DNA content in <i>P. aeruginosa</i> cultures.

<p>DNA (PI) staining of A - Aggregate harvested from a 48-h old stationary culture stained with PI. B – 3 day old biofilm grown in flow-cell stained with PI. C – GFP-tagged planktonic cells (OD – 0.5) stained with PI. Length of size bar: 15 µm.</p

Matrix production by mutants.

<p>We tested whether if mutants not able to produce the two distinct extracellular polysaccharides (Pel and Psl) could form intercellular fibers and if these fibers could be removed by DNAse treatment. WT - wild type PAO1, ΔpelA – PAO1 not able to produce Pel polysaccarides, ΔpslBCD - PAO1 not able to produce Psl polysaccarides, ΔpslBCDpelA - PAO1 not able to produce both Psl and Pel polysaccarides.</p

Localization of lections and DNA in aggregates.

<p>HHA-FITC and PI staining of aggregate harvested from a 48-h old stationary culture. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027943#pone-0027943-g001" target="_blank">Figure 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027943#pone-0027943-g002" target="_blank">2</a> represent two independent experiments with A, B and C representing HHA-FITC, PI and HHA-FITC+PI. To test whether if the fibers are made from more than one polymer, we co-stained the aggregates with PI (red) and a mannose-specific lectin stain (HHA-FITC) to visualize DNA and any present Psl polymers. Length of size bar; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027943#pone-0027943-g001" target="_blank">Figure 1</a>: 5 µm; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027943#pone-0027943-g002" target="_blank">Figure 2</a>: 10 µm.</p

Effect of DNase on aggregate formation.

<p>Stationary cultures grown with 90 U/ml DNase I in the medium. After 48-h there was a clear difference of the visible aggregation in the culture. The control was left untreated. The top and bottom panels represent two independent experiments.</p

Growth rates of flow-cell biofilms and aggregates over time.

<p>Growth rates were estimated by quantifying rRNA molecules per rDNA molecules by RT-PCR.</p

Antibiotic tolerance of maturing flow-cell biofilms of <i>P. aeruginosa</i> and aggregates harvested from a static <i>P. aeruginosa</i> culture.

<p>Biofilms and aggregates were grown for 24 h to 72-h prior to tobramycin (100 ug/ml) treatment for 24-h. For visualization by CLSM a GFP-tagged PAO1 strain (green) was used and stained with the DNA stain PI (red) for visualizing dead bacteria. Panels A, B and C represent biofilm tolerance on day 1, 2 and 3 respectively andpanels D and E are aggregates on day 1 and 2. Length of size bars: 20 µm.</p

Tolerance towards PMNs.

<p>Flow-cell biofilms (A+B) were grown for 72-h before addition of PMNs and the aggregates (C+D) were grown for 48-h before the addition of PMNs. The images shows that aggregates are not phagocytosed or penetrated by PMNs. For visualization a GFP-tagged PAO1 strain (green) was used and SYTO62 was used to stain the PMNs (red). Arrows point at paralyzed PMNs. Length of size bars: 20 µm.</p

Antibiotic tolerance of 48-h old aggregates harvested from a static <i>P. aeruginosa</i> culture and the effect of disruption by whirly mixing.

<p>Aggregates were grown for 48-h prior to tobramycin (100 ug/ml) treatment for 24-h or whirly mix (20 seconds) followed by tobramycin treatment for 24 h. 24 h. Bars represent the median. Mann-Whitney test were chosen to compare the medians of the different treatment regimens. ns indicates no significant differences between treatments (p>0.05). * Indicates significant difference (p<0.05). *** Indicates a very significant difference (p<0.0001).</p

Effect of prolonged whirly mix.

<p>Aggregates were grown for 48-h prior to tobramycin (100 ug/ml) treatment for 24-h, followed by whirly mix and another round of tobramycin treatment. Number indicates seconds of whirly mix. Bars represent the median. Kruskal-Wallis with Dunn's post test was chosen to compare the effect of whirly mix and subsequent treatment with tobramycin. The post test found the increased killing to be significant (P<0.05 when compared to 0 seconds) after 30 seconds of whirly mix.</p