Methods Bacterial isolates and isolation of isogenic
morphotypes Five B. pseudomallei isolates were examined in this study. Isolates 153, 164 and the reference isolate K96243 were cultured from cases of human melioidosis in Thailand, and isolates B3 and B4 were cultured from uncultivated land in northeast Thailand [19]. The colony morphology of all five parental isolates was type I, and isogenic types II and III were generated from type I of each strain using nutritional limitation [11]. Briefly, a single colony of type I on Ashdown agar was inoculated into 3 ml of TSB and JAK inhibitor incubated at 37°C in air in static conditions for 21 days. Bacterial culture was diluted and spread plated onto Ashdown agar. Morphotypes were identified using a morphotyping algorithm [11]. Isogenic types II and III generated from each parental type I were isolated from the plates of each strain. Growth curve analysis JQ1 concentration Growth curves were performed for the 3 isogenic morphotypes of each of the 5 B. pseudomallei isolates. A colony of B. pseudomallei was suspended in sterile phosphate buffered saline GSK2245840 concentration (PBS). The bacterial suspension
was adjusted to an optical density (OD) at 600 nm of 0.15 and diluted 100 times. One hundred microlitres of bacterial suspension was added to 10 ml of TSB and incubated at 37°C in air with shaking at 200 rpm for 28 h. At 2 h intervals, 100 μl of bacterial culture was removed, serially diluted 10-fold in PBS, and the bacterial count determined by plating on Ashdown agar in duplicate and performing a colony count following incubation at 37°C in air for 4 days. Doubling time
was calculated. Cell line and culture conditions Human monocyte-like cell line U937 (ATCC CRL-1593.2) originating from a histiocytic lymphoma was maintained in RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (PAA Laboratories), 100 units/ml of penicillin and 100 μg/ml of streptomycin (Invitrogen) and cultured at 37°C in a 5% CO2 humidified incubator [20]. Before exposure to B. pseudomallei, 1 × 105 U937 cells per well were transferred to a 24 well-tissue culture plate (BD Falcon) and activated by the addition of 50 ng/ml of phorbol 12-myristate 13-acetate (PMA) (Sigma) over 2 days [20]. The from medium was then replaced with 1 ml of fresh medium without PMA and incubated for 1 day. The differentiated macrophage was assessed by macrophage-like morphology [21]. Following washing 3 times with 1 ml of Hank’s balance salt solution (HBSS) (Sigma), 1 ml of fresh medium was gently added to the macrophages. Interaction of B. pseudomallei isogenic morphotypes with human macrophages The interaction assay was performed as previously described [11]. B. pseudomallei from an overnight culture on Ashdown agar was suspended in PBS, the bacterial concentration adjusted using OD at 600 nm and then diluted in PBS and inoculated into wells containing differentiated U937 cells to obtain an MOI of approximately 25 bacteria per cell.