Spatial-temporal imaging of bacterial infection and antibiotic response in intact animals

Abstract

We describe imaging the luminance of green fluorescent protein (GFP)-expressing bacteria from outside intact infected animals. This simple, nonintrusive technique can show in great detail the spatial-temporal behavior of the infectious process. The bacteria, expressing the GFP, are sufficiently bright as to be clearly visible from outside the infected animal and recorded with simple equipment. Introduced bacteria were observed in several mouse organs including the peritoneal cavity, stomach, small intestine, and colon. Instantaneous real-time images of the infectious process were acquired by using a color charge-coupled device video camera by simply illuminating mice at 490 nm. Most techniques for imaging the interior of intact animals may require the administration of exogenous substrates, anesthesia, or contrasting substances and require very long data collection times. In contrast, the whole-body fluorescence imaging described here is fast and requires no extraneous agents. The progress of Escherichia coli-GFP through the mouse gastrointestinal tract after gavage was followed in real-time by whole-body imaging. Bacteria, seen first in the stomach, migrated into the small intestine and subsequently into the colon, an observation confirmed by intravital direct imaging. An i.p. infection was established by i.p. injection of E. coli-GFP. The development of infection over 6 h and its regression after kanamycin treatment were visualized by whole-body imaging. This imaging technology affords a powerful approach to visualizing the infection process, determining the tissue specificity of infection, and the spatial migration of the infectious agents.

Figure 1

Whole-body imaging of E. coli-GFP infection in various organs. (A) E. coli-GFP infection in the stomach immediately after gavage of 10¹¹ E. coli-GFP. (B) E. coli-GFP infection in the small intestine 10 min after gavage. (C) E. coli-GFP infection in the small intestine 20 min after gavage. (D) E. coli-GFP infection in the small intestine 30 min after gavage. (E) E. coli-GFP infection in the small intestine 40 min after gavage. (F) E. coli-GFP infection in the small intestine 50 min after gavage. (G) E. coli-GFP infection in the small intestine 60 min after gavage. (H) E. coli-GFP infection in the colon 120 min after gavage. (I) E. coli-GFP infection in the colon immediately after enema of 10¹¹ E. coli-GFP.

Figure 2

Intravital imaging of E. coli-GFP infection in the stomach, small intestine, and colon after gavage. (A) E. coli-GFP infection in the stomach and the duodenum immediately after gavage of 10¹¹ E. coli-GFP. (B) E. coli-GFP infection in the small intestine 40 min after gavage. (C) E. coli-GFP infection in the colon 120 min after gavage.

Figure 3

Whole-body and intravital imaging of E. coli-GFP infection in the stomach, small intestine, and colon after gavage. (A) Whole-body image of E. coli-GFP infection in the stomach (arrowhead), the small intestine (fine arrows), and the colon (thick arrow) after multiple gavage of aliquots 3 × 10¹¹ E. coli-GFP. (B) Intravital image of E. coli-GFP infection in the stomach (arrowhead), the small intestine (fine arrows), and the colon (thick arrow) after multiple gavage of aliquots of 3 × 10¹¹ E. coli-GFP.

Figure 4

Whole-body imaging of E. coli-GFP peritoneal cavity infection and antibiotic response. (A and C) E. coli-GFP infection in the peritoneal cavity immediately after i.p. injection of 10⁹ E. coli-GFP. (B) Untreated mouse 6 h after i.p. injection. Animal died at this time point. (D) Kanamycin-treated mouse 6 h after i.p. injection. Animal survived. Arrows indicate the fluorescent images.

Figure 5

Intravital imaging of E. coli-GFP peritoneal cavity infection. E. coli-GFP infection in the peritoneal cavity immediately after i.p. injection of 10⁹ E. coli-GFP. The wall of the abdominal cavity was removed.