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 realtime 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.green fluorescent protein ͉ external optical imaging ͉ Escherichia coli ͉ antibiotic response ͉ mice M any biological research techniques depend on detecting uniquely distinguishable cells, e.g., cells exhibiting unique or aberrant morphologies. Far greater sensitivity is achieved by adding exogenous markers, especially those that are optically visible. The techniques of early embryologists marking developmental pathways with India ink injections have evolved into modern methods that include genetically transducing cells with reporters such as dye-activating enzymes. However, such methods still could not detect small, rare targets and usually could not be visualized in living systems. A qualitative advance in sensitivity is afforded by the new class of cell marking reagents exemplified by green fluorescent protein (GFP) and its derivatives. Genetically based, these can be permanent and nontoxic, and are very strongly fluorescent. Most importantly, because their presence seems to have little effect on the marked cell, observations in living tissue are now possible. We show here that GFP enables previously impossible, noninvasive tracking of bodily disease agents in space and time.GFP has been used as a reporter gene for numerous biological processes (1). Its freedom from required substrates or cofactors allows it to be expressed and visualized in living cells with no apparent...

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