Figure 10.

Washed TIGR4 bacteria rapidly kill preformed Sau biofilms. (A) Sau was inoculated (Sau) in microtiter plates containing THY and incubated for 4 h, after which planktonic cells were removed and fresh THY medium was added. Another set of wells were inoculated with TIGR4 and incubated for 4 h at 37°C. Planktonic cells, biofilms, or supernatants from this TIGR4 4 h culture were separated as specified in Material and Methods. Preformed Sau biofilms were left uninoculated (Sau), or inoculated with ~1 × 10⁶ cfu/ml of an early-log phase culture of planktonic TIGR4 cells (+Spn), or 4 h cultures of washed bacteria (+Plank/Bio), washed planktonic bacteria (+Plank), washed biofilms (+Bios) or supernatant (+Sup) and incubated for 2 h at 37°C. Cultures were harvested, serially diluted and plated onto salt mannitol agar plates to obtain Sau (cfu/ml). Error bars represent the standard errors of the means calculated using data from at least three independent experiments. Statistical significance in comparison to wells inoculated with (^*, p < 0.004) Sau or (♦, p < 0.001) +Plank/Bio. (B–E) Sau was inoculated into an eight-well slide and incubated for 4 h at 37°C. Sau Biofilms were challenged with 4 h cultures of washed TIGR4 bacteria and incubated for 30 min (B), 1 h (C), 1.5 h (D), and 2 h (E). At the end of incubation, biofilms were fixed with 2% PFA and stained with an anti-Sau antibody followed by an Alexa 555-labeled anti-rabbit secondary antibody (red) and then an anti-Spn antibody labeled with Alexa 488 (green). DNA was stained with DAPI. Preparations were analyzed by confocal microscopy. A representative xy optical section is shown. Bar = 20 μm.