Prophage Pf4, quorum sensing, and metabolic state shape phage responses in Pseudomonas aeruginosa biofilms

Bacteriophages have been shown as promising adjuvants or alternatives to antibiotics in treatment of infections with Pseudomonas aeruginosa, considered by WHO as a “priority pathogen” due its capacity to develop antimicrobial resistance and to form antibiotic tolerant biofilms. The observed differences in biofilm-phage interactions need to be resolved to develop efficient and predictable phage-based antibacterial strategies. We have studied the phage- P. aeruginosa biofilm interactions focusing on the role of profage Pf4, quorum-sensing proficiency (QS) and biofilm metabolism. We have investigated two different lytic phages (NP3 and vB_Pae-TbilisiM32 (M32)) on P. aeruginosa biofilms of following strains: reference strains PA14, PAO1, MPAO1 lineage and a clinical cystic fibrosis isolate. The role of Pf4 prophage was explored by comparisons of the lytic phages efficacy on biofilms of PAO1 (MPAO1 lineage) and its ΔPf4 mutant. We have further inquired the role of AHL-dependent QS and Pseudomonas quinolone signal (PQS) in protection against lytic phages by investigating biofilms of lasR , rhlR , lasRrhlR as well as pqs ( pqsC , pqsR , pqsH) PAO1 mutants. The role of the metabolic state for the effect of phage treatment in biofilms of different ages and after repeated phage treatment has been investigated. We document differences in P. aeruginosa biofilms in response to phage exposure and demonstrate the protective effect of prophage Pf4 and QS against lytic phages as well as the increased phage protection offered by low metabolic activity in older biofilms.