Linking phylogenetic identities of bacteria to starch fermentation in an in vitro model of the large intestine by RNA-based stable isotope probing

Carbohydrates, including starches, are an important energy source for humans, and are known for their interactions with the microbiota in the digestive tract. Largely, those interactions are thought to promote human health. Using 16S ribosomal RNA (rRNA)-based stable isotope probing (SIP), we identified starch-fermenting bacteria under human colon-like conditions. To the microbiota of the TIM-2 in vitro model of the human colon 7.4 g l⁻¹ of [U-¹³C]-starch was added. RNA extracted from lumen samples after 0 (control), 2, 4 and 8 h was subjected to density-gradient ultracentrifugation. Terminal-restriction fragment length polymorphism (T-RFLP) fingerprinting and phylogenetic analyses of the labelled and unlabelled 16S rRNA suggested populations related to Ruminococcus bromii, Prevotella spp. and Eubacterium rectale to be involved in starch metabolism. Additionally, 16S rRNA related to that of Bifidobacterium adolescentis was abundant in all analysed fractions. While this might be due to the enrichment of high-GC RNA in high-density fractions, it could also indicate an active role in starch fermentation. Comparison of the T-RFLP fingerprints of experiments performed with labelled and unlabelled starch revealed Ruminococcus bromii as the primary degrader in starch fermentation in the studied model, as it was found to solely predominate in the labelled fractions. LC-MS analyses of the lumen and dialysate samples showed that, for both experiments, starch fermentation primarily yielded acetate, butyrate and propionate. Integration of molecular and metabolite data suggests metabolic cross-feeding in the system, where populations related to Ruminococcus bromii are the primary starch degrader, while those related to Prevotella spp., Bifidobacterium adolescentis and Eubacterium rectale might be further involved in the trophic chain.     

Effects of the in vitro fermentation of oligofructose and inulin by bacteria growing in the human large intestine

The in vitro fermentability of oligofructose and inulin was compared with a range of reference carbohydrates by measuring bacterial end-product formation in batch culture. Short chain fatty acid and gas formation indicated that these substrates, which occur naturally in the diet and reach the colon in a largely intact form, were utilized by mixed populations of gut bacteria. Bacterial growth data showed that oligofructose and inulin exerted a preferential stimulatory effect on numbers of the health-promoting genus Bifidobacterium , whilst maintaining populations of potential pathogens ( Escherichia coli , Clostridium ) at relatively low levels. Pure culture studies confirmed the enhanced ability of bifidobacteria to utilize these substrates in comparison with glucose. Batch culture experiments demonstrated that the growth of Bifidobacterium infantis had an inhibitory effect towards E. coli and Clostridium perfringens. Potentially, an increase in the concentration of these substrates in the diet may therefore improve the composition of the large intestinal microflora and have positive effects on the quality of the Western diet.     

Contribution of the microflora to proteolysis in the human large intestine

Protease activities in human ileal effluent were approximately 20-fold greater than in normal faeces. Comparative studies with faeces from a person who did not have a pancreas suggested that a substantial proportion of the proteolytic activity in normal faeces was of bacterial origin. Thimerosal, iodoacetate, EDTA and cysteine significantly inhibited proteolysis in faeces, but not in small intestinal contents, showing that cysteine and metalloproteases were produced by bacteria in the large gut. These results, together with results from studies using p -nitroanilide substrates, demonstrated that faecal proteolysis was both qualitatively and quantitatively different from that in the small intestine. Studies with pure cultures of proteolytic gut bacteria indicated that the cell-bound proteases of Bacteroides fragilis -type organisms were likely to contribute significantly towards proteolytic activity associated with the washed cell fraction and washed particulate fraction of faeces. Extracellular proteases were formed by Streptococcus faecalis ST6, Propionibacterium acnes P6, Clostridium perfringens C16, Cl. bifermentans C21 and Cl. sporogenes C25. Inhibition results suggested that these bacteria, and similar organisms, may be partly responsible for the extracellular proteolytic activity found in the cell-free supernatant fraction of faeces.     

Contribution of the microflora to proteolysis in the human large intestine

Protease activities in human ileal effluent were approximately 20-fold greater than in normal faeces. Comparative studies with faeces from a person who did not have a pancreas suggested that a substantial proportion of the proteolytic activity in normal faeces was of bacterial origin. Thimerosal, iodoacetate, EDTA and cysteine significantly inhibited proteolysis in faeces, but not in small intestinal contents, showing that cysteine and metalloproteases were produced by bacteria in the large gut. These results, together with results from studies using p-nitroanilide substrates, demonstrated that faecal proteolysis was both qualitatively and quantitatively different from that in the small intestine. Studies with pure cultures of proteolytic gut bacteria indicated that the cell-bound proteases of Bacteroides fragilis-type organisms were likely to contribute significantly towards proteolytic activity associated with the washed cell fraction and washed particulate fraction of faeces. Extracellular proteases were formed by Streptococcus faecalis ST6, Propionibacterium acnes P6, Clostridium perfringens C16, Cl. bifermentans C21 and Cl. sporogenes C25. Inhibition results suggested that these bacteria, and similar organisms, may be partly responsible for the extracellular proteolytic activity found in the cell-free supernatant fraction of faeces.     

