The differences in colonic mucosal microbiota between normal individual and colon cancer patients by polymerase chain reaction-denaturing gradient gel electrophoresis.

OBJECTIVE: The aim of this study was to analyze the differences in the intestinal composition between normal individuals and colon cancer patients. METHODS: To establish the criteria for screening a normal individual for colon cancer, human colonic biopsies were obtained at routine colonoscopy. For patients with colon cancer, samples were obtained from cancerous regions. For normal individuals, colonic biopsies were taken from 3 sites of large intestine (descending, transverse, and ascending colon). Thereafter, a comparison of the microbiota structure by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was carried out. At last, bacterial species were identified by sequencing special bands from DGGE gels and comparing data with sequence databases. RESULT: With PCR-DGGE, we have discovered that the diversity and richness of the bacterial community from colon cancer patient's colonic mucosa were lower than that of the normal individual's sample. Then, a special DGGE band was found in the colon cancer patients. After sequencing, we confirmed that it had a high level of similarity with bacteroides. CONCLUSIONS: Colon cancers are closely related with the alteration of intestinal flora such as the reduction of biodiversity and richness of the bacterial community. Furthermore, the increase in proportion of bacteroides may be directly associated with colon cancer.

Characterization of mucosa-associated bacterial communities of the mouse intestine by terminal restriction fragment length polymorphism: Utility of sampling strategies and methods to reduce single-stranded DNA artifacts.

Terminal restriction fragment length polymorphism (T-RFLP) is a molecular technique used for comparative analysis of microbial community structure and dynamics. We evaluated three sampling methods for recovering bacterial community DNA associated with intestinal mucosa of mice (i.e. mechanical agitation with PBS, hand washing with PBS containing Tween 80, and direct DNA extraction from mucosal plugs). In addition, the utility of two methods (i.e. Klenow fragment and mung-bean nuclease) to reduce single-stranded DNA artifacts was tested. T-RFLP analysis indicated that diverse communities of bacteria are associated with mucosa of the ileum, cecum, and descending colon of mice. Although there was no significant difference in bacterial community structure between the mechanical agitation and direct DNA extraction methods regardless of intestinal location, community diversity was reduced for the hand wash method in the colon. The use of Klenow fragment and mung-bean nuclease have been reported to eliminate single-stranded DNA artifacts (i.e. pseudo-T-restriction fragments), but neither method was beneficial for characterizing mucosa-associated bacterial communities of the mouse cecum. Our study showed that the mechanical agitation and direct plug extraction methods yielded equivalent bacterial community DNA from the mucosa of the small and large intestines of mice, but the latter method was superior for logistical reasons. We also applied a combination of different statistical approaches to analyze T-RFLP data, including statistical detection of true peaks, analysis of variance for peak number, and group significance test, which provided a quantitative improvement for the interpretation of the T-RFLP data.

Heterogeneity in enterohemorrhagic Escherichia coli O157:H7 fecal shedding in cattle is related to Escherichia coli O157:H7 colonization of the small and large intestine.

In the last decade, Escherichia coli O157:H7 have emerged as important pathogens of the gastrointestinal tract of humans. Healthy cattle have been identified as the primary reservoir, however, the factors affecting heterogeneous E. coli O157:H7 fecal shedding are not fully understood. The aim of this study was to investigate the contribution of E. coli O157:H7 colonization of small and large intestinal sites to the heterogeneity of fecal shedding in cattle. There was a dose-dependant E. coli O157:H7 E318N colonization of duodenum, jejunum, ileum, cecum, ascending colon, spiral colon, descending colon, and the rectoanal junction in vitro with no difference in E. coli O157:H7 colonization of the rectoanal junction and other intestinal sites. There were 10-100 times greater E. coli O157:H7 colonization of intestinal sites from persistent shedding cattle compared with non persistent shedding cattle. Novel pathologies were associated with E. coli O157:H7 colonization sites in the small and large intestine. The first pathology, focal petechiae, was present throughout the intestinal tract of cattle that ceased shedding E. coli O157:H7 for 5-12 weeks or in the jejunum, ileum, cecum, and ascending colon of cattle shedding E. coli O157:H7 for 4-5 months. The second pathology, mucosal hemorrhages, was present in the same sites as the focal petechiae in cattle shedding for 5 months and these hemorrhages were in the final stages of repair. Several features of these hemorrhages support this conclusion including the brown appearance, low amount of classic E. coli O157:H7 induced A/E lesions, flattened epithelium, and blunted villi. Although mucosal hemorrhages were present in the jejunum, ileum, cecum, and ascending colon in cattle shedding for 4 months, many other pathologies were also present that were indicative of hemorrhagic enteritis as evidenced by the blood red appearance of hemorrhages, severe edema, and dark red erythema. Escherichia coli O157:H7 were associated with both pathologies suggesting it is the causative agent. The current study supports a relationship between the amount of E. coli O157:H7 colonization in intestinal sites and heterogeneous fecal shedding by cattle.

Inflammatory bowel disease in rats: bacterial and chemical interaction.

