Studies Of The Structural Changes Of Gut Microbiota In Response To Various Perturbations

The host and the microbiota harboured in the host buildup a whole superorganism. The whole host and microbiota interacts with the environmental factors and affects the health status of the superorganism. Exogenous compounds such as diet, drug or carcinogen may change the structure of intestinal microbiota and the host also may respond to these environmental perturbations. Monitoring the changes of the intestinal microbiota along with the changes of the health status of the superorganism in response to various perturbations will help us understand the role of the intestinal microbiota in the health of the superorganism.In the first part of this study, we monitored the gradual shift of intestinal microbiota during the formation of the precancerous lesion of colon cancer induced by the carcinogen 1,2-dimethylhydrazine (DMH) and observed the change of intestinal microbiota when the host was protected with two kinds of Chinese herbs. 28 male Wistar rats weighted 80-100g were divided randomly into four groups. Rats in model group (n=7), Coptidis Rhizoma-Evodiae Fructus herb treated group (n=7) and an oriental prescription consisting of twelve herbs named JFK treated group (n=7) received subcutaneous (s.c.) injection with 1,2-dimethylhydrazine (DMH) and the healthy control group (n=7) with carrier. Coptidis Rhizoma-Evodiae Fructus treated group and JFK treated group were orally dosed with corresponding herbs each day. Fresh feces of the animals in the four groups were collected at the last day of the 3rd, 5th and 9th week of the entire experimental period. V3 region of 16S rRNA gene PCR-DGGE and Bacteroides spp., Clostridium leptum subgroup, Lactic acid bacteria (LAB), Bifidobacterium spp. group specific PCR-DGGE followed by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used to monitor the structure of the intestinal microbiota of all rats.Fingerprints from V3 region of 16S rRNA gene and Clostridium leptum subgroup PCR-DGGE of fecal samples showed that structures of intestinal microbiota between control and only DMH treated rats were similar at 3rd and slightly different at 5th week, but significantly different from each other at 9th week when (37.7±2.6) aberrant crypt foci(ACF)developed in the only DMH treated rats'colons. Martens'uncertainty test followed by ANOVA test (p<0.05) identified two bands of V3 region of 16S rRNA gene PCR-DGGE which were Ruminococcus-like and Allobaculum-like bacteria by sequence and five bands of Clostridium leptum subgroup PCR-DGGE as key variables for discrimination of ACF rats from controls at the 9th week. Species-specific real-time PCR confirmed the significant abundance increase of Ruminococcus-like and Allobaculum-like bacteria in ACF rats at the 9th week. Fingerprints from Bacteroides spp., Lactic acid bacteria (LAB) and Bifidobacterium spp. group specific PCR-DGGE does not show obvious difference between ACF rats and controls at the 9th week. This structural segregation analysis of intestinal microbiota may become a new strategy for assessing heath status of the host. When the structure of the intestinal microbiota away from the controls, the heath status of the host maybe also change.In addition, histopathological examination confirmed formation of (16.7±1.2) ACF in Coptidis Rhizoma-Evodiae Fructus herb treated group and (15.1±2.9) ACF in JFK treated group at 9th week and both of the two formulations of Chinese herbal medicine could significantly reduce the number of ACF in colons. PCA score plot of V3 region of 16S rRNA gene PCR-DGGE fingerprint indicated that structure of intestinal microbiota of the two herbal medicine treated groups were shifted away from the only DMH treated group at the 9th week. Result of species-specific real-time PCR indicated that the abundance of the Ruminococcus obeum and Allobaculum-like bacteria in the two herbal treated groups decreased to the level of healthy control group with the decreased number of ACF, while it is significantly different from the only DMH treated group. Quantifying the decreased number of ACF has been used for evaluating chemoprevention efficiency to colon cancer, but it is an invasive method because it need execute the rats. This work indicates that monitoring the amount of Ruminococcus obeum and Allobaculum-like bacteria may be employed as a non-invasive method for evaluating chemoprevention efficiency of these kinds of complex drugs to colon cancer.In the second part of this study, we studied the modulation of fructo-oligosaccharides to mucosa associated bacteria and lumen bacteria of different parts of large intestine and fecal bacteria. Samples were taken from mucosa and content of cecum, proximal colon and distal colon and feces of 10 human flora-associated (HFA) piglets (5 piglets receive prebiotic treatment and 5 piglets as control). V3 region of 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) and Bifidobacterium spp, Lactic acid bacteria (LAB), Clostridium leptum subgroup specific PCR-DGGE followed by PCA and PLS-DA were used to analyze the structure of lumen and mucosa associated bacteria in the different parts of the large intestine and the effect of prebiotic on intestial microbiota.Clostridium sartagoforme had higher intensity in the mucosa of distal colon and Streptococcus pasteuri and Faecalibacterium prausnitzii had lower intensity in mucosa of distal colon when compared with other parts. Structure of the predominant bacteria among lumen contents in different parts of large intestine and feces was similar, but, significantly different from mucosa associated bacteria. Escherichia coli and an uncultured bacterium clone E308 (DQ326831)-like bacteria had higher intensity in the content when compared with which in the mucosa sample. Clostridiaceae bacterium NML-like bacteria appeared in the mucosa of distal colon of all piglets but not in the cecum and proximal colon. Structure of Bifidobacterium spp. and LAB had no obvious difference among different sampling positions. The prebiotic supplementation in this study showed modulating effects on the structure of C. leptum in the content of cecum and proximal colon. The intensity of Ruminococcus bromii decreased but Ruminococcus bromii strain YE282-like bacteria increased in the prebiotic treated group. Structure of predominant bacteria, Bifidobacterium spp. and LAB had no obvious difference between the prebiotic treated and control group.The last part of of this study is about one problem we met during the HFA piglets establishment and how we solved it. For establishment the HFA piglets, human fecal suspension should be orally inoculated to the newborn piglets. 17 of 24 piglets in two litters died in the first two weeks after the first administration of the human fecal suspension and one strain of opportunistic pathogen Kl. pneumoniae from fecal suspension of an apparently healthy human donor caused the fatal infection in HFA piglets. In most previous studies of establishing HFA rats/mice models, fecal suspension was usually obtained from apparently healthy human donor who had no history of either gastrointestinal or metabolic disorders and did not get any prescriptive supplementary diets and drug treatments for 3 months prior to the fecal collection. However, in these two HFA piglets'trials, our donor according to the current standard still cause death of piglets. This study suggests the previous standard for donor is not safe enough for establishment the HFA animals. Here, we recommend a new protocol for evaluating the safety of the human donor's flora for HFA animal. First, screening for pathogens and some opportunistic pathogens common for neonatal animal infection should be performed with culture or PCR-based methods. Second, preliminary tests should be performed, in which a large amount of the whole fecal flora (5-10 times of normal dosage) prepared for HFA animals should be orally inoculated to naturally born neonatal animals for at least three days (once a day). Only the whole fecal flora, which does not cause problems in the preliminary test, should be used in the following HFA experiment. According to this standard, until now, we did not meet serious problems during the establishment of HFA piglets.