Sandra Macfarlane

Regulation of short-chain fatty acid production

Short-chain fatty acid (SCFA) formation by intestinal bacteria is regulated by many different host, environmental, dietary and microbiological factors. In broad terms, however, substrate availability, bacterial species composition of the microbiota and intestinal transit time largely determine the amounts and types of SCFA that are produced in healthy individuals. The majority of SCFA in the gut are derived from bacterial breakdown of complex carbohydrates, especially in the proximal bowel, but digestion of proteins and peptides makes an increasing contribution to SCFA production as food residues pass through the bowel. Bacterial hydrogen metabolism also affects the way in which SCFA are made. This outcome can be seen through the effects of inorganic electron acceptors (nitrate, sulfate) on fermentation processes, where they facilitate the formation of more oxidised SCFA such as acetate, at the expense of more reduced fatty acids, such as butyrate. Chemostat studies using pure cultures of saccharolytic gut micro-organisms demonstrate that C availability and growth rate strongly affect the outcome of fermentation. For example, acetate and formate are the major bifidobacterial fermentation products formed during growth under C limitation, whereas acetate and lactate are produced when carbohydrate is in excess. Lactate is also used as an electron sink in Clostridium perfringens and, to a lesser extent, in Bacteroides fragilis. In the latter organism acetate and succinate are the major fermentation products when substrate is abundant, whereas succinate is decarboxylated to produce propionate when C and energy sources are limiting.

Dietary prebiotics: current status and new definition

In November 2008, a group of scientists met at the 6th Meeting of the International Scientific Association of Probiotics and Prebiotics (ISAPP) in London, Ontario, Canada, to discuss the functionality of prebiotics. As a result of this, it was concluded that the prebiotic field is currently dominated by gastrointestinal events. However, in the future, it may be the case that other mixed microbial ecosystems may be modulated by a prebiotic approach, such as the oral cavity, skin and the urogenital tract. Therefore, a decision was taken to build upon the current prebiotic status and define a niche for ‘dietary prebiotics’. This review is co-authored by the working group of ISAPP scientists and sets the background for defining a dietary prebiotic as ‘‘a selectively fermented ingredient that results in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health’’.

Comparison of Compositions and Metabolic Activities of Fecal Microbiotas in Young Adults and in Antibiotic-Treated and Non-Antibiotic-Treated Elderly Subjects

ABSTRACT The colonic microbiota mediates many cellular and molecular events in the host that are important to health. These processes can be affected in the elderly, because in some individuals, the composition and metabolic activities of the microbiota change with age. Detailed characterizations of the major groups of fecal bacteria in healthy young adults, in healthy elderly people, and in hospitalized elderly patients receiving antibiotics were made in this study, together with measurements of their metabolic activities, by analysis of fecal organic acid and ammonia concentrations. The results showed that total anaerobe numbers remained relatively constant in old people; however, individual bacterial genera changed markedly with age. Reductions in numbers of bacteroides and bifidobacteria in both elderly groups were accompanied by reduced species diversity. Bifidobacterial populations in particular showed marked variations in the dominant species, with Bifidobacterium angulatum and Bifidobacterium adolescentis being frequently isolated from the elderly and Bifidobacterium longum, Bifidobacterium catenulatum, Bifidobacterium boum, and Bifidobacterium infantis being detected only from the healthy young volunteers. Reductions in amylolytic activities of bacterial isolates in healthy elderly subjects and reduced short-chain fatty acid concentrations supported these findings, since bifidobacteria and bacteroides are important saccharolytic groups in the colon. Conversely, higher numbers of proteolytic bacteria were observed with feces samples from the antibiotic-treated elderly group, which were also associated with increased proteolytic species diversity (fusobacteria, clostridia, and propionibacteria). Other differences in the intestinal ecosystem in elderly subjects were observed, with alterations in the dominant clostridial species in combination with greater numbers of facultative anaerobes.

