5% of the bacterial inoculum (range 0.4-3.4% for different isolates) was recovered. There was no significant difference in this value between 3 isogenic morphotypes for all 5 isolates. The intracellular replication of B. pseudomallei between 4 to 8 h within macrophages is summarized in Figure 1. The replication rates for the 3 isogenic morphotypes of each strain obtained from two independent experiments were comparable (data not shown). Percent replication

at 8 h was defined in relation to the 4 h time point, which was used as the reference SC79 count. CA4P Analysis of pooled data for 5 isolates demonstrated that type I had a significantly higher rate of intracellular replication than either type II or III. The mean intracellular replication of type Temsirolimus molecular weight I at 8 h was 2.0 (95%CI 1.5-2.6, P = 0.004) times higher than that of type II, and 1.9 (95%CI 1.4-2.5, P = 0.004) times higher

than that of type III (Figure 1A). However, this pattern was not uniformly observed for each of the 5 isolates, as shown in Figure 1B-F. The higher replication fitness for type I based on the summary data was largely accounted for strains 164 and K96243. Other strains demonstrated a different pattern. For example, strain 153 type III had a higher intracellular replication than type I, a finding that replicates those of a previous study [11]. The mean intracellular bacterial count also varied between individual isolates. These differences were not due to the relative sensitivities of 3 isogenic morphotypes to 250 μg/ml kanamycin, as this experimental condition removed 99.9% of extracellular bacteria independent of type for all isolates (data not shown). Figure 1 Intracellular replication of 3 isogenic morphotypes of B. pseudomallei in human macrophages. Differentiated U937 cells were incubated for 2 h with B. pseudomallei at a MOI of 25:1, after which non-adherent bacteria were removed by washing and incubation for a further 2 h with kanamycin. At this 4 h time point, fresh medium containing kanamycin was added and incubation continued

for Palbociclib manufacturer a further 4 h. The bacterial count and colony morphology were enumerated at 4, 6 and 8 h by cell lysis and plating onto Ashdown agar. The data shown in Figure 1A represent mean values for each isogenic morphotype derived from 5 B. pseudomallei isolates and is expressed as the bacterial proportion at 6 and 8 h compared with the number at 4 h (which was defined as 100%). Figure 1B-1F shows the number of intracellular bacteria in CFU/ml for individual isolates. Data plots are means ± standard deviations. Susceptibility of isogenic morphotypes to acid To examine the effect of acid, growth of 3 isogenic morphotypes in LB at pH 4.0, 4.5, 5.0 and 7.0 was compared at each of 5 time points over 24 h of incubation. No growth difference was observed between morphotypes at any time point for pH 4.5, 5.0 or 7.0 (P > 0.10 for all time points). When cultured in LB broth at pH 4.