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

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

Single and dual species batch growth curves and competition index values.

<p><i>S. aureus</i> strain (Newman) and <i>P. aeruginosa</i> strains (PA14 and two clinical early and late isolates from a CF patient AA2 and AA43) were grown for 24 hours in BHI in single culture and in co-culture after inoculation at equal ratio from mid-exponential phase pure cultures. Growth rate was monitored by colony count after plating on selective media for both species. Results are represented as the mean of values obtained from three independent experiments. The error bars indicate the standard deviations. A nonlinear mixed-effect model was fitted, using a four-parameters logistic regression function. Panel A: growth curves of Newman in pure culture and in co-culture with PA14; Panel B: Competition index (CI) and Relative Increase Ratio (RIR) calculated from single and dual cultures of Newman and PA14; Panel C: growth curves of Newman in pure culture and in co-culture with AA2; Panel D: CI and RIR calculated from single and dual cultures of Newman and AA2; Panel E: growth curves of Newman in pure culture and in co-culture with AA43; Panel F: CI and RIR calculated from single and dual cultures of Newman and AA43. Each value represents the mean of CI and RIR values from three independent experiments and the bars indicate standard deviation. Statistically significant differences in Student's t test and in nonlinear mixed-effect model are indicated by symbols when present: *: p<0.05; **: p<0.01; ***: p<0.001.</p

<i>In vitro</i> growth inhibition of <i>S. aureus</i> and <i>P. aeruginosa</i>.

<p>Twenty-four <i>P. aeruginosa</i> isolates were collected from eight individuals with CF (SG, NN, BT, AA, TR, MF, KK, BST) at the onset of chronic colonization (numbered 1-2) or after 4.5–16.3 years of colonization (numbered 43-83). PAO1 and PA14 were included as reference strains. 5 µl spots of <i>P. aeruginosa</i> overnight cultures, normalized to 0.5 OD, were added to <i>S. aureus</i> lawn (normalized to 0.5 OD) on Mueller-Hinton agar and incubated overnight at 37°C. The table summarizes the results obtained: “weak inhibition” indicates an inhibition halo ≤15 mm; “strong inhibition” indicates an inhibition halo >15 mm and ≤25 mm; “very strong inhibition” indicates an inhibition halo >25 mm; “no inhibition“ indicates absence of inhibition halo (9 mm is the diameter of the <i>P. aeruginosa</i> spot).</p><p>* Indicates mucoid phenotype.</p>#<p>Indicates hypermutable phenotype. For statistical analysis see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089614#s2" target="_blank">Results</a>”.</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

Single and dual species batch growth curves and competitive index values.

<p>The two species were individually cultured or co-cultured at a 1∶1 ratio and grown for 24 h in NB medium at 37°C with vigorous aeration. Colony-forming unit counts (CFU) were determined at 0, 2, 4, 6, 8 and 24 h of bacterial growth. The results are the mean of Log (CFU ml⁻¹) values of three separated assays. Key: (A) Growth of clinical <i>P. aeruginosa</i> RP73 and <i>B. cenocepacia</i> LMG16656 strains in single and dual cultures; (B) Competitive index (CI) and relative increase ratio (RIR) generated from single and dual cultures of clinical <i>P. aeruginosa</i> RP73 and <i>B. cenocepacia</i> LMG16656 strains; (C) Growth of environmental <i>P. aeruginosa</i> E5 and <i>B. cenocepacia</i> Mex1 strains in single and dual cultures; (D) Competitive index (CI) and relative increase ratio (RIR) generated from single and dual cultures of environmental <i>P. aeruginosa</i> E5 and <i>B. cenocepacia</i> Mex1 strains. CI and RIR were calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052330#s4" target="_blank"><i>Materials and Methods</i></a>. Each value represents the mean of RIR and CI values from three separate assays, and the bars indicate standard deviations. * = <i>P</i><0.05, ** = <i>P</i><0.01 in the Student's t test.</p

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

<p>Bacteria were grown overnight in 96-well polyvinyl chloride flat-bottomed microtiter plates in NB medium at 37°C either individually cultured or co-cultured at a 1∶1 ratio or when individually cultured supplemented with sterile concentrated supernatant of the second organism at a final concentration of 1×. Biofilm biomass was quantified by staining with crystal violet and absorbance measurements at OD ₅₉₅. The values are means of three separated assays, and the bars indicate standard deviation. * = <i>P</i><0.05, ** = <i>P</i><0.01, *** = <i>P</i><0.001 in Student's t test. S = supernatant.</p

Cytokines and chemokines in lungs homogenates of C57BL/6NCrlBR mice after chronic infection.

a<p>Cytokines and chemokines are expressed as pg/ml (mean±SEM).</p>b<p>Significant difference of <i>P. aeruginosa</i> RP73 single infection <i>vs</i> co-infected mice (^#<i>P</i><0.05;^{# #}<i>P</i><0.01 Two-tailed student's t test).</p>c<p>Significant difference of <i>B. cenocepacia</i> LMG16656 single infection <i>vs</i> co-infected (^Δ<i>P</i><0.05; ^ΔΔ<i>P</i><0.01 Two-tailed student's t test).</p

<i>P. aeruginosa</i> and <i>B. cenocepacia</i> planktonic and sessile cells in single and dual cultures.

<p>Bacteria were grown overnight in 96-well polyvinyl chloride flat-bottomed microtiter plates in NB medium at 37°C either individually cultured or co-cultured at a 1∶1 ratio. CFU counts were determined at 24 h of bacterial growth in both planktonic and sessile fraction. Key: (A, left) Sessile cells of clinical pair (<i>P. aeruginosa</i> RP73 and <i>B. cenocepacia</i> LMG16656) in single and dual cultures; (A, right) Sessile cells of environmental pair (<i>P. aeruginosa</i> E5 and <i>B. cenocepacia</i> Mex1) in single and dual cultures; (B, left) Planktonic cells of clinical pair (<i>P. aeruginosa</i> RP73 and <i>B. cenocepacia</i> LMG16656) in single and dual cultures; (B, right) Planktonic cells of environmental pair (<i>P. aeruginosa</i> E5 and <i>B. cenocepacia</i> Mex1) in single and dual cultures; (C) CI and RIR mean values of sessile growth of <i>P. aeruginosa</i> versus <i>B. cenocepacia</i> (RP73 <i>versus</i> LMG16656, E5 <i>versus</i> Mex1); (D) CI and RIR of planktonic growth of <i>P. aeruginosa</i> versus <i>B. cenocepacia</i>. Each value represents the mean of RIR and CI values from three separate assays, and the bars indicate standard deviations. * = <i>P</i><0.05, ** = <i>P</i><0.01, *** = <i>P</i><0.001 in Student's t test.</p