In vivo bioluminescence imaging of the murine pathogen Citrobacter rodentium

Abstract

Citrobacter rodentium is a natural mouse pathogen related to enteropathogenic and enterohemorrhagic Escherichia coli. We have previously utilized bioluminescence imaging (BLI) to determine the in vivo colonization dynamics of C. rodentium. However, due to the oxygen requirement of the bioluminescence system and the colonic localization of C. rodentium, in vivo localization studies were performed using harvested organs. Here, we report the detection of bioluminescent C. rodentium and commensal E. coli during colonization of the gastrointestinal tract in intact living animals. Bioluminescence was dependent on intact blood circulation, suggesting that the colonic environment is not anaerobic but nanaerobic. In addition, BLI revealed that C. rodentium colonizes the rectum, a site previously unreported for this pathogen.

FIG. 1.

BLI in the gastrointestinal tract requires a functional blood supply. Mice were orally gavaged with 10⁹ bacteria, and in vivo bacterial localization was determined by BLI in live animals (A, D, and G), immediately after cervical dislocation (B, E, and H), and on harvested colons and cecums (C, F, and I). Mice were imaged at 5 h postgavage with C. rodentium ICC180 (A to C), at 5 h postgavage with E. coli ICC214 (D to F), or at the peak of C. rodentium ICC180 infection (day 8 postgavage) (G to I) with an integration time of 1 min (except panels marked with , which required a 10-min integration). Images were acquired using an IVIS50 system and are displayed as pseudocolor images of peak bioluminescence, with variations in color representing light intensity at a given location. Red represents the most intense light emission, while blue corresponds to the weakest signal. The color bar indicates relative signal intensity (as photons s⁻¹ cm⁻² sr⁻¹). For each time point, at least three animals were analyzed and a representative animal is shown.

FIG. 2.

In vivo growth dynamics of luminescent C. rodentium ICC180. Mice were orally gavaged with 10⁹ bacteria, and in vivo growth dynamics were determined by viable counts (given as CFU per gram of stool) (A) and BLI (B). Images were acquired using an IVIS50 system and are displayed as pseudocolor images of peak bioluminescence, with variations in color representing light intensity at a given location. Red represents the most intense light emission, while blue corresponds to the weakest signal. The color bar indicates relative signal intensity (as photons s⁻¹ cm⁻² sr⁻¹). Mice were imaged at various time points postgavage with an integration time of 1 min. If no luminescence was detected at 1 min, then a 10-min exposure was used (indicated by ).

FIG. 3.

Colonization of the rectum by C. rodentium. Frozen sections of rectum harvested from uninfected mice and mice at 6 days post-C. rodentium infection were cut and stained for immunohistochemistry with a rabbit antibacterial antibody as described previously (9). A section showing representative rectal tissue from an uninfected mouse is given in the top panel, while that from a C. rodentium-infected animal is given in the bottom panel. Bacteria are clearly evident, adhering to the surface epithelium and crypts of the rectum with resulting hyperplasia (bottom panel). Magnification, ×84.