In our group's prior studies of inhalational anthrax, we found that the infection proceeded asynchronously among animals (21
). Despite being exposed to spores on the same day and receiving the same dose, mice exhibited a time to death that varied from 2 to 8 days after infection. Moreover, BLI data and CFU counts showed variation in the kinetics of progression of the infection among animals. These observations led us to hypothesize the existence of a barrier to the progression of infection and that passage of bacteria beyond such a barrier is a stochastic process. To investigate this possibility, we engineered three strains of B. anthracis
Sterne that could be easily differentiated following a mixed infection. Results of mixed infections indicated a lack of a bottleneck in the lungs, NALT, and mLNs but the presence of a bottleneck in the pathway leading to infection of the cLNs, kidneys, and liver.
Dissemination of inhalational anthrax may occur via both the NALT and the lungs. We previously observed that, in this animal model of the illness, the first sites along these two pathways where vegetative bacilli can be detected are the NALT and mLNs (22
). We note with interest that the bottleneck(s) occurs downstream of these two sites where germination first occurs. We found previously that dissemination beyond the NALT requires functional lethal toxin (LT) (22
). Our interpretation of this result is that during an infection with a toxin-producing strain, the host response is engaged in the NALT but is unable to contain the infection in the face of LT activity. We speculate that, in the present study, the observed dominance of one strain in the cLNs results from replication of the first spore or bacillus to successfully break through this point in the host defenses.
The apparent bottleneck in this model of inhalational anthrax helps explain the observed asynchronous progression following inhalational infection. Studies using animal models of infection with pathogens such as Streptococcus pneumoniae
) and Yersinia pestis
) have reported similar results. In the latter case, the variability in the dissemination of Y. pestis
among individual animals was attributed to variability in the timing of progression of the infection beyond the site of intraperitoneal injection. The stochastic nature of passage beyond a bottleneck (or multiple bottlenecks) could lead to the wide variability seen in the kinetics of progression among animals. Another implication of this finding is that methods commonly used to identify genes required in vivo
(e.g., signature-tagged mutagenesis or transposon-site hybridization) are unlikely to be useful to study dissemination in this animal model. These methods require infection with a pool of mutant strains of bacteria, recovery of strains passing through the animal, and comparison of input and output pools. If only one or a few bacterial cells are responsible for the establishment of disseminated disease past the bottleneck, then identification of genes required for dissemination would not be possible using these methods (6
The results described here have implications regarding the independent versus cooperative action of bacteria in this animal model (19
). According to the independent action hypothesis, each bacterium acts alone and can cause infection; as the size of the inoculum is increased, the probability of infection occurring increases, because the probabilities of each bacterium causing infection are additive. Such a hypothesis contrasts with the cooperative action hypothesis, under which bacteria work together to cause infection; as the size of the inoculum is increased, the average probability per bacterium of causing infection increases, because of cooperation between individual bacteria. Recent reports supporting the independent action hypothesis include studies of a baculovirus-insect larva system, in which the frequency of dual-genotype infections was found to match predictions of the independent action model (36
), and a mouse urinary tract infection caused by uropathogenic Escherichia coli
, in which intracellular bacterial communities were determined to have arisen from a single bacterium (32
). The presence of a bottleneck is consistent with independent action of individual bacteria in this model of inhalational anthrax, at least at some point during the progression to systemic disease, because apparently a single bacterium can be the founder of the disseminated infection. Therefore, prophylaxis, detection, and early intervention are extremely important for the prevention and treatment of this serious illness.