Histopathology, <i>P. aeruginosa</i> load and localization in murine lungs after infection with clonal pair of early/late <i>P. aeruginosa</i> isolates.

<p>C57Bl/6NCrl were infected with 5×10⁶ cfu/lung of <i>P. aeruginosa</i> strains from AA lineage for 24 hours. Control mice were not infected. Lungs were stained with H&E and in immunofluorescence with specific antibody against <i>P. aeruginosa</i> (red) (<b>A, E, I</b>: AA2; <b>B, F, L</b>: AA43; <b>C, G, M</b>: AA44; <b>D, H, N</b>: not infected). Counterstaining was performed with 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (blue). <b>I–N</b>) Bacterial cells of <i>P. aeruginosa</i> are visible in the bronchia and pulmonary parenchyma. O) Severity of lesions and lung involvement is heterogeneous in different lobes of the same mice. Quantification of infiltrated and preserved areas as percentage of total tissue area with mean ± SEM is shown (n = 3 mice each/strain). Statistical analysis was calculated for pair wise comparisons between early and late strains (* p<0.05; ** p<0.01; *** p<0.001, Mann–Whitney). <b>P</b>) Dots represent individual measurements of the no. of cfu per lung, and horizontal lines represent median values after 12, 24 and 48 h. Two independent experiments were pooled. Statistical analysis was calculated for pair wise comparisons between early and late strains (* p<0.05, Student's t-test).</p

Correlation between survival percent and initial infection dose of clonal pair of early/late <i>P. aeruginosa</i> isolates in C57Bl/6NCrl.

<p>C57Bl/6NCrl mice were infected with different doses of <i>P. aeruginosa</i> strains from AA (<b>A</b>) and KK (<b>B</b>) clonal lineages. Survival of infected mice was followed over a period of 4 days and is indicated as a cumulative percent. Higher doses of late <i>P. aeruginosa</i> strains (AA43, AA44, KK71, KK72) are required for mortality when compared to early strains (AA2, KK1 and KK2). Two to three independent experiments were pooled (nr of mice: 5–18 as detailed in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035648#pone.0035648.s003" target="_blank">table S2</a></b>). Statistical analysis of pair wise comparisons for early and late strains are indicated *** p<0.001 (Mantel-Cox test).</p

Genotypic and phenotypic characteristic of <i>P. aeruginosa</i> sequential isolates from CF patients.

<p>Three clonal lineages (AA, KK and MF) of <i>P. aeruginosa</i> strains were isolated at the onset of chronic colonization (early: AA2, KK1, KK2, MF1) or several years after acquisition and before patient's death (late: AA43, AA44, KK71, KK72, MF51). Clonality of strains was assessed by Pulsed Field Gel Electrophoresis and was reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035648#pone.0035648-Bragonzi1" target="_blank">[4]</a>. Multiple phenotypic traits changed during genetic adaptation to the CF lung and included <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035648#pone.0035648-Bragonzi2" target="_blank">[14]</a>: (a) motility defect, (b) mucoid phenotype, (c) protease reduction, (d) siderophore reduction, (e) hemolysis reduction, (f) LasR phenotype, (g) growth rate reduction. In addition, lipopolysaccharide (LPS) lipid A (h) and peptidoglycan (PGN) muropeptides (i) were analysed exclusively in the lineage AA showing specific structural modifications temporally associated with CF lung infection as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035648#pone.0035648-Cigana1" target="_blank">[5]</a>. Additional data were reported in the online data supplement (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035648#pone.0035648.s002" target="_blank">Table S1</a></b>).</p

C57Bl/6NCrl and BALB/cAnNCrl inbred mouse strains exhibit a similar susceptibility after infection with clonal pair of early/late <i>P. aeruginosa</i> isolates.

<p>C57Bl/6NCrl (<b>A, B</b>) and BALB/cAnNCrl (<b>C, D</b>) mice were infected with 5×10⁶ cfu/lung of <i>P. aeruginosa</i> strains from AA (<b>A, C</b>) and 1×10⁷ cfu/lung KK (<b>B, D</b>) clonal lineages. Survival of infected mice was followed over a period of 4 days. Early strains (AA2, KK1 and KK2) were lethal while late strains (AA43, AA44, KK71, KK72) were attenuated in acute virulence. Two to three independent experiments were pooled (nr of mice: 5–18 as detailed in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035648#pone.0035648.s004" target="_blank">table S3</a></b>). Statistical analysis was calculated for pair wise comparisons between early and late strains (* p<0.05; ** p<0.01; *** p<0.001, Mantel-Cox test).</p

LD₅₀ of longitudinal clonal <i>P. aeruginosa</i> lineages in <i>G. mellonella</i> larvae 24 hours post infection.

