Competition in Biofilms between Cystic Fibrosis Isolates of Pseudomonas aeruginosa Is Shaped by R-Pyocins

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen and the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa infections are difficult to treat due to a number of antibiotic resistance mechanisms and the organism's propensity to form multicellular biofilms. Epidemic strains of P. aeruginosa often dominate within the lungs of individual CF patients, but how they achieve this is poorly understood. One way that strains of P. aeruginosa can compete is by producing chromosomally encoded bacteriocins, called pyocins. Three major classes of pyocin have been identified in P. aeruginosa: soluble pyocins (S types) and tailocins (R and F types). In this study, we investigated the distribution of S- and R-type pyocins in 24 clinical strains isolated from individual CF patients and then focused on understanding their roles in interstrain competition. We found that (i) each strain produced only one R-pyocin type, but the number of S-pyocins varied between strains, (ii) R-pyocins were generally important for strain dominance during competition assays in planktonic cultures and biofilm communities in strains with both disparate R- and S-pyocin subtypes, and (iii) purified R-pyocins demonstrated significant antimicrobial activity against established biofilms. Our work provides support for a role played by R-pyocins in the competition between P. aeruginosa strains and helps explain why certain strains and lineages of P. aeruginosa dominate and displace others during CF infection. Furthermore, we demonstrate the potential of exploiting R-pyocins for therapeutic gains in an era when antibiotic resistance is a global concern.IMPORTANCE A major clinical problem caused by Pseudomonas aeruginosa, is chronic biofilm infection of the lungs in individuals with cystic fibrosis (CF). Epidemic P. aeruginosa strains dominate and displace others during CF infection, but these intraspecies interactions remain poorly understood. Here we demonstrate that R-pyocins (bacteriocins) are important factors in driving competitive interactions in biofilms between P. aeruginosa strains isolated from different CF patients. In addition, we found that these phage-like pyocins are inhibitory against mature biofilms of susceptible strains. This highlights the potential of R-pyocins as antimicrobial and antibiofilm agents at a time when new antimicrobial therapies are desperately needed.

Keywords: Pseudomonas aeruginosa; bacteriocins; biofilms; cystic fibrosis; pyocins.

FIG 1

Spot test showing the biological activities of five representative clinical P. aeruginosa strains. Each plate is labeled with the indicator strain, while the numbers 1 to 5 represent test strains: 1 is A026, 2 is A014, 3 is P003, 4 is P013, and 5 is A018. In panel A, the cell extracts of the wild-type strains were used for the spot assay, while in panel B, the cell extracts of the null-R-pyocin mutant strains were used.

FIG 2

Planktonic cell competition assay in Transwell plates. (A) The A018 strain was grown alongside 3 different treatments (A026 wild type, A026 ΔR, or plain LB), and the percentage of surviving A018 cells in the population was determined over time. (B) The A026 strain was grown alongside 3 different treatments (A018 wild type, A018 ΔR, or plain LB), and the percentage of surviving A026 cells in the population was determined over time. Hourly estimation of the percentages was achieved by staining with BacLight LIVE/DEAD stain and viewing with a confocal laser scanning microscope. Live cell counts were performed in three fields for each reading. *, P < 0.001.

FIG 3

Strain competition in biofilms. Shown are 15-h biofilms developed in a microfluidic BioFlux device as a result of competition between A026 and A018. A026 dominated in the biofilm competition of A018 versus A026 and A018 ΔR versus A026 regardless of the fluorophore (mCherry or GFP) used to tag either strain (A to D). However, A018 dominated when in competition with the null-R mutant of A026 (A026 ΔR) (E and F). (A) A026_mCherry versus A018_GFP. (B) A026_GFP versus A018_mCherry. (C) A026_GFP versus A018 ΔR_mCherry. (D) A026_mCherry versus A018 ΔR_GFP. (E) A026 ΔR_GFP versus A018_mCherry. (F) A026 ΔR_mCherry versus A018_GFP.

FIG 4

Fifteen-hour biofilms showing the distribution of two strains. (A and B) Biofilms of mixed cultures of A018 ΔR_GFP and A026 ΔR_mCherry. Panel A is a split image of the fluorescent green and red channels at ×10 magnification showing a wider distribution of the strains as they form microniches, while panel B shows a closer view of the same biofilms at ×63 magnification. Panel C shows a biofilm of wild-type strains A018_GFP and P013_mCherry, which both have the same subtypes of S- and R-pyocins.

FIG 5

Single static treatment of biofilms of A018 or A026 grown on polystyrene beads using R-pyocins of a competitor (A026 or A018, respectively). Hour 0 readings are the CFU/ml values of the 24-h biofilm grown on the beads. In one set of beads, growth was allowed to proceed unhindered (unbroken lines), while in the other set, three beads were harvested every hour, their biofilms were treated for 1 h using purified R-pyocins, and the CFU count was recorded after treatment (broken lines). Beads in panel A were treated with R-pyocins from the wild type of competitor, while the ones in panel B were treated with cell extracts from R-pyocin mutants. *, P < 0.05.

FIG 6

Antibiofilm efficacy of R-pyocins. A 15-h biofilm of A018 was treated with R-pyocins extracted from A026 (A) and A026 ΔR (B). A significant portion of the biomass was killed after 2 h and full-depth lethal effects on the biomass was achieved after 4 h. This effect was absent in the control experiment, which utilized the R-pyocins of A026 ΔR.