Isolation of mucosa-associated microbiota dysbiosis in the ascending colon in hepatitis C virus post-sustained virologic response cirrhotic patients.

Background: Achieving sustained virologic response (SVR) in patients infected with hepatitis C virus (HCV) reduces all-cause mortality. However, the mechanisms and risk factors for liver fibrosis and portal hypertension post-SVR remain incompletely understood. In the gut-liver axis, mucosa-associated microbiota (MAM) substantially influence immune and metabolic functions, displaying spatial heterogeneity at the anatomical intestinal site. We analyzed MAM composition and function to isolate the locoregional MAM involved in chronic liver disease progression in HCV post-SVR patients. Methods: We collected MAM samples from three intestinal sites (terminal ileum, ascending colon, and sigmoid colon) via brushing during colonoscopy in 23 HCV post-SVR patients and 25 individuals without liver disease (controls). The 16S rRNA of bacterial DNA in specimens collected with a brush and in feces was sequenced. The molecular expression of intestinal tissues and hepatic tissues were evaluated by quantitative real-time PCR. Results: In the post-SVR group, the microbial ß-diversity of MAM, especially in the ascending colon, differed from the control group and was associated with liver fibrosis progression. In PICRUSt analysis, MAM in the ascending colon in the liver cirrhosis (LC) group showed compromised functions associated with the intestinal barrier and bile acid production, and FGF19 expression was markedly decreased in the terminal ileum biopsy tissue in the LC group. At the genus level, six short-chain fatty acid (SCFA)-producing bacterial genera, Blautia, Alistipes, Roseburia, Agathobaculum, Dorea, and Pseudoflavonifractor were reduced in the ascending colon of post-SVR LC patients. Conclusion: In patients of HCV post-SVR, we identified the association between the degree of liver fibrosis and dysbiosis of mucosa-associated SCFA-producing bacterial genera that may be related to intestinal barrier and bile acid production in the ascending colon.

Spatial and temporal modulation of enterotoxigenic E. coli H10407 pathogenesis and interplay with microbiota in human gut models.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) substantially contributes to the burden of diarrheal illnesses in developing countries. With the use of complementary in vitro models of the human digestive environment, TNO gastrointestinal model (TIM-1), and Mucosal Simulator of the Human Intestinal Microbial Ecosystem (M-SHIME), we provided the first detailed report on the spatial-temporal modulation of ETEC H10407 survival, virulence, and its interplay with gut microbiota. These systems integrate the main physicochemical parameters of the human upper digestion (TIM-1) and simulate the ileum vs ascending colon microbial communities and luminal vs mucosal microenvironments, captured from six fecal donors (M-SHIME). RESULTS: A loss of ETEC viability was noticed upon gastric digestion, while a growth renewal was found at the end of jejunal and ileal digestion. The remarkable ETEC mucosal attachment helped to maintain luminal concentrations above 6 log10 mL-1 in the ileum and ascending colon up to 5 days post-infection. Seven ETEC virulence genes were monitored. Most of them were switched on in the stomach and switched off in the TIM-1 ileal effluents and in a late post-infectious stage in the M-SHIME ascending colon. No heat-labile enterotoxin production was measured in the stomach in contrast to the ileum and ascending colon. Using 16S rRNA gene-based amplicon sequencing, ETEC infection modulated the microbial community structure of the ileum mucus and ascending colon lumen. CONCLUSIONS: This study provides a better understanding of the interplay between ETEC and gastrointestinal cues and may serve to complete knowledge on ETEC pathogenesis and inspire novel prophylactic strategies for diarrheal diseases.

Seasonal Variation Influences on Intestinal Microbiota in Rats.

Recently, several studies have indicated that the intestinal microbiota can be regulated by the individual attributes, and even the alternation of circadian rhythm. Inspired by this, we speculated that seasonal variation might also have some effect on the intestinal microbiota. A total of 11 Sprague-Dawley male rats, weighing 250-280 g, were divided into summer group (n = 5) and winter group (n = 6). Cages were individually ventilated at 20 ± 2 °C and 45-65% relative humidity with a circadian rhythm of 12/12 h. After 1 week of adaptively feeding, mucosal contents of jejunum, terminal ileum, and ascending colon were collected and analyzed by 16S rRNA Gene Amplicon Pyrosequencing. The results showed that intestinal microbiota of rats for the same strain were affected by season change under the same feeding condition and living environment. We should take seasonal factor into account in the future experimental design based on intestinal microbiome.

Alterations in microRNA expression profiles in inflamed and noninflamed ascending colon mucosae of patients with active Crohn's disease.

