**2.4 Colonic irrigations**

Each patient underwent 3 colonic irrigations over a period 2 weeks. Same trained physician performed colonic irrigations for all subjects. Irrigations using an intestinal irrigation devide (Colon Hydromat Comfort: Herrmann Apparatebau GmbH, Kleinwallstadt, Germany) that circulated approximately 30–50 L of purified warm water (38°C) through a filter (Uchiyama-Tanaka, 2009). During an irrigation process, which lasts for approximately 1 hour, a subject is laid supine on a bed and given abdominal massage. Because colonic irrigations utilize a large amount of water introduced using a tube and abdominal massage, there are some contraindications, such as renal failure, heart failure, liver cirrhosis, severe hemorrhoids, after post-intestinal polypectomy, post-abdominal surgery and pregnacy. The principle of the Colon Hydromat Comfort's function is shown in Figure1.

Filtered hot water and ordinary water are mixed and passed into a patient's colon through the speculum. The sewage water flows out due to the pressure in the water-filled colon. Some pictures of fecal residues and intestinal epithelium during colon irrigations are shown below.

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

Blood samples were collected from the subjects in a sitting position before the start and at 10

Each patient underwent 3 colonic irrigations over a period 2 weeks. Same trained physician performed colonic irrigations for all subjects. Irrigations using an intestinal irrigation devide (Colon Hydromat Comfort: Herrmann Apparatebau GmbH, Kleinwallstadt, Germany) that circulated approximately 30–50 L of purified warm water (38°C) through a filter (Uchiyama-Tanaka, 2009). During an irrigation process, which lasts for approximately 1 hour, a subject is laid supine on a bed and given abdominal massage. Because colonic irrigations utilize a large amount of water introduced using a tube and abdominal massage, there are some contraindications, such as renal failure, heart failure, liver cirrhosis, severe hemorrhoids, after post-intestinal polypectomy, post-abdominal surgery and pregnacy. The principle of

Filtered hot water and ordinary water are mixed and passed into a patient's colon through the speculum. The sewage water flows out due to the pressure in the water-filled colon. Some pictures of fecal residues and intestinal epithelium during colon irrigations are shown

min after the third irrigation. Serum samples were analyzed using a commercial kit.

**2.2 Analysis of fecal microbiotauction** 

Analyzer (Applied Biosystems, Tokyo, japan).

the Colon Hydromat Comfort's function is shown in Figure1.

**2.3 Laboratory determinations** 

**2.4 Colonic irrigations** 

below.

The Influence of Colonic Irrigation on Human Intestinal Microbiota 453

Results are given as means ± standard deviations (SD). Student's paired t-test was used to compare results before and after irrigations. P < 0.05 was considered statistically significant.

Patient Age Gender Underlying disease Symptom change No. 1 44 Male Pollen allergy Symptom-free (after 7 times)

No. 4 47 Male Atopic dermatitis Decreased itching

No. 5 28 Male Psoriasis Better (still undergoing

No. 10 29 Male Allergic Rhinitis Decreased blowing of nose

Each patient underwent 3 colonic irrigations over a period of 2 weeks. Fecal samples were collected before the first colon irrigation and at 1 week after the third irrigation. The relative proportions of bacteria found in the fecal samples of each patient before and after colonic

Presumed bacterium b a b a b a b a *Bifidobacterium* 18.6 16.5 22.9 17.1 8.7 14.9 1.8 14.5 Lactobacillales 2.2 4.5 4.1 0 6.1 4 0 0 *Bacteroides* 23 17 21.3 20.9 31.7 31.6 44.4 38.3 *Prevotella* 9.1 2.8 7.8 8.9 0 0 0 0 *Clostridium* cluster IV 11.6 15.4 14.5 19.1 25.5 23 22.7 13.9 *Clostridium* subcluster XIVa 13 16.3 17.7 24.7 21.3 19.4 14.6 22.4 *Clostridium* cluster XI 2.6 4.2 0 0 0 0 2.4 2.3 *Clostridium* cluster XVIII 7 4.4 2 1.4 0 0 5.1 0 others 13 18.8 9.6 8 6.7 7.1 9.1 8.6

1 2 3 4

Table 1. Subject's clinical characteristics and symptom changes after irrigations

No. 3 45 Female Ovarian cysts Size Smaller (8 cm to 6 cm, after

3 times)

irrigation)

Patient clinical characteristics are summarized in Table 1.

No. 2 47 Female Constipation Better

No. 6 38 Female Constipation Better No. 7 38 Female Constipation Better No. 8 32 Female Face eruption Better No. 9 39 Female Constipation Better

**2.5 Statistics** 

**3. Results** 

irrigation are shown in Table 2.

Table 2. Continued

Fig. 1. Principle of the Colon Hydromat Comfort

## **2.5 Statistics**

452 New Advances in the Basic and Clinical Gastroenterology

Fig. 1. Principle of the Colon Hydromat Comfort

Results are given as means ± standard deviations (SD). Student's paired t-test was used to compare results before and after irrigations. P < 0.05 was considered statistically significant.
