Characterization of B.cenocepacia BCAM0223 Mutant Figure 3. Effect of the BCAM0223 mutation on adherence to extracellular matrix proteins and biofilm formation. A) Adherence of wild- type B. cenocepacia K56-2 (filled bars) and BCAM0223::Tp mutant (open bars) to ECM proteins, laminin, fibronectin, collagen type I and IV and vitronectin. Only the binding capacity to vitronectin was significantly decreased in the mutant (*P,0.001). B) Static biofilm formation in polystyrene microtiter plates by wild-type B. cenocepacia K56-2 (filled bars) and BCAM0223::Tp mutant (open bars) at 24 and 48 h. Biofilm growth was quantified by the solubilization of crystal violet-stained cells with ethanol. Either after 24 h or 48 h incubation, the BCAM0223 mutant strain exhibited a statistically significant reduction in biofilm formation (*P,0.001 and **P,0.05). All the results are from three independent experiments; bars indicate SD. doi:10.1371/journal.pone.0041747.g003 Figure 4. Effect of the BCAM0223 mutation on serum resistance. The serum bactericidal assay performed using 30% normal human serum (NHS) incubated with B. cenocepacia K56-2 (black bars) and BCAM0223 mutant (open bars) showed significant sensitivity of the mutant to serum killing (*P,0.001). Heat inactivated NHS (hNHS) was used as control of the experiment showing no killing activity. Determination of the pathway involved in serum-killing of BCAM0223::Tp mutant, was performed using three distinct sera: NHS containing 10 mM MgCl2 plus 10 mM EGTA that block both, the lectin and classical pathways (LC-NHS), a C1q-depleted serum (C-NHS) (blocks classical pathway) (C-NHS) and factor B-depleted human serum (B-NHS) which contains solely the alternative complement pathway blocked. The wild-type B. cenocepacia K56-2 (filled bars) showed resistance to all three sera, whereas BCAM0223::Tp mutant (open bars) was resistant to LC-NHS and C-NHS but sensitive to B-NHS (*P,0.001), suggesting that the killing of the serum-sensitive BCAM0223::Tp mutant is mediated by the classical complement pathway. All the results are from three independent experiments. doi:10.1371/journal.pone.0041747.g004 PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e41747