Adaptation of Pseudomonas aeruginosa in Cystic Fibrosis airways influences virulence of Staphylococcus aureus in vitro and murine models of co-infection

Abstract

Cystic fibrosis (CF) airways disease represents an example of polymicrobial infection whereby different bacterial species can interact and influence each other. In CF patients Staphylococcus aureus is often the initial pathogen colonizing the lungs during childhood, while Pseudomonas aeruginosa is the predominant pathogen isolated in adolescents and adults. During chronic infection, P. aeruginosa undergoes adaptation to cope with antimicrobial therapy, host response and co-infecting pathogens. However, S. aureus and P. aeruginosa often co-exist in the same niche influencing the CF pathogenesis. The goal of this study was to investigate the reciprocal interaction of P. aeruginosa and S. aureus and understand the influence of P. aeruginosa adaptation to the CF lung in order to gain important insight on the interplay occurring between the two main pathogens of CF airways, which is still largely unknown. P. aeruginosa reference strains and eight lineages of clinical strains, including early and late clonal isolates from different patients with CF, were tested for growth inhibition of S. aureus. Next, P. aeruginosa/S. aureus competition was investigated in planktonic co-culture, biofilm, and mouse pneumonia model. P. aeruginosa reference and early strains, isolated at the onset of chronic infection, outcompeted S. aureus in vitro and in vivo models of co-infection. On the contrary, our results indicated a reduced capacity to outcompete S. aureus of P. aeruginosa patho-adaptive strains, isolated after several years of chronic infection and carrying several phenotypic changes temporally associated with CF lung adaptation. Our findings provide relevant information with respect to interspecies interaction and disease progression in CF.

Figure 1. Single and dual species batch growth curves and competition index values.

S. aureus strain (Newman) and P. aeruginosa 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.

Figure 2. Biofilm formation by S. aureus and P. aeruginosa strains in single and dual cultures.

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.

Figure 3. S. aureus and P. aeruginosa planktonic and sessile cells in single and dual cultures.

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 S. aureus strain Newman in pure culture and in co-culture with P. aeruginosa strains PA14, AA2 and AA43. Statistically significant differences are referred to Newman in pure culture. Panel B: planktonic (left) and sessile (right) cells of P. aeruginosa strains PA14, AA2 and AA43 in pure culture and in co-culture with S. aureus 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.

Figure 4. Percentage of planktonic and sessile cells in single and dual cultures.

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.

Figure 5. Competition between P. aeruginosa and S. aureus strains in a murine model of pneumoniae.

Planktonic S. aureus strain Newman and P. aeruginosa 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 S. aureus and P. aeruginosa 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 S. aureus that outcompetes P. aeruginosa; a CI value significantly >1 indicates a competitive advantage of P. aeruginosa that outcompetes S. aureus. 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.