0001 in each case). By contrast when fim2 was expressed in the Mrk- and Fim-deficient strain C3091∆fim∆mrk using this same system, no statistically significant accentuation in biofilm formation MEK inhibitor on either surface was observed (data not shown). Deletion of fim2 does not affect adhesion to human HCT-8 ileocaecal or 5637 bladder epithelial cells In vitro adhesion assays were performed to

further investigate whether KR2107 and its three isogenic mutants (KR2107∆fim, KR2107∆fim2 and KR2107∆fim∆fim2) exhibited differing cell adhesion properties. Human HCT-8 ileocecal and human 5637 bladder epithelial cell lines were chosen to investigate adherence to intestine- and bladder-derived cells, respectively. No significant differences were detectable by these in vitro tissue culture assays (Figure 5). Furthermore, despite the previously reported impaired urovirulence of a

fim-negative K. pneumoniae strain [22], the KR2107∆fim and KR2107∆fim∆fim2 mutants examined in this p38 MAPK phosphorylation study did not display any defect in adherence to bladder epithelial cells relative to KR2107 or KR2107∆fim2. It is possible that fim and/or fim2 expression was insignificant under the in vitro conditions used or that the K. pneumoniae capsule interfered with fimbrial function [38, 39]. Figure 5 Cell-adherence properties of K. pneumoniae KR2107 and its isogenic fim and/or fim2 mutants. (A) In vitro adhesion assays to human HCT-8 ileocaecal cells. (B) In vitro adhesion assays to human 5637 bladder epithelial cells. In ZD1839 chemical structure both cases percentages of bacteria that remained adherent to cell

monolayers after 3 h of incubation at 37°C followed by careful AP26113 washing are shown. Bars represent means and standard deviations. Deletion of fim2 does not affect murine intestinal colonization Epidemiological studies have elucidated that the first step in the majority of K. pneumoniae infections is gastrointestinal tract colonization [18]. To investigate whether fim2 influences this initial step, a 1:1 mixture of KR2107 and KR2107∆fim2 was fed to three mice and faecal CFU counts were monitored for 13 days. To exclude potential type 1 fimbriae-related masking, a competition experiment between KR2107∆fim and KR2107∆fim∆fim2 was also performed. As assessed by faecal CFU counts, no strain exhibited an obvious competitive advantage and all four strains were found to readily colonize the large intestine in similar numbers (~108 – 109 CFU/g) throughout the experiment (Figure 6). Apart from confirming that fim does not affect intestinal colonization [22], these results also suggested that fim2 does not play a significant role in murine intestinal colonization by K. pneumoniae. Figure 6 Murine intestinal colonization of K. pneumoniae KR2107 and its isogenic fim and/or fim2 mutants. (A) Intestinal co-colonization following oral feeding with a 1:1 mixture of KR2107 and KR2107∆fim2.