Sequencing Yersinia pestis, the bacterium that caused the Black Plague in Europe during 1348–50, is an amazing accomplishment. Y. pestis infection still occurs sporatically and causes fatalities despite the Age of Antibiotics. Even with animal models, there are questions remaining about the progression of infection. Nham et al. used in vivo imaging to examine the course of infection in a mouse animal model using a bioluminescent clone of Y. pestis.
To construct the bioluminescent clone, the Y. pestis strain CO92 was transformed with a plasmid carrying an lux operon from the bacterium Photorhabdus luminescens. This operon synthesized both enzyme and substrate so no exogenous substrate (e.g., luciferin for firefly luciferase) needed to be injected into the animal. After confirming the operon was functional in Y. pestis, the researchers compared replication of the transformed Y. pestis CO92 strain with the unaltered strain and found no differences. Nham et al. also examined the maintenance of the plasmid as well as the stability of the luminescent signal over time. They confirmed the signal was stable, and the plasmid was maintained over 13 generations.
After these in vitro tests, they next tested expression in a mouse model. The mice were injected subcutaneously with 100 colony-forming units (cfu) of the bioluminescent strain and monitored each day using an in vivo imaging instrument. After 24 hours, only the injection site was luminescent. However, as the days passed, luminescence increased. Not only did the researchers observe the location of luminescent sites on the mouse’s body; they also wanted to correlate the signal to internal organs. Therefore, animals were injected with the bioluminescent Y. pestis strain again, monitored, euthanized, dissected, and luminescence was monitored on the exposed organs. In all cases, the predicted locations matched the actual bioluminescent organs: injection site, liver, spleen, axillary (underarm) and inguinal (groin) lymph nodes.
The authors carried out a second confirmatory experiment by not only monitoring the spread of bioluminescence but also homogenizing the organs to correlate bacterial burden with luminescence in liver, spleen and inguinal lymph node. The cfu from the bacteria extracted from the organs did correspond to the intensity of the light emitted from the same location, indicating the in vivo imaging was a reliable test for the spread of infection in the three organs. These assessments also allowed the researchers to standardize the settings used and delineate the limits of detection (cfu) for each organ.
With this imaging tool validated for tracking Y. pestis infection in mice, Nham et al. used 46 BALB/cByJ female mice in six independent experiments, injecting the animals with 100cfu of luminescent bacteria into an abdominal structure called the linea alba and monitoring them daily. On the first day, light appeared only around the injection site, but by the second day, luminescence could be detected in at least one inguinal lymph node followed by the ipsilateral (same side) axillary lymph nodes. Interestingly, sometimes the luminescence visualization showed a line connecting the inguinal and axillary lymph node. To confirm that this was a lymph vessel draining from one node to the other, a blue dye was injected into the linea alba in four mice and the structure examined 1, 10, 20 or 30 minutes later. The lymphatic vessel that runs between the two lymph nodes was colored blue in the mice.
The luminescent bacteria progressed to the liver and spleen, infection likely following the lymph as demonstrated between the two nodes. The bacteria found their way into the blood (septicemia). By the time luminescence was detected in the entire mouse, the infection was terminal. In 26% of animals, there were exceptions to this lymph spreading scenario, likely a result of the injection introducing the bacteria into the bloodstream.
The authors worked out the kinetics of infection using 24 of the mice tested for which the data from these specimens were complete from day 1 to death. On day 1, all mice showed luminescence at the injection site. Light was detected in the inguinal lymph node at day 3.3 followed shortly by the axillary lymph node (day 3.6). The next organs to luminesce were the liver (3.9 days) and spleen (4.1 days). On day 5, the entire body was luminescent and death occurred at day 5.8. The authors noted while this was the average among the animals, individual animals could vary (e.g., seeing light in the inguinal lymph node appeared anywhere from day 1 to day 7). Regardless of the differences, once light was found in the inguinal lymph node, the time it took to spread to other organs were consistent: one day to reach the liver and spleen, two days to spread throughout the animal with death quickly to follow. The authors concluded that most of the differences in time were due to the time the luminescent bacteria took to migrate from the injection site to the inguinal lymph node, and once there, the infection spread rapidly throughout the mouse.
The technique used in this article blended the use of a bacterial luminescent operon express from a plasmid with in vivo imaging to discern the spread of Y. pestis in an animal model of bubonic plague. This was a novel approach to learning how the disease progressed rather than a simple snapshot at a moment in time. Interestingly, the results both reflected differences in the responses of individuals by how long it took for the bacteria to spread from the initial site, but showed that progression was predictable once the bacteria reached the inguinal lymph node. This experiment was performed in mice, which may not exactly reflect disease progression in humans, but there are interesting insights we can derive and apply to further study of a disease that humans equipped with antibiotics are unable to eliminate.
Nham T, Filali S, Danne C, Derbise A, & Carniel E (2012). Imaging of Bubonic Plague Dynamics by In Vivo Tracking of Bioluminescent Yersinia pestis. PLoS ONE, 7 (4) PMID: 22496846