FIG 5.

Confocal microscopy images depicting biofilm thickness and SagA localization in a semistatic biofilm model with or without proteinase K treatment. (a) Biofilms of six E. faecium strains—three clade A1 (E470, E1162, E1904) and three clade B (E980, E3548, E1590) strains—were grown for 24 h on poly-l-lysine-coated glass, in TSBg with or without proteinase K, at 120 rpm and 37°C. The ecological origins and clade assignments of the strains are listed in Table S1 in the supplemental material. Biofilms were incubated with a rabbit polyclonal anti-SagA antiserum and an Alexa Fluor 488-conjugated goat anti-rabbit antibody (green). Bacterial membranes were stained with FM 95-5 (red). (b) The presence of SagA in the biofilm supernatants of E. faecium strains incubated with (+) or without (−) proteinase K was analyzed by Western blotting using a rabbit polyclonal anti-SagA antiserum. (c) The thicknesses of biofilms were analyzed with Comstat/Matlab software at five random positions. Asterisks represent significant differences (*, P < 0.05; **, P < 0.01) by an unpaired two-tailed Student t test between thicknesses with and without proteinase K treatment. Pictures were taken at ×63 magnification with 2.0 optical zoom.