Expression of fim2 in E. coli HB101 appears to enhance biofilm formation K. pneumoniae readily colonizes and forms biofilms on abiotic surfaces such as urinary catheters and tracheal tubes [21, 37]. As surface-expressed structures play a key role in biofilm formation, the ability of KR2107 and its isogenic mutants to form biofilms was examined. However, absence of fim2 and/or fim had no effect on biofilm formation as assayed at 24 h under static growth conditions in LB or M9 media at either 37°C or 30°C mTOR inhibitor (Figure 4A; data not shown). To detect a potential contribution to biofilm formation that may have

been masked by low-level fim2 expression or capsule-related physical hindrance of fimbrial function [38], fim2 was over-expressed from pFim2-Ptrc Small molecule library solubility dmso in E. coli HB101 using 0.05 mM IPTG induction. Compared to HB101 carrying the empty pJTOOL-7

vector, HB101/pFim2-Ptrc exhibited similar biofilm formation at 48 h on polystyrene wells as assessed by post-washing crystal violet staining (Figure 4B). On the other hand, expression of fim2 in HB101 resulted in marginally denser biofilm in polyvinyl chloride wells as compared to the vector-only control, but this was not statistically significant (P = 0.464; Figure 4B). Figure 4 The fim2 locus appears to contribute to biofilm formation when expressed in E. coli HB101. (A) Results for biofilm formation assay on polystyrene for KR2107 and its three fim and/or fim2 isogenic mutants as determined by crystal violet absorbance data. Equivalent results, suggestive of no strain-to-strain differences, were obtained for assays on polyvinyl chloride plates (data not shown). (B) Biofilm Arachidonate 15-lipoxygenase formation assay based on heterologous expression of fim2 in E. coli HB101/pFim2-Ptrc. HB101 and HB101 carrying an empty pJTOOL-7 served as controls. Biofilm formation was quantified using crystal violet staining and absorbance was measured at 595 nm. Non-normalized crystal violet absorbance data are shown. (C) Biofilm formation assay results shown in (B) were normalized to take account

of pre-wash total cell numbers based on OD595 readings performed at 48 h, just prior to washing off non-surface adherent cells and crystal violet staining. Data shown in all cases represent means and standard deviations of three biological replicates, each assayed in eight wells (n = 24). An asterisk indicates a highly significant difference (P < 0.0001) from HB101 and HB101/pJTOOL-7. As HB101/pFim2-Ptrc grew to a much lower OD595 at 48 h than the other two strains, we also analysed the biofilm data as a ratio of crystal violet staining intensity to the pre-wash OD595 measurement that reflected total growth. This analysis suggested that the proportion of HB101/pFim2-Ptrc cells comprising biofilm growth as opposed to total growth (biofilm and planktonic cells) was almost twice that of HB101 and the vector only control strain (Figure 4C).