BACKGROUND AND AIM: The microRNA (miRNA) expression profiles of the terminal ileum, sigmoid colon, and rectal mucosa of adult patients with active Crohn's disease (CD) have been previously reported. The purpose of this study was to identify dysregulated miRNAs in the mucosa of the ascending colon. METHODS: Biopsy tissue samples were taken from the mucosae of inflammatory (iCD) or noninflammatory (niCD) areas of the ascending colons of adult patients with active CD. miRNA and mRNA expression profiles were detected using microarray analyses. miRNAs and messenger RNAs (mRNAs) demonstrating significant differences were validated via quantitative real-time polymerase chain reaction. Luciferase reporter genes were used to measure two miRNAs inhibition of potential target genes in human 293T cells in vitro. RESULTS: Compared with the healthy control group, the ascending colon miRNA expression profiles revealed that 43 miRNAs were significantly upregulated and 35 were downregulated in the iCD group. The mRNA expression profiles indicated that 3370 transcripts were significantly differentially expressed in the ascending colon, with 2169 upregulated and 1201 downregulated mRNAs in the iCD group, and only 20 miRNAs demonstrated significant differential expression in the niCD group. In contrast, nearly 100 miRNAs significantly varied between the iCD and niCD groups. Finally, luciferase reporter gene assays showed that hsa-miR-16-1 directly regulated the human C10orf54 gene and that they were negatively correlated. CONCLUSIONS: Our results indicated that the differentially expressed miRNAs and mRNAs were related to immune inflammation and intestinal flora. The data provide preliminary evidence that the occurrence of CD involves the inhibition of C10orf54 expression by hsa-miR-16-1.

The effect of experimental fusarium mycotoxicosis on microbiota diversity in porcine ascending colon contents.

The objective of the study was to determine the effect of exposure of pigs to the Fusarium mycotoxins zearalenone (ZEN) and deoxynivalenol (DON), administered together and separately, on the colon microbiota. An experiment was conducted for 42 days on gilts, randomly assigned to four groups and administered either ZEN, DON, ZEN+DON, or a placebo. The number of aerobic mesophilic bacteria, yeasts, molds, anaerobic Clostridium perfringens, fecal streptococci, Enterobacteriaceae, Escherichia coli, and lactic acid bacteria (LAB) were determined in the contents of the ascending colon. The influence of mycotoxins on the functional diversity of the colonic microbiota was assessed using EcoPlate tests (Biolog). Analysis revealed the predominance of LAB in all groups of pigs. Zearalenone, administered separately and together with DON, was found to have an adverse effect on mesophilic aerobic bacteria, but only after long exposure to this mycotoxin. During the six weeks of the experiment, the concentration of C. perfringens, E. coli, and other bacteria in the family Enterobacteriaceae was most considerably reduced in the experimental groups exposed to zearalenone, both separately and together with DON. Mycotoxins also affected the functional biodiversity of microorganisms. Both Shannon's diversity index and the number of catabolized substrates in Biolog plate (the R index) were much higher in the group subjected to mixed mycotoxicosis.

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.

Mode of delivery and early nutrition modulate microbial colonization and fermentation products in neonatal piglets.

Colonization of the intestinal microbiota after birth plays an important role in development of the neonatal gastrointestinal and immune systems. Two key environmental factors that influence the colonization pattern are delivery mode and nutrition. In this study, the impact of delivery mode and nutrition on microbial colonization and metabolic activity was investigated in the pig model. Vaginally (VD) or caesarean- (CD) delivered piglets were sow-reared (SR) or fed formula alone (FF) or with 4 g/L prebiotics [1:1 ratio of short-chain fructo-oligosaccharides (scFOS) and polydextrose (PDX); FP]. Intestinal contents were collected on d 7 and 14. SR piglets harbored different microbial populations from FF and FP piglets in ileum and ascending colon (AC). On d 7, FF piglets had a greater abundance of Clostridium XIVa in AC, but lower total bacteria, Clostridium XIVa, and Lactobacillus spp. in ileum and Fecalibacterium prausnitzii in AC compared with FP piglets. On d 14, total bacteria were more abundant in FP than FF piglets. Butyrate, isobutyrate, valerate, and isovalerate concentrations in AC were greater in SR piglets compared with FF or FP piglets. At both sampling days, acetate concentrations in AC were similar between the SR and FF groups, whereas propionate was higher in the SR compared with FF group. Delivery mode also significantly affected microbial populations. Bacterial densities differed in AC for Bacteroides-Prevotella at d 7 and Clostridium XIVa at d 14, being higher in VD piglets. Correspondingly, VD piglets had higher propionate in ileum and propionate and butyrate in AC compared with CD piglets. Our results indicate that both delivery mode and nutrition affect microbial composition and metabolic activity. Supplementation of scFOS/PDX to formula modulates microbial colonization and produces a SCFA pattern closer to that of SR piglets.

