001; ANOVA-test followed by Newman-Keuls multiple comparison post-test). B. Biofilm formed

by S. maltophilia on IB3-1 cell monolayers. Strain OBGTC37 formed the highest amount of biofilm, significantly higher (** P < 0.001; ANOVA-test followed by Newman-Keuls multiple comparison post-test) than other strains tested. Results are expressed as means + SDs. With regard to biofilm formation, as judged by the check details number of cfu recovered after 24 hours of incubation, S. maltophilia strain OBGTC37 produced the highest amount of biofilm (5.4 ± 0.8 × 107 cfu chamber-1) (Figure 1B), a value significantly higher if compared to the other strains tested (P < 0.001). No significant correlation was found between adhesiveness and the amount of biofilm formed (Pearson r, 0.158; P > 0.05). CLSM observation

of IB3-1 cell monolayers infected for 2 or 24 hours with S. maltophilia showed no significant differences in cellular detachment with respect to control, thus confirming the integrity of exposed IB3-1 monolayers. Furthermore, after 24 hours of infection, learn more both SEM and CLSM analysis revealed clusters of S. maltophilia cells scattered across almost all IB3-1 cells (Figures 2 and 3). CLSM analysis showed that microcolonies were embedded in extracellular Proteasome inhibitor drugs matrix whose amount was significantly increased following infection (Figure 3B). These morphological observations are strongly suggestive of S. maltophilia biofilm formation on IB3-1 cells. Figure 2 SEM observation of 24 hours-biofilm formed byclinical isolate S. maltophilia OBGTC9 on IB3-1 cell monolayer. Scanning

electron micrographs showing cell cluster morphology (microcolony) strongly suggestive of biofilm formation. Bacterial cells lose their outlines for the presence of extracellular matrix (arrows). Magnification: ×2.500 (Figure 2A), ×5.000 (Figure 2B). Figure 3 CLSM observation of 24 hours-biofilm formed byclinical isolate S. maltophilia OBGTC9 on IB3-1 cell monolayer. A-B. CLSM micrographs of not fixed specimens of unexposed (control; Figure 3A) and OBGTC9-exposed (Figure 3B) IB3-1 cell monolayer stained with Syto-9 (green fluorescence, indicating live cells), propidium iodide (red Non-specific serine/threonine protein kinase fluorescence, indcating dead cells), and Con-A (blue fluorescence, indicating extracellular matrix). Image capture was set for visualization of: (a) green fluorescence only; (b) red fluorescence only; (c) blue fluorescence only (3) or; (d) co-localization of all three fluorescence signals. Note the formation of a S. maltophilia microcolony embedded in matrix whose formation is significantly increased in infected vs control IB3-1 cell monolayers. C. CLSM examination of fixed IB3-1 monolayer exposed to S. maltophilia OBGTC9 for 24 hours: three-dimensional representation. Green fluorescence indicates autofluorescence of IB3-1 cytoplasm following exposure to fixation mixture; red fluorescence indicates binding of propidium iodide to nucleic acids of both IB3-1 and S. maltophilia cells.