BCAM0223-deficient B. cenocepacia K56-2, amplifying the entire gene. Hemagglutination Assay Cultures of B. cenocepacia K56-2 and BCAM0223::Tp mutant (initial OD640 0.2) were grown for 17 h at 37uC, 60 rpm, under the above established conditions. Cells were harvested by centrifugation, washed once in phosphate buffered saline (PBS) pH 7.4 and resuspended in PBS to an OD640nm of 12 units. The cells (30 mL volumes) were serially diluted (OD640 = 1/2, 1/3, 1/4, 1/5, 1/6) and placed in 96-well microtitre plates. To each dilution an equal volume of sheep red blood cells (3% v/v in PBS) was added. The plates were mixed gently with a pipette tip and incubated for 1 h at room temperature. PBS solution was used as a negative control. Each assay consisted of three to five replicates. Hemagglutination was detected by visual inspection of the suspension with direct comparison to the negative control. Each assay consisted of three to five replicates. Results are median values of 3 independent experiments. Adherence and Biofilm Formation Assays Bacterial adherence to extracellular matrix proteins (laminin, fibronectin, vitronectin, collagen types I and IV), was tested as described before with some modifications [28]. Briefly, 96-well polystyrene microplates were coated with 10 mg/mL collagen type IV (in PBS) and 0.5 of mg/mL vitronectin (in water) and placed at 4uC, overnight. Cultures of B. cenocepacia K56-2 and BCAM0223::Tp mutant (initial OD640 0.2) were grown for 17 h at 37uC, 60 rpm, in microaerophilic conditions, as previous described. Equal amounts (200 mL) were added to the ECM- coated wells. After incubation for 2 h at room temperature, non- bound bacteria were removed and adherent bacteria were fixed with 4% (w/v) paraformaldehyde for 20 min and stained with 1% (w/v) crystal violet for 10 min. After washing twice with PBS, the bound crystal violet was dissolved in 95% ethanol and the absorbance was measured at 595 nm in a microplate reader (Versamax, Molecular devices). Results are median values of at least 5 repeats from 3 independent experiments. Biofilm formation assays were based on the methodology described by Toole and Kolter [33]. Bacterial cultures were grown under the conditions described above, diluted in LB to an OD640nm of 0.05 and 200 mL of these cell suspensions were inoculated into wells of a 96-well polystyrene microtiter plate. Plates were statically incubated at 37uC for 24 or 48 hours. For biofilm quantification, the culture medium and unattached bacterial cells was removed and wells were rinsed three times with deionized water. Adherent bacteria were stained with 200 ml of a 1% (wt/v) crystal violet solution for 15 minutes at room temperature. After three gentle rinses with deionized water the dye was solubilised with 95% ethanol and measurement of the solution absorbance at 590 nm using a microplate reader. Results are median values of at least 5 repeats from 3 independent experiments. Serum Resistance Tests The resistance of B. cenocepacia K56-2 to Normal Human Serum (NHS) was assessed by use of a liquid-phase assay based on a previously described methodology [34]. Briefly, bacterial cultures were grown in microaerophilic conditions previously described. After 17 h of growth, cells were harvested and diluted in PBS to a final concentration of 106 CFU/mL. Then, 10 mL (104 CFU) of cell suspension was added to 30 mL of commercial NHS (Sigma-Aldrich) and 60 mL of PBS, resulting in a final serum concentration of 30%. The mixture was Characterization of B.cenocepacia BCAM0223 Mutant incubated at 37uC for 1 h and then placed on ice. Sub- sequently, 10 mL of each sample was serially diluted and plated to determine the colony-forming units (CFU). The initial bacterial loading was achieved replacing the NHS for PBS, and 10 mL of the bacterial suspension was serially diluted and plated. As control, we performed all experiments in parallel using heat-inactivated NHS (30 min at 56uC). The percentage of survival was determined as the quantity of bacteria that survived relatively to the initial bacterial loading. To block the alternative complement activation pathway, factor B-depleted serum (B-NHS) (Quidel) was used. To obstruct both the classical and lectin pathways, NHS was equilibrated in PBS containing 10 mM MgCl2 and 10 mM EGTA (LC-NHS) for 15 min on ice before adding the bacteria. Finally to selectively block the classical pathway a C1-q depleted serum (C-NHS) was used. Adhesion to and Invasion of Human Bronchial Epithelial Cells Adhesion and invasion experiments were carried out on non-CF 16HBE14o- and CF CFBE41o- bronchial epithelial cells using an adaptation of the method described by Martin and Mohr [35]. Firstly, cells were seeded on 24-well plates (Orange scientific) (56105 cells/well) in MEM in MEM medium supplemented and cultured for 24 h at 37uC, in a humidified atmosphere with 5% CO2. Then, the cell monolayers were washed with PBS and maintained in MEM medium without supplements. Parental and BCAM0223::Tp strains were used to infect host cells at a multiplic- ity of infection (MOI) of 50:1 (bacteria per epithelial cell). For adhesion assays, the infected monolayers were incubated at 37uC in 5% CO2 for 30 min to allow bacterial adherence. After this, cells were washed three times with PBS and lysed with 200 mL of the buffer (10 mM EDTA, 0.25% Triton X-100) for 30 min at room temperature. The adhered bacteria were quantified by plating serial dilutions of the cell lysates. Results are expressed as a ratio of the wild type and corrected with the initial bacterial dose applied. For invasion assays, the infected monolayers were incubated at 37uC in 5% CO2 for 2 h to allow bacterial entry. After incubation, the monolayers were washed three times with PBS and a combination of amikacin and ceftazidime (2 mg/ml, each) was added and incubated for two hours. The supernatants were then plated to confirm the effectiveness of antibiotic treatment. Finally, the cell monolayers were washed three times with PBS and intracellular bacteria were released using the same lysis buffer used in adhesion experiments. Bacteria were quantified by plating serial dilutions of the cell lysates. The results are expressed as a ratio of the wild type and corrected with the initial bacterial dose applied. Confocal Microscopy Examination B. cenocepacia K56-2 and BCAM0223::Tp mutant cells were electroporated with pIN29, a plasmid that encodes the red fluorescent protein DsRed [36]. Bacterial adhesion assays were performed as described above except coverslips were introduced in the 24-well plates before the cells seeding. Cells were fixed with 3.7% paraformaldehyde for 20 min before being quenched with 50 mM NH4Cl (Sigma) for 10 min and immersed in 0.2% triton X-100 (Sigma) for 5 min. Thereafter, the coverslips were saturated with 0.5% bovine serum albumin (BSA) for 30 min. The immunostaining was achieved by incubation with the primary mouse E-cadherin antibody (clone HECD-1, Takara Bio Inc. Shiga, Japan) (1:100 dilution) followed by the secondary polyclonal goat anti-rabbit serum coupled to Alexa Fluor 488 (Invitrogen) PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e41747