Histoplanimetrical study on the relationship between invasion of indigenous bacteria into intestinal crypts and proliferation of epithelial cells in rat ascending colon.

The relationship between the invasion of indigenous bacteria into intestinal crypts and the proliferation of epithelial cells was histoplanimetrically investigated in the rat ascending colon. Indigenous bacteria preferentially adhered to the intestinal superficial epithelial cells in the mesenterium-attached mucosa (MAM) compared to those in the mesenterium-non-attached mucosa (MNM). Intestinal crypts with indigenous bacteria were also significantly more frequently found in MAM than in MNM. Total epithelial cells, columnar epithelial cells and goblet cells were significantly more abundant in the intestinal crypts with no-indigenous bacteria in MAM (MAM-C) than those in MNM (MNM-C), whereas the columnar epithelial cells were less abundant in MAM-C than in the intestinal crypts with indigenous bacteria in MAM (MAM-C-B). Columnar epithelial cells and goblet cells immuno-positive for proliferating cell nuclear antigen (PCNA) in MAM-C were more abundant than those in MNM-C, but less abundant than those in MAM-C-B. Toll-like receptor (TLR)-2, -4 and -9 were immuno-positive in the striated borders of the intestinal superficial epithelial cells, but their positive intensities were weaker in MAM than in MNM. From these findings, indigenous bacteria were confirmed to preferentially settle on the intestinal superficial epithelium of MAM in the rat ascending colon, and low TLRs-expression might contribute to the preferential settlement of indigenous bacteria in MAM. The increase of proliferating epithelial cells is probably induced by the invasion of indigenous bacteria into the intestinal crypts of MAM.

Microbial composition and in vitro fermentation patterns of human milk oligosaccharides and prebiotics differ between formula-fed and sow-reared piglets.

The microbial composition and in vitro fermentation characteristics of human milk oligosaccharides (HMO), lacto-N-neotetraose (LNnT), a 2:1 mixture of polydextrose (PDX) and galactooligosaccharides (GOS), and short-chain fructooligosaccharides (scFOS) by pooled ascending colonic microbiota from 9- and 17-d-old formula-fed (FF) and sow-reared (SR) piglets were assessed. pH change and gas, SCFA, and lactate production were determined after 0, 2, 4, 8, and 12 h of incubation. In most donor groups, the pH change was greater for scFOS fermentation and lower for PDX/GOS than for other substrates. LNnT fermentation produced larger amounts of gas, total SCFA, acetate, and butyrate than did the other substrates, whereas HMO and scFOS produced higher amounts of propionate and lactate, respectively. In general, pH change, total SCFA, acetate, and propionate production were greater in pooled inoculum from FF and 9-d-old piglets, whereas SR-derived inoculum produced higher amounts of butyrate and lactate after 4 h fermentation. Gut microbiota were assessed by 16S ribosomal RNA V3 gene denaturing gradient gel electrophoresis analysis and real-time qPCR. Microbial structures differed among the 4 groups before fermentation, with higher counts of Bifidobacterium in SR piglets and higher counts of Clostridium cluster IV, XIVa, and Bacteroides vulgatus in FF piglets. Lactobacillus counts were higher in 9-d-old piglets than in 17-d-old piglets, regardless of diet. Bifidobacterium, Bacteroides, and clostridial species increased after 8 and 12 h fermentation on most substrates. In summary, piglet diet and age affect gut microbiota, leading to different fermentation patterns. HMO have potential prebiotic effects due to their effects on SCFA production and microbial modulation.

Degradation kinetics of metronidazole and olsalazine by bacteria in ascending colon and in feces of healthy adults.

PURPOSE: To compare the degradation kinetics of metronidazole and olsalazine by the bacteria of ascending colon and the bacteria of feces of healthy adults. METHODS: Contents of the ascending colon of seven healthy adults were collected under conditions simulating the bioavailability/bioequivalence studies in the fasted and in the fed states on a crossover basis. Material from the contents of the ascending colon was prepared by ultracentrifuging and diluting the precipitate with a volume of normal saline equivalent to that of the supernatant. Fecal material was prepared from feces of three healthy adults collected at two occasions that were separated by at least 6 months. Ex vivo drug degradation kinetics were evaluated under anaerobic conditions. RESULTS: Mean half-lives of metronidazole degradation in material from the contents of the ascending colon collected in the fasted state and in fecal material were 16.1 and 2.4 min, respectively (p<0.001). The corresponding numbers for olsalazine were 57.8 and 9.2 min, respectively (p<0.001). Both compounds were stable in material from the contents of ascending colon collected in the fed state. CONCLUSIONS: Compared with data in fecal material, degradation of metronidazole and olsalazine in material from the contents of the ascending colon is significantly slower and it becomes non-significant during the arrival of fresh food remnants in the region.