Microbes involved in dissimilatory nitrate reduction in the human large intestine

Nitrate-limited batch cultures, incorporating 20 different fermentation substrates and inoculated with human faeces, mainly selected for the growth of enterobacteria. The microbial diversity involved was determined by a combination of phenotypic and genotypic procedures. Continuous culture with lactate as the sole electron donor selected for similar micro-organisms, but when antibiotics were incorporated to inhibit Escherichia coli and lactate was replaced with choline, there was a wider microbial diversity recovered. Clostridium ramosum and Bacteroides vulgatus were then isolated as well as enterobacteriaceae.     

Changes in physico-chemical properties of human large intestine tumour cells membrane

Tumour cells produce and excrete to blood many substances which are present in the cell itself in trace amounts only. Our work has been aimed at the determination of changes in electric charge and in phospholipid composition of large intestine normal mucosa and colorectal cancer cells.Surface charge density of tumour unaffected mucosa and of tissue sections from tumours, was measured by electrophoresis. The measurements were carried out at various pH of solution. Membrane isoelectric point was determined by measuring its electric charge in function of pH as well as total positive charge at low pH and total negative charge at high pH. Qualitative and quantitative composition of phospholipids in the membrane was determined by HPLC. Four phospholipid classes were identified: PI, PS, PE and PC and their surface concentrations were determined.The electric charge calculated from phospholipid concentrations is by three orders of magnitude higher than that determined electrophoretically. It indicates that the groups present in the membrane surface are involved in equilibria in which the charge is neutralized.The electric charge calculated from phospholipid concentrations is by three orders of magnitude higher than that determined electrophoretically. It indicates that the groups present in the membrane surface are involved in equilibria in which the charge is neutralized.Tumour changes provoke an increase in surface charge density of large intestine membrane, whereas the content of individual phospholipids increased or decreased depending on a patient.     

Ontogeny and distribution of certain endocrine cells in the human fetal large intestine

Summary In 9 fetuses, 9 to 24 weeks-old, the occurrence and relative distribution of argentaffin cells, as well as of cells immunoreactive to somatostatin (SRIF), glucagon-like polypeptide (GLI), pancreatic polypeptide (PP) and substance P (SP) were studied in five segments of the colon (appendix, cecum, ascending colon, descending colon, and rectosigmoid). For each colonic segment, data concerned with the occurrence of endocrine cells were expressed either as mean absolute numbers of specific cells per entire mucosal section, or as cell densities per mm³ of mucosa after calculation of the mucosal volume of the sections. Argentaffin, GLI, SRIF and PP immunoreactive cells are all present in relatively large numbers, scattered along the entire length of the colonic mucosa as early as the 9th–10th week of gestation, whereas substance P-containing cells occur sporadically and first appear during the 14th–17th week. Until the 20th week, with progressing embryonic development, an increase was determined in absolute numbers per section of all types of endocrine cells in all segments of the colon. This observation is clearly related to the general growth of the colonic mucosa, since cell densities per mm³ of mucosa do not greatly change or even decrease during gestation. However, it is possible that densities of argentaffin, GLI and BPP cells increase in the appendix around the 14th–17th week of gestation. Between the 20th and 24th week, absolute numbers of cells per section remain stable or slightly increase, while cell densities tend rather to decrease in all segments. These data demonstrate that some endocrine cells are present very early in the human fetal colon, but their functional significance remains to be elucidated.This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM)     

c-kit immunoreactive interstitial cells of Cajal in the human small and large intestine

 c-kit immunohistochemistry was performed on unfixed frozen sections of human small (duodenum, jejunum, and ileum) and large intestine (ascending, transverse, descending, and sigmoid colon). The c-kit immunoreactive cells in the muscularis externa of the intestinal wall were identified as interstitial cells of Cajal (ICC) and mast cells. ICC were identified by their morphology, localization, and organization based on previous light and electron microscopic studies. In the small intestine, ICC were located primarily in relation to the myenteric plexus of Auerbach, but also in septa between circular muscle lamellae. In the large intestine, ICC were seen in relation to Auerbach’s plexus, but also and in great numbers in the circular muscle layer and in teniae of the longitudinal muscle layer. The morphology of the ICC was similar in the small and large intestine, but the pattern of distribution was obviously different. c-kit immunoreactive mast cells were found predominantly in the inner part of the circular muscle layer. The anti-c-kit method is found to be an easy and reliable method to study at least most of the interstitial cells of Cajal and thereby contribute to further normal and pathological studies. Accepted: 31 July 1997     

Extracellular and cell-associated glycosidase activities in different regions of the human large intestine

Viable counts of anaerobic bacteria increased distally from the right to left regions of the human colon. A wide variety of cell-bound glycosidases were detected in the bowel and their specific activities were generally greater in the right colon. High levels of enzymes associated with the degradation of endogenously produced glyco-proteins were found throughout the large intestine without significant regional differences.     

Eicosanoids and the large intestine

During the past three decades, many studies have been conducted to determine the precise role of eicosanoids in colorectal physiology and pathophysiology. This research has increased our understanding of bioactive lipid signaling, and may contribute to the development of more effective therapeutic modalities for digestive diseases in the future. The purpose of this report is to provide a brief overview of the role of eicosanoids in the colon and rectum. This information has been organized according to both functional and disease-related categories. The role of eicosanoids in colonic secretion, motility, inflammatory bowel disease, and colorectal neoplasia will be discussed.