<p>>LD₅₀ is higher than the maximum infection dosage used. Data represent mean values of at least three independent experiments.</p

Invasion of clonal pair of early/late <i>P. aeruginosa</i> isolates in IB3-1 and C38 cells.

<p><b>A</b>) Fold of invasion relative to AA2 after 1 h of stimulation with AA clonal lineage. <b>B</b>) Fold of invasion relative to KK1 after 1 h of stimulation with KK clonal lineage. Measurements were performed in triplicate. Statistical analysis was calculated for pair wise comparisons between early and late strains (* p<0.05, ** p<0.01, *** p<0.001, Student's t-test).</p

Lung inflammatory response in susceptible A/J and resistant C3H/HeOuJ <i>P</i>. <i>aeruginosa</i>-infected mice.

<p>The number of neutrophils (<b>A</b>), macrophages (<b>B</b>), lymphocytes (<b>C</b>) and epithelial cells (<b>D</b>) recruited in the airways were determined in the BALF from A/J (n = 12) (blue bar) and C3H/HeOuJ (n = 12) (green bar) mice by cytospin after 6, 12 and 18 hours of <i>P. aeruginosa</i> infection with 5×10⁶ CFU of AA2 clinical isolate. Bars represent mean values and the error bars the standard error of the mean (SEM). The data are pooled from two independent experiments. Statistical significance by Mann-Whitney U test is indicated: *p<0.05, **p<0.01, ***p<0.001.</p

<i>P. aeruginosa</i> load and leukocytes recruitment in BALF and lung of A/J and C3H/HeOuJ mice.

<p>A/J (n = 12, for each time) and C3H/HeOuJ (n = 12, for each time) mice were challenged with 5×10⁶ CFU of AA2 clinical stain and analysed during a time course post-infection. Bacterial loads in the BALF+Lung (<b>A</b>), BALF (<b>B</b>) and lung (<b>C</b>) and were counted at 6, 12 and 18 hours in surviving mice. Dots represent CFUs in individual mice and horizontal lines represent median values reported in log scale. Total leukocytes were analyzed in BALF of <i>P. aeruginosa</i> infected mice (<b>D</b>). Bars represent median values and the error bars indicate the standard error of the mean (SEM). Blue is referred to A/J and green to C3H/HeOuJ. The data are pooled from two independent experiments. Statistical significance by Mann-Whitney U test is indicated: **p<0.01, ***p<0.001, ****p<0.0001.</p

Histopathology in susceptible A/J and resistant C3H/HeOuJ <i>P</i>. <i>aeruginosa</i>-infected mice.

<p>The lungs of A/J (<b>A–D</b>) and C3H/HeOuJ (<b>E–H</b>) were stained with H&E (<b>A–C, E–H</b>) and in immunofluorescence with specific antibody against <i>P. aeruginosa</i> (red) (<b>D, H</b>). Counterstaining was performed with 49,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (blue). After time course analysis, extensive infection and inflammation were visible in murine lungs with major differences between A/J and C3H/HeOuJ. Arrows indicate aggregates of lynphocytes infiltrates. Bars, 200 µm for H&E images, 100 µm for immunofluorescence images. Severity of lesions and lung involvement was scored as reported in <b>Fig S4</b>.</p

Survival, body weight and mean survival time after <i>P. aeruginosa</i> infection in inbred mouse strains.

<p>A/J (n = 22), BALB/cJ (n = 9), BALB/cAnNCrl (n = 8), BALB/cByJ (n = 12), C3H/HeOuJ (n = 26), C57BL/6J (n = 10), C57BL/6NCrl (n = 15), DBA/2J (n = 12), and 129S2/SvPasCRL (n = 12) mice were inoculated with 5×10⁶ CFU of <i>P. aeruginosa</i> clinical isolate AA2, and monitored for survival (<b>A</b>) and weight change for a period of seven days after infection (<b>C, D</b>). In addition, mean survival time was calculated based on the survival curve (<b>B</b>). Bar represent mean values and the error bars the standard error of the mean (SEM). The data are pooled from two to four independent experiments. Statistical significance by Mantel-Cox test for survival (<b>A</b>), One- way ANOVA with Bonferroni's Multiple comparison test (<b>B</b>) for mean survival time and Two-way ANOVA with Bonferroni's Multiple comparison test (<b>C, D</b>) is reported in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106873#pone.0106873.s005" target="_blank">Table S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106873#pone.0106873.s007" target="_blank">S3</a></b>.</p