**4. Discussion**

454 New Advances in the Basic and Clinical Gastroenterology

b a b a b a b a b a b a 1.9 2 17.9 2.6 22.6 28.2 9.9 1 0 28.5 5.6 26.7 0 0 1.5 0 2.7 2.5 0 0 3.3 3.2 1.6 1.5 28.3 14.7 13.5 14.4 15.6 25.4 29.8 12.3 59.8 30.3 32.9 33 38.3 31.6 23.9 30.3 0 0 0 40.3 0 0 0 0 2.8 8.1 3.8 1.5 12.6 11.7 0 19.7 0 19.1 0 4.8 3.9 15.8 19 11.2 22.8 17.4 26.5 11.6 7.7 13 24.4 15.1 1.7 0 1 3.4 11.1 3 13.8 2.5 3.3 3.3 0 0 1.1 0 1.1 0.6 0 1.5 0 1.2 1.7 0 0 0 22 27.8 18.3 35.9 12.6 10.3 20 11.3 24.3 2.4 35.5 18.9

5 6 7 8 9 10

No 1-10: Patients in Table 1. b: Before colonic irrigations. a: After 3 times colonic irrigations

proportions of these bacterial orders are shown in Table 3.

experience any difficulties.

Table 2. The changes in the fecal microbiota between prior to and after 3 clonic irrigations

Presumed microbiota % prior to (mean ± SD) after p

*Bifidobacterium* 10.99±8.85 15.29±10.74 p>0.05 Lactobacillales 2.15±1.99 1.57±1.84 p>0.05 *Bacteroids* 30.08±13.86 23.8±9.2 p>0.05 *Prevotella* 7.88±13.07 11.39±16.09 p>0.05 *Clostridium* IV 9.4±9.62 13.62±7.04 p>0.05 *Clostridum* XIVa 17.07±7.33 16.64±4.42 p>0.05 *Clostridum* XI 3.61±4.9 1.87±1.69 p>0.05 *Clostridum* XVIII 1.8±2.4 0.91±1.38 p>0.05 Others 17.13±8.85 14.92±10.42 p>0.05

Table 3. The mean changes in the microbiota between prior to and after 3 colonic irrigations

According to Collins et al. (1994), *Clostridium* clusters and subclusters cannot revide the unknown pole in intestine. For example, *Faecalibacterium prausnitzil* is an important bacteria as butyrate-producing bacterium in *Clostridium* cluster IV. In contrast, *Clostridium perfringens* is a well known as harmful bacterium in *Clostridium* cluster I. Lactobacillales and *Bifidobacterium* are considered to be healthy, beneficial fecal bacteria. In this study, beneficial bacteria decreased in some patients. Serum electrolytes after irrigations (sodium, potassium, and chlorine) exhibited no significant changes from their values before irrigation (data not shown). Patient symptoms were improved after irrigations (Table 1), and they did not

There were no significant differences in the overall quantities of fecal bacteria in samples collected before and after irrigations. There was also no tendency for changes int the proportions of Lactobacillales and *Bifidobacterium* and *Clostridium* subclusters. The This study showed that colonic irrigations are safe in terms of serum electrolytes for subjects with normal renal function and had a positive impact on these subjects' symptoms. However, these irrigations showed no tendency for any effects on the intestinal microbiota. Colonic irrigation was developed at the National Aeronautics and Space Administration and has been used worldwide in the care of allergic and pollen diseases, skin disorders, and constipation. I previously reported that colonic irrigation may induce lymphocyte transmigration from GALT into the circulation, which may improve the function of both the colon and the immune system functions (Uchiyama-Tanaka, 2009). The increase in the lymphocytes was suspected to be the result of lymphocytes transmigrating as intraepithelial lymphocytes from Peyer's patched and lymph nodes around the intestine as a result of irrigation and abdominal massage.

