Figure 6

Uptake and growth of B. pseudomallei strains in J774A.1 murine macrophages. J774A.1 cells (duplicate wells in each of two 24-well tissue culture plates) were infected with B. pseudomallei strains at an MOI of 10 and incubated for 1-hr to allow phagocytosis of the organisms. Following incubation, the monolayers were incubated for 2-hr in medium containing gentamicin to kill extracellular bacteria. After gentamicin treatment (i.e. 3-hr post infection), the wells of one plate were washed, lysed, serially diluted, and spread onto agar plates to determine the number of bacteria phagocytosed by macrophages. The results of this first part of the experiments (i.e. bacterial uptake) are shown in panel A and are expressed as the percentage of bacteria (± standard error) used to infect macrophages that were phagocytosed. The wells of the other tissue culture plate inoculated with B. pseudomallei strains were washed once, fresh medium without antibiotics was added to wells, and the plate was incubated for an additional 5-hr. Following this incubation (i.e. 8-hr post-infection), the wells were processed as described above in order to enumerate bacterial numbers. The results of this second part of the experiments (i.e. intracellular growth of phagocytosed bacteria) are shown in panel B and are expressed as a growth/uptake ratio (± standard error) obtained by dividing the number of bacteria/well at 8-hr post infection by the number of bacteria/well at the 3-hr post infection time point. These experiments were repeated on at least 3 separate occasions. The asterisk indicates that the difference between the intracellular growth of the double mutant strain DD503.boaA.boaB and that of its parent isolate DD503 is statistically significant (P < 0.05). Panel C shows the total number of bacteria in the inoculum (grey bars), the number of phagocytosed bacteria (open bars, 3-hr post infection) and the total number of bacteria/well at the end point of the experiment (black bars, 8-hr post infection).