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.

FIG. 1.

The two-stage continuous culture system used to simulate bacterial colonization of mucin under nutrient-excess (vessel 1) and nutrient-limited (vessel 2) conditions.

FIG. 2.

Development of bacterial populations in mucin biofilms. Closed circles, vessel 1; open circles, vessel 2. The solid line shows steady-state planktonic populations in vessel 1, and large broken lines show those in vessel 2. Small broken lines indicate bacterial numbers in the chemostat wall biofilm in vessel 1.

FIG. 3.

Development of bacteroides populations in mucin biofilms. Closed circles, vessel 1; open circles, vessel 2. The solid line shows steady-state planktonic populations in vessel 1, and large broken lines show those in vessel 2. Small broken lines indicate bacterial numbers in the chemostat wall biofilm in vessel 1.

FIG. 4.

Colonization of mucin gels in vessel 2 by fecal bacteria. (A) Scanning electron micrograph of mucin gel at zero time. (B) Scanning electron micrograph of mucin gel after 24-h immersion in the chemostat.

FIG. 5.

Fluorescent light micrograph of mucin gels labeled with 16S rRNA oligonucleotide probes. (A) Mucin gel at 12 h, showing enterobacteria (FITC; green), bacteroides (Cy3; red), and bifidobacteria (Cy5; blue). (B) Bacteroides microcolonies at 12 h, labeled with a genus-specific oligonucleotide probe (Cy3). Magnification, ×1,000.