Based on my personal experience, patient symptoms can improve after colonic irrigations. However, it has been proposed that colon irrigations with large volumes of water may obliterate the microbiota and induce electrolyte abnormalities, but no studies support these claims. The results of this study suggests that after 3s colonic irrigations, the composition of the microbiota changes, but there is no tendency for the changes in the bacterial components.

Colonic irrigations are different from enemas for the following reasons: (a) they are not selfadministered, but are administered by a professionally trained person; and (b) they are administered using a device that controls water flow and infuses the entire colon with water, in contrast to the more limited infusion of warm filtered water into the rectum. The water circulates throughout the colon and removes its contents while the patient lies on a bed. The temperature and pressure of water are closely monitored and regulated during a series of fills and releases to aid colonic peristalsis. Because this method involves a closed system, the waste materials are removed without any unpleasant odor or discomfort, which are usually associated with enemas.

The intestine is an important site of local immunity and nutrition (Iiai et al., 2002). It is a major site of extrathymic T cell differentiation, and numerous activated and quiescent lymphocytes are produced within GALT. The very important role of the intestine, as a part of the immune system is due to the intestinal microbiota. Thus, it has been speculated in peoplewho suffer from constipation and who harbor fecal residues, the intestine may have a diminished function in the immune system (Alveres, 2001, 1924).

The intestinal epithelium is the first line of defense system to encounter intestinal pathogens and dietary antigens. It has been speculated that when the intestine is filled with feces, there may be a reduced function of this immune system caused by toxins leaking from the gut, in addition to bacterial translocation from the gut to the systemic circulation caused by a breakdown of the intestinal wall. This breakdown can be caused by a variety of injuries to the body at further locations far from the gut.

It has been reported that increased gut permeability and bacterial translocation play a role in multiple organ failure (MOF: Swank & Deitch, 1996). Failure of the gut barrier is central to the hypothesis that toxins escaping from the gut lumen contribute to the activation of a host's immune inflammatory defense mechanisms, which subsequently leads to auto-

The Influence of Colonic Irrigation on Human Intestinal Microbiota 457

Collins, MD.; Lawson, PA., Willems ,A., Cordoba, JJ., Fernandez-Garayzabal, J., Garcia, P.,

Garcia-Tsao, G.; Lee, FY., Bardeb, GE., Cartun, R., West, AB. (1995) Bacterial translocation to

Guy-Grand, D.; Cerf-Bensussan, N., Malissen, B., Malassis-Seris ,M., Briottet, C., Vassalli, P.

Hayashi, H.; Sakamoto, M. & Benno, Y. (2002). Phylogenetic analysis of human gut

Hayashi, H.; Sakamoto, M., Bennno, Y. (2002). Fecal microbial diversity in a strict vegetarian

Iiai, T.; Watanabe, H., Suda, T., Okamoto, H., Abo, T., Hatakeyama, K. (2002). CD161+T

Langendijk, PS.; Schut, F., Jansen, GJ., Raangs, GC., Kamphuius, GR., Wilkison ,MH.,

Nagashima, K.; Hisada ,T., Sato, M., Mochizuki, J. (2002). Application of new primer-

Nagashima, K.; Mochizuki, J., Hisada, T., Suzuki, S., Shimomura, K. (2006). Phylogenetic

Purohit, V.; Bode, JC., Bode, C., Brenner, DA., Choudhry, MA., Hamilton, F., Kang, YJ.,

Suau, A.; Bonnet, R., Sutren, M., Godon, JJ., Gibson, G.R., Collins, MD., Dore, J. (1999).

Swank, GM.; Deitch, EA. (1996) Role of the gut in multiple organ failure. Bacterial translocation and permeability changes. *World J Surg* 20: pp. 411-417 Takahashi, S.; Kawamura ,T., Kanda, Y., Taniguchi, T., Nishizawa, T., Iiai, T., Hatakeyama,

consequences: Summary of a symposium. *Alcohol* 42: pp. 349-361

application in fecal samples. *Appl Environ Microbiol* 61: pp. 3069-3075 Moor, WE.; Holdeman, LV.(1974). Human fecal flora: the normal flora of 20 Japanese-

*Bacteriol.* 44: pp. 812-826.

methods. *Microbiol Immunol,* 46: pp. 535-548

Hawaiians. *Appl Environ Microbiol*. 27: pp. 961-979

*Clin Exp Immunol* 129: pp. 92-98.

