This study demonstrates a method for in vivo imaging of bacterial infections using genetically engineered bacteria that produce bioluminescence. The technique allows for the quantification of metabolically active bacteria at infection sites in anesthetized mice.
Take an anesthetized mouse with skin incisions infected with a pathogenic bacterium.
The bacterium is genetically engineered to carry a plasmid containing the luxABCDE reporter system, which enables autonomous bioluminescence.
Place the mouse in a prone position on the bioluminescence imaging platform.
At the infection site, the bacterium’s luxA and luxB genes encode the luciferase enzyme, while the luxCDE genes encode enzymes that synthesize long-chain fatty aldehydes, which serve as a substrate for luciferase.
In the presence of oxygen, luciferase catalyzes the oxidation of its cofactor, reduced flavin mononucleotide, which then reacts with the long-chain fatty aldehyde, producing blue-green light.
This bioluminescence indicates the presence of metabolically active bacteria at the infection site.
Using the imaging system, image the infection site and quantify the bioluminescence signal intensity.
For in vivo imaging of the infected wounds, follow biosafety level two containment protocols for transport of the mice to and from the imaging instrument and place the anesthetized mouse in the prone position within the imaging chamber.
In the imaging software, set the exposure time, binning, f-stop, and field of view as appropriate for the experiment.
Next, create a region of interest at the wound site and measure the average detected flux in photons per second.
To measure the background, create a region of interest over a random area on the imaging platform and subtract the background number of photons per second.
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