Activity of two Pseudomonas aeruginosa virulent phages on biofilms

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium responsible for various types of infections like bloodstream, urinary tract, eyes, wounds and chronic airways (especially in cystic fibrosis patients) infections. As a member of the ESKAPE group of pathogens, P. aeruginosa infections are difficult to treat due to antibiotic resistance and immune evasion mechanisms such as biofilm formation.

Biofilms are microbial communities encased in an extracellular matrix (ECM). In P. aeruginosa biofilms, ECM is mainly composed by three exopolysaccharides (EPS): alginate, Psl and Pel with different proportion depending on the strain, but also, by extracellular DNA and proteins. ECM forms a physical barrier which protects bacteria against the immune system and antimicrobial compounds like antibiotics.

Here, we explored the efficacity of two P. aeruginosa virulent phages on biofilms: a Pbunavirus LS1 which has O-antigen chain of the LPS as primary receptor, and a Bruynoghevirus LUZ24 whose receptor was unknown at the onset of this work. We firstly characterized the B. LUZ24 primary receptor as polysaccharide Psl, the main EPS in P. aeruginosa PAO1 biofilms. We next explored, by confocal laser microscopy, the ability of LUZ24 and LS1 phages to degrade PAO1 biofilms over a 48 hours period. Single phage application during the establishment phase of the biofilms (6 hours pre-culture) delayed biofilm growth by 18 hours, while the two phages combination prevented growth completely over 48 hours. In mature biofilms (16 hours pre-culture), at the end of the kinetics, single phage treatment led to a 2-fold reduction of viable bacteria, while the two phages combination led to a 4-fold reduction.

Overall, even if mature biofilms remain challenging to eradicate, these two phages present complementary killing activities, so that combining them is a promising strategy for phage therapy.