Additional file 3: of Mapping genetic determinants of host susceptibility to Pseudomonas aeruginosa lung infection in mice

Effect of the Pairl1 locus on survival in A/J, C3H/HeOuJ and heterozygotes. (DOCX 106 kb

Additional file 5: of Mapping genetic determinants of host susceptibility to Pseudomonas aeruginosa lung infection in mice

List of the most promising candidates genes for susceptibility to acute P. aeruginosa pulmonary infection. (DOCX 27 kb

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

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

Biofilm formation by <i>S. aureus</i> and <i>P. aeruginosa</i> strains in single and dual cultures.

<p>Bacteria were grown overnight in 96-well flat-bottom microtiter plates in NB medium at 37°C either individually cultured or co-cultured at a 1∶1 ratio. Biofilm biomass was quantified by staining with crystal violet and absorbance measurements at OD 595 nm. The values represent the means of three independent experiments, and the bars indicate standard deviation. Statistically significant differences in Student's t test are indicated by symbols when present: **: p<0.01; ***: p<0.001.</p

<i>S. aureus</i> and <i>P. aeruginosa</i> planktonic and sessile cells in single and dual cultures.

<p>Bacteria were grown overnight in 96-well flat-bottom microtiter plates in NB medium at 37°C either individually cultured or co-cultured at a 1∶1 ratio. CFU counts were determined in both planktonic and sessile fractions. Panel A: planktonic (left) and sessile (right) cells of <i>S. aureus</i> strain Newman in pure culture and in co-culture with <i>P. aeruginosa</i> strains PA14, AA2 and AA43. Statistically significant differences are referred to Newman in pure culture. Panel B: planktonic (left) and sessile (right) cells of <i>P. aeruginosa</i> strains PA14, AA2 and AA43 in pure culture and in co-culture with <i>S. aureus</i> strain Newman. The values represent the means of three independent experiments, and the bars indicate standard deviation. Statistically significant differences in non-parametric Mann–Whitney test are indicated by symbols when present: **: p<0.01; ***: p<0.001.</p

Competition between <i>P. aeruginosa</i> and <i>S. aureus</i> strains in a murine model of pneumoniae.

<p>Planktonic <i>S. aureus</i> strain Newman and <i>P. aeruginosa</i> clinical isolates AA2 and AA43 and reference strain PA14 were used to infect C57BL/6NCrlBR mice at a ratio of 1∶1. After 18 hours of acute infection lungs homogenates were plated on selective plates to determine <i>S. aureus</i> and <i>P. aeruginosa</i> CFU. Each circle represents the CI for a single animal in each group. A CI value equal to 1 indicates equal competition of the two species; a CI value significantly <1 indicates a competitive advantage of <i>S. aureus</i> that outcompetes <i>P. aeruginosa</i>; a CI value significantly >1 indicates a competitive advantage of <i>P. aeruginosa</i> that outcompetes <i>S. aureus</i>. Wilcoxon signed rank test of the null hypothesis that the distribution of CI is symmetric about 1 was performed. Statistically significant differences are indicated by symbols when present: *: p<0.05; **: p<0.01. The data are pooled from two or three independent experiments.</p

Percentage of planktonic and sessile cells in single and dual cultures.

<p>Bacteria were grown overnight in 96-well flat-bottom microtiter plates in NB medium at 37°C either individually cultured or co-cultured at a 1∶1 ratio. CFU counts were determined in both planktonic and sessile fractions and the percentage of S. aureus and P. aeruginosa in the two fractions of single and dual cultures was calculated. Panel A: percentages of planktonic and sessile cells of Newman in single culture (first histogram), PA14 in single culture (second histogram), Newman and PA14 in ideal co-culture if the 2 species would not interfere each other (third histogram, percentages have been calculated considering the values of the first and second histograms), and Newman and PA14 in co-culture (fourth histogram). Panel B: percentages of planktonic and sessile cells of Newman in single culture (first histogram), AA2 in single culture (second histogram), Newman and AA2 in ideal co-culture if the 2 species would not interfere each other (third histogram, percentages have been calculated considering the values of the first and second histograms), and Newman and AA2 in co-culture (fourth histogram). Panel C: percentages of planktonic and sessile cells of Newman in single culture (first histogram), AA43 in single culture (second histogram), Newman and AA43 in ideal co-culture if the 2 species would not interfere each other (third histogram, percentages have been calculated considering the values of the first and second histograms), and Newman and AA43 in co-culture (fourth histogram). SA: S. aureus; PA: P. aeruginosa.</p