Toll-like receptors-2 ,- 3 and -4 expression patterns on human colon and their regulation by mucosal-associated bacteria

The colonic epithelium provides an interface between the host and micro‐organisms colonising the gastrointestinal tract. Molecular recognition of bacteria is facilitated through Toll‐like receptors (TLR). The colonic epithelium expresses relatively high levels of mRNA for TLR3 and less for TLR2 and ‐4. Little is known of the expression patterns and mode of induction of expression for these pattern recognition receptors in human colon. The aim of this study was to investigate their localization in the gut and induction of expression in epithelial cell lines by mucosal bacteria. TLR2 and ‐4 were expressed only in crypt epithelial cells, expression was lost as the cells matured and moved towards the gut lumen. In contrast, TLR3 was only produced in mature epithelial cells. HT29 and CACO‐2 had different levels of expression for TLR1–4. Co‐culture of HT29 cells with different mucosal isolates showed that they were highly responsive to bacterial challenge, with up‐regulation of mRNA for TLR1–4. In contrast, CACO‐2 cells were refractive to bacterial challenge, showing little difference in mRNA levels. TLR3 was induced in HT29 only by Gram‐positive commensals with up‐regulation of both mRNA and protein and an enhancement of the antiviral immune response. This pattern of expression allows induction of responsiveness to bacteria only by the crypt epithelium so that tolerance to commensal organisms can be maintained. In contrast, mature columnar epithelium is able to respond to viral pathogens, which are not part of the normal gut commensal microbiota.

Fermentation in the human large intestine: its physiologic consequences and the potential contribution of prebiotics.

The human large intestine harbors a complex microbiota containing many hundreds of different bacterial species. Although structure/function relationships between different components of the microbiota are unclear, this complex multicellular entity plays an important role in maintaining homeostasis in the body. Many of the physiologic properties of the microbiota can be attributed to fermentation and the production of short-chain fatty acids (SCFAs), particularly acetate, propionate, and butyrate. In healthy people, fermentation processes are largely controlled by the amounts and different types of substrate, particularly complex carbohydrates that are accessible to bacteria in the colonic ecosystem. However, other factors impact on bacterial metabolism in the large gut, including large bowel transit time, the availability of inorganic terminal electron acceptors, such as nitrate and sulfate, and gut pH. They all affect the types and levels of SCFA that can be formed by the microbiota. This is important because to a large extent, acetate, propionate, and butyrate have varying physiologic effects in different body tissues. Prebiotics such as galactooligosaccharides together with inulins and their fructooligosaccharide derivatives have been shown to modify the species composition of the colonic microbiota, and in various degrees, to manifest several health-promoting properties related to enhanced mineral absorption, laxation, potential anticancer properties, lipid metabolism, and anti-inflammatory and other immune effects, including atopic disease. Many of these phenomena can be linked to their digestion and SCFA production by bacteria in the large gut.

FAO technical meeting on prebiotics

Recognizing the possible beneficial effect of prebiotics in food, the Food and Agriculture Organization of the United Nations (FAO) convened a Technical meeting to start work on the evaluation of the functional and health properties of prebiotics. A group of international experts agreed on guidelines, recommended criteria, and methodology for conducting a systematic approach for the evaluation of prebiotics leading to its safe use in food. It was recommended that a full expert consultation be convened under the auspices of FAO. This work provides governments, industry, and consumers with scientific advice in relation to functional and health aspects of prebiotics and general guidance for the assessment of prebiotics in relation to their nutritional properties or safety. These guidelines may also be used by Member Countries and Codex Alimentarius to identify and define what data need to be available to accurately substantiate health and nutrition claims.

Colonization of mucin by human intestinal bacteria and establishment of biofilm communities in a two-stage continuous culture system.

ABSTRACT The human large intestine is covered with a protective mucus coating, which is heavily colonized by complex bacterial populations that are distinct from those in the gut lumen. Little is known of the composition and metabolic activities of these biofilms, although they are likely to play an important role in mucus breakdown. The aims of this study were to determine how intestinal bacteria colonize mucus and to study physiologic and enzymatic factors involved in the destruction of this glycoprotein. Colonization of mucin gels by fecal bacteria was studied in vitro, using a two-stage continuous culture system, simulating conditions of nutrient availability and limitation characteristic of the proximal (vessel 1) and distal (vessel 2) colon. The establishment of bacterial communities in mucin gels was investigated by selective culture methods, scanning electron microscopy, and confocal laser scanning microscopy, in association with fluorescently labeled 16S rRNA oligonucleotide probes. Gel samples were also taken for analysis of mucin-degrading enzymes and measurements of residual mucin sugars. Mucin gels were rapidly colonized by heterogeneous bacterial populations, especially members of the Bacteroides fragilis group, enterobacteria, and clostridia. Intestinal bacterial populations growing on mucin surfaces were shown to be phylogenetically and metabolically distinct from their planktonic counterparts.