AIM: To develop a novel model of colitis in rats, using a combination of iodoacetamide and enteropathogenic E. coli (EPEC), and to elucidate the pathophysiologic processes implicated in the development of ulcerative colitis (UC). METHODS: Male Sprague-Dawley rats (n = 158) were inoculated intrarectally on a weekly basis with 4 different combinations: (a) 1% methylcellulose (MC), (b) 100 microL of 6% iodoacetamide (IA) in 1% MC, (c) 200 microL containing 4 x 10(8) colony factor units (CFU) of EPEC, and (d) combined treatment of (IA) followed by bacteria (B) after 2 d. Thirty days post treatment, each of the four groups was divided into two subgroups; the inoculation was stopped for one subgroup and the other subgroup continued with biweekly inoculation until the end of the experiment. Colitis was evaluated by the clinical course of the disease, the macroscopic and microscopic alterations, activity of myeloperoxidase (MPO), and by TNF-alpha gene expression. RESULTS: Findings indicative of UC were seen in the combined treatment (IA + B) as well as the IA continued treatment groups: the animals showed slow rate of increase in body weight, diarrhea, bloody stools, high colonic ulcer score, as well as histological alterations characteristic of UC, with an extensive inflammatory reaction. During the course of the experiment, the MPO activity was consistently elevated and the TNF-alpha gene expression was upregulated compared to the control animals. CONCLUSION: The experimental ulcerative colitis model used in the present study resembles, to a great extent, the human disease. It is reproducible with characteristics indicative of chronicity.

Mycobacterium avium subspecies paratuberculosis infection in cases of irritable bowel syndrome and comparison with Crohn's disease and Johne's disease: common neural and immune pathogenicities.

Mycobacterium avium subsp. paratuberculosis causes Johne's disease, a systemic infection and chronic inflammation of the intestine that affects many species, including primates. Infection is widespread in livestock, and human populations are exposed. Johne's disease is associated with immune dysregulation, with involvement of the enteric nervous system overlapping with features of irritable bowel syndrome in humans. The present study was designed to look for an association between Mycobacterium avium subsp. paratuberculosis infection and irritable bowel syndrome. Mucosal biopsy specimens from the ileum and the ascending and descending colon were obtained from patients with irritable bowel syndrome attending the University of Sassari, Sassari, Sardinia, Italy. Crohn's disease and healthy control groups were also included. Mycobacterium avium subsp. paratuberculosis was detected by IS900 PCR with amplicon sequencing. Data on the potential risk factors for human exposure to these pathogens and on isolates from Sardinian dairy sheep were also obtained. Mycobacterium avium subsp. paratuberculosis was detected in 15 of 20 (75%) patients with irritable bowel syndrome, 3 of 20 (15%) healthy controls, and 20 of 23 (87%) people with Crohn's disease (P = 0.0003 for irritable bowel syndrome patients versus healthy controls and P = 0.0000 for Crohn's disease patients versus healthy controls). One subject in each group had a conserved single-nucleotide polymorphism at position 247 of IS900 that was also found in isolates from seven of eight dairy sheep. There was a significant association (P = 0.0018) between Mycobacterium avium subsp. paratuberculosis infection and the consumption of hand-made cheese. Mycobacterium avium subsp. paratuberculosis is a candidate pathogen in the causation of a proportion of cases of irritable bowel syndrome as well as in Crohn's disease.

Impact of orally administered microcapsules on gastrointestinal microbial flora: in-vitro investigation using computer controlled dynamic human gastrointestinal model.

Oral administration of artificial cell microcapsules has been proposed for various therapy procedures using biologically active materials. Recently we have designed novel APPPA microcapsules using alginate, poly-L-lysine, pectin, poly-L-lysine and alginate that have shown superior oral delivery features. This article investigates, in-vitro using a computer controlled dynamic gastrointestinal (GI) model, effects of APPPA microcapsules on health of gastrointestinal (GI) microbial flora. The impact of APPPA microcapsules on GI bacterial population, total anaerobes, total aerobes, Escherichia coli, Lactobacillus sp. and Staphylococcus sp. has been analyzed. In addition, the effects of microcapsules on GI microbial extracellular enzymatic activities have been investigated. Result shows the altered activities of microbial flora and enzymes due to the use of APPPA microcapsule. The most disparity is observed in the colon ascendans microbial activities. This study would have significant impact on future microcapsule design. However, further in-vivo studies are required.

Bifidobacterial diversity determined by culturing and by 16S rDNA sequence analysis in feces and mucosa from ten healthy Spanish adults.

This study aimed to identify the numerically predominant bifidobacterial species in feces and mucosa of healthy Spanish people and to determine their phenotypic and genetic diversity. To this end, both traditional culturing and molecular methods were used. A set of 196 bifidobacterial colonies was identified from the counting plates by sequencing of a stretch of their 16S rRNA gene. Representative isolates were phenotypically characterized by their carbohydrate fermentation profile and genotypically typed by RAPD-PCR. Four 16S rDNA libraries composed of 113 clones from two fecal and two mucosal samples were independently analyzed. Seven bifidobacterial species were identified by culturing, and six by 16S rDNA analysis. Both methodologies showed Bifidobacterium longum and B. pseudocatenulatum to predominate in feces and mucosa, although high interindividual variability was noted. High phenotypic variation was observed in the fermentation profile of different isolates from the same species. RAPD analysis showed that two to five strains made up the subjects' personal bifidobacterial communities. The identification of the dominant bifidobacterial species could be useful for the rational design, use, and evaluation of probiotics in our community.