Figure 5

Competitive co-colonization and colonization displacement experiments in vivo . (A) Competitive colonization study comparing GI retention of the glycogen-deficient ?glgA mutant with the parent strain. Antibiotic-resistant derivatives of parent and ?glgA mutant were generated for the studies as described in the text. Germ-free 129S6/SvEv mice (2 males, 2 females; 1226 weeks old) were used in the co-colonization experiment carried out at the North Carolina State University Gnotobiotic Core Facility. Animal use protocols were approved by the Institutional Animal Care and Use Committee of North Carolina State University. Mice were maintained in cages in germ-free flexible film isolators housed in a room with cycles of 12h of light and darkness, and were provided access to a standard diet (Prolab RMH 3500, LabDiet, St. Louis, MI) and water ad libitum. The mice were verified germ-free by culturing of fecal samples aerobically and anaerobically on plate count agar and MRS agar prior to experiments. On Day 0, both parent and ?glgA mutant cells grown overnight in MRS broth were harvested, washed once with phosphate-buffered saline (PBS, pH7.4; Life Technologies) and resuspended in fresh PBS. The cultures were each titered in PBS and combined in 1:1 ratio to obtain 1 x 10⁷ cells in total per 200?L of gavage volume. Following gavage (200?L/mouse), fecal samples were collected periodically, weighed, homogenized in PBS, diluted and plated onto antibiotic selective media for enumeration of the parent and mutant populations. (B) Germ-free mice (3 males, 27weeks old) were first gavaged with the glgA mutant. A second gavage with the parent strain was subsequently performed when the glgA mutant population reached 6 x 10⁸ 8 x 10⁸CFU/g of fecal samples (indicated by a red arrow). Preparation of bacterial cells for gavage and sampling of fecal samples were carried out as described above.