Altered Virulence of Biofilm Pneumococci Figure 4. Modifications of polysaccharide capsule during biofilm growth contribute to hyper-adhesive phenotype. (A) Direct ELISA technique, using sera to type 4 polysaccharide capsule, comparing the amount of capsule present in three dilutions of whole cell lysates from planktonic or biofilm-derived bacteria. (B). Bacterial adhesion assay comparing planktonic and biofilm cultures of T4R and R6, an unencapsulated derivative of TIGR4 and serotype 2 respectively, to A549 cells. Values are expressed as fold increase adhesion relative to the planktonic counterparts. Statistical analysis was performed using a two-tailed Student’s t-test. doi:10.1371/journal.pone.0028738.g004 production (Figure 6). Immunodot blot was used because PsrP is glycosylated and separates at a molecular weight of .4000 kDa, thus it does not readily enter the SDS-PAGE gels normally used to separate proteins [26]. Thus we confirmed changes increased biofilm production of three adhesins and reduced production of the toxin pneumolysin. Figure 5. Selection for the transparent phenotype occurs during biofilm growth. (A) Representative images (n = 6) of individual bacterial colonies grown from either seed cultures (planktonic) or after 48 h of biofilm growth on blood agar plates. (B) Phase variation during biofilm growth as visualized by oblique, transmitted illumination of the planktonic culture and biofilm-derived bacteria. Percentage of phase variants in planktonic and biofilm cultures as determined by random counts of .100 individual bacterial colonies. (C) Relative levels of phosphorylcholine (ChoP) measured in whole cell lysates of planktonic or biofilm-derived bacteria were measured by Direct ELISA and by western blot analysis using monoclonal antibodies to ChoP (TEPC-15) (D) Immunoblot analysis of teichoic acids were performed using whole cell lysates of planktonic and biofilm-derived bacteria separated on 15% SDS-PAGE (E). doi:10.1371/journal.pone.0028738.g005 PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e28738