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

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.The porcine intestinal microbiota change in response to dietary carbohydrate composition due to specific substrate preferences of bacteria (6). Therefore, inclusion of specific nonstarch polysaccharides (NSP) in the diet of pigs allows manipulation of the composition of the intestinal microbiota. The NSP can also reduce digestibility of nutrients in the small intestine (8). The resulting changes in nutrient flow alter the availability of fermentable substrate in the different sections of the gut and thus may modify the bacterial community structure. Differences in the fermentability levels of individual NSP may not only affect the kinetics of their degradation by intestinal bacteria but may also change the composition of the fermentation end products (49). Particularly, butyrate is an important metabolite because of its potential to affect gene expression and to improve cellular development in enterocytes (38). The ability of gut microbiota to produce butyrate can vary considerably in response to environmental factors, such as diet composition (3). However, the number of butyrate-producing bacteria in complex fecal samples has been difficult to estimate by targeting the 16S rRNA gene, because these bacteria do not form a homogeneous phylogenetic group, and both butyrate producers and non-butyrate producers are found within the same phylogenetic clusters belonging to Clostridium clusters I, III, IV, XI, XIVa, XV, and XVI (27). Two alternative pathways for butyrate formation in bacteria harboring the rumen and human colon have been described (7, 26). The majority of human colonic butyrate producers use butyryl-coenzyme A (CoA) CoA transferase, whereas soil bacteria mostly utilize the butyrate kinase for the last step of butyrate formation (26, 27). However, information about the butyrate pathways used by intestinal bacteria in pigs is not available.In addition to the effects of the functional properties of NSP on intestinal physiology and fermentation processes, selection of specific NSP fractions may also prevent or stimulate overgrowth of pathogenic bacteria. For instance, dietary inclusion of highly viscous carboxymethylcellulose (CMC) has been shown to increase fecal shedding of enterotoxigenic Escherichia coli in weaned pigs (15). There is a need to identify those dietary NSP fractions that may either increase or reduce the numbers of potential pathogenic bacteria to formulate diets exerting beneficial effects on gut health, which is particularly important in antibiotic-free feeding regimens.Most studies pertaining on the effect of diet composition on the bacterial community in pigs have employed natural NSP sources and cereal-based diets, thereby resulting in a mixture of different soluble and insoluble NSP showing considerable interactions and modification of intestinal bacterial ecophysiology (6, 36, 37). Purified NSP fractions are increasingly available from the bioprocessing industry for use in food preparation and potentially in animal feeds, where economics and possible health benefits warrant this use. However, less is known about the fermentative properties of purified NSP fractions than about those of NSP in the grain matrix (37), which may also differ according to their origins.The aim of the present study was to examine the effects of four purified NSP fractions differing in their functional properties, i.e., viscosity and fermentability, on the ileal and fecal bacterial community, butyrate production pathway genes, and the occurrence of virulence factor genes of swine-pathogenic E. coli, including enterotoxigenic and enteroaggregative E. coli (11, 13).(This study was presented in part at the 11th Digestive Physiology in Pigs Symposium, Reus, Spain, 19 to 22 May 2009.)