**2.2 Analysis of fecal microbiotauction**

Fecal samples were collected before the first colonic irrigation and at 1 week after the third irrigation. These samples were analyzed using a kit from TechnoSuruga Laboratory Co., Ltd. (Shizuoka Japan). Fecal microbiota analysis targeted bacterial 16S rRNA genes with a terminal restriction fragment length polymorphism (T-RFLP) analysis program (Nagashima's method) (Ando et al., 2007; Nagashima et al. 2002, 2006). T-RFLP was performed as previously described by Nagashima et al. (2002, 2006). The 16S rRNA genes were amplified using a forward primaer, 516f [5'-TGCCAGCAGCCGCGGTA-3'] and a reverse primer, 1510r [5'-GGTTACCTTGTTACGACTT-3']. The 5' ends of the forward primer, 516f were labeled with 6'-carboxyfluorescein, which was synthesized by Applied Biosystems Japan (Tokyo, Japan). The purified PCR products (2 μL) were digested with 10 U of either BslI (New England BioLabs, Inc., Ipswich, MA, USA) at 55°C for 3 h. The lengths of terminal restriction fragments were determined with the ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems, Tokyo, japan).
