Conventional techniques to study microbial pathogenesis in mammalian models have primarily relied on end point assays (e.g., survival analysis or lethal dose 50 [LD₅₀]) or on sacrificing animals at designated time points to characterize bacterial dissemination to specific tissues. The development of noninvasive approaches to image disease progression, without having to sacrifice the animal, allows for a temporal, real-time analysis of host–pathogen interactions. The use of such noninvasive techniques also facilitates reduction of animal numbers required for these studies—one of the three R’s of good laboratory animal practice [1].

In this chapter, we describe the use of optical diagnostic imaging to characterize bacterial disease progression within a small animal host. For optical imaging, animals are infected with bacteria that emit light or photons. Currently, two light-emitting modalities exist: fluorescent or bioluminescent bioreporters. Bioluminescent bioreporters have been favored because they tend to be more sensitive than equivalent fluorescent bioreporters and demonstrate a dynamic relationship to bacterial viability, which is lacking in fluorescent systems because of the long half-life of fluorescent proteins in inactivated bacteria [2]. Since first described by Contag et al. [3], bioluminescent bioreporters using bacterial luxCDABE luciferase operons have been developed for many different pathogens. A major advantage of the bacterial luciferase systems over eukaryotic luciferase systems is that the bacterial lux operon also synthesizes the substrate for the luciferase enzyme [2]. Therefore, bioreporters using a bacterial system do not require addition of exogenous substrate (usually through injection prior to imaging) to facilitate optical imaging. This also eliminates the potential complication of bioavailability issues with an exogenously added substrate. Furthermore, bacterial and eukaryotic luciferases have different spectral emissions, allowing for the potential to use both in the same system (i.e., monitor bacteria with a bacterial luciferase bioreporter and immune response with a eukaryotic luciferase bioreporter).

Bioluminescent bioreporters for specific pathogens may be available from commercial sources, requests from other laboratories, or designed and engineered in advance in house. Technical details on the development of specific bioreporters will not be discussed here, but several important criteria should be considered when selecting or designing an appropriate system. First, the reporter should be as sensitive as possible without altering the growth of the bacterium. This can be achieved by increasing the copy number of the reporter (i.e., expression from a plasmid vs. chromosome) or by engineering a highly active promoter to drive the expression of the luxCDABE operon. Second, the reporter must be maintained by the bacterium throughout the entire course of the animal infection. Integration of the luxCDABE reporter into the genome or onto a plasmid with a toxin–antitoxin selection system can improve stability of the reporter genes. Finally, the expression of the reporter should be constitutive during infection so that bioluminescence closely correlates with the number of viable bacteria regardless of the time or location being imaged. This can also be controlled through empirical selection of a promoter to control the transcription of the luciferase operon. However, as each new model is being developed, extensive in vivo and ex vivo characterization of the bioreporter in regard to bioluminescence and actual bacterial numbers needs to be determined to establish if bioluminescence directly correlates to bacterial load.

The following technique focuses on using optical imaging and subsequent data analysis to characterize bacterial infection in small animal models using intranasal instillation of Yersinia pestis as an example. However, similar approaches can be applied to a variety of bacterial pathogens and routes of inoculation.