Mucosa-Associated Bacterial Diversity in Relation to Human Terminal Ileum and Colonic Biopsy Samples

ABSTRACT Little is known about bacterial communities that colonize mucosal surfaces in the human gastrointestinal tract, but they are believed to play an important role in host physiology. The objectives of this study were to investigate the compositions of these populations in the distal small bowel and colon. Healthy mucosal tissue from either the terminal ileum (n = 6) or ascending (n = 8), transverse (n = 8), or descending colon (n = 4) of 26 patients (age, 68.5 ± 1.2 years [mean ± standard deviation]) undergoing emergency resection of the large bowel was used to study these communities. Mucosa-associated eubacteria were characterized by using PCR-denaturing gradient gel electrophoresis (DGGE), while real-time PCR was employed for quantitative analysis. Mucosal communities were also visualized in situ using confocal laser scanning microscopy. DGGE banding profiles from all the gut regions exhibited at least 45% homology, with five descending colon profiles clustering at ca. 75% concordance. Real-time PCR showed that mucosal bacterial population densities were highest in the terminal ileum and that there were no significant differences in overall bacterial numbers in different parts of the colon. Bifidobacterial numbers were significantly higher in the large bowel than in the terminal ileum (P = 0.006), whereas lactobacilli were more prominent in the distal large intestine (P = 0.019). Eubacterium rectale (P = 0.0004) and Faecalibacterium prausnitzii (P = 0.001) were dominant in the ascending and descending colon. Site-specific colonization in the gastrointestinal tract may be contributory in the etiology of some diseases of the large intestine.

Degradation of Cross-Linked and Non-Cross-Linked Arabinoxylans by the Intestinal Microbiota in Children

ABSTRACT In humans, nonstarch polysaccharides (NSP), such as arabinoxylans (AX), are not digested in the upper gut and provide fermentable carbon sources for bacteria growing in the large bowel. Despite the ubiquity of AX in nature, the microbiologic and physiologic consequences of AX digestion in the gut are poorly understood. In this study, we investigated the breakdown of ferulic acid-cross-linked AX (AXF) and non-cross-linked AX in childrens intestinal microbiotas, using starch as a readily fermentable polysaccharide for comparative purposes. The experiments were performed using pH-controlled fermentation vessels under anaerobic conditions. The results demonstrated that there was variation in the metabolism of these polysaccharides by colonic microbiotas. AX was always degraded more slowly than starch, while ferulic acid cross-linking reduced the rate of AX fermentation, as shown by fermentation product measurements. Starch digestion was associated with significant acetate and butyrate production, whereas AX breakdown resulted in increased propionate formation. In general, the presence of fermentable carbohydrate significantly increased the total anaerobe counts and eubacterial rRNA concentrations (P < 0.01), while non-cross-linked AX digestion was principally associated with increased viable counts of Bacteroides fragilis group organisms, which was supported by increases in Bacteroides-Porphyromonas-Prevotella group rRNA (P < 0.01). Starch was considerably more bifidogenic than AX in these fermentations. In conclusion, in this study we found that the effects of AX and AXF on the microbial ecology and metabolism of intestinal microbiotas are similar in children and adults.

Chemotaxonomic Analysis of Bacterial Populations Colonizing the Rectal Mucosa in Patients with Ulcerative Colitis

The etiology of ulcerative colitis (UC) is unknown, but evidence links it to bacteria belonging to the normal colonic microbiota. The aims of this study were to characterize bacteria colonizing the rectal epithelium, and to investigate whether significant differences existed in UC. Rectal biopsy specimens were obtained via endoscopy from 9 patients with active colitis and 10 patients without inflammatory bowel disease. Complex bacterial communities colonized the rectal mucosa in all subjects. Overall, 72 bacterial taxa (18 genera) were detected. Twenty species were common to both groups, but only differences in bifidobacteria were statistically significant (P=.005). Peptostreptococci were only detected in patients with UC. Microscopy showed that bacteria in mucosal biofilms often occurred in microcolonies. Interindividual variations in mucosal biofilms made it difficult to assign a role for specific bacteria in UC etiology. However, differences in bifidobacteria and peptostreptococci may implicate these organisms in this disease.