Phenotypes of Non-Attached Pseudomonas aeruginosa Aggregates Resemble Surface Attached Biofilm

For a chronic infection to be established, bacteria must be able to cope with hostile conditions such as low iron levels, oxidative stress, and clearance by the host defense, as well as antibiotic treatment. It is generally accepted that biofilm formation facilitates tolerance to these adverse conditions. However, microscopic investigations of samples isolated from sites of chronic infections seem to suggest that some bacteria do not need to be attached to surfaces in order to establish chronic infections. In this study we employed scanning electron microscopy, confocal laser scanning microscopy, RT-PCR as well as traditional culturing techniques to study the properties of Pseudomonas aeruginosa aggregates. We found that non-attached aggregates from stationary-phase cultures have comparable growth rates to surface attached biofilms. The growth rate estimations indicated that, independently of age, both aggregates and flow-cell biofilm had the same slow growth rate as a stationary phase shaking cultures. Internal structures of the aggregates matrix components and their capacity to survive otherwise lethal treatments with antibiotics (referred to as tolerance) and resistance to phagocytes were also found to be strikingly similar to flow-cell biofilms. Our data indicate that the tolerance of both biofilms and non-attached aggregates towards antibiotics is reversible by physical disruption. We provide evidence that the antibiotic tolerance is likely to be dependent on both the physiological states of the aggregates and particular matrix components. Bacterial surface-attachment and subsequent biofilm formation are considered hallmarks of the capacity of microbes to cause persistent infections. We have observed non-attached aggregates in the lungs of cystic fibrosis patients; otitis media; soft tissue fillers and non-healing wounds, and we propose that aggregated cells exhibit enhanced survival in the hostile host environment, compared with non-aggregated bacterial populations

Identification and Characterization of an N-Acylhomoserine Lactone-Dependent Quorum-Sensing System in Pseudomonas putida Strain IsoF

Recent reports have shown that several strains of Pseudomonas putida produce N-acylhomoserine lactones (AHLs). These signal molecules enable bacteria to coordinately express certain phenotypic traits in a density-dependent manner in a process referred to as quorum sensing. In this study we have cloned a genomic region of the plant growth-promoting P. putida strain IsoF that, when present in trans, provoked induction of a bioluminescent AHL reporter plasmid. Sequence analysis identified a gene cluster consisting of four genes: ppuI and ppuR, whose predicted amino acid sequences are highly similar to proteins of the LuxI-LuxR family, an open reading frame (ORF) located in the intergenic region between ppuI and ppuR with significant homology to rsaL from Pseudomonas aeruginosa, and a gene, designated ppuA, present upstream of ppuR, the deduced amino acid sequence of which shows similarity to long-chain fatty acid coenzyme A ligases from various organisms. Using a transcriptional ppuA::luxAB fusion we demonstrate that expression of ppuA is AHL dependent. Furthermore, transcription of the AHL synthase ppuI is shown to be subject to quorum-sensing regulation, creating a positive feedback loop. Sequencing of the DNA regions flanking the ppu gene cluster indicated that the four genes form an island in the suhB-PA3819 intergenic region of the currently sequenced P. putida strain KT2440. Moreover, we provide evidence that the ppu genes are not present in other AHL-producing P. putida strains, indicating that this gene cluster is so far unique for strain IsoF. While the wild-type strain formed very homogenous biofilms, both a ppuI and a ppuA mutant formed structured biofilms with characteristic microcolonies and water-filled channels. These results suggest that the quorum-sensing system influences biofilm structural development

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

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

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

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

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