Pseudomonas aeruginosa isolates from dental units waterlines can be divided in two distinct groups, including one displaying phenotypes similar to isolates from cystic fibrosis patients.

Pseudomonas aeruginosa displays broad genetic diversity, giving it an astonishing capacity to adapt to a variety of environments and to infect a wide range of hosts. While many P. aeruginosa isolates of various origins have been analyzed, isolates from cystic fibrosis (CF) patients have received the most attention. Less is known about the genetic and phenotypic diversity of P. aeruginosa isolates that colonize other environments where flourishing biofilms can be found. In the present study, 29 P. aeruginosa isolates from dental unit waterlines and CF patients were collected and their genetic and phenotypes profiles were compared to determine whether environmental and clinical isolates are related. The isolates were first classified using the random amplified polymorphic DNA method. This made it possible to distribute the isolates into one clinical cluster and two environmental clusters. The isolates in the environmental cluster that were genetically closer to the clinical cluster also displayed phenotypes similar to the clinical isolates. The isolates from the second environmental cluster displayed opposite phenotypes, particularly an increased capacity to form biofilms. The isolates in this cluster were also the only ones harboring genes that encoded specific epimerases involved in the synthesis of lipopolysaccharides, which could explain their increased ability to form biofilms. In conclusion, the isolates from the dental unit waterlines could be distributed into two clusters, with some of the environmental isolates resembled the clinical isolates. Keywords: Pseudomonas aeruginosa, cluster, RAPD, elastase, biofilm, Dictyostelium discoideum, cell lysi

Comparative genomics of isolates of a pseudomonas aeruginosa epidemic strain associated with chronic lung infections of cystic fibrosis patients

Pseudomonas aeruginosa is the main cause of fatal chronic lung infections among individuals suffering from cystic fibrosis (CF). During the past 15 years, particularly aggressive strains transmitted among CF patients have been identified, initially in Europe and more recently in Canada. The aim of this study was to generate high-quality genome sequences for 7 isolates of the Liverpool epidemic strain (LES) from the United Kingdom and Canada representing different virulence characteristics in order to: (1) associate comparative genomics results with virulence factor variability and (2) identify genomic and/or phenotypic divergence between the two geographical locations. We performed phenotypic characterization of pyoverdine, pyocyanin, motility, biofilm formation, and proteolytic activity. We also assessed the degree of virulence using the Dictyostelium discoideum amoeba model. Comparative genomics analysis revealed at least one large deletion (40-50 kb) in 6 out of the 7 isolates compared to the reference genome of LESB58. These deletions correspond to prophages, which are known to increase the competitiveness of LESB58 in chronic lung infection. We also identified 308 non-synonymous polymorphisms, of which 28 were associated with virulence determinants and 52 with regulatory proteins. At the phenotypic level, isolates showed extensive variability in production of pyocyanin, pyoverdine, proteases and biofilm as well as in swimming motility, while being predominantly avirulent in the amoeba model. Isolates from the two continents were phylogenetically and phenotypically undistinguishable. Most regulatory mutations were isolate-specific and 29% of them were predicted to have high functional impact. Therefore, polymorphism in regulatory genes is likely to be an important basis for phenotypic diversity among LES isolates, which in turn might contribute to this strain's adaptability to varying conditions in the CF lung

Non-synonymous SNPs and DIPs among <i>P. aeruginosa</i> LES isolates found in regulatory genes.

<p>*indicates that the polymorphism introduces a stop codon or a frame shift in the protein sequence.</p>1<p>Genes selected have a PseudoCAP annotation that includes terms “Transcriptional regulators” and/or “Two-component regulatory systems” (pseudomonas.com).</p>2<p>Virulence factor database annotation is indicated between parentheses when applicable.</p>3<p>LESL: LESlike.</p

Summary of SNPs and DIPs among <i>P. aeruginosa</i> LES isolates and between LES and laboratory strain PAO1.

1<p>Polymorphisms were identified with CLC Genomics Workbench (CLCbio) with the following criteria: default parameters for base quality, minimum coverage = 6, minimum frequency  = 0.8 for each new genome against reference LESB58 and 0.95 for all LES or LESlike against reference PAO1.</p

Circular genome map of <i>P. aeruginosa</i> LESB58 compared to 7 LES isolates and strain PAO1.

<p>Map and underlying analysis were performed with the CGView Comparison Tool <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087611#pone.0087611-Grant1" target="_blank">[23]</a>. Rings from the outside inward: LESB58 plus-strand, LESB58 minus-strand, 308 unique non-synonymous polymorphisms among LES isolates, LES400, LES431, LESB65, LESlike1, LESlike4, LESlike5, LESlike7, PAO1 and GC content plot. LES prophages (Pro), genomic islands (GI) and LES specific mini island (MI) are indicated on the outside of the map <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087611#pone.0087611-Winstanley1" target="_blank">[10]</a>. Orange colored BLAST hits: 90–98% identity, blue colored BLAST hits: 0–90% identity.</p

<i>P. aeruginosa</i> LES accessory genome dendrogram and heatmap.

<p>Strain PAO1 was included as an outgroup. Using Panseq <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087611#pone.0087611-Laing1" target="_blank">[24]</a>, genomes were fragmented in 500 bp segments (default parameters), which were considered as accessory genome if absent from at least 1 of the 9 genomes analysed. Shades of grey represent percent identity, with white = 0% and black = 100%. The dendrogram is based on a hierarchical cluster analysis (Euclidean distance, method = average, r-project version 2.15.1). * Position 1,720,917 in LESB58 (15 kb gap), adjacent to LES prophage 4; ** Position 3,607,181 in LESB58 (32 kb gap).</p

Pseudomonas Community Annotation Project (PseudoCAP) annotation of polymorphic genes among <i>P. aeruginosa</i> LES isolates.

<p>P-values are from Fisher’s exact tests comparing genes with non-synonymous polymorphisms with LESB58 whole genome composition (* p<0.05, ** p<0.01 & q <0.05).</p

Assembled genomes for 7 <i>P. aeruginosa</i> isolates of the Liverpool epidemic strain.

1<p>Shotgun libraries of the first 3 strains (UK) were sequenced individually on a quarter (LES400, LESB65) or a half (LES431) 454 plate. Shotgun libraries of the last 4 strains (Ontario, Canada) were barcoded and sequenced on a full 454 plate.</p>2<p><i>De novo</i> assembly was performed with GS De Novo Assembler (Roche); genome finishing was done with Consed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087611#pone.0087611-Gordon1" target="_blank">[20]</a>.</p

Pyocyanin and pyoverdine production.

<p>A. Measurements of pyocyanin from overnight culture supernatants, error bars: standard deviation from 3 independent experiments. B. Pyoverdine production on Pseudomonas agar F (Difco) visualized under long wavelength UV light, results shown are from 1 of 3 replicates.</p