Pseudomonas aeruginosa is an opportunistic pathogen possessing a large genome with high level of plasticity, allowing the bacteria to adapt to hostile environments. Bacteriophages have been shown to drive the emergence of P. aeruginosa variants playing a significant role in bacterial survival, activity, and evolution. Although phage therapy has proven to be promising in several animal case studies and in some human clinical trials, it has always been considered with skepticism by the medical community because of the emergence of phage-resistant variants.
The purpose of this study was to investigate the mechanisms and frequency of resistance in response to infection by cocktails of virulent phages belonging to different genera. Phage Ab05, a podovirus (ФKMV-like), phage Ab09, a podovirus (N4-like), phage Ab27, a myovirus (PB1-like) and phage Ab17, a myovirus (KPP10-like), isolated from waste water in Abidjan (Ivory Coast), were used to perform single or multiple infection of P. aeruginosa strain PAO1 allowing the selection of several phage resistant variants whose genome was, then, fully sequenced through Illumina technology.
Several phenotypic alterations were observed in the phage-resistant variants; defects in bacterial motility and in biofilm formation due to mutations in type IV pilus biosynthesis genes, characterized variants selected by Ab05 alone or as a component of a cocktail. Mutations in genes involved in the LPS production and in the alginate regulation were also identified. Cross resistance was systematically observed for phages using LPS as a receptor. Pseudolysogeny was a frequent outcome of infections and in some case was stable enough to allow the emergence of new mutations. The use of cocktail does not lower significantly the frequency of phage-resistance and in addition we show that pseudolysogeny is a major actor in the selection of mutations.

We thank Direction Générale de l’Armement (DGA) and ANR “Resisphage” for financial support to this project.