We obtained similar results when we repeated the experiments using TSBDC, a growth medium that supports planktonic growth of both organisms C646 nmr (Figure 10B). These results suggest that similar to other previous observations, P. aeruginosa eliminates S. aureus, when the two are grown together at the same time. Figure 10 PAO1 inhibits AH133 in co-culture. Overnight LB cultures of AH133 and PAO1/pMRP9-1 were pelleted, washed, and resuspended in PBS. Resuspended cells of each species were inoculated into ASM+ (A) or TSBDC (B) at an initial OD600 of selleck kinase inhibitor approximately 0.015. The CFU/ml of each species was determined at the time of inoculation (0-time) and after 48 h of growth using selective

media (Methods). The graphs show CFU/ml obtained from

BLS in ASM+ (A) and planktonic growth in TSBDC (B). Values represent the means of at least three independent experiments ± SEM. To simulate the scenario in which S. aureus colonizes the CF lung first and P. aeruginosa follows, we then examined the effect of PAO1 on partially developed (8 h) AH133 BLS. As AH133 expresses GFP, we transformed PAO1 with pMP7605, a plasmid from which RFP is expressed constitutively [34], to allow visualization of each strain within the BLS. Individually, the strains produced well developed BLS in ASM+ (Figures 2, 10A). At 8 h post inoculation, AH133 formed a defined structure (Figure 11A). We then added PAO1/pMP7605 (at a starting density similar to that used to initiate the www.selleckchem.com/products/midostaurin-pkc412.html AH133 culture) and continued incubation for 56 h. The cultures were analyzed at 8- and 16-h intervals to 64 h for the

AH133 alone and 56 h post addition of PAO1/pMP7605 for changes in the BLS produced by AH133 and the development of any PAO1 BLS (Figure 11A, B). At 16 and 24 h post-initiation, AH133 produced well-developed mature BLS (Figure 11A). The AH133 BLS changed in appearance over the rest of the time course, but did not disappear (Figure 11A). In contrast, in the dual culture, the Pyruvate dehydrogenase AH133 structure was considerably reduced at the corresponding time points (Figure 11B). By 32 h only remnants of the AH133 BLS remained, and by 40–56 h, the AH133 BLS appeared to be completely replaced by well-developed PAO1 BLS (Figure 11B). The regression of AH133 structure appears to be due to the expansion of the PAO1 structure at 8, 16, and 32 h post-initiation (Figure 11B). Figure 11 Elimination of AH133 BLS is due to the bactericidal effect of PAO1. PAO1/p7605 (red) gradually eliminates previously-formed AH133 (green) BLS. ASM+ was inoculated with AH133 and the cultures were grown for 8 h to allow for the partial development of AH133 BLS. (A) One culture was continued without addition of PAO1 for a total of 64 h. (B) The other culture was inoculated with PAO1/p7605 (starting density similar to that used to initiate the AH133 culture).