Effect of acute predation with bacteriophage on intermicrobial aggression by Pseudomonas aeruginosa

Abstract

In persons with structural lung disease, particularly those with cystic fibrosis (CF), chronic airway infections cause progressive loss of lung function. CF airways can be colonized by a variety of microorganisms; the most frequently encountered bacterial and fungal pathogens are Pseudomonas aeruginosa and Aspergillus fumigatus, respectively. Co-infection with P. aeruginosa and A. fumigatus often results in a more rapid loss of lung function, indicating that interactions between these pathogens affect infection pathogenesis. There has been renewed interest in the use of viruses (bacteriophage, mycoviruses) as alternatives to antibiotics to treat these infections. In previous work, we found that filamentous Pf bacteriophage produced by P. aeruginosa directly inhibited the metabolic activity of A. fumigatus by binding to and sequestering iron. In the current study, we further examined how filamentous Pf bacteriophage affected interactions between P. aeruginosa and A. fumigatus. Here, we report that the antifungal properties of supernatants collected from P. aeruginosa cultures infected with Pf bacteriophage were substantially less inhibitory towards A. fumigatus biofilms. In particular, we found that acute infection of P. aeruginosa by Pf bacteriophage inhibited the production of the virulence factor pyoverdine. Our results raise the possibility that the reduced production of antimicrobials by P. aeruginosa infected by Pf bacteriophage may promote conditions in CF airways that allow co-infection with A. fumigatus to occur, exacerbating disease severity. Our results also highlight the importance of considering how the use of bacteriophage as therapeutic agents could affect the behavior and composition of polymicrobial communities colonizing sites of chronic infection.

Fig 1. Enumeration of bacteria and Pf phage in Pseudomonas aeruginosa (Pa) supernatants.

(A) After overnight growth, bacterial density was estimated by absorbance (OD₆₀₀). (B) Pf phage in filtered supernatants collected from overnight cultures of the indicated P. aeruginosa strains were measured by enumerating plaque-forming units (PFU/ml) as described [14]. In all non-infected cultures, Pf phage levels were below the limit of detection of 2000 PFU/ml. N = 6 +/- SD mean.

Fig 2. Effect of phage infection on formation of Af biofilm, as studied in an agar assay (BCAM).

Black bars show inhibition of Af biofilm by planktonic supernatants of Pa after acute infection with phage, white bars show inhibition of Af biofilm by planktonic supernatants of the same strains of Pa cultured in the absence of phage. Grey bar shows negative control (RPMI medium only). Horizontal asterisks compare the bars at the ends of the horizontal brackets; two and three asterisks represent differences of p ≤ 0.01, p ≤ 0.001, respectively, comparing each Pa isolate uninfected vs. after phage infection. Comparison of all vs RPMI (the Af control, grey bar): PAO1+Pf4 or PAK+Pf1 p>0.05. ΔpvdD+Pf4 or ΔlasR/rhlR+Pf4: p ≤ 0.01. All other bars: p≤0.001. Comparison of PAO1 vs. its mutants: ΔcoaB: p≤0.05, ΔlasR/rhlR: p ≤ 0.01. ΔpqsA: p>0.05. All other bars: p≤0.001. Comparison PAO1 vs PAK: p≤0.001.

Fig 3. Effect of phage infection on formation of Af biofilm, as studied in liquid in wells.

Black bars show inhibition of Af biofilm by planktonic supernatants of Pa after acute infection with phage, white bars show inhibition of Af biofilm by planktonic supernatants of the same strains of Pa cultured in the absence of phage. Grey bar shows negative control (RPMI medium only). Horizontal asterisks compare the bars at the ends of the horizontal brackets; two and three asterisks represent differences of p ≤ 0.01, p ≤ 0.001, respectively, comparing each Pa isolate uninfected vs. after phage infection. Comparison of all vs RPMI (the Af control, grey bar): ΔcoaB+ Pf4: p ≤ 0.01; PAO1+Pf4, ΔpvdD+Pf4 and ΔPAO728 + Pf4: p>0.05. All other bars: p≤0.001. Comparison PAO1 vs. its mutants: ΔpvdD and ΔPAO728: p≤0.001. All other bars: p>0.05. Comparison PAO1 vs PAK: p≤0.001.

Fig 4. Effect of phage infection on preformed Af biofilm, as studied in an agar assay (BHAM).

Black bars show inhibition of Af biofilm by planktonic supernatants of Pa after acute infection with phage, white bars show inhibition of Af biofilm by planktonic supernatants of the same strains of Pa cultured in the absence of phage. Grey bar shows negative control (RPMI medium only). Horizontal asterisks compare the bars at the ends of the horizontal brackets; two and three asterisks represent differences of p ≤ 0.01, p ≤ 0.001, respectively, comparing each Pa isolate uninfected vs. after phage infection. Comparison of all vs RPMI (the Af control, grey bar): ΔlasR/rhlR or ΔPA0728: p≤0.05; ΔpvdD or ΔcoaB + Pf4 or ΔpqsA + Pf4 or PAK + Pf1: p ≤ 0.01; PAK: p>0.05. All other bars: p≤0.001. Comparison of PAO1 vs. its mutants: ΔlasR/rhlR: p≤0.05; ΔpvdD: p ≤ 0.001; All other bars: p>0.05. Comparison PAO1 vs PAK: p≤0.01.

Fig 5. Effect of phage infection on preformed Af biofilm, as studied in liquid in wells.

Black bars show inhibition of Af biofilm by planktonic supernatants of Pa after acute infection with phage, white bars show inhibition of Af biofilm by planktonic supernatants of the same strains of Pa cultured in the absence of phage. Grey bar shows negative control (RPMI medium only). Horizontal asterisks compare the bars at the ends of the horizontal brackets; three asterisks represent differences of p ≤ 0.001, respectively, comparing each Pa isolate uninfected vs. after phage infection. Comparison of all vs RPMI (the Af control, grey bar): All bars: p≤0.001. Comparison PAO1 vs. its mutants: ΔPAO728: p≤0.001. All other bars: p>0.05. Comparison PAO1 vs PAK: p≤0.001.

Fig 6. Effect of phage infection on the production of pyoverdine by Pa.

Black bars show pyoverdine production after phage infection, white bars show basal pyoverdine production by planktonic supernatants of the same strains of Pa not infected by phage. One and three asterisks make comparisons within each pair framed by the horizontal bracket; p ≤ 0.05, p ≤ 0.001, respectively.