Glass capillary flow reactors were inoculated with the GFP-P. aeruginosa 17 isolate and the biofilm formation was followed with CLSM. Following 48 h growth, the capillary RG-7388 cost reactor was inoculated with isolate 80 and the flow was stopped for 3 h to allow attachment. The MK5108 in vivo bacterial biofilms were stained with rhodamine

B (reference colour) and observed with CLSM 24 h after inoculation with isolate 80 (Fig. 4). Isolate gfp-17 was identified by green fluorescence due to the production of GFP, and isolate 80 was identified by rhodamine B. The excitation and emission wavelengths were distant between the fluorophores and did not overlap. Isolate gfp-17 established a green lawn that colonised the reactor surface, while isolate 80 was observed as spatially distributed red cell clumps within the established biofilm. Furthermore, cross sectional analysis of the biofilm (Fig. 5) showed that isolate 80 was not only attached to the surface of the isolate 17 biofilm, but that the cells were incorporated into the three dimensional structure Givinostat of the established biofilm, suggesting that isolate 80 was able to migrate into the established biofim

despite its lack of twitching and swimming motility. Figure 4 CSLM images of mixed biofilm produced by Pseudomonas aeruginosa isolates gfp -17 (green) and isolate 80 (red) in a glass capillary flow reactor. Isolate gfp-17 was allowed

to establish a biofilm for 48 h and then isolate 80 was inoculated into the flow reactor. After 24 h incubation the mixed biofilm was stained and GFP and rhodamine B were excited at 488 nm and 567 nm respectively. Figure 5 Cross section of the mixed Pseudomonas aeruginosa biofilm. Isolate gfp-17 was allowed to establish a biofilm for 48 h and then isolate 80 was inoculated into the flow reactor. After 24 h incubation the mixed biofilm was stained and GFP and rhodamine B were excited at 488 nm and 567 nm respectively. As can be seen from the cross section, isolate 80 became PAK6 incorporated into the biofilm body and was not simply attached to the surface of the isolate gfp-17 biofilm. Discussion The CF lung can be colonised by P. aeruginosa isolates that display heterogeneity in both motility and biofilm phenotype. We evaluated the association between types of motility and biofilm formation using a set of 96 clinical isolates of P. aeruginosa. Several studies have reported that motility is required to initiate cell attachment [8, 37–39] although there is still no consensus as to the contribution of each type of motility to the overall process of biofilm development. While P. aeruginosa is a motile bacterium, the lack of motility in CF isolates has been previously reported [15] and here some 47% of the isolates were non-motile.