minerals. After 15 days, the pigs were killed, and digesta collected from ileum and colon were analyzed for proximate nutrients, short-chain fatty acids (SCFAs), lactic acid (LA) and ammonia. Ileal and total tract digestibility of proximate nutrients and non-starch polysaccharides (NSPs) were determined using HCl-insoluble ash as a marker. Organic matter (OM) ileal digestibility was greater (P < 0.05) for diets based on hulless barley (77% ± 1.1% on average), as compared with hulled barley (64% ± 1.4%) and oat (58% ± 1.5%). Similar trends were found for total tract OM digestibility, varying from 90% ± 0.3% for hulless barley to 67% ± 0.4% for oat, on average. NSP digestibility differed (P < 0.05) within and between cereal types, ranging from 20% (hulled barley plus 163 g/kg BBG or 40 g/kg β-glucan) to 51% (SB94893 hulless barley cultivar with high β-glucan and high amylose ratio) at the ileum and from 44% (hulled barley) to 84% (SB94893 cultivar) at the total tract level. No dietary effect (P > 0.05) was found for SCFA concentration in ileal contents, whereas in colonic contents, SCFA was lower in pigs fed oat (P < 0.001). LA concentration was greater (P < 0.001) in the colon of pigs fed hulless barley than in pigs fed hulled barley and oat. Expressed per kg carbohydrate (NSP + starch) fermented, the ammonia concentration at the colon was lowest for hulled barley diets (supplemented with β-glucan) and the highest for oat diets. In conclusion, the interaction of both form and level of β-glucan impacted nutrient digestibility and fermentation. Hulless barleys with high soluble NSP such as β-glucan and resistant starch yielded, in general higher SCFA and LA and lower ammonia. Hulless barleys may, therefore, have potential for use in feeding strategies designed to improve gut health in pigs.

Effect of carbohydrate composition in barley and oat cultivars on microbial ecophysiology and proliferation of Salmonella enterica in an in vitro model of the porcine gastrointestinal tract.

ABSTRACT The influence of the carbohydrate (CHO) composition of cereal cultivars on microbial ecophysiology was studied using an in vitro model of the porcine gastrointestinal tract. Ten hull-less barley cultivars, six barley cultivars with hulls, six oat cultivars, and six oat groats that differed in β-glucan, nonstarch polysaccharide (NSP), and starch contents and starch type were hydrolyzed enzymatically and incubated for 72 h with pig feces. Fermentation kinetics were modeled, and microbial compositions and short-chain fatty acid (SCFA) profiles were analyzed using terminal restriction fragment length polymorphism and gas chromatography. Cluster analysis and canonical ordination revealed different effects on fermentation and microbial ecology depending on the type of CHO and cultivar. First, in cultivars of barley with hulls and oats, the cellulose and insoluble NSP contents (i) increased Ruminococcus flavefaciens-like and Clostridium xylanolyticum-like phylotypes, (ii) increased acetate production, and (iii) decreased fermentation activity. Second, in hull-less barley cultivars the β-glucan, amylose, amylopectin, crude protein, and soluble NSP contents determined the microbial community composition and activity as follows: (i) the amylose contents of the hull-less barley varieties increased the butyrate production and the abundance of Clostridiumbutyricum-like phylotypes, (ii) the β-glucan content determined the total amounts of SCFA, and (iii) the amylopectin and starch contents affected the abundance of Clostridiumramosum-like phylotypes, members of Clostridium cluster XIVa, and Bacteroides-like bacteria. Finally, the effect of CHO on proliferation of Salmonella enterica in the model was determined. Salmonella cell counts were not affected, but the relative proportion of Salmonella decreased with hull-less barley cultivars and increased with oat cultivars as revealed by quantitative PCR. Our results shed light on the complex interactions of cereal CHO with intestinal bacterial ecophysiology and the possible impact on host health.