FIG 4.

Biofilm consortia formed by invasive strains TIGR4 and S19F. (A and B) S19F (strain 5131) (A) or S19F (strain 4924) (B) was inoculated alone or with TIGR4 and incubated for 8 h, after which biofilms were harvested and DNA was extracted. Serotype-specific qPCRs targeting serotype 19F were used to quantify the specific bacterial load of S19F strains. (C to Q). Strains were incubated (as described for panels A and B) for 4 or 8 h, after which pneumococcal strains were stained with an anti-S19 antibody conjugated to Alexa 555 (red channel) or an anti-S4 antibody conjugated to Alexa 488 (green channel). Pneumococcal DNA was stained with DAPI (blue channel). Shown are confocal micrographs of S19F stains inoculated alone (C to F) or with TIGR4 and only showing red and blue channels (RB) (G to J) or showing all channels (RGB) (K to N). Results for strain TIGR4 incubated alone for 4 h (O) or 8 h (P) are also shown. (Q) An enlargement of the indicated area in panel P. Arrows point to zones where capsule staining concentrated in poles, and asterisks show pneumococci that lost their capsule. Bars, 10 μm (shown in panels L and N and valid for all other panels except panel Q, in which the bar equals 5 μm).