template in a reaction containing 26 QuantiTect SYBR Green mix (Qiagen), RNase-free water and 20 gene-specific primers. The gene-specific primers were designed using Primer3 and synthesized by Eurofins MWG Operon (Alabama, USA). The qRT-PCR was conducted using an ABI 7900HT machine (Applied Biosystems). The reactions were denatured at 95uC for 15 min followed by amplification with 45 cycles of 94uC for 15 s, 55uC for 30 s and 72uC for 30 s. Reactions were followed by a melt curve analysis that starts at 55uC, with a dissociation step at 95uC for 1 min plus 0.5uC/cycle for 80 cycles. The qRT-PCR data was analyzed using a comparative cycle threshold (DCt) method [33]. The DCt was normalized to genes that did not exhibit any significant change in expression as identified by the microarray experiments. Each sample from each biological replicate was analysed twice (technical replicates). Quantification of CPS and teichoic acid Levels of CPS and teichoic acid containing ChoP were determined using ELISA as previously described with minor modifications [34]. Stocks of pneumococci grown under biofilm and planktonic conditions were suspended in 1 ml of PBS and sonicated for three 10 s intervals on ice prior to storage at 220uC. Total cellular protein was determined by bicinchoninic acid (BCA) assay (Sigma) as per the manufactures instructions. Bacterial sonicates at the designated protein concentrations were diluted in a sodium bicarbonate/carbonate buffer (100 mM, pH 9.6) and fixed overnight to 96-well polystyrene plates (Nunc Maxisorp, Apogent, USA) at 4uC. The plates were blocked with 1% BSA in PBS (blocking buffer) for 2 h at room temperature then washed 3 times with PBS. For detection of serotype 4 CPS, type specific rabbit antiserum against serotype 4 capsule (Statens Serum Institut, Denmark) was added at 1:20,000 in blocking buffer, and incubated at room tempera- ture for 1 h. Binding of type-specific antiserum, was detected by washing, incubation with an HRP- conjugated goat anti rabbit antibody at 1:10,000 in blocking buffer, washing, and development by standard methods using tetramethylbenzidine and hydrogen peroxide as the substrate reagent. Plates were read using a plate reader at 450 nm. Relative amounts of ChoP were detected as above with some modifications. For quanti- fication of teichoic acid mouse monoclonal IgA antibody (Clone ID TEPC-15, Cat# M1421, Sigma) specific ChoP was used at 1:5,000 and binding of primary antibody was detected using an alkaline phosphatase conjugated goat anti-mouse IgA (1:10,000) (Cat# 1040-04, Southern Biotech, USA). Levels of teichoic acid were also confirmed by Western blot analysis. Equal amounts of whole bacterial cell sonicates (15 mg) as measured by BCA assay were separated on a 15% SDS-PAGE gel and electrophoretically transferred to nitrocellulose mem- branes using standard methods. Membranes were blocked with 4% bovine serum albumin (BSA) in PBS for 2 h at room temperature then incubated overnight at 4uC with mouse monoclonal antiserum (TEPC-15) specific for ChoP at a dilution of 1:10,000. Alkaline phosphatase conjugated goat anti-mouse IgA at 1:5,000 was used to detect the primary antibody and NBT/BCIP Ready-to Use Tablets (Roche) were used as the alkaline phosphatase substrate for development. Equal loading of sonicates was confirmed by Coomassie brilliant blue staining of parallel loaded gels, and the staining of membranes with Ponceau stain (Sigma) following immuno- blot. For ELISA experimental values are expressed as the average of a minimum of three independent experiments performed in triplicate. For consistency immunoblots were performed three times. Altered Virulence of Biofilm Pneumococci Immunoblot assays Whole cell bacterial lysates were prepared by sonication of the samples on ice for three 10 s intervals. Total cellular protein was determined by bicinchoninic acid (BCA) assay (Sigma) as per the manufacturer’s instructions. Whole cells lysates (25 mg) were separated by 12% SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. For PsrP, samples were directly blotted onto nitrocellulose membranes. Membranes were blocked with PBS containing 4% bovine serum albumin (BSA) and 0.1% Tween-20 for 1 h and incubated overnight at 4uC with rabbit sera to PsrP or CbpA (a gracious gift from Elaine Tuomanen, Memphis TN), or mouse monoclonal sera to or pneumolysin (NCL-SPN; Novocastra Laboratories) or ChoP. A HRP-conjugated secondary antibody was used for detection of the proteins by chemilumine- sence. Equal loading of sonicates was confirmed by Coomassie brilliant blue staining of parallel loaded gels, and the staining of membranes with Ponceau stain (Sigma) following immunoblot. Determination of phase variant phenotype Phase variation in pneumococci was assessed as previously described [24]. Pneumococci were streaked onto tryptic soy broth (TSB) plates supplemented with 1% agar onto which 100 ml of catalase (6300 U) (Sigma) was added. Plates were incubated at 37uC in 10% CO2 for 16 h. Following incubation colony morphologies (i.e. phase variation) were assessed under magnifi- cation and oblique transmitted illumination by differential interference contrast (DIC) microscopy using a Zeiss Axiovision Imager Z1. Frequency of phase variants was determined by counting and determining the phenotype of $100 random colonies from at lest 6 plates with either seed culture or biofilm derived TIGR4. Colony images were captured using a Leica S6D light microscope and digital camera. Statistical analysis For pair-wise comparisons of groups statistical analyses were performed using a two-tailed Student’s t-test. For multivariate analyses a 1-Way ANOVA followed by a post-priori test using Sigma Stat software was used. For survival studies a Kaplan Meier Log-Rank test was used. Values were determined to be statistically significant if the P,0.05. Results Verification of mature pneumococcal biofilm formation We first utilized electron microscopy to verify that pneumococci growing within the bioreactor lines were indeed in a mature biofilm state. SEM of TIGR4 revealed that biofilm formation occurred in a series of steps including initial attachment, formation of a ‘‘bacteria lawn’’, then the striking incremental formation of a large biofilm matrix (Figure 1A–D). By 24 h, pneumococci had begun to form an extracellular polymeric matrix (EPM). At 48 h, EPM was the main component of the biofilm, encompassing most but not all pneumococci (Figure 1E). TEM analysis determined that the mature biofilm was predominantly acellular and primarily composed of dead pneumococci (Figure S1A). At 24 h only 67% of biofilm pneumococci were presumably viable (having an intact membrane and discernible electron dense body) and by 48 h only 27% were intact (Figure S1B). Thus, we determined that a major component of the mature biofilm and by extension the EPM was remnants of dead bacteria. A key characteristic of biofilms is their recalcitrance to antimicrobials [35,36]. We tested the resistance of pneumococci isolated from the biofilm reactor using a modified version of the broth antimicrobial microdilution assay (Figure 1F) [29]. Typically PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e28738