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.Epithelial surfaces in the human gastrointestinal tract are covered by a layer of mucus, which prevents most microorganisms reaching and persisting on the mucosal surface (13). This viscoelastic gel (1) is protective against adhesion and invasion by many pathogenic microorganisms, bacterial toxins, end products of metabolism, pancreatic endopeptidases, microbial antigens, and other damaging agents present in the lumen of the bowel. The mucus gel mainly consists of water (ca. 95%) and glycoproteins (1 to 10%), which are responsible for its viscosity and gel-forming properties (18), as well as electrolytes, proteins, antibodies, and nucleic acids (1).Mucins are chemically and structurally diverse molecules, but they invariably contain large quantities of galactose and hexosamines with lesser amounts of fucose. Strongly polar groups such as neuraminic (sialic) acids and sulfate are also present, although to a highly variable degree (49). The carbohydrate moieties occur as linear and branched oligosaccharides, which can constitute up to 85% of the molecule by weight (53). These oligosaccharides are attached to a protein core via serine or threonine residues. The attachment of sulfate and sialic acids to terminal mucin oligosaccharides confers resistance to digestion by glycosidases (8).

Summaries

“…Treatment vessels were dosed with 0. Results Total VFA, ammonia and lactate levels were found to be consistent between all vessels and values similar to previous studies (Macfarlane et al, 2005), with no significant treatment effects. qPCR analysis indicated a stable number of bacterial cells surviving in culture, comparable to in vivo values (Suchodolski, 2011).…”

“…Anaerobic conditions were maintained by bacterial fermentation and redox continuously measured. A sterile nutritive culture medium (Macfarlane et al, 2005) provided the fermentation substrates, supplied continuously by peristaltic pump to the vessels at 0.5 ml/min, with equal outflow. Treatment vessels were dosed with 0.…”

Mucin-bacterial interactions in the human oral cavity and digestive tract

“…53 Luminal mucosa-associated bacteria varied inter-individually, and showed intra-individual similarity on different sites of the colon. [54][55][56] The mucosa-associated bacteria are in close contact with/proximity to the epithelial cells, and therefore can have a major impact on these cells.…”

“…In an in vitro fermentation system mimicking the human intestine, Macfarlane et al reported that a heterogeneous bacterial community was colonizing mucin. 53 These authors used a combination of cultivation and visualization of the bacterial community by FISH, based on specific and general 16S rRNA probes. They observed that this community consisted of members of the Bacteroides fragilis group, bifidobacteria, enterobacteria and clostridia.…”

Crosstalk between the microbiome and cancer cells by quorum sensing peptides

“…signaling molecules produced by gram-negative bacteria, were identified in human feces of gastrointestinal disease patients as well as healthy subjects [13]. Moreover, bacterial quorum sensing molecules are likely to play a role in bacterial colonization of mucosa, thus requiring quorum sensingmediated biofilm formation [14]. Finally, Casula and Cutting showed the germination of Bacillus subtilis spores in the murine gastrointestinal tract, thereby probably requiring signaling peptides for quorum sensing pathway activation [15].…”