*Bioscience Microflora.* 25: pp. 99-107.

108: pp. 1835-1841

471-481.

831.

1251-1262

4799-4807

Cai, J., Hippe, H., Farrow, JAE.(1994). The Phylogeny of the Genus Clostridium: Proposal of Five New Genera and Eleven New Species Combinations. *Int J Syst* 

mesenteric lymph nodes is increased in cirrhotic rats with ascites. *Gastroenterology* 

(1991). Two gut intraepithelial CD8+ lymphocyte population with different T cell receptors. A role for the gut epithelium in T cell differentiation. *J Exp Med*. 173: pp.

microbiota using 16S rDNA clone libraries and strictly anaerobic culture-based

as determined by molecular analysis and cultivation. *Microbiol Immunol* 46: pp.819-

(NT) cells exist predominantly in human intestinal epithelium as well as in liver.

Welling, GW. (1995). Quantitative fluorescence in situ hybridization of Bifidobacterium spp. With genus-specific 16S rRNA-targeted probes and its

enzyme combinations to terminal restriction fragment length polymorphism profiling of bacterial populations in human feces. *Appl Environ Microbiol* 69: pp.

analysis of 16S ribosomal RNA gene sequences from human fecal microbiota and improved utility of terminal restriction fragment length polymorphism profiling.

Keshavarzin, A., Rao, R., Sartor, RB., Swanson, C., Turner, JR. (2008) Alcohol, intestinal bacterial growth, intestinal permeability to endotoxin, and medical

Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut. *Appl Environ Microbiol* 65: pp.

K., Abo, T.(2005). Multipotential acceptance of Peyer's patches in the intestine for

intoxication and tissue destruction that are seen in the septic response characteristics of MOF (Swank & Deitch,1996; Garcia-Tsao et al., 1995; Purohit et al., 2008). Thus, colonic irrigation is useful for removing fecal residues.

Although irrigation is useful for establishing a "good" status in the intestine in terms of removing fecal residues, we should not expect too much from these irrigations. Factors like proper nutrition and food intake and a stress-free life style are also important in improving the microbiota, rather than colonic irrigations alone. One report showed that the microbiota of people on a strict vegetarian diet was very different from those on normal diets (Hayashi et al. 2002b). The removal of residual fecal matter in the colon may provide a break from a bad dietary cycle and highlight the importance of intestinal care in everyday life.

Colonic irrigation is relatively safe and is a good method for impressing upon patients the importance of intestinal care. But according to the results of this study, some patients' microbiota deteriorated. Although safe in terms of serum electrolytes, it should be noted that colonic irrigations should not be performed for patients with renal failure, heart disease, liver cirrhosis with ascites, recent abdominal surgery, pregnancy, and other conditions. In addition, excessive therapy such as everyday irrigations should be avoided. This may result in the loss of massive amount of digestive fluid. Except for the first 3 times of colonic irrigation, subsequent irrigations should be performed within a minimum 1 month interval.

This study was limited by its small study population. Some subjects showed reduced proportions in beneficial fecal bacteria, although their symptoms including allergic rhinorrhea, constipation, skin itching, and eczema, were improved. We should be careful with regard to the duration and number of irrigations administered and preferably take probiotics after colonic irrigation. If the patient is in a "good" status in terms of stool analysis, a single trial with an adequate duration would be sufficient.

Another limitation of this study was that the actual numbers of bacteria were not determined using these methods. T-RFLP is only useful for estimating the proportions of bacteria. Hence, we need a more efficient quantitative method for bacterial analysis in addition to the existing one with an advantageous cost versus performance.

In conclusion, colonic irrigation has no influence on serum electrolytes and may induce improvements in symptoms without any effects on the intestinal microbiota.
