For S. mutans, a calibration curve using isopropyl alcohol-killed and live cells in varying proportions resulted in a linear correlation

between the ratio of green to red fluorescence and the amount of live cells (data not shown). For carolacton treated cells, Figure 3 shows that the extent of biofilm damage calculated from fluorescence staining was much smaller than that obtained by CFU counting. Thus, the green/red ratio of fluorescence is a conservative estimate of biofilm damage in S. mutans. Dose-dependent damage of biofilms of S. mutans by carolacton Biofilm damage was determined for 24 h old biofilms of S. mutans grown under anaerobic conditions, which predominate in dental plaque, using concentrations of carolacton between 0.0053 μM and 106.5 μM. As shown in Figure 4, carolacton decreased biofilm viability over a concentration Romidepsin supplier range of three orders of magnitude (from 0.053 μM to 53 μM) to approximately the same degree (55 – 65%). At a concentration of 0.01 μM (5 ng/ml) carolacton, biofilm damage was already

35%. This type of dose-response relationship is typical for quorum sensing controlled processes. A very low inducing threshold concentration is followed by a broad saturation range, resulting in the lack of a linear relationship between signal concentration and response [33]. Figure 4 Effect of carolacton concentration on the membrane damage of S. mutans biofilms. Biofilms were grown for 24 h under anaerobic conditions and stained using BTK inhibitor supplier the LIVE/DEAD BacLight Bacterial Viability kit. Green and red fluorescence was determined, and biofilm damage was calculated as reduction of the fluorescence ratio green/red compared to untreated controls. Each data point is the average of triplicate samples. Standard deviations are given for data points determined ifenprodil in at least three independent experiments. Time course of biofilm damage by carolacton We next investigated the dynamics of biofilm growth and its disturbance by carolacton during the first

24 h under anaerobic conditions. Green fluorescence of biofilms stained with SYTO9 alone is a measure of the total amount of biofilm cells, both alive and membrane damaged, and was applied here to study the growth of S. mutans biofilms with and without carolacton. Figure 5A shows two typical time courses for biofilm growth. In the untreated control, the amount of biofilm cells reached its maximum after 8 – 12 h, followed by a plateau and sometimes by a slow decrease, presumably due to detachment of biofilm fragments in the mature biofilm. During these first 12 h of biofilm growth, carolacton (5.3 μM) reduced the total amount of biofilm cells, as determined by total green fluorescence, up to 54%, but this effect was not observed any more after 24 h. Figure 5 Time course of biofilm growth of S. mutans in the presence and absence of carolacton.