212 intestinal microorganisms, and faecal material seems to represent the colonic microbiota composition best (Eck- burg et al. 2005). In contrast, there is only limited insight in the composition of the microbiota that resides in the small intestine. Especially the lower part of the small intestine, the ileum, has received minimal attention, mainly due to sampling difﬁculties caused by the inaccessibility of this region (Booijink et al. 2007). The composition of the small intestinal microbiota is largely inﬂuenced by a combination of gastric acid, bile and pancreatic secretions that enter the GI tract in the duodenum, and which together create a harsh environment for most microorganisms (Booijink et al. 2007). Hence, compared with other regions, few microorganisms are able to inhabit the upper part of the GI tract. In addition, the antegrade peristaltic movements as part of the migrating motor complex (MMC) ensure a relatively short passage time through the small intestine (3–5 h) by pushing the microbiota towards the large intestine, thus leaving limited time for microorganisms to replicate and increase in numbers (Booijink et al. 2007). The short passage time allows transitioning bacteria to retain viability. Furthermore, cellular enzymes such as glutamate decarboxylase and bile resistance systems offer protection against the low pH and bile salts encountered in this upper part of the GI tract, respectively (Audia et al. 2001; Merritt and Donaldson 2009). The small intestine is the part of the GI tract where most of the host enzymatic digestion of the food occurs. The products of these digestive activities are absorbed in more distal parts of the small intestine, the jejunum and espe- cially the ileum. The conditions in the ileum are more favourable for microbial growth compared with the proximal part of the small intestine, as for example the pH is less acidic and bile acids are reabsorbed. Therefore, the number of microorganisms in the ileum can be higher compared with the duodenum (Booijink et al. 2007). Most of the knowledge about the small intestinal mic- robiota has been derived from studies with ileal biopsies collected during surgical intervention (Ahmed et al. 2007; Baumgart et al. 2007; Wang et al. 2003, 2005; Willing et al. 2009) or from samples obtained from elderly indi- viduals at autopsy (Hayashi et al. 2005). In addition, ileal efﬂuent from ileostomy patients has been used to study the diversity of the luminal microbiota of the human ileum (Booijink et al. 2010; Hartman et al. 2009). It was shown that the composition of the microbiota in ileostomy efﬂuent clearly differs from that of the faecal microbiota. Com- pared with faecal microbiota, ileostomy efﬂuent microbiota is less diverse and less stable, since large ﬂuctuations in ileal microbiota proﬁles per individual were observed over time (Booijink et al. 2010). One of the main ﬁndings of this study by Booijink and colleagues was that ileostomy efﬂuent showed a higher relative abundance of species 123 Genes Nutr (2011) 6:209–240 within the orders Lactobacillales and Clostridiales, espe- cially Veillonella- and Streptococcus-related phylotypes (Booijink et al. 2010). In addition, species belonging to Clostridium cluster I were detected in high levels, in con- trast with the reduced levels of species belonging to the Bacteroidetes and Clostridium clusters III, IV and XIVa. More recently, it was demonstrated that the microbiota composition of ileostomy efﬂuent, which is characterised by an abundance in Streptococcus and Veillonella species, is more similar to the proximal small intestinal microbiota and clearly differs from that of the ileum (Zoetendal et al. 2011). In addition to the variation in microbial composition along the GI tract, the microbiota present in the intestinal lumen also differs signiﬁcantly from that attached to and imbedded in the intestinal mucus layer. Since mucosa- associated microorganisms live in close contact with host cells, it is likely they execute different functions within the GI ecosystem compared with luminal microorganisms. Several studies have reported a signiﬁcant difference in dominant microbial community composition between colonic biopsies and faecal samples in humans (Eckburg et al. 2005; Lepage et al. 2005; Zoetendal et al. 2002). It should be kept in mind, however, that in these studies colonic biopsies were obtained from humans undergoing standard colonoscopy, which in general is preceded by a laxative preparation in order to clean the GI tract. The inﬂuence of this procedure on the luminal and mucosa- associated microbiota is still largely unknown (Mai et al. 2006). Animal models could provide a means to study both the microbial composition along the GI tract as well as the difference in luminal and mucosa-associated microbiota, without the need for physiological alterations during sam- pling. It has been demonstrated in rodents that intestinal microorganisms are able to survive and even proliferate in the outer loose mucus layer since the glycans present in this layer are accessible as energy source for these micro- organisms (Kim and Ho 2010; Johansson et al. 2010). In contrast, the inner stratiﬁed ﬁrmly attached mucus layer probably prevents the intestinal bacteria from coming in contact with the colonic epithelial cells (Johansson et al. 2008). The organisation of the mucus layers varies amongst the different parts of the GI tract, as it has been observed that the mucus layers in the stomach and the colon are well deﬁned, in contrast to the small intestine where the mucus is less evenly distributed (Atuma et al. 2001; Johansson et al. 2010). Most likely, such differences in mucus layer organisation will be associated with variation in the mucosa-associated microbiota along the GI tract. A recent study in mice has shown that the dominant microbiota composition of proximal colonic mucosa-associated and faecal microbiota are very similar to each other, but differ