J774A.1 macrophages were exposed to Burkholderia www.selleckchem.com/products/epz-6438.html strains at an MOI of 10 and the mean numbers of intracellular

bacteria were determined at 2, 4, 6, 8 and 12 hrs post infection. (A) Uptake of bacteria by macrophages as determined by bacterial counts 2 hrs post infection relative to the input numbers. (B-D) Intracellular survival and replication of B. pseudomallei (Bps; panel B), B. thailandensis (Bt; panel C) and B. oklahomensis (Bo; panel D) in J774A.1 macrophage cells. Error bars represent the standard selleck chemical error of the mean. All infections were performed as three independent experiments, each with three technical replicates. The insert in panel C represents individual bacterial counts and the mean value at 12 hrs post infection with different B. thailandensis strains. High virulence isolates of B. pseudomallei grow more rapidly in J774A.1 macrophages than low virulence isolates, B. thailandensis or B. oklahomensis Next, intracellular replication was measured at 2, 4, 6, 8 and 12 hrs post infection. There was a significant difference between the numbers of intracellular

B. pseudomallei strains 576 and K96243 at 12 hrs post infection (P = 0.002; Figure 1B) and both were significantly higher than numbers of intracellular B. pseudomallei strain 708a and any of the B. thailandensis or B. oklahomensis strains tested (P < 0.002, both). Bacterial numbers were over 10-fold AR-13324 molecular weight lower with any of the B. thailandensis or B. oklahomensis strains tested (compare Figure 1B to Figure 1C & 1D). To test whether the low numbers of intracellular bacteria observed with B. pseudomallei 708a, which is more sensitive to kanamycin, was a consequence of low levels of antibiotic crossing the eukaryotic cell membrane, J774A.1 cells were infected with B. thailandensis DW503 (an amrAB-oprA efflux pump mutant and therefore highly

sensitive to kanamycin) and intracellular bacterial numbers were compared to its parental strain E264. The numbers of bacteria isolated at each time point were not significantly different between strains E264 and DW503 (data not shown). Our results also showed variance between the patterns of growth in macrophages of different isolates of B. thailandensis. The B. thailandensis strains previously isolated from cases ifenprodil of human disease, CDC272 and CDC301, showed increased numbers at 12 hrs post infection relative to B. thailandensis E264 (P < 0.004, both; see insert in Figure 1C) and the two B. oklahomensis strains C6786 and E0147 (P < 0.009, both), but not B. thailandensis strain Phuket (P > 0.05). To show that these differences in bacterial numbers were due to differences in intracellular replication and survival rather than a difference in bacterial fitness, growth rates of bacteria in antibiotic free media were compared. There was no significant difference between any of the strains tested (data not shown).