Polysaccharide degradation by human intestinal bacteria during growth under multi-substrate limiting conditions in a three-stage continuous culture system

Abstract

Human faecal microorganisms were grown on mixtures of chemically diverse polymerised C‐sources (starch, pectin, xylan, mucin, arabinogalactan, inulin, guar gum) in a three‐stage continuous culture model of the colon. The effects of retention time (R= 27.1 h, R= 66.7 h) on bacterial populations, their expression of hydrolytic enzymes involved in substrate depolymerisation, carbohydrate utilisation and short chain fatty acid formation were investigated. Eleven bacterial marker groups were studied in the fermenters. Strictly anaerobic bacteria predominated including bacteroides, bifidobacteria, clostridia and anaerobic Gram‐positive cocci. Changing system retention time from 27.1 to 66.7 h did not significantly affect the predominant bacterial populations in V1, however, enterobacterial cell numbers increased in V3, while saccharolytic anaerobe recoveries declined markedly, reflecting their greater dependence on polymerised carbon sources in the fermentation system. The majority of polysaccharide degrading activity in the colon model was cell‐associated, under all culture conditions. Increasing R from 27.1 to 66.7 h did not substantially change overall polysaccharidase (amylase, polygalacturonanase, xylanase, arabinogalactanase, galactomannanase) profiles, however, synthesis of some glycosidases was enhanced (e.g. α‐glucosidase, N‐acetyl‐β‐glucosaminidase, neuraminidase), whereas reduced expression of other enzymes such as β‐galactosidase, N‐acetyl‐α‐galactosaminidase, α‐fucosidase and α‐arabinofuranosidase occurred. These observations demonstrate that catabolite regulation is an important control process in the colonic microbiota, with respect to the induction and repression of enzyme synthesis, and that substrate availability plays a major role in regulating bacterial metabolism. Measurements of carbohydrate utilisation demonstrated that while all polysaccharides in the feed medium were digested extensively by bacteria growing in the fermentation system, specific rates of carbohydrate utilisation were maximal at R= 27.1 h. These data also provided evidence of bacterial substrate preferences in the colon model, particularly in relation to xylan and inulin digestion, demonstrating that catabolite regulatory mechanisms were also involved in controlling the assimilation of carbohydrate in the microbiota. Short chain fatty acid measurements showed that fermentation was more efficient at R= 27.1 h compared to R= 66.7 h, with putative conversion of carbohydrate to short chain fatty acids being approximately 60% and 40%, respectively. This was probably due to increased maintenance energy requirements at low bacterial growth rates. Differences were also observed with respect to short chain fatty acid molar ratios, with more propionate, branched chain and longer chain fatty acid production at R= 66.7 h, demonstrating the increasing importance of amino acid fermentation under these culture conditions.

Colonization of Mucin by Human Intestinal Bacteria and Establishment of Biofilm Communities in a Two-Stage Continuous Culture System

“…Interestingly, N-acetyl neuraminic acid was assimilated rapidly, even though initial neuraminidase activities were low in the mucin, while a marked lag was observed with galactose and fucose uptake and with N-acetyl galactosamine in vessel 2, despite the existence of significant glycosidase activities. These observations show that microbial catabolite regulatory mechanisms were affecting the way mucin oligosaccharides were being utilized (10,27,40).…”

“…The two-stage continuous fermentation system used in this investigation for studying the formation of mucin-degrading biofilms enabled colonization of the glycoprotein gels to be examined under nutrient and energy-rich conditions characteristic of the proximal bowel (vessel 1) and the same bacteria to be investigated under carbon and energy-limited conditions similar to those in the distal bowel (vessel 2) (40). Inoculation of the chemostat with fecal bacteria has been shown previously to effectively simulate colonic populations that occur in vivo (33).…”

“…Isolation media for strictly anaerobic bacteria were Wilkins-Chalgren agar (WCA), for total anaerobes, eubacteria, anaerobic gram-positive cocci, and clostridia; WCA with addition of nonsporing anaerobe supplements (to prevent growth of spore-forming species); WCA with gram-negative anaerobe supplements, which was selective for gram-negative organisms; Mann-Rogosa-Sharpe medium, for lactobacilli. Bacteria belonging to the Bacteroides fragilis group were isolated and enumerated using Bacteroides mineral salts agar (31), bifidobacteria were counted on Beerens agar (4), and fusobacteria were counted on fusobacterium agar (40). Plates for aerobic incubation were removed from the anaerobic chamber and incubated at 37°C.…”

Modulation of genotoxic enzyme activities by non-digestible oligosaccharide metabolism in in-vitro human gut bacterial ecosystems

“…The multi-stage fermentation system has been validated employing bacteriological and physiological criteria obtained from studies on material obtained from different regions of the large bowel at autopsy [43,44]. The model has been used previously in several experiments, in studies on the breakdown of pancreatic endopeptidases [45], mucin metabolism [46], distribution of bacterial populations in different regions of the bowel [44], bi®dobacterial ecology [34] and polysaccharide breakdown [33].…”

Nondigestible Oligosaccharides Enhance Bacterial Colonization Resistance against Clostridium difficile In Vitro

“…Bacterial enumeration. Viable counts were determined as described previously (23,25). Bacteria from the fermentation vessels were suspended and serially diluted (10-fold) by using anaerobic, half-strength peptone water (5 g of peptone per liter and 2.5 g of NaCl per liter in distilled water).…”