Spatial organization of intestinal microbiota in the mouse ascending colon.

Complex microbial populations are organized in relation to their environment. In the intestine, the inner lining (mucosa) is a potential focal point for such organization. The proximal murine colon contains mucosal folds that are known to be associated with morphologically distinct microbes. To identify these microbes, we used the technique of laser capture microdissection (LCM) to sample microbes associated with these folds (interfold region) and within the central lumen (digesta region). Using 16S rRNA gene tag pyrosequencing, we found that microbes in the interfold region were highly enriched for the phylum Firmicutes and, more specifically, for the families Lachnospiraceae and Ruminococcaceae. Other families such as Bacteroidaceae, Enterococcaceae and Lactobacillaceae were all enriched in the digesta region. This high-resolution system to capture and examine spatial organization of intestinal microbes should facilitate microbial analysis in other mouse models, furthering our understanding of host-microbial interactions.

Increased inflammation and lethality of Dusp1-/- mice in polymicrobial peritonitis models.

The mitogen-activated protein kinase phosphatase Dusp1 (also known as MKP-1) is essential for control of the inflammatory response to systemic challenge with the lipopolysaccharide of Gram-negative bacteria. Here, we have investigated the consequences of Dusp1-deficiency in colon ascendens stent peritonitis (CASP) and caecal ligation and puncture (CLP), two mouse models of septic peritonitis. Following CASP, Dusp1(-/-) mice had increased serum levels of CCL4, interleukin-10 (IL-10) and IL-6, with differences from wild-type mice being dependent on severity of sepsis. These cytokines, along with inducible nitric oxide synthase messenger RNA, were also expressed at higher levels in spleen and liver. Similar over-production of these cytokines was detected in the CLP model, with even larger differences from wild-type mice. Despite the increased inflammatory response, bacterial clearance was impaired in Dusp1(-/-) mice subjected to CASP and CLP. Dusp1(-/-) mice suffered increased lethality in both peritonitis models. Together our data indicate that exaggerated inflammatory responses to gut bacteria introduced into the peritoneum in the absence of Dusp1 do not help to control bacterial replication but are detrimental for the host.

Colon ascendens stent peritonitis (CASP)--a standardized model for polymicrobial abdominal sepsis.

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for establishing new therapeutic approaches in this field. Therefore, animal models are required that are able to closely mimic the human disease and also sufficiently deal with scientific questions. The Colon Ascendens Stent Peritonitis (CASP) is a highly standardized model for polymicrobial abdominal sepsis in rodents. In this model, a small stent is surgically inserted into the ascending colon of mice or rats leading to a continuous leakage of intestinal bacteria into the peritoneal cavity. The procedure results in peritonitis, systemic bacteraemia, organ infection by gut bacteria, and systemic but also local release of several pro- and anti-inflammatory cytokines. The lethality of CASP can be controlled by the diameter of the inserted stent. A variant of this model, the so-called CASP with intervention (CASPI), raises opportunity to remove the septic focus by a second operation according to common procedures in clinical practice. CASP is an easily learnable and highly reproducible model that closely mimics the clinical course of abdominal sepsis. It leads way to study on questions in several scientific fields e.g. immunology, infectiology, or surgery.

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.

A new concept in colonic drug targeting: a combined pH-responsive and bacterially-triggered drug delivery technology.

BACKGROUND: Current approaches to colonic drug delivery exploit one of two main physiological characteristics: the pH change or increase in bacterial numbers along the gastrointestinal tract. Here, we describe a new concept in targeted delivery, which combines these triggers to improve colonic delivery. AIM: To assess the in-vivo targeting performance of a novel colonic delivery coating comprising a mixture of pH-responsive enteric polymer (Eudragit S) and biodegradable polysaccharide (resistant starch) in a single layer matrix film. METHODS: Tablets (radio-labelled) were film-coated with the dual-mechanism coating and administered in a three-way crossover study to eight healthy volunteers (i) without food, (ii) with breakfast or (iii) 30 min before breakfast. The site of intestinal disintegration was assessed using gamma scintigraphy. RESULTS: The coated tablets were able to resist breakdown in the stomach and small intestine. Consistent disintegration of the dosage form was seen at the ileocaecal junction/large intestine. The site of disintegration remained unaffected by feeding. CONCLUSIONS: The dual-mechanism (pH/bacterial) coating provides colon-specificity. Each trigger mechanism has the capacity to act as a failsafe, ensuring appropriate targeting in the gastrointestinal tract. This platform technology has potential for systemic applications or the treatment of local disorders of the large intestine, such as inflammatory bowel disease.

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.