A novel bacteriophage cocktail reduces and disperses Pseudomonas aeruginosa biofilms under static and flow conditions

Abstract

Pseudomonas aeruginosa is an opportunistic human pathogen that forms highly stable communities - biofilms, which contribute to the establishment and maintenance of infections. The biofilm state and intrinsic/acquired bacterial resistance mechanisms contribute to resistance/tolerance to antibiotics that is frequently observed in P. aeruginosa isolates. Here we describe the isolation and characterization of six novel lytic bacteriophages: viruses that infect bacteria, which together efficiently infect and kill a wide range of P. aeruginosa clinical isolates. The phages were used to formulate a cocktail with the potential to eliminate P. aeruginosa PAO1 planktonic cultures. Two biofilm models were studied, one static and one dynamic, and the phage cocktail was assessed for its ability to reduce and disperse the biofilm biomass. For the static model, after 4 h of contact with the phage suspension (MOI 10) more than 95% of biofilm biomass was eliminated. In the flow biofilm model, a slower rate of activity by the phage was observed, but 48 h after addition of the phage cocktail the biofilm was dispersed, with most cells eliminated (> 4 logs) comparing with the control. This cocktail has the potential for development as a therapeutic to control P. aeruginosa infections, which are predominantly biofilm centred.

Figure 1

Electron micrograph images of phage (A) DL52, (B) DL54, (C) DL60, (D) DL62, (E) DL64 and (F) DL68 infecting P . aeruginosa negatively stained with 1% uranyl acetate. Scale is indicated by the bars.

Figure 2

Comparative genomic analysis of the annotated M yoviridae phages DL52, DL60 and DL68. Nucleotide sequences were compared using the Artemis Comparison Tool (ACT). Predicted reading frames are denoted by arrows and genes encoding proteins with at least 73% amino acid identity between the genomes are indicated by shaded regions.

Figure 3

Representation of the annotated P odoviridae phages DL54, DLA62 and DL64. Nucleotide sequences were analysed using the Artemis tool and predicted reading frames are denoted by arrows.

Figure 4

Dynamic of P . aeruginosa  PAO1 bacteria with single phage and phage cocktail in liquid cultures over 24 h of incubation at 37°C. Absorbance readings at 600 nm were taken in a microtitre plate reader. P seudomonas aeruginosa  PAO1 isolate growing with only SM buffer (), with single DL52 (), DL54 (), DL60 (), DL62 (), DL64 () DL68 () and with the phage cocktail in SM buffer () and also a negative‐control SM‐only buffer () are shown in the figure. Assays were performed three times, and OD ₆₀₀ was expressed as the mean ± standard deviation.

Figure 5

P seudomonas aeruginosa  PAO1 biofilm treated with the phage cocktail over 48 h. (A) Normalized biofilm biomass treated with the phage cocktail at two different MOIs (OD ₆₀₀ reading after CV staining). (C) Normalized cell metabolic activity in the biofilm treated with the phage cocktail at MOI 1 (OD ₆₀₀ reading after XTT assay). (B) and (D) show the estimation of numbers of viable bacterial cells and phage particles in the biofilms treated with the phage cocktail. Assays were performed three times, and the means ± standard deviations are indicated. Statistical significance of biofilm reduction was assessed by performing Student's t‐test. P ‐values are indicated (*P < 0.05).

Figure 6

P seudomonas aeruginosa  PAO1 48 h biofilm grown under flow conditions in the Robbins device and treated with a phage in emulsion suspension or PBS in emulsion suspension (control) for 48 h. (A) Number of viable cells recovered from the disruption of the biofilms; (B) number of phage particles recovered from the disputed biofilms; (C) 3D images of the biofilms observed under the confocal microscope stained with LIVE/DEAD stain at a magnification of 20×. Live (green) and dead (red) bacterial cells can be observed on the surface of the stainless steel disc. Enumeration assays were performed three times, and the means ± standard deviations are indicated. Statistical significance of biofilm reduction was assessed by performing Student's t‐test. P ‐values are indicated (*P < 0.05; ***P < 0.001).