For S. typhimurium infection, mice were gavaged with 20 mg streptomycin (Sigma) in 100 μl sterile water, 24 h before infection. S. typhimurium strain SL1344 was grown overnight in LB with streptomycin (50 μg/ml) and gavaged to mice at 5×108 CFU in 100 μl volume.
Citrobacter rodentium strains DBS100 or DBS120 pler-lux were grown in LB overnight at 37 °C, then diluted 1:100 and grown for 2.5 hours, centrifuged and resuspended in 0.01 volumes PBS, and mice were gavaged with ~5×109 bacteria in 100 μl LB. To determine mouse colonization levels, a fresh fecal pellet or SI contents (gently squeezed to remove, except last 3 cm of ileum) was weighed, mashed in 500 μl of PBS, serially diluted, and plated on MacConkey (Becton, Dickinson, and Co.) or LB agar with 50 μg/ml kanamycin. For luciferase measurements, the fecal homogenate was adjusted to 10 mg in 1 ml PBS in an eppendorf tube and light measured in a Triathler scintillation counter (Hidex, Turku, Finland), before plating dilutions in PBS on agar with kanamycin. Colon-adherent bacteria were measured in a 1 cm piece from the middle of the colon. The piece was opened longitudinally, washed in 1 mM DTT/PBS by vortexing for 10 s, then washed in PBS, and placed in an eppendorf in 500 μl PBS for light measurement as before. The piece was then mashed and dilutions plated on agar with kanamycin.
Citrobacter rodentium strains DBS100 or DBS120 pler-lux were grown in LB overnight at 37 °C, then diluted 1:100 and grown for 2.5 hours, centrifuged and resuspended in 0.01 volumes PBS, and mice were gavaged with ~5×109 bacteria in 100 μl LB. To determine mouse colonization levels, a fresh fecal pellet or SI contents (gently squeezed to remove, except last 3 cm of ileum) was weighed, mashed in 500 μl of PBS, serially diluted, and plated on MacConkey (Becton, Dickinson, and Co.) or LB agar with 50 μg/ml kanamycin. For luciferase measurements, the fecal homogenate was adjusted to 10 mg in 1 ml PBS in an eppendorf tube and light measured in a Triathler scintillation counter (Hidex, Turku, Finland), before plating dilutions in PBS on agar with kanamycin. Colon-adherent bacteria were measured in a 1 cm piece from the middle of the colon. The piece was opened longitudinally, washed in 1 mM DTT/PBS by vortexing for 10 s, then washed in PBS, and placed in an eppendorf in 500 μl PBS for light measurement as before. The piece was then mashed and dilutions plated on agar with kanamycin.