The PCR products were cut with HinfI and separated on a 1.2% agarose gel. Due to asymmetric

location of the HinfI cleavage site inside the invertible element, different sized DNA 17-AAG manufacturer fragments ACP-196 purchase are obtained depending on the orientation of the phase switch. Results Role of fimbriae in K. pneumoniae biofilm formation by investigating monoculture biofilms To investigate the role of type 1 and type 3 fimbriae in K. pneumoniae biofilm formation a well-defined isogenic type 1 fimbriae mutant (C3091Δfim), a type 3 fimbriae mutant (C3091Δmrk), and a type 1 and 3 fimbriae double mutant (C3091ΔfimΔmrk) of the clinical UTI isolate C3091 were used. The wild type and its fimbriae mutants were found to have similar growth rates in the modified FAB medium used for biofilm experiments

(results not shown). Biofilm formation was observed four hours after inoculation of bacteria and after one, two, and three days. Four hours after inoculation of the flow-system, single cells of the wild type strain and its type 1 fimbriae mutant were observed adhering to the substratum SB203580 in vitro whereas only very few cells of the type 3 fimbriae and the type 1 and 3 fimbriae double mutant were detected (results not shown). After 24 hours the wild type and the type 1 fimbriae mutant were found to form characteristic biofilms on the substratum observed as long extended colonies in the flow direction (Figure 1). Figure 1 One-day old biofilms of K. pneumoniae C3091 and its isogenic fimbriae mutants at flow 0.2 mm/s. Biofilm formation was examined in three independent experiments with similar results. Box sides 230 μm × 230 μm. In contrast, the type 3 fimbriae mutant and the type 1 and 3 fimbriae double mutant only formed distinct microcolonies. Thus type 3 fimbriae, but not type 1 fimbriae, are important for attachment to the substratum as well as the initial stages of biofilm formation. Effect of flow on biofilm formation To investigate the influence of shear forces on biofilm about formation, a similar experiment

was performed, except the media flow speed was raised from 0.2 mm/s to 0.8 mm/s. Under higher flow speed, the influence of type 3 fimbriae was even more pronounced (Figure 2). The two mutants unable to express type 3 fimbriae (C3091Δmrk and C3091ΔfimΔmrk) formed even fewer and smaller colonies. Also the biofilm formation of the wild type and the type 1 fimbriae mutant was influenced by the higher flow speed. Both cell types formed flat biofilms compared to biofilms under lower flow velocity, likely due to increased removal of loosely attached cells. However, the biofilms were significantly more pronounced and continuous and covered most of the surface compared to the biofilms of the type 3 fimbriae mutant and the type 1 and 3 fimbriae double mutant (Figure 2).